Heterologous expression of gentian MYB1R transcription factors

J.Agric.Food Chem.2010,58,4819–48244819DOI:10.1021/jf1000502Heterologous Expression and Biochemical Characterization ofr-Glucosidase from Aspergillus niger by Pichia pastrorisD ONG-L I C HEN,†,‡X ING T ONG,†,‡S HANG-W EI C HEN,†,‡S HENG C HEN,†,‡D AN W U,†,‡S HU-G UANG F ANG,§J ING W U,*,†,‡AND J IAN C HEN*,†,‡†State Key Laboratory of Food Science and Technology,Jiangnan University,1800Lihu Avenve,Wuxi214122,People’s Republic of China,‡School of Biotechnology,Jiangnan University,1800Lihu Avenue,Wuxi214122,People’s Republic of China,and§Leipu Biotechnology LimitedCompany,720-1-216Chailun Road,Shanghai201203,People’s Republic of ChinaThe aglu of Aspergillus niger encodes the pro-protein of R-glucosidase,and the mature form of wild-typeenzyme is a heterosubunit protein.In the present study,the cDNA of R-glucosidase was cloned andexpressed in Pichia pastoris strain KM71.The activity of recombinant enzyme in a3L fermentor reached2.07U/mL after96h of induction.The recombinant R-glucosidase was able to produce oligoisomaltose.The molecular weight of the recombinant enzyme was estimated to be about145kDa by SDS-PAGE,and it reduced to106kDa after deglycosylation.The enzymatic activity of recombinant R-glucosidasewas not significantly affected by a range of metal ions.The optimum temperature of the enzyme was60°C,and it was stable below50°C.The enzyme was active over the range of pH3.0-7.0withmaximal activity at ing p NPG as substrate,the K m and V max values were0.446mM and43.48U/mg,respectively.These studies provided the basis for the application of recombinant R-glucosidase inthe industry of functional oligosaccharides.KEYWORDS:R-Glucosidase;Aspergillus niger;Picha pastoris;expression;characterizationINTRODUCTIONR-Glucosidases(EC3.2.1.20)are a group of typical exotype carbohydrases,which catalyze the liberation of R-glucose from non-reducing terminals of substrates(1).Various types of R-glucosidases are widespread in nature,distributed in microorganisms,insects, plants and mammals(2).Some R-glucosidases,e.g.those from Aspergillus niger(3),Bacillus stearothermophilus(4),Saccharomyces cerevisiae(5)etc.,not only hydrolyze glycosides but also can catalyze transglucosidation reaction.For example,A.niger R-glucosidase can transfer a glucosyl residue to the6-OH of the accepting glucose unit and yield isomaltose,panose,isomaltotriose and tetrasacchar-ides from maltose(6,7).Isomaltose,panose,isomaltotriose and tetrasaccharides are defined as isomaltooligosaccharides(IMOs)(8). Since IMOs are stimulating materials to Bifidobacteria and total lactic acid bacteria of human intestines(7),they are of special interest to the food industry.In addition,IMOs are also utilized in animal feed to increase dry matter and calcium digestibility(9).It has been reported that,among all the R-glucosidases,A.niger R-glucosidase gives a notable yield of total transglycosylating pro-ducts(10),therefore,the preparation of R-glucosidase from A.niger has been long investigated by many researchers.However,low yield of R-glucosidase from wild-type A.niger makes it uneconomical for large-scale industrial application,thus genetic engineering approach is highly necessary for the expression of this enzyme in a new host of efficiency.Previously,A.niger R-glucosidase gene aglu was identi-fied by Kimura(1).Biochemical studies showed that aglu was a pro-protein(11).It was suggested that the pro-protein was subjectedto limited proteolysis after secretion from the A.niger cells and14residues inside the polypeptide were cleaved,which resulted in aheterosubunit protein of the mature form of R-glucosidase(11).Probably due to this special phenomena,expression of aglu has beenattempted only in fungal species,such as Aspergillus nidulans(11)andEmericella nidulans(12).Increasing copy number of aglu by chro-mosomal integration could also improve the yield of R-glucosidase inA.niger(13).However,the yield obtained by these efforts was still low and could not reach the requirement of industrial applications.The yeast Pichia pastoris expression system,as a eukaryoticexpression system,has been a favorite system for expressingheterologous proteins due to its many advantages,such as highproduction and high secretion efficiency(14).The Pichia expres-sion system has been extensively used in the enzyme industry forthe production of recombinant proteins(15).In our previous work,the cDNA of A.niger(No.SG136)R-glucosidase was clonedby overlap-PCR and expressed in P.pastoris(16).However,itwas found that the biological activity of the recombinant enzymewas low.This study described the cloning of the cDNA of R-glucosidase from another strain of A.niger(CICIM F0620)by RT-PCR and its expression in P.pastoris.In addition,thecharacterization of the recombinant enzyme from P.pastoriswas also investigated.These studies provide the basis for theapplication of recombinant R-glucosidase in the industry ofIMOs production.MATERIALS AND METHODSStrains,Vectors and Materials.The strain of A.niger(No.CICIM F0620)was from China Center for Type Culture Collection(CCTCC).*Corresponding author.Phone:þ86-510-85327802.Fax:þ86-510-85327802.E-mail:jingwu@(J.W.);jchen@jiangnan.(J.C.)./JAFCPublished on Web04/06/2010©2010American Chemical Society4820J.Agric.Food Chem.,Vol.58,No.8,2010Chen et al.P.pastoris KM71and the plasmid pPIC9K were obtained from Invitro-gen.The EZ-10Spin Column Plasmid Mini-Preps kit,agarose gel DNA purification kit,restriction enzymes,and T4DNA ligase were obtained from TakaRa(Dalian,China).p-Nitrophenyl-R-D-glucopyranoside (p NPG)was obtained from Seebio Biotech.Inc.(Shanghai,China).Endo H f was obtained from New England Biolabs(Beijing,China).Other chemicals were obtained from Sinopharm Chemical Reagent Co.Ltd. (Shanghai,China).DNA primers were synthesized by Shanghai Sangon Biological Engineering Technology&Services Co.Ltd.(Shanghai, China).DNA sequencing was performed by Shanghai Sangon Biological Engineering Technology&Services Co.Ltd.(Shanghai,China).Gene Cloning of A.niger r-Glucosidase.The cDNA of A.niger R-glucosidase exclude the fragment of its signal peptide was cloned by RT-PCR.Total RNA of A.niger was extracted and purified as described previously(17).cDNA was synthesized using SuperScript III First-Strand Synthesis System for RT-PCR(Invitrogen,USA).The obtained first-strand cDNA was served as template for PCR.The cloning primer sequences were designed according to GenBank(GeneID:4991096)as follows:ATTAATGCGGCCGCGTCCACCACTGCCCCT TCC(for-ward primer)and AGCACTAGCGGCCGCCCATTCCAATACC-CAGT TTTCC(reverse primer).The Not I restriction site(underlined) was designed into the primers.The amplification was carried out under the following conditions:the first step was at95°C for4min,followed by 30cycles of94°C for45s,58°C for45s,and72°C for4min,and the final extension was carried out at72°C for10min.The PCR product was digested with Not I,gel-purified and then ligated into pPIC9K which was subjected to a similar treatment.The recombinant plasmid,pPIC9K/aglu, was identified by restriction analysis and sequencing.Expression of r-Glucosidase in P.pastoris.The recombinant plasmid pPIC9K/aglu was linearized with Bgl II and then electroporated into P.pastoris KM71.The transformants were selected at30°C on the MD agar plates for2-4days.The presence of the R-glucosidase gene in the transformants was confirmed by PCR using yeast genomic DNA as template.For expression,the colonies were grown in10mL of YPD medium at30°C for24h,then inoculated into50mL of BMGY medium and shaken (200rpm)at30°C until OD600of5-6was reached.The cells were collected by centrifugation at5000rpm for5min at4°C,resuspended in25mL of BMMY.To maintain induction,methanol was supplemented every24h to a final concentration of1.0%(v/v)throughout the induction phase.The recombinant P.pastoris with the highest R-glucosidase yield was used to scale up fermentation in a3L fermentor(BIOFLO110,America). The fermentation began at batch growth phase in1.5L BSM at30°C and pH5.5,and the pH was maintained with ammonium hydroxide.After the level of dissolved oxygen increased,continuous glycerol feeding was carried out until the OD600reached100.When the dissolved oxygen increased again,a methanol solution was added to the fermentor.The level of dissolved oxygen was maintained above30%throughout the induction phase.DO-stat methanol feeding strategies were applied.Hydrolytic Activity of Enzyme.The hydrolytic activity of R-gluco-sidase was measured as the amount of p-nitrophenol(p NP)released from p NPG(18).The reaction mixture contained1mL of100mM sodium acetate buffer(pH5.5),0.05mL of10mM p NPG,and0.05mL of appropriately diluted enzyme.The reaction was incubated at50°C for 15min and terminated by1mL of1M sodium carbonate solution.One unit(U)of enzyme activity was defined as the amount of1μmol p NP produced per min under the above conditions.Transglycosylation Activity of Enzyme.The reaction mixture (2.5mL),which consisted of2mL of10%(w/v)maltose solution in 100mM sodium acetate buffer(pH5.5)and500μL of enzyme(to reach a final activity of0.4U/mL),was incubated at60°C.At different intervals, 100μL reaction mixtures were taken and incubated at100°C for5min to inactivate the enzyme.Samples were centrifuged at12,000rpm for5min and analyzed by HPLC.The HPLC analysis was performed on an Agilent separation module(model1200)equipped with quaternary pump,using a Hypersil NH2column(4.6Â250mm).The mobile phase consisted of75% acetonitrile and25%water used at a flow rate of1mL/min.A refractive index detector(Agilent model1200)was used(5).Purification of Recombinant r-Glucosidase.The culture super-natant of engineered P.pastoris was obtained by centrifugation at10000g for20min and then concentrated by ultrafiltration(30kDa cutoff membrane,Amicon).R-Glucosidase was precipitated with70%(v/v)ethanol and collected by centrifugation(10000g,20min).The precipitate was dissolved in50mL of buffer A(20mM sodium acetate buffer,pH5.5), and dialyzed against two liters of buffer A at4°C overnight.Solid (NH4)2SO4was added to the dialyzed sample to a final concentration of 20%(w/v).The sample was filtered(0.22μm)and loaded onto a Phenyl HP Sepharose FF column pre-equilibrated with20%(NH4)2SO4in buffer A.A reverse gradient from20%to0%(NH4)2SO4in buffer A was applied at a flow rate of1.0mL/mim over60min.The fractions containing p NPG hydrolase activity were pooled and dialyzed against1L of buffer A overnight.The purified enzyme was stored at-80°C.Molecular Weight Determinations.The subunit molecular weight of recombinant R-glucosidase was determined by SDS-PAGE.The native molecular weight of recombinant R-glucosidase was determined by gel filtration utilizing a Superdex20010/300GL column.The elution volume was determined in triplicate for all samples and standards.Deglycosylation of Recombinant r-Glucosidase.Ten micrograms of recombinant R-glucosidase was denatured with glycoprotein denaturing buffer at100°C for10min.After the addition of G5reaction buffer,Endo H f was added and the reaction mixture was incubated for1h at37°C(19).HPLC Analysis of Purified Recombinant Enzyme.The purified enzyme was analyzed by gel filtration HPLC on an Agilent separation module(model1200)equipped with quaternary pump,using a TSK-gel G3000SWXL column(7.5Â300mm).The mobile phase consisted of 10mM sodium phosphate buffer(pH6.8)used at a flow rate of0.6mL/ min.In addition,the enzyme was also subjected to a reverse-phase HPLC with a C18column(3.9Â150mm)(Delta-Pak).The reverse-phase HPLC analysis was performed according to the previous report(2).Temperature Optimum and Thermostability.The optimal tempera-ture of the recombinant enzyme was measured at temperatures ranging between30and90°C at pH5.5,using p NPG as substrate in100mM acetate buffer.Since the pH of acetate buffer is temperature-dependent, the pH of the buffers was adjusted to5.5at the desired temperatures.At each temperature,the buffer and p NPG were preincubated for5min.The reaction was initiated by the addition of the enzyme and allowed to proceed for an additional15min.The thermostability was determined by incubating the enzyme in100mM acetate buffer(pH5.5)at the various temperatures(50-70°C).At different intervals,samples were taken and assayed for residual activity.The experiments were carried out in three independent experiments.pH Optimum and Stability.pH optimum of the recombinant enzyme was measured over a pH range of3.0-7.0by using acetate buffer (pH3.0-5.0),sodium phosphate buffer(pH5.0-6.0)and Tris-HCl buffer (pH6.0-7.0),respectively.To determine the pH stability,the enzyme was preincubated in the various buffers described above at4°C for24h,and then assayed for residual activity at pH5.5.The experiments were carried out in three independent experiments.Determination of Kinetic Parameters.Enzyme assays were per-formed in acetate buffer(pH5.5)at50°C using p NPG as substrate.Sub-strate concentrations were in the range of0.1-2.0mM.The Michaelis-Menten parameters,V max and K m,were calculated from double reciprocal plots of reaction curve(20).The experiments were carried out in three independent experiments.Effect of Ions on Enzyme Activity.To determine the effect of metal ions(Ca2þ,Co2þ,Mg2þ,Mn2þ,Pb2þ,Zn2þ,Fe2þ,Ba2þ,Ni2þ,Cu2þ, Al3þ,and Pb2þ)on the recombinant R-glucosidase activity,the enzyme was preincubated with each metal ion at a1mM concentration in100mM acetate buffer(pH5.5)at50°C for1h.The residual enzyme activity was assayed at50°C.The experiments were carried out in three independent experiments.RESULTSCloning and Expression of r-Glucosidase in P.pastoris.The cDNA of R-glucosidase excluding the fragment of signal peptide was reverse transcribed from the total RNA of A.niger(CICIM F0620)and cloned into P.pastoris expression vector pPIC9K. Nucleotide sequence analysis showed that the gene length was 2883bp encoding a protein consisting of960amino acids.The cDNA sequence and amino acid sequence were compared with those of A.niger CBS513.88R-glucosidase.It was shown that the cDNA sequence shared99.8%homology with that of A.nigerArticle J.Agric.Food Chem.,Vol.58,No.8,20104821CBS513.88R -glucosidase (GenBank Accession No.4991096)and differed by four nucleotides.The amino acid sequence shared 100%identity with that of A.niger CBS513.88R -glucosidase (NCBI Accession No.XP_001402053).For expression,the recombinants were screened in MD and YPD/G418plates,and the insert was identified by PCR.The P.pastoris KM71transformed with vector pPIC9K was used as control.After 120h of induction on methanol in a shake flask,the p NPG hydrolase activity in the culture supernatant of recombi-nant P.pastoris KM71/pPIC9K-aglu reached 1.15U/mL,which was 18.2-fold higher than that of native R -glucosidase extracted from A.niger (CICIM F0620).The protein concentration in the culture medium was 517μg/mL.SDS -PAGE analysis showed that there was one major band of protein,approximately 145kDa,secreted into the culture medium (Figure 1).No p NPG hydrolase activity was detected in the culture supernatant of the control strain under the same culture conditions.The expression efficiency of the engineered P.pastoris was further explored in a 3L fermentor.As shown in Figure 2,after methanol induction for 5days,R -glucosidase activity and protein concentration in the culture supernatant were 2.07U/mL and 918μg/mL,which were 1.80-fold and 1.78-fold higher than those in shake condition,respectively.The yield of recombinant enzyme in the culture media of engineered P.pastoris was 32.8-fold higher than that of native R -glucosidase extracted from A.niger (No.CICM F0620).Transglycosylation Activity of Recombinant r -Glucosidase.To determine whether the expressed R -glucosidase has transglycosy-lation activity,the culture supernatant of recombinant P.pastoris KM71/pPIC9K-aglu was incubated with 10%(w/v)maltose solution at 60°C.The progress of the reaction was monitored by HPLC.To identify the components in the reaction mixture,theretention time of each peak from HPLC was compared with those of IMOs standards.As shown in Figure 3,isomaltose,panose,isomaltotriose were able to be detected in the reaction mixture by HPLC.The results showed that recombinant R -glucosidase had transglycosylation activity.Purification of the Recombinant r -Glucosidase.The recombi-nant R -glucosidase was purified from culture supernatant by ultrafiltration,ethanol fraction and hydrophobic interaction chromatography.The purified enzyme was homogeneous by SDS -PAGE with a specific activity of 2.52U/mg.Physical Properties.The molecular weight of recombinant enzyme as determined by SDS -PAGE was 145kDa (Figure 4).The molecular weight of the native enzyme determined by gel filtration chromatography was 277kDa (Figure 5),1.9times the molecular weight determined by SDS -PAGE.Therefore,recom-binant R -glucosidase is predicted to have a dimeric structure in solution.In addition,the calculated molecular weight of the mature R -glucosidase was 106kDa (http://www.expasy.ch/tools/pi_tool.html),which is different from that estimated from SDS -PAGE.This difference is probably caused by glycosylation.Sequence analysis showed that there were 18potential N-glycosylation sites in A.niger R -glucosidase (http://www.cbs.dtu.dk/services/).In order to confirm whether the recombinant enzyme is glycosy-lated,the purified enzyme was subjected to be treated by deglycosylase (Endo H f ).As shown in Figure 4,the molecular weight of the expressed enzyme was reduced after the treatment.These results indicated that the recombinant protein was glyco-sylated in the host of P.pastoris .Furthermore,the purified enzyme showed one single peak by HPLC with a TSK-gel column,while it showed two peaks by HPLC with a reverse phase column (Figure 6).Temperature Optimum and Thermostability.The optimum temperature curve of the purified R -glucosidase showed that the enzyme activity increased with increasing temperature from 30to 60°C and decreased from 60to 90°C (Figure 7A ).At the optimum temperature of 60°C,the activity was 5-fold higher than that at 30°C.The thermostable experiment showed that the enzyme retained 50%of activity after 72h at 50°C or 3h at 60°C,but was rapidly inactivated above 70°C (Figure 7B ).Since the stability of the recombinant R -glucosidase is similar to the wild-type enzyme (2),which was stated to be stable,the recombinant enzyme from the yeast expression system in the present study is also considered to be stable against heat.pH Optimum and Stability.The optimum pH of recombinant R -glucosidase was 4.5(Figure 8A ).The enzyme exhibited the highest enzymatic activity (>90%of maximum)between pH 3.5and 5.5.It retained more than 50%of its maximal activity between pH 3.0and 8.0after incubation at 4°C for 24h (Figure 8B ).Kinetic Studies.The kinetics of the recombinant enzyme was analyzed using p NPG as substrate.At 50°C,the K m and V max of the recombinant enzyme were 0.446mM and 43.48U/mg,respectively.The K m value was similar to that of the native enzyme,which was 0.620mM (12).Metal Requirement.Previously,it was reported that the activity of R -glucosidases from Escherichia coli (21),Mucor racemosus (22)and Thermotoqa maritima (23)was able to be stimulated in the presence of Al 3þ,Ca 2þ,K þ,Mg 2þor Mn 2þ(1-10mM),while there is no metal requirement information for R -glucosidase of A.niger .In the present study,the metal requirement of the recombinant R -glucosidase was analyzed by using 1mM metal ions (Ca 2þ,Co 2þ,Mg 2þ,Mn 2þ,Pb 2þ,Zn 2þ,Fe 2þ,Ba 2þ,Ni 2þ,Cu 2þ,Al 3þ,and Pb 2þ).The enzyme was preincubated witheachFigure 1.SDS -PAGE analysis of culture supernatant of engineered P.pastoris .Lane 1marker;lanes 2-6culture supernatant of recombinant P.pastoris after methanol induction from 1to 5days at the shaker flask level;lane 7control.Figure 2.Time profiles for batch cultivations of recombinant P.pastoris in 3L fermentor.4822J.Agric.Food Chem.,Vol.58,No.8,2010Chen et al.metal ion and then assayed for the activity.The results showed that none of these metals significantly affected the activity of recombinant enzyme,which suggested that the structure and the catalysis of the enzyme are not sensitive to the metal ions in the experimental condition.DISCUSSIONIn the industry of functional oligosaccharides,the transglyco-sylation activity of A.niger R -glucosidase has been applied to produce IMOs (7).In order to improve the yield of enzyme production,in the present study,cDNA of A.niger R -glucosidase was cloned and expressed in P.pastoris KM71.Previously,A.niger R -glucosidase was expressed in A.nidulans (11)and E.nidulans (12),and the expression level was reported to be 0.04U/mg and 0.96U/mg,respectively.In addition,the yield of recombinant R -glucosidase obtained from E.nidulans was re-ported to be 0.65U/mL (11).In the present study,the enzyme activity in the culture supernatant of a 3L fermentor could reach 2.07U/mL and 2.21U/mg protein.The high expression level obtained in the present study probably due to that,compared with filamentous fungal expression systems,the P.pastoris expression system has several advantages for expression of heterologous proteins,including use of the strong and regulated AOX1promoter,the effectively selectable markers,secretion signals and methods for coping with proteases (24).Previously,cDNA of A.niger (No.SG136)R -glucosidase was cloned and expressed in P.pastoris in our laboratory;however,the yield of recombinant R -glucosidase was only 0.08U/mL.The low expression level compared to that in the present study of A.niger (CICIM F0620)might be caused by nonidentical gene sequences in both cases as well as the low copy number of the target gene in the host cell.Thus,to the best of our knowledge,the production of recombinant R -glucosidase obtained in the present study represents the highest yield reported sofar.Figure 3.Analysis of transglucosidation products by HPLC.(A )Standard (commercial IMOs ).Peak 1,maltose;peak 2,isomaltose;peak 3,panose;peak 4,isomaltotriose.(B )Transglucosidation products by recombinant R -glucosidase.Peak 5,glucose.Figure 4.SDS -PAGE analysis for deglycosylation of recombinant R ne 1,marker;lane 2,recombinant R -glucosidase;lane 3,deglycosylated recombinant R -glucosidase;lane 4,endoglycosidase H f (Endo H f ).Figure 5.Molecular weight determination of native recombinant R -gluco-sidase by Superdex 20010/300gel filtration chromatography.1,standard;2,β-amylase (M r 200,000);3,alcohol dehydrogenase (M r 150,000);albumin bovine serum (M r 66,000);carbonic anhydrase (M r 29,000);cytochrome C (M r 12,400);a,recombinant R -glucosidase.Error bars correspond to the standard deviation of threedeterminations.Figure 6.Reverse-phase HPLC of recombinant R -glucosidase.Article J.Agric.Food Chem.,Vol.58,No.8,20104823Chemical characterization of recombinant A.niger R -glucosi-dase has been performed previously when the gene of A.niger R -glucosidase (including introns and extrons)was cloned and expressed using fungal species as host cell (11-13).Although in our previous study,the cDNA of A.niger R -glucosidase was cloned and expressed in yeast,only the condition of transglyco-sylation reaction of the recombinant enzyme was investi-gated (16).In order to demonstrate if the R -glucosidase from the fungal source A.niger has been successfully post-translational modified in the yeast expression system,in the present study,the biochemical properties of the recombinant enzyme were char-acterized in detail.Comparative biochemical characterization of native R -gluco-sidase from A.niger and recombinant enzyme from P.pastoris indicated that they had similar optimal pH and temperature.The recombinant R -glucosidase remained active at a wide pH range and was stable against thermal denaturation.These results showed that the recombinant R -glucosidase was potentially to be effectively useful in the preparation of IMOs.Wild-type R -glucosidase from A.niger is a glycoprotein con-taining 25.5-27.6%carbohydrate (25).Deglycosylation analysis of the recombinant enzyme indicated that it was glycosylated in P.pastoris .Considering the similar catalytic properties between the wild-type and recombinant enzyme,it seems like the recom-binant enzyme has the correct glycosylation patterns to ensure its biological activity.Previously,it was reported that the gene length of aglu is 3124bp,containing three introns and four extrons.It encodes 985amino acids,and the N-terminal sequence from Met-1to Leu-25is predicted to be a signal peptide which is similar to the typical eukaryotic signal sequence (11).In addition,it was found that the mature wild-type R -glucosidase was actually a heterosubunit protein,in which the two heterosubunits were composed of residues 26-252and 267-985of aglu respectively.Even though the mechanism of this mature protein formation process has not been demonstrated,it was suggested that the pro-protein of aglu was subjected to a limited proteolysis after secretion from the cells (11).Interestingly,it was found that the two heterosubunits had a very tight interaction and could not be separated by SDS -PAGE,while they were able to be separated by reverse-phase HPLC (2).In the present study,a similar phenomenon was also observed.The recombinant enzyme exhibitedoneFigure 7.Effects of temperature on activity and stability of recombinant R -glucosidase.(A )Temperature optimum.The activity of recombinant R -glucosidase at 60°C was defined as 100%.(B )Thermostability of the enzyme,50°C (]),60°C (0)and 70°C (4).The activity of recombinant R -glucosidase without heat treated was defined as 100%.Error bars correspond to the standard deviation of three independentdeterminations.Figure 8.Effects of pH on activity and stability of recombinant R -glucosidase.(A )pH optimum.The activity of recombinant R -glucosidase at pH 4.5was defined as 100%.(B )pH stability.The activity of recombinant R -glucosidase at pH 4.5was defined as 100%.Error bars correspond to the standard deviation of three independent detrminations.4824J.Agric.Food Chem.,Vol.58,No.8,2010Chen et al.band on SDS-PAGE,one peak by gel filtration HPLC,and two peaks by reverse-phase HPLC.Attempting to N-terminal sequence the recombinant enzyme failed probably due to glycosylation issues.Considering that the biochemical pro-perties of recombinant enzyme in the present study are similar to those of the wild-type,it is assumed that the recombinant enzyme was also proteolyzed after synthesis and formed a heterosubunit protein.If this is correct,it may be presumed that the protease which hydrolyzes the pro-R-glucosidase has no strict substrate specificity.It not only exists in A.niger but also exists in P.pastoris.Further information is needed to support this hypothesis.In summary,cDNA of A.niger R-glucosidase was cloned, expressed and characterized in detail.The expression level of2.07 U/mL and2.21U/mg protein obtained in the culture media in the present study represents the highest yield of A.niger R-glucosi-dase reported so far.Detailed biochemical characterization demonstrated that the recombinant enzyme from P.pastoris was similar to that of native R-glucosidase from A.niger, suggesting that the recombinant enzyme has been successfully post-translationally modified in the P.pastoris expression system. Further enhancement of the yield of the recombinant R-glucosi-dase by fermentation technology is currently underway in our laboratory,and these studies will provide the basis for the application of recombinant R-glucosidase in the industry of IMOs production.LITERATURE CITED(1)Kimura, A.;Takata,M.;Sakai,O.;Matsui,H.;Takai,N.;Takayanagi,T.;Nishimura,I.;Uozumi,T.;Chiba,plete amino acid sequence of crystalline alpha-glucosidase from Aspergil-lus niger.Biosci.,Biotechnol.,Biochem.1992,56(8),1368–1370. 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(12)Ogawa,M.;Nishio,T.;Minoura,K.;Uozumi,T.;Wada,M.;Hashimoto,N.;Kawachi,R.;Oku,T.Recombinant alpha-glucosi-dase from Aspergillus niger.Overexpression by Emericella nidulans, purification and Characterization.J.Appl.Glycosci.2006,53,13–16.(13)Lee,D.G.;Nishimura-Masuda,I.;Nakamura,A.;Hidaka,M.;Masaki,H.;Uozumi,T.Overproduction of aphla-glucosidase in Aspergillus niger transformed with the cloned gene aglA.J.Gen.Appl.Microbiol.1998,44,177–181.(14)Ruanglek,V.;Sriprang,R.;Ratanaphan,N.;Tirawongsaroj,P.;Chantasigh,D.;Tanapongpipat,S.;Pootanakit,K.;Eurwilaichitr, L.Cloning,expression,characterization and high cell-density pro-duction of recombinant endo-1,4-β-xylanase from Aspergillus niger in Pichia pastoris.Enzyme Microb.Technol.2007,41(1-2),19–25.(15)Macauley-Patrick,S.;Fazenda,M.L.;McNeil,B.;Harvey,L.M.Heterologous protein production using the Pichia pastoris expres-sion system.Yeast2005,22(4),249–270.(16)Tong,X.;Tang,Q.;Wu,Y.;Wu,J.;Chen,J.Cloning of the geneencoding aphla-glucosidase from Aspergillus niger and its expression in Pichia pastoris.Acta Microbiol.Sin.2009,49(2),262–268. (17)Chantasingh,D.;Pootanakit,K.;Champreda,V.;Kanokratana,P.;Eurwilaichitr,L.Cloning,expression and characterization of a xylanase10from Aspergillus terreus(BCC129)in Pichia pastoris.Protein Expression Purif.2006,46(1),143–149.(18)Nashiru,O.;Koh,S.;Lee,S.-Y.;Lee,D.-S.Novel R-glucosidasefrom extreme thermophile Thermus caldophilus GK24.J.Biochem.Mol.Biol.2001,34(4),347–354.(19)Chen,X.;Cao,Y.;Ding,Y.;Lu,W.;Li,D.Cloning,functionalexpression and characterization of Aspergillus sulphureus beta-mannanase in Pichia pastoris.J.Biotechnol.2007,128(3),452–461.(20)Tseng,S.J.;Hsu,J.P.A Comparison of the Parameter EstimatingProcedures for the Michaelis-Menten Model.J.Theor.Biol.1990, 145(4),457–464.(21)Olusanya,O.;Olutiola,P.O.Characterisation of maltase fromenteropathogenic Escherichia coli.FEMS Microbiol.Lett.1986,36 (2-3),239–244.(22)Yamasaki,Y.;Suzuki,Y.;Ozawa,J.Certain properties of alpha-glucosidase from Mucor racemosus.Agric.Biol.Chem.1997,41, 1559–1565.(23)Raasch,C.;Streit,W.;Schanzer,J.;Bibel,M.;Gosslar,U.;Liebl,W.Thermotoga maritima Agla,an extremely thermostable NADþ, Mn2þ,and thiol-dependent alpha-glucosidase.Extremophiles2000, 4(4),189–200.(24)Cregg,J.M.;Cereghino,J.L.;Shi,J.;Higgins,D.R.Recombinantprotein expression in Pichia pastoris.Mol.Biotechnol.2000,16(1), 23–52.(25)Lee,S.S.;He,S.;Withers,S.G.Identification of the catalyticnucleophile of the Family31alpha-glucosidase from Aspergillus niger via trapping of a5-fluoroglycosyl-enzyme intermediate.Bio-chem.J.2001,359,381–386.Received for review January6,2010.Revised manuscript received March17,2010.Accepted March23,2010.This work was supported financially by the National Outstanding Youth Foundation of China (20625619),Research Program of State Key Laboratory of Food Science and Technology(SKLF-MB-200802,SKLF-TS-200910, SKLF-KF-200909),Program of Innovation Team of Jiangnan University(2008CXTD01),Public Topic of Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University (KLIB-KF200904)and the Key Program of National Natural Science Foundation of China(No.20836003).。

第一页A band|A带A chromosome|A染色体[二倍体染色体组中的正常染色体（不同于B染色体）] A site|[核糖体]A部位ABA|脱落酸abasic site|脱碱基位点，无碱基位点abaxial|远轴的abequose|阿比可糖，beta脱氧岩藻糖aberrant splicing|异常剪接aberration|象差；畸变；失常abiogenesis|自然发生论，无生源论ablastin|抑殖素（抑制微生物细胞分裂或生殖的一种抗体）abnormal distrbution|非正态分布abnormality|异常，失常；畸形，畸变ABO blood group system|ABO血型系统aboriginal mouse|原生鼠abortin|流产素abortion|流产，败育abortive egg|败育卵abortive infection|流产（性）感染abortive transduction|流产（性）转导ABP|肌动蛋白结合蛋白abrin|相思豆毒蛋白abscisic acid|脱落酸abscission|脱落absolute|绝对的absolute configuration|绝对构型absolute counting|绝对测量absolute deviation|绝对偏差absolute error|绝对误差absorbance|吸收，吸光度absorbed dose|吸收剂量absorbent|吸收剂absorptiometer|吸光计absorptiometry|吸光测定法absorption|吸收absorption band|吸收谱带absorption cell|吸收池absorption coefficient|吸收系数absorption spectroscopy|吸收光谱法absorption spectrum|吸收光谱；吸收谱absorptive endocytosis|吸收（型）胞吞（作用） absorptive pinocytosis|吸收（型）胞饮（作用） absorptivity|吸光系数；吸收性abundance|丰度abundant|丰富的，高丰度的abundant mRNAs|高丰度mRNAabzyme|抗体酶acaricidin|杀螨剂accedent variation|偶然变异accelerated flow method|加速流动法accepting arm|[tRNA的]接纳臂acceptor|接纳体，（接）受体acceptor site|接纳位点，接受位点acceptor splicing site|剪接受体acceptor stem|[tRNA的]接纳茎accessible|可及的accessible promoter|可及启动子accessible surface|可及表面accessory|零件，附件；辅助的accessory cell|佐细胞accessory chromosome|副染色体accessory factor|辅助因子accessory nucleus|副核accessory pigment|辅助色素accessory protein|辅助蛋白（质）accommodation|顺应accumulation|积累，累积accuracy|准确度acenaphthene|二氢苊acene|并苯acentric|无着丝粒的acentric fragment|无着丝粒断片acentric ring|无着丝粒环acetal|缩醛acetaldehyde|乙醛acetalresin|缩醛树脂acetamidase|乙酰胺酶acetamide|乙酰胺acetate|乙酸盐acetic acid|乙酸，醋酸acetic acid bacteria|乙酸菌，醋酸菌acetic anhydride|乙酸酐acetification|乙酸化作用，醋化作用acetin|乙酸甘油酯，三乙酰甘油酯acetoacetic acid|乙酰乙酸Acetobacter|醋杆菌属acetogen|产乙酸菌acetogenic bacteria|产乙酸菌acetome body|酮体acetome powder|丙酮制粉[在-30度以下加丙酮制成的蛋白质匀浆物] acetomitrile|乙腈acetone|丙酮acetyl|乙酰基acetyl coenzyme A|乙酰辅酶Aacetylcholine|乙酰胆碱acetylcholine agonist|乙酰胆碱拮抗剂acetylcholine receptor|乙酰胆碱受体acetylcholinesterase|乙酰胆碱酯酶acetylene|乙炔acetylene reduction test|乙炔还原试验[检查生物体的固氮能力] acetylglucosaminidase|乙酰葡糖胺糖苷酶acetylglutamate synthetase|乙酰谷氨酸合成酶acetylsalicylate|乙酰水杨酸；乙酰水杨酸盐、酯、根acetylsalicylic acid|乙酰水杨酸acetylspiramycin|乙酰螺旋霉素AchE|乙酰胆碱酯酶achiral|非手性的acholeplasma|无胆甾原体AchR|乙酰胆碱受体achromatic|消色的；消色差的achromatic color|无色achromatic lens|消色差透镜achromatin|非染色质acid catalysis|酸催化acid fibroblast growth factor|酸性成纤维细胞生长因子acid fuchsin|酸性品红acid glycoprotein|酸性糖蛋白acid hydrolyzed casein|酸水解酪蛋白acid medium|酸性培养基acid mucopolysaccharide|酸性粘多糖acid phosphatase|酸性磷酸酶acid protease|酸性蛋白酶acid solvent|酸性溶剂acidic|酸性的acidic amino acid|酸性氨基酸acidic protein|酸性蛋白质[有时特指非组蛋白]acidic transactivator|酸性反式激活蛋白acidic transcription activator|酸性转录激活蛋白 acidification|酸化（作用）acidifying|酸化（作用）acidolysis|酸解acidophilia|嗜酸性acidophilic bacteria|嗜酸菌acidophilous milk|酸奶aclacinomycin|阿克拉霉素acoelomata|无体腔动物acomitic acid|乌头酸aconitase|顺乌头酸酶aconitate|乌头酸；乌头酸盐、酯、根aconitine|乌头碱aconitum alkaloid|乌头属生物碱ACP|酰基载体蛋白acquired character|获得性状acquired immunity|获得性免疫acridine|吖啶acridine alkaloid|吖啶（类）生物碱acridine dye|吖啶燃料acridine orange|吖啶橙acridine yellow|吖啶黄acriflavine|吖啶黄素acroblast|原顶体acrocentric chromosome|近端着丝染色体acrolein|丙烯醛acrolein polymer|丙烯醛类聚合物acrolein resin|丙烯醛树脂acropetal translocation|向顶运输acrosin|顶体蛋白acrosomal protease|顶体蛋白酶acrosomal reaction|顶体反应acrosome|顶体acrosome reaction|顶体反应acrosomic granule|原顶体acrosyndesis|端部联会acrylamide|丙烯酰胺acrylate|丙烯酸酯、盐acrylic acid|丙烯酸acrylic polymer|丙烯酸（酯）类聚合物acrylic resin|丙烯酸（酯）类树脂acrylketone|丙烯酮acrylonitrile|丙烯腈actidione|放线（菌）酮[即环己酰亚胺]actin|肌动蛋白actin filament|肌动蛋白丝actinin|辅肌动蛋白[分为alfa、beta两种，beta蛋白即加帽蛋白] actinmicrofilament|肌动蛋白微丝actinometer|化学光度计actinomorphy|辐射对称[用于描述植物的花]actinomycetes|放线菌actinomycin D|放线菌素Dactinospectacin|放线壮观素，壮观霉素，奇霉素action|作用action current|动作电流action potential|动作电位action spectrum|动作光谱activated sludge|活性污泥activated support|活化支持体activating group|活化基团activating transcription factor|转录激活因子activation|激活；活化activation analysis|活化分析activation energy|活化能activator|激活物，激活剂，激活蛋白activator protein|激活蛋白active absorption|主动吸收active biomass|活生物质active carbon|活性碳active center|活性中心active chromatin|活性染色质active dry yeast|活性干酵母active dydrogen compounds|活性氢化合物active ester of amino acid|氨基酸的活化酯active hydrogen|活性氢active immunity|主动免疫active oxygen|活性氧active site|活性部位，活性中心active transport|主动转运active uptake|主动吸收activin|活化素[由垂体合成并由睾丸和卵巢分泌的性激素]activity|活性，活度，（放射性）活度actomyosin|肌动球蛋白actophorin|载肌动蛋白[一种肌动蛋白结合蛋白]acute|急性的acute infection|急性感染acute phase|急性期acute phase protein|急性期蛋白，急相蛋白acute phase reaction|急性期反应，急相反应[炎症反应急性期机体的防御反应] acute phase reactive protein|急性期反应蛋白，急相反应蛋白acute phase response|急性期反应，急相反应acute toxicity|急性毒性ACV|无环鸟苷acyclic nucleotide|无环核苷酸acycloguanosine|无环鸟苷，9-（2-羟乙氧甲基）鸟嘌呤acyclovir|无环鸟苷acyl|酰基acyl carrier protein|酰基载体蛋白acyl cation|酰（基）正离子acyl chloride|酰氯acyl CoA|脂酰辅酶Aacyl coenzyem A|脂酰辅酶Aacyl fluoride|酰氟acyl halide|酰卤acylamino acid|酰基氨基酸acylase|酰基转移酶acylating agent|酰化剂acylation|酰化acylazide|酰叠氮acylbromide|酰溴acyloin|偶姻acyltransferase|酰基转移酶adamantanamine|金刚烷胺[曾用作抗病毒剂]adamantane|金刚烷adaptability|适应性adaptation|适应adapter|衔接头；衔接子adapter protein|衔接蛋白质adaptin|衔接蛋白[衔接网格蛋白与其他蛋白的胞质区]adaptive behavior|适应性行为adaptive enzyme|适应酶adaptive molecule|衔接分子adaptive response|适应反应[大肠杆菌中的DNA修复系统]adaptor|衔接头；衔接子adaxial|近轴的addition|加成addition compound|加成化合物addition haploid|附加单倍体addition line|附加系additive|添加物，添加剂additive effect|加性效应additive genetic variance|加性遗传方差additive recombination|插入重组，加插重组[因DNA插入而引起的基因重组] addressin|地址素[选择蛋白(selectin)的寡糖配体，与淋巴细胞归巢有关]adducin|内收蛋白[一种细胞膜骨架蛋白，可与钙调蛋白结合]adduct|加合物，加成化合物adduct ion|加合离子adenine|腺嘌呤adenine arabinoside|啊糖腺苷adenine phosphoribosyltransferase|腺嘌呤磷酸核糖转移酶adenoma|腺瘤adenosine|腺嘌呤核苷，腺苷adenosine deaminase|腺苷脱氨酶adenosine diphoshate|腺苷二磷酸adenosine monophosphate|腺苷（一磷）酸adenosine phosphosulfate|腺苷酰硫酸adenosine triphosphatase|腺苷三磷酸酶adenosine triphosphate|腺苷三磷酸adenovirus|腺病毒adenylate|腺苷酸；腺苷酸盐、酯、根adenylate cyclase|腺苷酸环化酶adenylate energy charge|腺苷酸能荷adenylate kinase|腺苷酸激酶adenylic acid|腺苷酸adenylyl cyclase|腺苷酸环化酶adenylylation|腺苷酰化adherence|粘着，粘附，粘连；贴壁adherent cell|贴壁赴 徽匙牛ㄐ裕┫赴 掣剑ㄐ裕┫赴?/P>adherent culture|贴壁培养adhering junction|粘着连接adhesin|粘附素[如见于大肠杆菌]adhesion|吸附，结合，粘合；粘着，粘附，粘连adhesion factor|粘着因子，粘附因子adhesion molecule|粘着分子，粘附分子adhesion plaque|粘着斑adhesion protein|粘着蛋白，吸附蛋白adhesion receptor|粘着受体adhesion zone|粘着带[如见于细菌壁膜之间]adhesive|粘合剂，胶粘剂adhesive glycoprotein|粘着糖蛋白adipic acid|己二酸，肥酸adipocyte|脂肪细胞adipokinetic hormone|脂动激素[见于昆虫]adipose tissue|脂肪组织adjust|[动]调节，调整；修正adjustable|可调的adjustable miropipettor|可调微量移液管adjustable spanner|活动扳手adjusted retention time|调整保留时间adjusted retention volume|调整保留体积adjuvant|佐剂adjuvant cytokine|佐剂细胞因子adjuvant peptide|佐剂肽adjuvanticity|佐剂（活）性adoptive immunity|过继免疫adoptive transfer|过继转移ADP ribosylation|ADP核糖基化ADP ribosylation factor|ADP核糖基化因子ADP ribosyltransferase|ADP核糖基转移酶adrenal cortical hormone|肾上腺皮质(激)素adrenaline|肾上腺素adrenergic receptor|肾上腺素能受体adrenocepter|肾上腺素受体adrenocorticotropic hormone|促肾上腺皮质(激)素adrenodoxin|肾上腺皮质铁氧还蛋白adriamycin|阿霉素，亚德里亚霉素adsorbent|吸附剂adsorption|吸附adsorption catalysis|吸附催化adsorption center|吸附中心adsorption chromatography|吸附层析adsorption film|吸附膜adsorption isobar|吸附等压线adsorption isotherm|吸附等温线adsorption layer|吸附层adsorption potential|吸附电势adsorption precipitation|吸附沉淀adsorption quantity|吸附量adult diarrhea rotavirus|成人腹泻轮状病毒advanced glycosylation|高级糖基化advanced glycosylation end product|高级糖基化终产物 adventitious|不定的，无定形的adverse effect|反效果，副作用aecidiospore|锈孢子，春孢子aeciospore|锈孢子，春孢子aequorin|水母蛋白，水母素aeration|通气aerator|加气仪，加气装置aerial mycelium|气生菌丝体aerobe|需氧菌[利用分子氧进行呼吸产能并维持正常生长繁殖的细菌] aerobic|需氧的aerobic bacteria|需氧（细）菌aerobic cultivation|需氧培养aerobic glycolysis|有氧酵解aerobic metabolism|有氧代谢aerobic respiration|需氧呼吸aerobic waste treatment|需氧废物处理aerobiosis|需氧生活aerogel|气凝胶aerogen|产气菌aerolysin|气单胞菌溶素Aeromonas|气单胞菌属aerosol|气溶胶aerosol gene delivery|气溶胶基因送递aerospray ionization|气喷射离子化作用aerotaxis|趋氧性[（细胞）随环境中氧浓度梯度进行定向运动]aerotolerant bacteria|耐氧菌[不受氧毒害的厌氧菌]aerotropism|向氧性aesculin|七叶苷，七叶灵aetiology|病原学B cell|B细胞B cell antigen receptor|B细胞抗原受体B cell differentiation factor|B细胞分化因子B cell growth factor|B细胞生长因子B cell proliferation|B细胞增殖B cell receptor|B细胞受体B cell transformation|B细胞转化B chromosome|B染色体[许多生物（如玉米）所具有的异染质染色体] B to Z transition|B-Z转换[B型DNA向Z型DNA转换]Bacillariophyta|硅藻门Bacillus|芽胞杆菌属Bacillus anthracis|炭疽杆菌属Bacillus subtillis|枯草芽胞杆菌bacitracin|杆菌肽back donation|反馈作用back flushing|反吹，反冲洗back mutation|回复突变[突变基因又突变为原由状态]backbone|主链；骨架backbone hydrogen bond|主链氢键backbone wire model|主链金属丝模型[主要反应主链走向的实体模型]backcross|回交backflushing chromatography|反吹层析，反冲层析background|背景，本底background absorption|背景吸收background absorption correction|背景吸收校正background correction|背景校正background gactor|背景因子background genotype|背景基因型[与所研究的表型直接相关的基因以外的全部基因]background hybridization|背景杂交background radiation|背景辐射，本底辐射backmixing|反向混合backside attack|背面进攻backward reaction|逆向反应backwashing|反洗bacmid|杆粒[带有杆状病毒基因组的质粒，可在细菌和昆虫细胞之间穿梭]bacteremia|菌血症bacteria|（复）细菌bacteria rhodopsin|细菌视紫红质bacterial adhesion|细菌粘附bacterial alkaline phosphatase|细菌碱性磷酸酶bacterial artificial chromosome|细菌人工染色体bacterial colony|（细菌）菌落bacterial colony counter|菌落计数器bacterial conjugation|细菌接合bacterial filter|滤菌器bacterial invasion|细菌浸染bacterial motility|细菌运动性bacterial rgodopsin|细菌视紫红质，细菌紫膜质bacterial vaccine|菌苗bacterial virulence|细菌毒力bactericidal reaction|杀（细）菌反应bactericide|杀（细）菌剂bactericidin|杀（细）菌素bactericin|杀（细）菌素bacteriochlorophyll|细菌叶绿素bacteriochlorophyll protein|细菌叶绿素蛋白bacteriocide|杀（细）菌剂bacteriocin|细菌素bacteriocin typing|细菌素分型[利用细菌素对细胞进行分型]bacterioerythrin|菌红素bacteriofluorescein|细菌荧光素bacteriology|细菌学bacteriolysin|溶菌素bacteriolysis|溶菌（作用）bacteriolytic reaction|溶菌反应bacteriophaeophytin|细菌叶褐素bacteriophage|噬菌体bacteriophage arm|噬菌体臂bacteriophage conversion|噬菌体转变bacteriophage head|噬菌体头部bacteriophage surface expression system|噬菌体表面表达系统bacteriophage tail|噬菌体尾部bacteriophage typing|噬菌体分型bacteriophagology|噬菌体学bacteriopurpurin|菌紫素bacteriorhodopsin|细菌视紫红质bacteriosome|细菌小体[昆虫体内一种含有细菌的结构]bacteriostasis|抑菌(作用)bacteriostat|抑菌剂bacteriotoxin|细菌毒素bacteriotropin|亲菌素bacterium|细菌bacteroid|类菌体baculovirus|杆状病毒bag sealer|封边机baking soda|小苏打BAL 31 nuclease|BAL 31核酸酶balance|天平balanced heterokaryon|平衡异核体balanced lethal|平衡致死balanced lethal gene|平衡致死基因balanced linkage|平衡连锁balanced pathogenicity|平衡致病性balanced polymorphism|平衡多态性balanced salt solution|平衡盐溶液balanced solution|平衡溶液balanced translocation|平衡易位balbaini ring|巴尔比亚尼环[由于RNA大量合成而显示特别膨大的胀泡，在多线染色体中形成独特的环]Balbiani chromosome|巴尔比亚尼染色体[具有染色带的多线染色体，1881年首先发现于双翅目摇蚊幼虫]ball mill|球磨ball mill pulverizer|球磨粉碎机ball milling|球磨研磨balloon catheter|气囊导管[可用于基因送递，如将DNA导入血管壁]banana bond|香蕉键band|条带，带[见于电泳、离心等]band broadening|条带加宽band sharpening|条带变细，条带锐化band width|带宽banding pattern|带型banding technique|显带技术,分带技术barbiturate|巴比妥酸盐barium|钡barly strip mosaic virus|大麦条纹花叶病毒barly yellow dwarf virus|大麦黄矮病毒barnase|芽胞杆菌RNA酶[见于解淀粉芽胞杆菌]barophilic baceria|嗜压菌baroreceptor|压力感受器barotaxis|趋压性barotropism|向压性barr body|巴氏小体barrel|桶，圆筒[可用于描述蛋白质立体结构，如beta折叠桶]barrier|屏障，垒barstar|芽胞杆菌RNA酶抑制剂[见于解淀粉芽胞杆菌]basal|基础的，基本的basal body|基粒basal body temperature|基础体温basal component|基本成分，基本组分basal expression|基础表达，基态表达basal granule|基粒basal heat producing rate|基础产热率basal lamina|基膜，基板basal level|基础水平，基态水平basal medium|基本培养基，基础培养基basal medium Eagle|Eagle基本培养基basal metabolic rate|基础代谢率basal metabolism|基础代谢basal promoter element|启动子基本元件basal transcription|基础转录，基态转录basal transcription factor|基础转录因子base|碱基；碱base analog|碱基类似物，类碱基base catalysis|碱基催化base composition|碱基组成base pairing|碱基配对base pairing rules|碱基配对法则,碱基配对规则base peak|基峰base pire|碱基对base ratio|碱基比base stacking|碱基堆积base substitution|碱基置换baseline|基线baseline drift|基线漂移baseline noise|基线噪声basement membrane|基底膜basement membrane link protein|基底膜连接蛋白basic amino acid|碱性氨基酸basic fibroblast growth factor|碱性成纤维细胞生长因子basic fuchsin|碱性品红basic medium|基础培养基basic number of chromosome|染色体基数basic protein|碱性蛋白质basic solvent|碱性溶剂basic taste sensation|基本味觉basidiocarp|担子果basidiomycetes|担子菌basidium|担子basipetal translocation|向基运输basket centrifuge|（吊）篮式离心机basket drier|篮式干燥机basket type evaporator|篮式蒸发器basonuclin|碱（性）核蛋白[见于角质形成细胞，含有多对锌指结构] basophil|嗜碱性细胞basophil degranulation|嗜碱性细胞脱粒basophilia|嗜碱性batch|分批；批，一批batch cultivation|分批培养batch culture|分批培养物batch digestor|分批消化器batch extraction|分批抽提，分批提取batch fermentation|分批发酵，（罐）批发酵batch filtration|分批过滤batch operation|分批操作batch process|分批工艺，分批法batch reactor|间歇反应器，分批反应器batch recycle cultivation|分批再循环培养batch recycle culture|分批再循环培养（物）bathochrome|向红基bathochromic shift|红移bathorhodopsin|红光视紫红质，前光视紫红质batrachotoxin|树蛙毒素[固醇类生物碱，作用于钠通道] baytex|倍硫磷BCG vaccine|卡介苗bead mill|玻珠研磨机bead mill homogenizer|玻珠研磨匀浆机bean sprouts medium|豆芽汁培养基beauvericin|白僵菌素becquerel|贝可（勒尔）bed volume|（柱）床体积bee venom|蜂毒beef broth|牛肉汁beef extract|牛肉膏，牛肉提取物beet yellows virus|甜菜黄化病毒Beggiatoa|贝日阿托菌属[属于硫细菌]behavior|行为；性质，性能behavioral control|行为控制behavioral isolation|行为隔离behavioral thermoregulation|行为性体温调节behenic acid|山yu酸，二十二（烷）酸belt desmosome|带状桥粒belt press|压带机belt press filter|压带（式）滤器bench scale|桌面规模，小试规模benchtop bioprocessing|桌面生物工艺[小试规模]benchtop microcentrifuge|台式微量离心机bend|弯曲；弯管；转折bending|弯曲；转折，回折beneficial element|有益元素bent bond|弯键bent DNA|弯曲DNA,转折DNAbenzene|苯benzhydrylamine resin|二苯甲基胺树脂benzidine|联苯胺benzilate|三苯乙醇酸（或盐或酯）benzimidazole|苯并咪唑benzodiazine|苯并二嗪，酞嗪benzoin|苯偶姻，安息香benzophenanthrene|苯并菲benzopyrene|苯并芘benzoyl|苯甲酰基benzoylglycine|苯甲酰甘氨酸benzyl|苄基benzyladenine|苄基腺嘌呤benzylaminopurine|苄基氨基嘌呤benzylisoquinoline|苄基异喹啉benzylisoquinoline alkaloid|苄基异喹啉（类）生物碱benzylpenicillin|苄基青霉素berberine|小檗碱Bertrand rule|贝特朗法则bestatin|苯丁抑制素[可抑制亮氨酸氨肽酶的一种亮氨酸类似物]C value|C值[单倍基因组DNA的量]C value paradox|C值悖理[物种的C值和它的进化复杂性之间无严格对应关系]C4 dicarboxylic acid cycle|C4二羧酸循环cachectin|恶液质素[即alfa肿瘤坏死因子]cadaverine|尸胺cadherin|钙粘着蛋白[介导依赖（于）钙的细胞间粘着作用的一类跨膜蛋白质，分为E-,N-,P-等若干种，E表示上皮（epithelia)，N表示神经（neural），P表示胎盘（placental）] cadmium|镉caerulin|雨蛙肽cage|笼cage compound|笼形化合物cage coordination compound|笼形配合物cage effect|笼效应cage structure|笼形结构[非极性分子周围的水分子所形成的有序结构]calbindin|钙结合蛋白calciferol|麦角钙化（固）醇calcimedin|钙介蛋白[钙调蛋白拮抗剂]calcineurin|钙调磷酸酶[依赖于钙调蛋白的丝氨酸—苏氨酸磷酸酶]calcionin|降钙素calcium binding protein|钙结合蛋白（质）calcium binding site|钙结合部位calcium channel|钙通道calcium chloride|氯化钙calcium influx|钙流入calcium mediatory protein|钙中介蛋白（质）calcium phosphate|磷酸钙calcium phosphate precipitation|磷酸盐沉淀calcium pump|钙泵calcium sensor protein|钙传感蛋白（质）calcium sequestration|集钙（作用）calcyclin|钙（细胞）周边蛋白calcyphosine|钙磷蛋白[是依赖于cAMP的蛋白激酶的磷酸化底物]caldesmon|钙调（蛋白）结合蛋白[主要见于平滑肌，可与钙调蛋白及肌动蛋白结合] calelectrin|钙电蛋白[最初发现于鳗鱼电器官的一种钙结合蛋白]calf intestinal alkaline phosphatase|（小）牛小肠碱性磷酸酶calf serum|小牛血清calf thymus|小牛胸腺calgranulin|钙粒蛋白calibration|校准，标准calibration curve|校正曲线calibration filter|校准滤光片calibration protein|校准蛋白calicheamycin|刺孢霉素[来自刺孢小单胞菌的抗肿瘤抗生素，带有二炔烯官能团] calicivirus|杯状病毒calli|（复）胼胝体，愈伤组织[用于植物]；胼胝[见于动物皮肤]callose|胼胝质，愈伤葡聚糖callose synthetase|愈伤葡聚糖合成酶callus|胼胝体，愈伤组织[用于植物]；胼胝[见于动物皮肤]callus culture|愈伤组织培养calmodulin|钙调蛋白calnexin|钙联结蛋白[内质网的一种磷酸化的钙结合蛋白]calomel|甘汞calomel electrode|甘汞电极calorie|卡calpactin|依钙（结合）蛋白[全称为“依赖于钙的磷脂及肌动蛋白结合蛋白”]calpain|（需）钙蛋白酶calpain inhibitor|（需）钙蛋白酶抑制剂calpastatin|(需）钙蛋白酶抑制蛋白calphobindin|钙磷脂结合蛋白calphotin|钙感光蛋白[感光细胞的一种钙结合蛋白]calprotectin|（肌）钙网蛋白[骨骼肌肌质网膜上的钙结合蛋白]calretinin|钙（视）网膜蛋白calsequestrin|（肌）集钙蛋白calspectin|钙影蛋白calspermin|钙精蛋白[睾丸的一种钙调蛋白结合蛋白]caltractin|钙牵蛋白[一种与基粒相关的钙结合蛋白]Calvin cycle|卡尔文循环，光合碳还原环calyculin|花萼海绵诱癌素[取自花萼盘皮海绵的磷酸酶抑制剂]calyptra|根冠calyx|花萼cambium|形成层[见于植物]cAMP binding protein|cAMP结合蛋白cAMP receptor protein|cAMP受体蛋白cAMP response element|cAMP效应元件cAMP response element binding protein|cAMP效应元件结合蛋白Campbell model|坎贝尔模型camphane|莰烷camphane derivative|莰烷衍生物camphore|樟脑camptothecin|喜树碱Campylobacter|弯曲菌属Campylobacter fetus|胎儿弯曲菌属Canada balsam|加拿大香脂，枞香脂canaline|副刀豆氨酸canalization|[表型]限渠道化，发育稳态[尽管有遗传因素和环境条件的干扰，表型仍保持正常]canavanine|刀豆氨酸cancer|癌症cancer metastasis|癌症转移cancer suppressor gene|抑癌基因cancer suppressor protein|抑癌基因产物，抑癌蛋白（质）candicidin|杀假丝菌素candida|念珠菌属Candida albicans|白色念珠菌candle jar|烛罐cannabin|大麻苷；大麻碱canonical base|规范碱基canonical molecular orbital|正则分子轨道canonical partition function|正则配分函数canonical sequence|规范序列cantharidin|斑蝥素canthaxanthin|角黄素canyon|峡谷[常用于比喻某些生物大分子的主体结构特征]cap|帽，帽（结构）cap binding protein|帽结合蛋白cap site|加帽位点capacitation|获能[特指镜子在雌性生殖道中停留后获得使卵子受精的能力]capacity|容量capacity factor|容量因子capillarity|毛细现象capillary|毛细管；毛细血管capillary absorption|毛细吸收capillary action|毛细管作用capillary attraction|毛细吸力capillary column|毛细管柱capillary culture|毛细管培养capillary electrode|毛细管电极capillary electrophoresis|毛细管电泳capillary free electrophoresis|毛细管自由流动电泳capillary gas chromatography|毛细管气相层析capillary isoelectric focusing|毛细管等电聚焦capillary isotachophoresis|毛细管等速电泳capillary membrane module|毛细管膜包capillary transfer|毛细管转移[通过毛细管作用进行核酸的印迹转移] capillary tube|毛细管capillary tubing|毛细管capillary zone electrophoresis|毛细管区带电泳capillovirus|毛状病毒组capping|加帽，加帽反应；封闭反应；帽化，成帽capping enzyme|加帽酶capping protein|[肌动蛋白]加帽蛋白caprin|癸酸甘油酯caproin|己酸甘油酯capromycin|卷曲霉素，缠霉素caproyl|己酸基caprylin|辛酸甘油酯capsid|（病毒）衣壳，（病毒）壳体capsid protein|衣壳蛋白capsidation|衣壳化capsomer|（病毒）壳粒capsular polysaccharide|荚膜多糖capsulation|包囊化（作用），胶囊化（作用）capsule|荚膜capsule swelling reaction|荚膜肿胀反应capture|捕捉，俘获capture antigen|捕捉抗原[酶免疫测定中用于捕捉抗体的抗原]capture assay|捕捉试验carbamyl|氨甲酰基carbamyl ornithine|氨甲酰鸟氨酸carbamyl phosphate|氨甲酰磷酸carbamyl phosphate synthetase|氨甲酰磷酸合成酶carbamyl transferase|氨甲酰（基）转移酶carbamylation|氨甲酰化carbanion|碳负离子carbanyl group|羰基carbene|卡宾carbenicillin|羧苄青霉素carbenoid|卡宾体carbocation|碳正离子carbodiimide|碳二亚胺carbohydrate|糖类，碳水化合物carbohydrate fingerprinting|糖指纹分析carbohydrate mapping|糖作图，糖定位carbohydrate sequencing|糖测序carbol fuchsin|石炭酸品红carboline|咔啉，二氮芴carbon assimilation|碳同化carbon balance|碳平衡carbon cycling|碳循环carbon dioxide|二氧化碳carbon dioxide compensation|二氧化碳补偿点carbon dioxide fertilization|二氧化碳施肥carbon dioxide fixation|二氧化碳固定carbon dioxide tension|二氧化碳张力carbon fiber|碳纤维carbon fixation|碳固定carbon isotope|碳同位素carbon isotope analysis|碳同位素分析carbon isotope composition|碳同位素组成carbon monoxide|一氧化碳carbon source|碳源carbonate|碳酸盐，碳酸酯carbonate plant|碳化植物carbonic anhydrase|碳酸酐酶carbonium ion|碳正离子carbonyl|羰基carbonylation|羰基化carboxydismutase|羰基岐化酶，核酮糖二磷酸羧化酶 carboxydotrophic bacteria|一氧化碳营养菌carboxyglutamic acid|羧基谷氨酸carboxyl|羧基carboxyl protease|羧基蛋白酶carboxyl terminal|羧基端carboxyl transferase|羧基转移酶carboxylase|羧化酶carboxylation|羧（基）化carboxylic acid|羧酶carboxymethyl|羧甲基carboxymethyl cellulose|羧甲基纤维素carboxypeptidase|羧肽酶[包括羧肽酶A、B、N等]carcinogen|致癌剂carcinogenesis|致癌，癌的发生carcinogenicity|致癌性carcinoma|癌carcinostatin|制癌菌素cardenolide|强心苷cardiac aglycone|强心苷配基，强心苷元cardiac cycle|心动周期cardiac glycoside|强心苷cardiac receptor|心脏感受器cardiohepatid toxin|心肝毒素[如来自链球菌]cardiolipin|心磷脂cardiotoxin|心脏毒素cardiovascular center|心血管中枢cardiovascular disease|心血管疾病cardiovirus|心病毒属[模式成员是脑心肌炎病毒]carlavirus|香石竹潜病毒组carmine|洋红carminomycin|洋红霉素carmovirus|香石竹斑驳病毒组carnation latent virus|香石竹潜病毒carnation mottle virus|香石竹斑驳病毒carnation ringspot virus|香石竹环斑病毒carnitine|肉碱carnitine acyl transferase|肉碱脂酰转移酶carnosine|肌肽[即beta丙氨酰组氨酸]carotene|胡萝卜素carotene dioxygenase|胡萝卜素双加氧酶carotenoid|类胡萝卜素carotenoprotein|胡萝卜素蛋白carpel|[植物]心皮carrageen|角叉菜，鹿角菜carrageenin|角叉菜胶carrier|载体，运载体，携载体；携带者，带（病）毒者，带菌者 carrier ampholyte|载体两性电解质carrier catalysis|载体催化carrier coprecipitation|载体共沉淀carrier DNA|载体DNAcarrier free|无载体的carrier phage|载体噬菌体carrier precipitation|载体沉淀（作用）carrier state|携带状态carriomycin|腐霉素，开乐霉素cartridge|[萃取柱的]柱体；软片，胶卷；子弹，弹药筒casamino acid|（水解）酪蛋白氨基酸，酪蛋白水解物cascade|串联，级联，级联系统cascade amplification|级联放大cascade chromatography|级联层析cascade fermentation|级联发酵casein|酪蛋白，酪素casein kinase|酪蛋白激酶[分I、II两种]Casparian band|凯氏带[见于植物内表皮细胞]Casparian strip|凯氏带cassette|盒，弹夹[借指DNA序列组件]cassette mutagenesis|盒式诱变casting|铸，灌制CAT box|CAT框[真核生物结构基因上游的顺式作用元件]catabolism|分解代谢catabolite gene activator protein|分解代谢物基因激活蛋白 catabolite repression|分解代谢物阻抑，分解代谢产物阻遏catalase|过氧化氢酶catalytic active site|催化活性位catalytic activity|催化活性catalytic antibody|催化性抗体，具有催化活性的抗体catalytic constant|催化常数[符号Kcat]catalytic core|催化核心catalytic mechanism|催化机理catalytic RNA|催化性RNAcatalytic selectivity|催化选择性catalytic site|催化部位catalytic subunit|催化亚基cataphoresis|阳离子电泳cataract|白内障catechin|儿茶素catechol|儿茶酚，邻苯二酚catecholamine|儿茶酚胺catecholamine hormones|儿茶酚胺类激素catecholaminergic recptor|儿茶酚胺能受体catenane|连环（体），连锁，链条[如DNA连环体]；索烃catenating|连环，连接catenation|连环，连锁，成链catenin|连环蛋白[一类细胞骨架蛋白，分alfa/beta/gama三种] catharanthus alkaloid|长春花属生物碱cathepsin|组织蛋白酶[分为A、B、C、D、E…H、L等多种]catheter|导管cathode layer enrichment method|阴极区富集法cathode ray polarograph|阴极射线极谱仪cation acid|阳离子酸cationic acid|阳离子酸cationic catalyst|正离子催化剂cationic detergent|阳离子（型）去污剂cationic initiator|正离子引发剂cationic polymerization|正离子聚合，阳离子聚合 cationic surfactant|阳离子（型）表面活性剂cationization|阳离子化cauliflower mosaic virus|花椰菜花叶病毒caulimovirus|花椰菜花叶病毒组caulobacteria|柄病毒Cavendish laboratory|（英国）卡文迪什实验室caveola|小窝，小凹caveolae|(复）小窝，小凹caveolin|小窝蛋白cavitation|空腔化（作用）cavity|沟槽，模槽，空腔dammarane|达玛烷dammarane type|达玛烷型Dane particle|丹氏粒[乙型肝炎病毒的完整毒粒]dansyl|丹（磺）酰，1-二甲氨基萘-5-磺酰dansyl chloride|丹磺酰氯dansyl method|丹磺酰法dantrolene|硝苯呋海因[肌肉松弛剂]dark current|暗电流dark field|暗视野，暗视场dark field microscope|暗视野显微镜，暗视场显微镜 dark field microscopy|暗视野显微术，暗视场显微术 dark reaction|暗反应dark repair|暗修复dark respiration|暗呼吸dark room|暗室，暗房dark seed|需暗种子data accumulation|数据积累data acquisition|数据获取data analysis|数据分析data bank|数据库data base|数据库data handling|数据处理data logger|数据记录器data logging|数据记录data output|数据输出data processing|数据处理data recording|数据记录dauermodification|持续饰变daughter cell|子代细胞daughter chromatid|子染色单体daughter chromosome|子染色体daughter colony|子菌落[由原生菌落续发生长的小菌落]daunomycin|道诺霉素daunorubicin|道诺红菌素de novo sequencing|从头测序de novo synthesis|从头合成deactivation|去活化（作用），失活（作用），钝化deacylated tRNA|脱酰tRNAdead time|死时间dead volume|死体积deadenylation|脱腺苷化DEAE Sephacel|[商]DEAE-葡聚糖纤维素，二乙氨乙基葡聚糖纤维素 dealkylation|脱烷基化deaminase|脱氨酶deamination|脱氨（基）death phase|死亡期[如见于细胞生长曲线]death point|死点deblocking|去封闭debranching enzyme|脱支酶，支链淀粉酶debris|碎片，残渣decahedron|十面体decane|癸烷decantation|倾析decanting|倾析decapacitation|去（获）能decarboxylase|脱羧酶decarboxylation|脱羧（作用）decay|原因不明腐败decay accelerating factor|衰变加速因子decay constant|衰变常数deceleration phase|减速期[如见于细胞生长曲线]dechlorination|脱氯作用deciduous leaf|落叶decline phase|[细胞生长曲线的]衰亡期decoagulant|抗凝剂decoding|译码，解码decomposer|分解者[可指具有分解动植物残体或其排泄物能力的微生物] decompression|降压，减压decondensation|解凝（聚）decontaminant|净化剂，去污剂decontaminating agent|净化剂，去污剂decontamination|净化，去污decorin|核心蛋白聚糖[一种基质蛋白聚糖，又称为PG-40]dedifferentiation|去分化，脱分化deep colony|深层菌落deep etching|深度蚀刻deep jet fermentor|深部喷注发酵罐deep refrigeration|深度冷冻deep shaft system|深井系统[如用于污水处理]defasciculation factor|解束因子[取自水蛭，可破坏神经束]defective|缺损的，缺陷的defective interfering|缺损干扰defective interfering particle|缺损干扰颗粒，干扰缺损颗粒defective interfering RNA|缺损干扰RNAdefective interfering virus|缺损干扰病毒defective mutant|缺损突变体，缺陷突变型，缺陷突变株defective phage|缺损噬菌体，缺陷噬菌体defective virus|缺损病毒，缺陷病毒defense|防御，防卫defense peptide|防卫肽defense response|防御反应，防卫反应defensin|防卫素[动物细胞的内源性抗菌肽]deficiency|缺乏，缺损，缺陷deficient|缺少的，缺损的，缺陷的defined|确定的defined medium|确定成分培养基，已知成分培养液defintion|定义defoliating agent|脱叶剂defoliation|脱叶deformylase|去甲酰酶[见于原核细胞，作用于甲酰甲硫氨酸]degasser|脱气装置degassing|脱气，除气degeneracy|简并；简并性，简并度degenerate|简并的degenerate codon|简并密码子degenerate oligonucleotide|简并寡核苷酸degenerate primer|简并引物degenerate sequence|简并序列degeneration|退化，变性degenerin|退化蛋白[与某些感觉神经元的退化有关]deglycosylation|去糖基化degradable polymer|降解性高分子degradation|降解degranulation|脱（颗）粒（作用）degree of acidity|酸度degree of dominance|显性度degree of polymerization|聚合度degron|降解决定子[决定某一蛋白发生降解或部分降解的序列要素] deguelin|鱼藤素dehalogenation|脱卤（作用）dehardening|解除锻炼dehumidifier|除湿器dehydratase|脱水酶dehydrated medium|干燥培养基dehydration|脱水（作用）dehydroepiandrosterone|脱氢表雄酮dehydrogenase|脱氢酶dehydrogenation|脱氢（作用）dehydroluciferin|脱氢萤光素deionization|去离子（作用）deionized|去离子的deionized water|去离子水deionizing|去离子（处理）delayed early transcription|（延）迟早期转录[可特指病毒]delayed fluorescence|延迟荧光delayed heat|延迟热delayed hypersensitivity|延迟（型）超敏反应delayed ingeritance|延迟遗传delayed type hypersensitivity|迟发型超敏反应deletant|缺失体deletion|缺失deletion mapping|缺失定位，缺失作图deletion mutagenesis|缺失诱变deletion mutant|缺失突变体deletion mutantion|缺失突变deletional recombination|缺失重组delignification|脱木质化（作用）deliquescence|潮解delivery flask|分液瓶delocalized bond|离域键。

食品化学模拟题含参考答案一、单选题（共85题，每题1分，共85分）1.乳脂的主要脂肪酸是( )？A、棕榈酸、油酸和软脂酸B、棕榈酸、油酸和硬脂酸C、硬脂酸、亚油酸和棕榈酸D、硬脂酸、软脂酸和亚油酸正确答案：B2.( )属于胡萝卜素类色素A、番茄红素B、玉米黄素C、柑橘黄素D、辣椒红素正确答案：A3.海产动物油脂中含大量__( )___脂肪酸,富含维生素A和维生素D。

A、长链饱和B、短链饱和C、长链多不饱和D、短链不饱和正确答案：C4.美拉德反应不利的一面是导致氨基酸的损失,其中影响最大的人体必需氨基酸 ( )。

A、LysB、PheC、ValD、Leu正确答案：A答案解析：有害的：为许多食品带来不良的风味和色泽,或导致氨基酸的结构被破坏，特别是赖氨酸5.花青素的基本母核为( )?A、2-苯基苯并吡喃酮B、异戊二烯C、2-苯基苯并吡喃阳离子D、卟啉环正确答案：C6.能水解淀粉分子a-1,4糖苷键,不能水解a-1,6糖苷键，但能越过此键继续水解的淀粉酶是( ) 。

A、葡萄糖淀粉酶B、脱枝酶C、a-淀粉酶D、β-淀粉酶正确答案：C7.( )呈绿色A、硫代肌红蛋白B、氧合肌红蛋白C、高铁肌红蛋白D、氧化氮高铁肌红蛋白正确答案：A8.下列过程中可能为不可逆的是( )?A、H3PO4 在水中的电离B、Na2S 的水解C、蛋白质的盐析D、蛋白质的变性正确答案：D9.某食品的水分活度为0.88,将此食品放于相对湿度为92%的环境中,食品的重量会 ( )。

A、先减小后增大B、增大C、减小D、不变正确答案：B10.被称为食品中第七大营养素的是？A、油脂B、蔗糖C、膳食纤维D、葡萄糖正确答案：C答案解析：营养素：指那些能维持人体正常生长发育和新陈代谢所必需的物质。

如糖类，蛋白质，脂质，维生素，矿物质，水11.以下指标用于衡量某种香气物质对整个食品香气的贡献程度的是( )?A、差别阈值B、香气值C、绝对阈值D、阈值正确答案：B12.油脂氢化后( )？A、不饱和度提高B、熔点提高C、稳定性降低D、抗氧化性减弱正确答案：B答案解析：油脂的氢化油脂中的不饱和脂肪酸双键在催化剂(Pt、Pd、Ni 等)作用下，与氢发生加成反应，使油脂不饱和程度降低的过程。

薛常鲁，张鹏飞，白丽君，等. 豆血红蛋白在酿酒酵母中的异源表达[J]. 食品工业科技，2023，44（20）：101−107. doi:10.13386/j.issn1002-0306.2022120029XUE Changlu, ZHANG Pengfei, BAI Lijun, et al. Heterologous Expression of Leghemoglobin in Saccharomyces cerevisiae [J]. Science and Technology of Food Industry, 2023, 44(20): 101−107. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022120029· 生物工程 ·豆血红蛋白在酿酒酵母中的异源表达薛常鲁1,2，张鹏飞1,2，白丽君1,2，肖功年1,2，魏培莲1,2,*（1.浙江科技学院生物与化学工程学院，浙江杭州 310023；2.浙江省农产品化学与生物加工技术重点实验室，浙江杭州 310023）摘　要：豆血红蛋白是一种植物源血红蛋白，在植物蛋白肉加工中可作为重要的风味催化剂和着色剂，大大增加植物蛋白肉的拟真性。

为实现豆血红蛋白在食品级微生物中的异源表达，将携带豆血红蛋白LBC2基因的质粒PESC-TRP 转入酿酒酵母CEN.PK2-1C 中，通过添加kozak 序列促进其翻译，并进一步对启动子序列进行选择，以提高豆血红蛋白表达量。

对重组菌株进行发酵，用Western blot 分析LegH 蛋白表达水平。

结果显示，诱导型启动子GAL1,10较三种组成型启动子TEF1、ADH1、GAP 在表达LegH 能力上具有显著优势，较产量最低的ADH1启动子提升了3.93倍。

在组成型启动子中，TEF1启动子能力较优，是ADH1启动子的2.91倍，是GAP 启动子的1.2倍。

百合分子生物学研究进展徐雷锋摘要百合具有很高的观赏价值，是世界著名的切花之一。

本文从百合ＤＮＡ分子标记及其初步应用、转基因和百合基因克隆等3方面综述了国内外在百合分子物学方面所取得的研究进展，并对分子生物学在百合应用上存在的问题与前景进行了讨论。

关键词百合DNA分子标记基因克隆转基因分子生物学Advance in Study on Lilium Molecular BiologyAbstract Lilium has high ornamental value, is one of world famous cut-flowers. A review was given in this paper with advance in study on Lilium molecular biology , such as DNA molecular marker ,gene cloning gene engineering. The questions and prospect of molecular biology research on Lilium were also discussed．Key words Lilium DNA molecular marker gene cloning gene engineering molecular biology百合(Lilium)是百合科百合属多年生草本球根植物,是世界重要的鲜切花之一，有2 000 余年栽培历史，具有较高的经济和观赏价值。

加强百合育种具有很高的经济效益和社会效益。

长期以来其品质的改良都是依赖于传统的遗传育种方法。

植物分子生物学的迅速发展,为改良植物品质和性状提供了全新的途径,利用转基因的手段改良百合性状和品质变得切实可行[1]。

目前，在百合分子生物学方面的研究主要包括百合DNA分子标记的研究和应用、利用转基因技术育种及百合相关的基因克隆等。

徐州2024年小学3年级英语第一单元期中试卷考试时间：100分钟（总分:140）A卷考试人：_________题号一二三四五总分得分一、综合题(共计100题)1、填空题：A ferret can scurry through tight ______ (空间).2、What sweet food is made from cocoa beans?A. CandyB. ChocolateC. CakeD. Ice cream答案:B3、听力题：The ______ is home to many fish.4、What do you call a place where you can buy food?A. LibraryB. Grocery storeC. MallD. Zoo答案:B5、听力题：We like to go _____ (钓鱼).6、填空题：My __________ (玩具名) is made of __________ (材料).7、What is the name of the story about a boy who never grew up?A. Alice in WonderlandB. Peter PanC. The Little MermaidD. Beauty and the Beast答案:B8、听力题：He is ________ (running) in the race.9、填空题：My ________ (老师) encourages us to do our best in school.10、What is the name of the famous explorer who sailed the ocean blue in 1492?A. Christopher ColumbusB. Ferdinand MagellanC. Vasco da GamaD. Hernán Cortés答案: A11、填空题：I have a toy _______ that can change colors.12、听力题：I like to watch ___. (movies)13、What is the capital of Egypt?A. CairoB. AlexandriaC. GizaD. Luxor答案: A14、填空题：My grandma makes the best ________ (汤) ever.15、听力题：A chemical formula shows the number and types of _____ in a compound.16、填空题：A _____ (草甸) is a grassy area with wildflowers.17、填空题：古代的________ (historians) 通过研究遗留物来了解过去。

PH4of Petunia Is an R2R3MYB Protein That Activates Vacuolar Acidification through Interactions withBasic-Helix-Loop-Helix Transcription Factors of the Anthocyanin Pathway WFrancesca Quattrocchio,1Walter Verweij,1Arthur Kroon,Cornelis Spelt,Joseph Mol,and Ronald Koes2Institute for Molecular and Cellular Biology,Vrije Universiteit,1081HV Amsterdam,The NetherlandsThe Petunia hybrida genes ANTHOCYANIN1(AN1)and AN2encode transcription factors with a basic-helix-loop-helix (BHLH)and a MYB domain,respectively,that are required for anthocyanin synthesis and acidification of the vacuole in petal cells.Mutation of PH4results in a bluerflower color,increased pH of petal extracts,and,in certain genetic backgrounds,the disappearance of anthocyanins and fading of theflower color.PH4encodes a MYB domain protein that is expressed in the petal epidermis and that can interact,like AN2,with AN1and the related BHLH protein JAF13in yeast two-hybrid assays. Mutation of PH4has little or no effect on the expression of structural anthocyanin genes but strongly downregulates the expression of CAC16.5,encoding a protease-like protein of unknown biological function.Constitutive expression of PH4 and AN1in transgenic plants is sufficient to activate CAC16.5ectopically.Together with the previousfinding that AN1 domains required for anthocyanin synthesis and vacuolar acidification can be partially separated,this suggests that AN1 activates different pathways through interactions with distinct MYB proteins.INTRODUCTIONIn plants,the vacuole occupies a large part(up to90%)of the cell volume and is important for a variety of physiological processes, such as pH homeostasis,osmoregulation,ion transport,and stor-age of metabolites.Moreover,it plays an important role in cell growth,because the enlargement of a cell is mostly attributable to an increase in the volume of the vacuole rather than of the cytoplasm(reviewed in Taiz,1992;Maeshima,2001;Gaxiola et al., 2002).The lumen of vacuoles is acidic compared with the cytoplasm, and in some cells(e.g.,in lemon[Citrus limon]fruit)it can reach pH values as low as1.Among the most abundant proteins on the vacuolar membrane are vacuolar ATPase(v-ATPase)and pyrophosphatase proton pumps(Szponarski et al.,2004)that transport protons from the cytoplasm into the vacuole,thereby contributing to the acidification of the vacuolar lumen.The resulting electrochemical gradient across the vacuolar mem-brane is the driving force for the transport of a variety of compounds(ions,sugars)via secondary symport and antiport transporters and channels(reviewed in Taiz,1992;Maeshima, 2001;Gaxiola et al.,2002).In most species,the coloration offlowers and fruits results from the accumulation offlavonoid pigments(anthocyanins)in the vacuoles of(sub)epidermal cells.Because the absorption spectrum of anthocyanins depends on the pH of their environ-ment(de Vlaming et al.,1983),the color of a tissue depends in part on the pH of the vacuolar lumen,thus makingflower color a convenient and reliable reporter to monitor alterations in vacuolar pH(Yoshida et al.,1995,2003)In morning glory(Ipomoea tricolor)petals,the vacuolar pH is relatively low when theflower bud opens,resulting in a red color, but upon further maturation,the vacuolar pH increases and the petals acquire a strong blue color(Yoshida et al.,1995).This color change and the increase of vacuolar pH require a putative Naþ/Hþexchanger encoded by the PURPLE gene(Fukada-Tanaka et al.,2000).Most likely,PURPLE transports sodium ions into and protons out of the vacuole,resulting in a less acidic vacuole and a bluer color.Petunia hybridaflowers normally have a lower pH than Ipo-moeaflowers,and the color of wild-typeflowers stays on the reddish(low pH)side of the color spectrum.By genetic analyses, seven loci(named PH1to PH7)have been identified that,when mutated,cause a more bluishflower color and an increase in the pH of crude petal extracts(Wiering,1974;de Vlaming et al.,1983; van Houwelingen et al.,1998),suggesting that these genes are required for acidification of the vacuole(de Vlaming et al.,1983). Mutations in the genes ANTHOCYANIN1(AN1),AN2,and AN11 cause,besides the loss of anthocyanin pigments,an increased pH of petal extracts.That this pH shift is at least in part attributable to an increased pH of the vacuolar lumen was evident from the bluishflower color specified by particular an1 alleles(formerly known as ph6)that lost the capacity to activate the vacuolar acidification function but could still drive anthocy-anin synthesis(Spelt et al.,2002).1These authors contributed equally to this work.2To whom correspondence should be addressed.E-mail ronald.koes@falw.vu.nl;fax31-20-5987155.The authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors()are:FrancescaQuattrocchio(francesca.quattrocchio@falw.vu.nl)and Ronald Koes(ronald.koes@falw.vu.nl).W Online version contains Web-only data.Article,publication date,and citation information can be found at/cgi/doi/10.1105/tpc.105.034041.The Plant Cell,Vol.18,1274–1291,May2006,ª2006American Society of Plant BiologistsAN1and AN11are required for transcriptional activation of a subset of structural anthocyanin genes,encoding the enzymes of the pathway,in all pigmented tissues(Quattrocchio et al., 1993)and encode a basic-helix-loop-helix(BHLH)transcription factor and a WD40protein,respectively(de Vetten et al.,1997; Spelt et al.,2000).AN2encodes a MYB-type transcription factor whose function appears to be(partially)redundant,because it is expressed only in petals and not in other pigmented tissues (Quattrocchio et al.,1999).Moreover,even in the an2null mutant, pigmentation of the petals is reduced,but not fully blocked,and the pH shift in an2petal homogenates is smaller than that in an1 or an11petals(Quattrocchio et al.,1993;Spelt et al.,2002).In addition,AN1and AN11play a role in the development of epidermal cells in the seed coat(Spelt et al.,2002).The anthocyanin pathway has been shown to be activated by similar MYB,BHLH,and WD40proteins in a wide variety of species,indicating that this function is well conserved(reviewed in Winkel-Shirley,2001;Koes et al.,2005).Several studies revealed that these MYB,BHLH,and WD40proteins could interact physically,indicating that they may operate in one transcription activation pathway and may activate their target genes as a(ternary)complex(Goff et al.,1992;Zhang et al.,2003; Baudry et al.,2004;Kroon,2004;Zimmermann et al.,2004). Besides petunia,Arabidopsis thaliana is the only other species in which these activators are known to control multiple processes. In Arabidopsis,the WD40protein TRANSPARENT TESTA GLABRA1(TTG1)(Walker et al.,1999)is required for the syn-thesis of anthocyanin and proanthocyanidin pigments,the pro-duction of seed mucilage,and the development of trichomes on stems and leaves(Koornneef,1981),whereas in roots it sup-presses the formation of root hairs in certain cells(Galway et al., 1994).During the regulation of anthocyanin synthesis,trichome development,and nonhair development in the root,TTG1coop-erates with two functionally equivalent BHLH proteins encoded by GLABRA3(GL3)and ENHANCER OF GLABRA3(EGL3) (Payne et al.,2000;Bernhardt et al.,2003;Ramsay et al.,2003; Zhang et al.,2003),whereas synthesis of proanthocyanidins in the seed coat depends on a distinct BHLH protein encoded by TRANSPARENT TESTA8(TT8)(Nesi et al.,2000).TTG1and GL3/ EGL3or TT8activate these distinct processes by associating with distinct MYB partners.During trichome development,they interact with R2R3MYBs encoded by GLABROUS1(GL1)or MYB23(Oppenheimer et al.,1991;Kirik et al.,2001,2005),and for the development of nonhair cells in the root they interact with a functionally equivalent MYB encoded by WEREWOLF(WER) (Lee and Schiefelbein,2001),whereas for anthocyanin synthesis, their MYB partner is probably PRODUCTION OF ANTHOCYA-NIN PIGMENT1(PAP1)or PAP2(Borevitz et al.,2000;Baudry et al.,2004;Zimmermann et al.,2004).The involvement of anthocyanin regulators in trichome and root hair development is seen only in Arabidopsis and not in other species for which regulatory anthocyanin mutants have been isolated,such as Antirrhinum majus,maize(Zea mays),or petunia. Nevertheless,RED(R)(BHLH)and PALE ALEURONE COLOR (PAC1)(WD40)from maize can restore the hair defects in Arabi-dopsis ttg1mutants(Lloyd et al.,1992;Carey et al.,2004), indicating that the functional diversification did not depend on alterations in these WD40and BHLH proteins but on the diver-gence of their MYB partners and/or their downstream target genes.Whether these MYB,BHLH,and WD40proteins also activate vacuolar acidification in species other than petunia is unclear.To unravel the mechanisms and the biochemical pathways by which AN1,AN11,and AN2control vacuolar pH,we set out to isolate the genetically defined PH loci by transposon-tagging strategies and the downstream structural genes by RNA profiling methods.Here,we describe the isolation and molecular char-acterization of PH4.We show that PH4is a member of the MYB family of transcription factors that is expressed in the petal epidermis and that can interact physically with AN1and JAF13,a functionally related BHLH protein that can also drive anthocyanin synthesis(Quattrocchio et al.,1998).Because PH4plays no apparent role in anthocyanin synthesis,we propose that AN1 activates anthocyanin synthesis and vacuolar acidification through interactions with distinct MYB proteins.RESULTSMutations That Alter pH in PetalsThe petunia line R27contains functional alleles for all of the regulatory anthocyanin genes that color the petal(AN1,AN2,and AN11)but contains mutations in the structural genes HYDROX-YLATION AT FIVE(HF1)and HF2,both encoding FLAVONOID 3959HYDROXYLASE(Holton et al.,1993),and RHAMNOSYLA-TION AT THREE(RT),encoding ANTHOCYANIN RHAMNOSYL-TRANSFERASE(Kroon et al.,1994);consequently,the major anthocyanins synthesized are cyanidin derivatives(de Vlaming et al.,1984;Wiering and de Vlaming,1984)(Figure1).In addition, R27is mutant for FLAVONOL(FL),which strongly reduces flavonol synthesis(Figure1)and increases the accumulation of cyanidin derivatives(de Vlaming et al.,1984;Wiering and de Vlaming,1984)(Figure1).Consequently,theflowers of R27have a bright red color(Figure2A).The lines W138and W137derive from R27by dTPH1insertions in AN1and AN11,respectively (alleles an1-W138and an11-W137),and,consequently,bear whiteflowers with red or pink revertant spots(Doodeman et al., 1984a,1984b)(Figure2B).Among progeny of W138and W137,we found several new mutations affectingflower color(van Houwelingen et al.,1998; Spelt et al.,2002).In one class of mutants,the color of the AN1or AN11revertant spots(in an an1-W138or an11-W137back-ground)(Figure2C)or of the whole corolla(in an AN1or AN11 germinal revertant)had changed from red to purplish(Figures2D and2E).Subsequent complementation analyses showed that these mutations represented new alleles of PH2(allele ph2-A2414),PH3(allele ph3-V2068),and PH4(alleles ph4-V2166, ph4-B3021,ph4-X2052,and ph4-V2153)(see Supplemental Table1online).Yet another unstable ph4allele(ph4-X2377)was recovered from the Syngenta breeding program in a family segregating3:1for wild types with red petals and mutants with purplish petals with an occasional revertant red spot.The alleles ph4-V2166,ph4-V2153,an1-W138,an1-W225, and an11-W134all cause a similar increase in petal extract pH (Figure2F)(Spelt et al.,2002).To determine at which stage AN1,The PH4Gene of Petunia1275AN11,PH3,and PH4are active,we analyzed flowers of different developmental stages.Figure 2G shows that wild-type petals start to acidify around developmental stage 4,when the bud is about to open.In an1,ph3,and ph4mutants,this acidification is reduced but not blocked completely.This finding suggests that multiple vacuolar acidification pathways operate in petals,some of which are independent of AN1,AN11,PH4,and PH3.Analysis of double mutant flowers showed that the pH of an1ph4or an11ph4petal homogenates is not significantly different from that of any of the single mutants,suggesting that these genes operate in the same vacuolar acidification pathway (Figure 2F).To test whether alterations in pigment synthesis also contrib-uted to the color change in ph4petals,we analyzed anthocyanins in wild-type (R27)and ph4-V2153petals of closed buds (stage 4)by HPLC.Figure 2H shows that both genotypes accumulate a nearly identical mixture of anthocyanins.R27petals synthesize only small amounts of flavonol copigments (quercetin deriva-tives)as a result of its fl/flgenotype,and HPLC analysis did not reveal clear differences in the accumulation of these compounds in ph4petals (see Supplemental Figure 1online).Thus,the ph4mutation has,at least in the R27genetic background,little or no effect on the synthesis and modification of anthocyanins.Early genetic work had shown that a mutation in PH4or a closely linked gene triggers the complete fading of flower colorand the disappearance of anthocyanins after opening of the flower bud,if combined with a dominant allele at the FADING locus (Wiering,1974;de Vlaming et al.,1982).Anthocyanins with a 3RGac5G substitution pattern are particularly sensitive to fading,whereas the 3-glucosides (as in rt mutants like R27and derived lines)and the 3-rutinosides show little or no fading (de Vlaming et al.,1982).When we crossed the unstable ph4-V2166allele into a genetic background that allows the synthesis of 3RGac5G-substituted anthocyanins,the flowers displayed upon opening a blue-violet color and were dotted with red-violet spots and sectors resulting from reversions of ph4(Figure 2H).In the next days,the color of the blue-violet (ph4)cells faded to nearly white,whereas the red-violet (PH4)revertant sectors retained their color.Control crosses with isogenic PH4plants (lines R27and W138)yielded only progeny with evenly red-colored,nonfading corollas,whereas crosses with a stable reces-sive ph4-V2153parent gave only progeny with evenly colored blue-violet,fading corollas (ph4).This finding demonstrates that it is the mutation of PH4,and not that of a linked gene,that triggers fading.The an1-G621allele expresses a truncated AN1protein that can drive anthocyanin synthesis but not vacuolar acidification (Spelt et al.,2002).When crossed into a background that allows the synthesis of 3RGac5G-substituted anthocyanins,the an1-G621allele also triggered fading (Figure 2I),and similar results were obtained with the an1-B3196allele in distinct crosses.Strikingly,the unstable ph2-A2414(Figure 2J)and ph5(data not shown)alleles did not induce fading when crossed into an FA background synthesizing 3RGAac5G-substituted anthocyanins,suggesting that fading in an1and ph4petals is not triggered by the upregulation of vacuolar pH alone and may depend on some other vacuolar defect.Isolation of PH4Using Transposon-Tagged AllelesBecause most mutant alleles that arose in W138were attribut-able to insertions of a 284-bp dTPH1transposon (Souer et al.,1996;de Vetten et al.,1997;van Houwelingen et al.,1998;Quattrocchio et al.,1999;Spelt et al.,2000;Toben˜a-Santamaria et al.,2002;Vandenbussche et al.,2003),we anticipated that the unstable alleles ph4-V2166,ph4-X2052,and ph4-B3021might also harbor insertions of dTPH1.To identify the dTPH1copy in ph4-B3021,we analyzed dTPH1flanking sequences in mutant and wild-type plants by transpo-son display (van den Broek et al.,1998)and found a 98-bp fragment that was amplified from the three ph4-B3021plants analyzed but not from the two wild-type plants homozygous for the parental PH4allele (Figure 3A).Subsequent isolation,clon-ing,and sequencing of this fragment showed that it contained 66bp of dTPH1sequence and 32bp of flanking sequence that was identical to a cDNA clone (MYBa )that had been isolated inde-pendently by yeast two-hybrid screen using an AN1bait (see below).PCR experiments with gene-specific primers showed that in ph4-B3021and ph4-V2166plants,MYBa was disrupted by a 284-bp dTPH1insertion in the 59and 39ends of the protein-coding region,respectively (Figure 3B).Analysis of PH4progeny plants that originated from germinal reversions ofph4-B3021Figure 1.Genetic Control of the Anthocyanin Pathway in Petunia Petals.The main anthocyanins and flavonols (gray boxes)are synthesized via a branched pathway.Genes that control distinct steps are indicated in boldface italics.Malonyl-CoA and p -coumaroyl-CoA are converted by the enzymes CHALCONE SYNTHASE (expressed from two distinct genes,CHSa and CHSj ),CHALCONE ISOMERASE (encoded by CHIa ),and FLAVONOID 3HYDROXYLASE (encoded by AN3)into dihydro-kaempferol (dHK).Hydroxylation of dHK on the 39or the 39plus 59position is controlled by HT (for HYDROXYLATION AT THREE )and the homologs HF1and HF2(for HYDROXYLATION AT FIVE )to yield dihydroquercitin (dHQ)and dihydromyricitin (dHM),respectively.The simplest anthocyanins in petunia flowers are 3-glucosides (3G).Through the action of RT (for RHAMNOSYLATION AT FIVE )and AAT (for ANTHOCYANIN-RUTINOSIDE ACYLTRANSFERASE )and others,antho-cyanins with a 3-rutinoside p -coumaroyl-5-glucoside (3RGac5G)substi-tution pattern are generated.The colors displayed by the various anthocyanins (in a flPH background)are shown in parentheses.1276The Plant CellFigure 2.Phenotypic Analysis of Flower Pigmentation Mutants.(A)Flower of the wild-type line R27(AN1,AN11,PH4).(B)Flower of the line W137(an11-W137,PH4)showing AN11-R revertant sectors,resulting from excisions of dTPH1,on a white (an11-W137)background.(C)Flower homozygous for the unstable alleles an11-W137and ph4-B3021.Reversion of an11-W137results in spots with a purplish color rather than red,as a result of the ph4-B3021mutation.Somatic reversions of ph4-B3021can be seen occasionally as red (PH4-R )spots within the purplish ph4-B3021sectors (inset).(D)Flower of line R154harboring the unstable ph4-V2166allele in an AN1-R AN11-R background.Note the red PH4revertant sectors on the purplish ph4background.(E)Flower of line R149harboring the stable recessive ph4-V2153allele in an AN1-R AN11-R genetic background.The PH4Gene of Petunia 1277(Figure3C)and ph4-V2166(Figure3D)showed that reversion of the ph4phenotype correlated with excision of dTPH1from MYBa.DNA analyses of plants containing ph4-X2052or ph4-C3540, either in homozygous or heterozygous condition,showed that these mutants also contained dTPH1insertions in MYBa, whereas the stable recessive ph4-V2153mutants contained an ;4-kb TPH6insertion that is nearly identical to the TPH6 element found in the alleles an1-W17and an1-W219(Spelt et al.,2002)(Figure3B).Plants harboring the weakly unstable ph4-X2377allele contained a177-bp insertion29bp down-stream from the translation start site.The insertion isflanked by an8-bp target site duplication and has a12-bp terminal inverted repeat with similarity to the terminal inverted repeats of hAT family transposons,including dTPH1and Activator from maize. Because the internal sequences of the insertion show no simi-larity to other transposons and are too short to encode a transposase,it apparently represents a new(sub)family of non-autonomous petunia transposons that we named dTPH7.The ph4lines V64and M60both contain a dTPH1insertion in MYBa in exactly the same position(Figure3B).In summary,these data show that mutations in the isolated gene(MYBa)fully correlate with the phenotype conferred by the ph4mutant,implying that PH4is identical to MYBa.Below,we refer to this gene as PH4.PH4Encodes a MYB Domain ProteinSequence analysis of a full-size PH4cDNA and the correspond-ing genomic region showed that the PH4mRNA is encoded by two exons separated by a715-bp intron(Figure3B).The cDNA contains a single large open reading frame encoding a291–amino acid protein.Database searches showed that a103–amino acid domain,located near the N terminus,is conserved in a large number of plant and animal proteins all belonging to the MYB family of transcription factors(Figures4A and4B).The MYB domain consists of one,two,or three helix-helix-turn-helix motifs that potentially bind DNA.PH4,like the majority of plant MYBs(Stracke et al.,2001),contains only the repeats R2and R3 (Figure4B).R2R3MYB genes constitute a large family of;125genes in Arabidopsis and>85genes in rice(Oryza sativa)and have been categorized into42subgroups based on similarities in the encoded proteins and intron–exon structures(Stracke et al., 2001;Jiang et al.,2004).Generally,sequence similarity between MYB proteins is restricted to the N-terminal MYB domain,but19 subgroups of R2R3MYBs share some conserved motifs in their C-terminal domains that may indicate similarities in function (Stracke et al.,2001;Jiang et al.,2004).PH4is most similar to the R2R3MYB proteins BNLGHi233 from upland cotton(Gossypium hirsutum),MYBCS1and MYB5 from grape(Vitis vinifera),MYB5from Arabidopsis,and MYB4from rice(Figure4A).The clustering of R2R3MYBs in Figure4is in good agreement with previous analyses based on a much larger set of MYBs(Stracke et al.,2001;Jiang et al.,2004) and extends the data at some points.For example,grape MYBA1and tomato(Solanum lycopersicum)ANT1,recently identified regulators of the anthocyanin pathway in grape (Kobayashi et al.,2004)and tomato(Mathews et al.,2003), respectively,cluster with known members(AN2of petunia,PAP1 and PAP2of Arabidopsis)of subgroups6and N9as defined by Stracke et al.(2001)and Jiang et al.(2004),respectively.Curi-ously,C1and Pl,regulators of the anthocyanin pathway in maize, cluster in a distinct group(5/N8)together with TT2,a regulator of proanthocyanidin and anthocyanin synthesis in Arabidopsis (Shirley et al.,1995),again consistent with previous results. ODO1,an activator of the synthesis of phenylpropanoid vol-atiles in petuniaflowers,clusters with Arabidopsis MYB42and MYB85,similar to previous results(Verdonk et al.,2005),but it does not contain the conserved motif found in the C termini of MYB42and MYB85(Jiang et al.,2004).Figure4B shows that PH4,Arabidopsis MYB5,grape MYBCS1 and MYB5,cotton BNLGHi233,and rice MYB4share two con-served motifs in their C-terminal domains.Such motifs have been used as signatures for the classification of subgroups(Stracke et al.,2001;Jiang et al.,2004).Given that these C-terminal motifs in PH4and related R2R3MYBs are much better conserved than those in proteins of subgroup N9,we propose that these MYBs form a new subgroup that we tentatively named G20,in accor-dance with the numbering of Jiang et al.(2004).This classificationFigure2.(continued).(F)pH values(means6SD;n¼7)of petal homogenates of different genotypes in the R27genetic background.Note that the absolute pH values that are measured show some variation in time,possibly in response to variable environmental conditions in the greenhouse,although the differences between mutants and the wild type are virtually constant.(G)Petal homogenate pH(means6SD;n¼5)duringflower development in wild-type,an1,ph3,and ph4petals.Developmental stages were defined as follows:stage2,30-to35-mm buds;stage3,35-to45-mm buds;stage4,buds of maximum size(45to50mm);stage5,unfoldingflowers;stage6,fully openflowers around anthesis.(H)HPLC analysis of methanol-extractable anthocyanins in petals of stage4flower buds from lines R27(PH4)and R149(ph4-V2153).The arrows denote the retention time of cyanidin3-glucoside.(I)Phenotype of ph4-V2166/ph4-V64flowers in a background that allows the synthesis of3RGac5G-substituted anthocyanins,resulting from the cross R1493V64,showing subsequent stages(from left to right)offlower color fading.Note that the blue-violet ph4cells fade,whereas the red-violet PH4 revertant sectors(white arrows)do not.(J)Phenotype of an1-G621/an1-W138flowers in a background that synthesizes3RGac5G-substituted anthocyanins,showing subsequent stages(from left to right)offlower color fading.Note that mutant(an1-G621)tissues fade,whereas full AN1revertant sectors(mostly originating from excisions of dTPH1from an1-W138)do not fade.(K)Phenotype of a mature ph2-A2414flower(comparable to the rightmostflowers in[I]and[J])in a background(R1603V26)that synthesizes 3RGac5G-substituted anthocyanins.Note that neither the PH2tissue(red-violet sectors)nor the ph2tissue(blue-violet background)displays fading. 1278The Plant Cellis supported by the finding that both Arabidopsis MYB5and PH4contain only one intron (Li et al.,1996)(Figure 3),whereas the majority of R2R3MYBs contain two (Jiang et al.,2004).Yeast two-hybrid assays showed that Arabidopsis MYB5(Zimmermann et al.,2004)and PH4(see below)can interact physically with functionally similar BHLH proteins,consistent with the idea that they have similar functions.However,the expression pattern of MYB5(Li et al.,1996)seems quite differentfrom that of PH4(see below).Rice MYB4induces freezing and chilling tolerance when constitutively expressed in Arabidopsis (Vannini et al.,2004),and grape MYB5induces the synthesis of anthocyanins and proanthocyanidins when overexpressed in tobacco (Nicotiana tabacum )(Deluc et al.,2006).Definite proof for functional equivalence and/or orthology will require the swapping of genes between species and/or the identification of (direct)target genes (see Discussion).Expression of PH4and Mutant AllelesTo determine the expression pattern of PH4,we measured the amount of PH4mRNAs in different tissues of the wild-type line V30by RT-PCR.We used line V30because it contains functional alleles of all regulatory pigmentation genes (Koes et al.,1986),whereas R27is mutant for an4,a regulator of anthocyanin synthesis and AN1expression in anthers (Quattrocchio et al.,1993;Spelt et al.,2000).Figure 5A shows that PH4is relatively strongly expressed in the limb of the petal,whereas in the tube only weak PH4expression is detected.Possibly,the low amount of transcripts in the tube sample originated from cells near the border of the limb and the tube.The expression in the limb reaches a maximum at developmental stages 5to 6(Figure 5A),when the bud is opening,which correlates with the moment that pH differences were first seen between wild-type and ph4petal limb extracts (Figure 2E).Ovaries are the only other tissue besides petals in which we detected clear PH4expression.This organ also expresses AN1(Figure 5A)(Spelt et al.,2000),which directs the activation of the anthocyanin biosynthetic gene DFR ,but for unknown reasons this does not result in the accumulation of anthocyanin pigments (Huits et al.,1994).Anthers of V30are pigmented by anthocyanins and express AN1mRNA during early stages of development (stages 1to 3).However,no PH4transcripts were detected in this tissue.The same holds for the stigma and style.AN1is weakly expressed in sepals,leaves,and stems of V30,which correlates with the synthesis of low amounts of anthocyanin;PH4,however,is not expressed in these tissues.Roots are normally not pigmented and do not express PH4or AN1.To examine to what extent the transposon insertions affected the expression of PH4,we analyzed ph4mRNAs in petal limbs of different mutants by RNA gel blot analysis,RT-PCR (data not shown),and rapid amplification of 39cDNA ends (39RACE).Figure 5B shows that the amount of PH4transcripts in petal limbs homozygous for the ph4alleles X2052,B3021,C3540,and V2166is strongly reduced compared with that of the isogenic wild-type line R27.The same holds for PH4transcripts ex-pressed from the X2377allele compared with an isogenic rever-tant (ph4-X2377R ).Presumably,the dTPH1insertions in these alleles result in highly unstable mRNAs that are rapidly degraded.The small amount of wild-type-size PH4transcripts in these corollas presumably originates from cells in which dTPH1was excised from PH4.The insertions in ph4-V64and ph4-V2153cause the accumu-lation of short ph4transcripts.Cloning and sequencing of these products revealed that they resulted from polyadenylation within the dTPH1and TPH6sequences,respectively,andencodeFigure 3.Molecular Analysis of PH4.(A)Transposon display analysis of plants homozygous for the parental wild type (þ/þ)or the mutable ph4-B3021allele (m/m).The rightmost lane contains a radiolabeled 123-bp size marker.The arrow indicates a fragment derived from PH4.(B)Map of the PH4gene and mutant alleles.Boxes represent exons,and the thin line represents an intron.Protein-coding regions are indicated by double height,and the region encoding the R2and R3repeats of the MYB domain is filled in black.The open and closed circles represent the start and stop codons,respectively.The triangles indicate transposon insertions in the indicated alleles:the large open triangle represents TPH6,the mid-size closed triangles represent dTPH1,and the small open triangle represents dTPH7.(C)PCR analysis of plants harboring ph4-B3021and derived stabl e ph4alleles.þindicates the parental wild-type allele,m indicates the mutable ph4-B3021allele,R1indicates a derived revertant allele,and –indicates a stable recessive ph4allele.The primers used were 583and 1060(Table 1).(D)PCR analysis of plants harboring ph4-V2166and derived germinal revertant alleles.m represents the mutable ph4-V2166allele,and R1,R2,and R3represent three independently isolated revertant alleles.The primers used were 690and 582.The PH4Gene of Petunia 1279。

专利名称：HETEROGENOUS/HOMOGENEOUSCOPOLYMER发明人：BROWN, Stephen, John,DOBBIN,Christopher, John, Brooke,ELSTON, Clayton,Trevor,AUBEE, Norman, Dorien,Joseph,ARNOULD, Gilbert,Alexander,MARSHALL, Sarah,KALE, Lawrence,Thomas,WEBER, Mark申请号：CA2003001585申请日：20031020公开号：WO04/041927P1公开日：20040521专利内容由知识产权出版社提供摘要：Novel polyethylene copolymer compositions prepared with a homogeneous catalyst system are characterized by having a unique high molecular weight, low comonomer (high density) fraction. These heterogeneous/homogeneous compositions may be prepared using a solution polymerization process in which the polymerization reactor contains a gradient in temperature, catalyst concentration or monomer concentration. The heterogeneous/homogeneous compositions of this invention are easily processed into films having excellent tear strengths and low hexane extractables.申请人：BROWN, Stephen, John,DOBBIN, Christopher, John, Brooke,ELSTON, Clayton, Trevor,AUBEE, Norman, Dorien, Joseph,ARNOULD, Gilbert, Alexander,MARSHALL, Sarah,KALE, Lawrence, Thomas,WEBER, Mark地址：CH,CA,CA,CA,CA,CA,CA,CA,CA国籍：CH,CA,CA,CA,CA,CA,CA,CA,CA 代理机构：CHISHOLM, Scott, P.更多信息请下载全文后查看。

大白菜钙网蛋白1b-BrCRT1b过表达诱导拟南芥器官再生的起始Over-expression of Chinese cabbage calreticulin 1b, BrCRT1b,induces initiation of organogenesis in ArabidopsisZheng-Lu Jin4, Sun Hwa Ryu3, Joon Ki Hong1, Chi Eun Song1, Chan Ju Lim1, Young Ju Choi2, Kyun Oh Lee1,Woo Sik Chung1, Sang Yeol Lee1 and Chae Oh Lim1*1Environmental Biotechnology National Core Research Center and PMBBRC, Gyeongsang National University, Jinju 660-701, Korea2Department of Food and Nutrition, Silla University, Busan 617-736, Korea3Korea Forest Research Institute, Seoul 130-712, Korea4Department of Life Science, Shangqiu University, Shangqiu 470-000, ChinaReceved May 8, 2007 ; accepted September 15, 2007摘要内质网内腔上CRT是一个主要的Ca2+结合蛋白。

已经从大白菜的花蕾中分离出来了蛋白表现出与45Ca2+结合和分子一个编码CRT的cDNA，BrCRT1b。

重组体BrCRT1b21-418伴侣的活性。

BrCRT1b转录产物在根，幼嫩叶片和花中得到积累。

在较低浓度的生长激素或细胞分裂素条件下，组成型BrCRT1b的过表达促进健康茎秆的生长和根的形成。

黑曲霉GOD基因在枯草芽孢杆菌中的表达于思颖程鹏张静博曹平华李元晓马彦博李旺*河南科技大学动物科技学院，河南洛阳471023摘要通过初筛和复筛，筛选出高产GOD的黑曲霉菌种（Aspergillus niger），提取黑曲霉的基因组DNA，以此为模板扩增GOD基因，将目的基因密码子优化后，插入表达载体pT7M，导入枯草芽孢杆菌7024E中，通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）检测表达情况，旨在研究黑曲霉葡萄糖氧化酶（GOD）基因在枯草芽孢杆菌中的表达。

试验结果显示，成功筛选出1株高产GOD的黑曲霉菌株（P-1），从其基因组中克隆了GOD基因，该基因序列全长1818bp，编码605个氨基酸；构建了表达载体并转化至宿主，构建重组GOD基因枯草芽孢杆菌，2%木糖诱导发酵，SDS-PAGE和Western-blot检测到重组枯草芽孢杆菌能够表达GOD。

表明GOD基因在野生型枯草芽孢杆菌中获得了成功表达。

关键词葡萄糖氧化酶；黑曲霉；基因重组；枯草芽孢杆菌葡萄糖氧化酶（glucose oxidase，GOD）是一类含有黄素腺嘌呤二核苷酸（flavine adenine dinucleo⁃tide，FAD）的二聚体蛋白酶，能够特异性催化β-D-葡萄糖被氧化成葡萄糖酸和过氧化氢[1]。

GOD基于其安全无毒等特性广泛地应用于饲料添加剂、食品、制药和化学等领域[2-7]。

枯草芽孢杆菌（B.subtilis）作为一种革兰氏阳性菌，已被美国食品药品监督管理局（FDA）评为生物安全（Generally regarded as safe，GRAS）菌株[8]，具有易于培养、繁殖速度快、遗传背景清晰等特点。

与大肠杆菌相比，其具有优异的蛋白分泌能力，方便下游表达产物的分离；同时，B.subtilis具有良好的发酵工艺技术，这为目的产物进一步扩大生产提供了基础。

B.subtilis表达系统已表达了多种产物并广泛应用于制药、食品和饲料等行业中[9-13]。