The CID1cyclin C-like gene is important for plant infection in Fusarium graminearum
Xiaoying Zhou,Christina Heyer,Yoon-E Choi,Rahim Mehrabi,Jin-Rong Xu *
Department of Botany and Plant Pathology,Purdue University,West Lafayette,IN 47907,United States
a r t i c l e i n f o Article history:
Received 3May 2009
Accepted 5November 2009
Available online 10November 2009Keywords:Mycotoxin
Deoxynivalenol Pathogenicity Gibberella zeae
a b s t r a c t
Head blight or scab caused by Fusarium graminearum is a destructive disease of wheat and barley.The pathogen can cause severe yield losses and contaminates infested kernels with harmful mycotoxins.In this study,we characterized the CID1gene in F.graminearum that is an ortholog of the Fusarium verticil-loides FCC1and yeast UME3genes.The protein encoded by CID1has typical structural features of C-type cyclins.Deletion of CID1resulted in a reduction in conidiation and vegetative growth but an increase in pigmentation.The D cid1mutant was female sterile but could outcross as a male.It was signi?cantly reduced in DON production and virulence on wheat heads and corn stalks.Only about 50%of inoculated spikelets developed scab symptoms and scab disease rarely extended to nearby ?orets,suggesting that the D cid1mutant was defective in colonizing and spreading in wheat heads.Deletion of CID1resulted in reduced expression levels of TRI5and TRI101but increased PKS12expression.When expressed in F.ver-ticillioides ,the CID1gene complemented the defects of the D fcc1mutant in conidiation,hyphal growth,and fumonisin production.Our data indicate that the CID1C-type cyclin gene plays multiple roles in the regulation of vegetative growth,sexual development,conidiation,DON production,and pathogenicity.
ó2009Elsevier Inc.All rights reserved.
1.Introduction
Fusarium graminearum (Schwabe)infects a broad range of crop plants including wheat,barley,and maize (Bai and Shaner,2004;Goswami and Kistler,2004).In addition to yield losses,infested cereals are often contaminated with mycotoxins,such as deoxyni-valenol (DON),that are harmful to humans and animals (Desjar-dins et al.,1996;McMullen et al.,1997).DON is a potent inhibitor of protein synthesis in eukaryotic organisms and causes a variety of acute and chronic toxicoses.Ascospores that are forc-ibly discharged from mature perithecia are the primary inoculum for the infection of ?owering wheat heads in the spring (McMullen et al.,1997;Trail et al.,2005).Infection of wheat can occur from the beginning of anthesis until the dough stage of kernel develop-ment,and is initiated when ascospores or conidia are deposited on or inside ?owering spikelets.The fungus penetrates through sto-mata or ?oral bracts (Pritsch et al.,2000).From the infected ?oret,the fungus can spread up and down the spike and cause severe damage.
Molecular mechanisms underlying pathogenesis in F.graminea-rum are complex and not fully understood.In addition to genes re-lated to well conserved signal transduction pathways (Hou et al.,2002;Jenczmionka et al.,2003;Jenczmionka and Schafer,2005;Urban et al.,2003;Yu et al.,2008),a number of pathogenicity
genes with diverse biological or biochemical functions have been characterized (Han et al.,2007;Lee et al.,2005;Lu et al.,2003;Seong et al.,2005,2006;Shim et al.,2006).The ?rst pathogenicity factor most thoroughly characterized by molecular studies in F.graminearum is the trichodiene synthase gene TRI5(Desjardins et al.,2000;Proctor et al.,1995).Although trichothecene mycotox-ins are not necessary for the initial infection,they are important for the spread of the fungus within colonized spikes (Bai et al.,2002;Desjardins et al.,2000;Harris et al.,1999;Proctor et al.,1995).In the past decade,the trichothecene biosynthetic gene cluster and biosynthesis pathway have been extensively studied.Most genes encoding enzymes for metabolic steps in DON biosynthesis have been characterized,including TRI3,TRI5,TRI6,TRI7,TRI10,and TRI101(Kimura et al.,2003,2007;Lee et al.,2002).TRI6and TRI10are pathway-speci?c transcription factors for the TRI genes (Hohn et al.,1999;Seong et al.,2009;Tag et al.,2001).However,global regulatory systems that control the expression of TRI genes and DON synthesis in F.graminearum are not well characterized.In F.verticilloides ,a close relative of F.graminearum ,the FCC1C-type cyclin-like gene was identi?ed by random insertional muta-genesis to be important for fumonisin biosynthesis (Shim and Woloshuk,2001).On corn kernels and de?ned minimal medium at pH 6,the fcc1deletion mutant was reduced in conidiation and blocked in fumonisin production.Further studies indicated that the Fck1cyclin-dependent kinase directly interacts with Fcc1.Deletion of FCK1also lead to a signi?cant reduction in conidiation and fumonisin production on corn kernels (Bluhm and Woloshuk,
1087-1845/$-see front matter ó2009Elsevier Inc.All rights reserved.doi:10.1016/j.fgb.2009.11.001
*Corresponding author.Fax:+17654960363.E-mail address:jinrong@https://www.doczj.com/doc/439986144.html, (J.-R.Xu).
Fungal Genetics and Biology 47(2010)
143–151
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i
2006).The Fcc1-Fck1system appears to play an important regula-tory role in conidiation and mycotoxin biosynthesis in F.verticillio-ides.A number of genes that are likely regulated by Fcc1have been identi?ed,including FZR1that encodes a zinc?nger transcription factor(Flaherty and Woloshuk,2004;Pirttila et al.,2004).
In this study initiated by a summer research student,we iden-ti?ed the F.verticilloides Fcc1ortholog in F.graminearum named CID1(for Cyclin C-like gene required for infection and DON produc-tion1).Deletion of CID1resulted in a reduction in conidiation but an increase in pigmentation.The D cid1mutant was reduced in vegetative growth and DON production.It was defective in spread-ing in wheat heads and nearly non-pathogenic.When expressed in F.verticillioides,the CID1gene complemented the defects of the D fcc1mutant in conidiation,hyphal growth,and fumonisin pro-duction.Our data indicate that this well-conserved cyclin C-and cyclin-dependent kinase system is involved in the regulation of trichothecene synthesis and plant infection processes in F. graminearum.
2.Materials and methods
2.1.Fungal strains and growth conditions
The Fusarium graminearum wide type strain PH-1and transfor-mants generated in this study were routinely maintained at25°C on V8juice agar and incubated in liquid CMC medium for conidium production as described(Hou et al.,2002;Trail et al.,2003).To as-say the effects of pH on conidiation,the CMC medium was adjusted to pH3.0,5.0,7.0,and9.0with phosphoric acid as described(Shim and Woloshuk,2001).Conidia were collected by?ltration through two layers of Miracloth(Calbiochem,La Jolla,CA)and counted with a hemacytometer(Hou et al.,2002).Protoplast preparation and fungal transformation were performed as described(Proctor et al.,1995).TB3medium(0.3%yeast extract,0.3%casamino acids, and20%sucrose)supplemented with200l g/ml hygromycin B (Calbiochem)or200l g/ml G418(Sigma,St.Louis,MO)was used for selection of transformants(Seong et al.,2006).Growth rate on V8juice agar was assayed with race tube cultures incubated at25°C for16days.For assaying defects of the D cid1mutant in stress responses,cultures grown on YEPD medium(0.3%yeast ex-tract,1%peptone,2%glucose,1.8%g agar)with different concen-trations of NiCl2,CdCl2,and H2O2was measured for colony radius after incubation for7days.Sel?ng and outcrossing with the nit1mutant11622were performed on carrot agar plates (Hou et al.,2002).DON and ergosterol production in infected wheat kernels were measured as previously described(Bluhm and Woloshuk,2005;Bluhm and Woloshuk,2006;Goswami and Kistler,2005).To assay the effect of acidic pH on DON production, the de?ned medium containing0.2%agmatine(Gardiner et al., 2009a)was buffered with phosphoric acid to different pH values as described(Shim and Woloshuk,2001).Culture conditions for DON production followed what were described by Gardiner and colleagues(2009a,b).
For F.verticillioides,the wild-type strain FT536,the D fcc1mu-tant(Shim and Woloshuk,2001),and the D fcc1/CID1comple-mented strain were cultured on V8juice agar for assaying vegetative growth.For conidium production, F.verticillioides strains were grown in the BSAL6.0medium(Shim et al.,2006)at 25°C.
2.2.The CID1gene replacement vector and mutant
A0.7-kb upstream sequence and a0.8-kb downstream?anking sequence of the CID1gene were ampli?ed with primer pairs CID1-F1/CID1-R2and CID1-F3/CID1-R4(Table4),respectively.Primers HYGF,HYR,YGF,and HYGR(Table4)were used to amplify PCR fragments for generating the D cid1deletion construct by the split-marker approach(Catlett et al.,2003).After fungal transfor-mation,hygromycin-resistant transformants were screened by PCR with primers CID1-F5and CID1-R6(Table4).The putative D cid1mutant was further con?rmed by Southern blot analysis fol-lowing standard molecular protocols(Sambrook and Russell, 2001).Genomic DNA was digested with Xho I and separated on 0.8%agarose gel before being transferred to a nylon membrane.
For complementation assays,a3.4-kb fragment containing the entire CID1gene,including the open reading frame,1.7-kb pro-moter region,and0.6-kb terminator region,was ampli?ed with primers CID1-CF and CID1-CR(Table4)and cloned between the Bam HI and Not I sites of pHZ100(Bluhm et al.,2007)to generate the complementation construct pCID1.In pHZ100,the neomycin-resistant cassette derived from pSM334was inserted into the vec-tor backbone.Plasmid pCID1was transformed into the D cid1mu-tant CH1as described(Proctor et al.,1995).
2.3.Corn stalk and wheat infection assays
Conidia were collected from5-day-old CMC cultures by?ltra-tion through two layers of Miracloth(Calbiochem)and resus-pended to5?104conidia/ml in sterile distilled water.For infection assays with corn plants of cultivar Pioneer2375,sterile wooden toothpicks were brie?y dipped in the spore suspension and then slowly droved into the stalk between the tassel and node immediate below.Stalk rot symptoms were examined14days-post-inoculation(dpi)by removing the toothpick and slicing corn stalks into halves along the inoculation sites.Six-week-old plants of wheat cultivar Norm were used for inoculation as described (Gale et al.,2002;Kang and Buchenauer,1999).The second,full-sized spikelet from the base of the in?orescence was injected with 10l l of the spore suspension.Inoculated wheat heads were cov-ered with a small plastic bag for2days.Infected plants were then cultured at ambient greenhouse conditions.Symptomatic spikelets in each head were counted14dpi to calculate the disease index as described(Gale et al.,2002;Seong et al.,2006).At least nine inoc-ulated wheat heads per treatment were used for each test and all tests were repeated four times.Diseased kernels were harvested from inoculated spikelets and assayed for DON and ergosterol as described(Bluhm et al.,2007).
2.4.Quantitative RT-PCR(qRT-PCR)
A total of108conidia of each strain was inoculated into100ml of YEPD medium and incubated at25°C for48h with shaking at 150rpm.Mycelia were harvested by?ltration through two layers of Miracloth.RNA was isolated from mycelia with the Trizol re-agent(Invitrogen,Carlsbad,CA).The StrataScript QPCR cDNA syn-thesis kit(Stratagene,La Jolla,CA)was used for cDNA synthesis following the instruction provided by the manufacturer.The pri-mer pairs used for qRT-PCR were Tri5QF/TRI5QR for TRI5, PKS12QF/PKS12QR for PKS12(Table4),TubQF/TubQR(Bluhm et al.,2007)for the beta-tubulin gene TUB2.PCR was performed with a MX3000(Stratagene)System with the program of2min at50°C,10min at95°C,and40cycles of15s at95°C followed by1min at60°C.Data were collected with the sequence detector software MXPro.All expression data were normalized to TUB2 expression and relative changes in gene expression levels were cal-culated by the comparative Ct method(Applied Biosystems,Foster City,CA).For each gene assayed by qRT-PCR,data from three bio-logical replicates were used to calculate the mean and standard deviation.
144X.Zhou et al./Fungal Genetics and Biology47(2010)143–151
https://www.doczj.com/doc/439986144.html,plementation of the F.verticilloides D fcc1mutant with the CID1gene
Plasmid pCID1containing the full length CID1gene was trans-formed into the F.verticillioides D fcc1mutant FT536as described (Shim and Woloshuk,2001).Neomycin-resistant transformants were isolated and veri?ed by PCR with primers CID-5F and CID-6R to contain the CID1gene integrated in the F.verticillioides gen-ome.Cracked corn cultures incubated at 25°C for 14days were used for assaying fumonisin B1(FB1)production as described (Shim and Woloshuk,2001).3.Results
3.1.The CID1gene is well conserved in ?lamentous ascomycetes BLASTP search revealed that the homolog of the F.verticilloides FCC1gene in F.graminearum is FGSG_04355.3,which was named
the CID1gene (for Cyclin C-like gene required for infection and DON production 1)in this study.CID1also is homologous to UME3of Saccharomyces cerevisiae that encodes a C-type cyclin or cyclin C-like protein.Ume3p is one component of the RNA polymerase II holoenzyme,and it is required for the repression of genes involved in meiosis and stress response (Kuchin et al.,1995).
The CID1gene is predicted to encode a 321amino acid peptide.Like Ume3and Fcc1,the Cid1protein had the cyclin box for cyclin-CDK interactions and the holoenzyme associating domain (Fig.1A).It also contains the PEST-rich region and RQLK motif that are known to be responsible for Ume3degradation in S.cerevisiae (Cooper et al.,1997).Cid1protein is 85%identical to Fcc1and its ortholog from F.oxysporum (FOXG_13879).All these Fusarium cy-clin C-like proteins are highly conserved in the cyclin box and the cyclin destruction domains.
3.2.The D cid1mutant is defective in hyphal growth and conidiation To determine the function of CID1in F.graminearum ,we gen-erated mutants in which the CID1gene was deleted with the split-marker approach (Catlett et al.,2003)(Fig.1B).After trans-formation of wild-type PH-1protoplasts,hygromycin-resistant transformants were isolated and screened by PCR with primers CID-F5and CID-R6(Fig.1B).One D cid1mutant named CH1was identi?ed and further con?rmed by Southern blot analysis (Fig.1C).When hybridized with the downstream ?anking frag-ment ampli?ed with primers F3and F4(probe 1),a 1.5kb band was detected in PH-1.The D cid1mutant had a 7.0kb band that was diagnostic for the gene replacement event.When hybridized with the CID1ORF region ampli?ed with primers F5and R6(probe 2),the 1.5kb band detected in PH-1was absent in the D cid1mutant (Fig.1C).
The growth rate of the D cid1mutant was measured with race tube cultures grown at 25°C for 16days.On average,it grew about 30%slower than the wild-type strain (Table 1).The mutant also was reduced about 50%in conidiation (Table 1).Conidia produced by the D cid1mutant had only four cells (three septa)instead of 5–6celled wild-type conidia (Fig.2A).Some of them also lacked the typical morphology of Fusarium conidia.In addition,conidium ger-mination was slightly delayed in the mutant but germ growth and hyphal branching appeared to be normal (Fig.2A).The D cid1mu-tant had increased pigmentation in aerial hyphae of aging cultures.Five-day-old V8agar cultures of the mutant appeared to be more reddish than those of the wild-type strain (Fig.2B).
To con?rm that deletion of CID1was responsible for observed defects in vegetative growth and conidiation,we transformed the full-length wild-type CID1allele into the D cid1mutant CH1.The resulting transformant CH1comp (D cid1/CID1)had the wild-type growth rate and was normal in conidiation (Table 1),indicating that the defects of the mutant were complemented by the ectopic copy of the CID1
gene.
Fig.1.The CID1gene and generation of the D cid1mutant.(A)CID1encodes a typical cyclin C-like protein.Filled box,cyclin domain;empty box,the ROLK motif;hatched box,PEST-rich region.(B)The split-marker approach was used to replace the entire CID1gene with the hygromycin resistance cassette.Probe 1and probe 2are PCR products ampli?ed with primers F3/R4and F5/R6,respectively.X:Xho I.(C)Southern blot analysis with Xho I-digested geonomic DNA of PH-1and the D cid1mutant.Two different blots were hybridized with probe 1(left)and probe 2(right).
Table 1
Vegetative growth,conidiation,and DON production in Fusarium graminearum strains.Strains
Growth rate a (mm/day)Conidiation b (5?104/ml)DON c (ppm)Erg c (ppm)DON/Erg ratio PH-1
12.5±0.3A *133.0±0.6A 318.4±1.2103.4±2.1 3.01A CH1(D cid1)
9.2±0.2D 67.0±0.6D 20.5±0.691.5±2.00.25H CH1comp (D cid1/CID1)
12.5±0.5A
132.0±0.6A
290.5±2.3
97.9±2.1
2.99A
a
Growth rate was measured with race tube cultures (V8juice agar)after incubating at 25°C for 16days.Means and standard errors were calculated from six replicates for each strain.b
Conidiation was assayed with 5-day-old CMC cultures as described (Hou et al.,2002).c
DON and ergosterol (Erg)was assayed with the wheat kernels that were harvested from the inoculated ?orets 14dpi.*
The same letter indicated that there was no signi?cant difference.Different letters were used to mark statistically signi?cant difference (P =0.05).
X.Zhou et al./Fungal Genetics and Biology 47(2010)143–151145
3.3.CID1is essential for female fertility
F.graminearum is a homothallic fungus but it can be forced to outcross.On sel?ng carrot agar plates,the D cid1mutant failed to form perithecia (Fig.2B).Close examination indicated that the mu-tant was blocked in the differentiation of protoperithecia.Although it was female sterile,the D cid1mutant was able to mate as the male.When mating with a nit1mutant,D cid1nit1recombinant progeny were isolated from outcross perithecia.The comple-mented transformant CH1comp (D cid1/CID1)produced fertile peri-thecia on sel?ng plates (Fig.2C),further indicating that CID1is essential for female fertility in F.graminearum
.
Fig.2.Defects of the D cid1mutant in conidium morphology,colony pigmentation,and mating.(A)Conidia of the wild-type PH-1,D cid1mutant,and D cid1/CID1complemented (comp)strains were examined after incubation in liquid CM for indicated times.Bar =10l m.(B)Colonies of PH-1,D cid1mutant,and comp strains grown on V8agar for 5days.(C)Sel?ng plates of PH-1,D cid1mutant,and D cid1/CID1complemented strain.Black dots on the surface were perithecia.
146X.Zhou et al./Fungal Genetics and Biology 47(2010)143–151
3.4.The D cid1mutant is defective in colonizing ?owering wheat heads To determine the function of CID1in plant infection,?owering wheat heads were point-inoculated with conidium suspensions.On wheat heads inoculated with the wild-type strain,typical scab symptoms were observed in the inoculated and nearby spikelets 14days post-inoculation (dpi).Under the same conditions,only about 50%of the spikelets inoculated with D cid1conidia developed scab symptoms.On heads with disease symptoms caused by the D cid1mutant,wheat scab was limited to the inoculated spikelets (Fig.3A).Spikelets nearby the inoculation site remained healthy 14dpi.On average,the D cid1mutant had a disease index score of 0.5(Fig.3B).In comparison with the disease index of the wild-type strain,the mutant had a 16-fold reduction in virulence.Re-introduction the wild-type CID1gene complemented the defects of the D cid1mutant.In wheat head infection assays,the D cid1/CID1complemented strain was as virulent as PH-1(Fig.3).These data indicated that CID1is important for full virulence on wheat.
3.5.The CID1gene also is important for full virulence on maize F.graminearum is also a corn stalk rot pathogen.In corn plants inoculated with the D cid1mutant,stalk rot symptoms were ob-served at the inoculation site (Fig.4).When measured at 14dpi,the average length of stalk rot lesions caused by the D cid1mutant was 1.6±0.2cm.Under the same conditions,the length of stalk rot
lesions were 6.3±0.5cm and 6.3±0.7cm (Table 1),respectively,for the wild-type and complemented strains.Therefore,the D cid1mutant had a 75%reduction in virulence on corn stalks.These re-sults suggest that the CID1gene might be important for F.grami-nearum to colonize and spread in the corn pith.3.6.The D cid1mutant is defective in stress responses
In yeast,the ume3mutant has increased sensitivities to various stresses (Cohen et al.,2003;Cooper et al.,1997).To test whether the D cid1mutant had similar defects,Ni 2+,Cd 2+,and H 2O 2were added to YEPD medium (Table 2).In the presence of 500l m Ni 2+,the growth rate of the mutant was about 30%of that of the wild-type strain.Colony growth was completely inhibited by 1mM Ni 2+in the D cid1mutant,but the wild-type strain (PH-1)still grew under the same conditions (Table 2).The D cid1mutant also was more sensitive to the CdCl 2and H 2O 2treatments than the wild-type strain.In the presence of 100l m Cd2+or 0.04%H 2O 2,it grew about 50%slower than the wild-type strain (Table 2).The D cid1mutant,but not the wild-type strain,failed to grow on med-ium with 500l m Ni 2+or 1%H 2O 2.These results suggest that the CID1gene may be important for responses to oxidative and heavy metal stresses.
3.7.DON production and TRI5expression are reduced in the D cid1mutant
To determine the role of CID1in DON production,we isolated the diseased wheat kernels from the inoculated spikelets and mea-sured DON content.On average,only about 20ppm DON (Table 1)was detected in wheat kernels infected by the D cid1mutant.Ker-nels inoculated with the wild-type and complemented strains con-tained approximately 300ppm DON.Because the D cid1mutant was reduced in growth rate,the same samples also were
assayed
Fig.3.The D cid1mutant was defective in disease spread in wheat heads.(A)Infection assays with ?owering wheat heads.Typical heads were photographed 14dpi.The spikelets inoculated with PH-1,D cid1mutant CH1,D cid1/CID1strain CH1comp and water are marked with arrows.(B)Disease index of PH-1,D cid1mutant CH1,and D cid1/CID1complemented strain CH1comp.Mean and standard error were calculated from four independent infection
assays.
Fig.4.Infection assays with corn stalks.Toothpicks carrying fungal spores were used to inoculate corn plants between the ?rst node and tassel.Stalk rot symptoms were examined 14dpi.The inoculation sites are marked with an arrow.
X.Zhou et al./Fungal Genetics and Biology 47(2010)143–151147
for ergosterol for a measure of fungal biomass.After normalization with ergosterol,it was clear that DON production was signi?cantly reduced by deletion of the CID1gene (Fig.5A).The DON/ergosterol
ratio in wheat kernels colonized by the D cid1mutant was about nine times less than that of the wild-type and complemented transformant (Fig.5A),indicating that CID1plays an important role in regulating DON synthesis in wheat kernels.
To further support the conclusion that the CID1gene affects DON synthesis,we assayed the expression of the trichodiene syn-thase gene TRI5by quantitative RT-PCR (qRT-PCR).RNA samples were isolated from hyphae grown in YEPD medium for 2–3days.In the D cid1mutant,the expression level of TRI5was reduced about four times in comparison with that of the wild-type or com-plemented strain (Fig 5B).We also assayed the expression of the TRI101gene that encodes a trichothecene acetyltransferase.The expression level of TRI101was reduced in the D cid1mutant but to a lesser extent than that of TRI5(Fig.5B).
Because of increased pigmentation in the mutant deleted for CID1,we also assayed the expression levels of two polyketide syn-thase genes,PKS12and PKS10.In comparison with the wild type,the expression level of PKS12,which is responsible for aurofusarin synthesis in F.graminearum was increased about three times in the D cid1mutant (Kim et al.,2005;Malz et al.,2005).Expression of PKS12in the complemented transformant was comparable to the wild-type level (Fig.6B).In contrast,PKS10was expressed at sim-ilar levels in all these three strains assayed (Fig.6B).These results indicated that deletion of CID1had different effects on the expres-sion of different PKS genes.Increased expression of PKS12might result in increased production of aurofusarin and contribute to en-hanced pigmentation observed in the D cid1mutant.
3.8.Acidic pH failed to suppress the defects of the D cid1mutant in conidiation and toxin production
Because acidic pH has a signi?cant impact on conidiation and fumonisin synthesis in F.verticillioides (Shim and Woloshuk,
Table 2
Differences between the wild-type and D cid1mutant in stress responses.Treatments
Concentration
Reduction in the growth rate (%)*PH-1
D cid1mutant Ni 2+(l M)
10015.6±3.117.4±1.225019.7±2.920.4±1.350033.2±2.166.5±1.8100079.4±1.0100Cd 2+(l M)
103±2.923.0±2.710031.5±0.960.9±3.025068.5±2.282.6±1.150084.4±0.8100H 2O 2(%)
0.0112.6±2.327.4±1.00.0439.1±3.162.2±0.30.0850.9±1.997.0±0.10.10
61.2±0.9
100
*
The radius of colonies was measured after incubating at 25°C for 7days.Reduction in the growth rate was estimated by comparing colonial growth on medium with each treatment with that of PH-1and the D cid1mutant on regular YPEG medium.Means and standard deviation were calculated from three
replicates.
Fig.5.DON production and TRI5expression.(A)The DON/ergosterol ratio in wheat kernels infected with PH-1,D cid1mutant,and D cid1/CID1complemented strain.Ergosterol was measured as the estimate for fungal biomass.DON production was reduced in the D cid1mutant.(B)The expression levels of TRI5and TRI101were measured by real-time qRT-PCR.After being normalized to TUB2expression,their relative expression levels in the D cid1mutant were presented as fold changes in comparison with those of the wild-type strain (arti?cially set to 1).Mean and standard deviation were calculated with data from three biological
replicates.
Fig.6.Pigmentation and PKS12expression.(A)Liquid cultures of the wild-type and D cid1mutant strains.Enhanced pigmentation was observed in the D cid1mutant after incubation in CMC medium for 5days.(B)The relative quantity of the PKS10and PKS12transcripts was compared between the wild-type strain (arbitrarily set to 1)and the D cid mutant.Mean and standard deviation were calculated with data from three replicates.The expression level of PKS12,but not PKS10,was increased in the D cid mutant.
148X.Zhou et al./Fungal Genetics and Biology 47(2010)143–151
2001),we also assayed conidiation and DON production in D cid1 cultures buffered at different pH.In F.graminearum,acidic pH ap-peared to have an inhibitory effect on conidiation(Table3).Both the wild-type and D cid1mutant strains produced fewer conidia at pH5than at pH7.At pH3,conidiation was completely blocked (Table3).Similar to what was reported recently(Gardiner et al., 2009b),we noticed that acidic pH had a stimulatory effect on DON production in F.graminearum.In both the wild-type and D cid1mutant strains,DON production was increased about two folds in cultures grown at pH3in comparison with those grown at pH7(Table3).However,the mutant still produced signi?cant less DON than the wild-type strain at either pH3or pH5(Table3). These results indicate that acidic pH failed to rescue the defects caused by deletion of the CID1gene in F.graminearum.
3.9.CID1functionally complements the F.verticillioides D fcc1mutant
To determine its function in F.verticillioides,we transformed the CID1gene(under the control of its native promoter)into the D fcc1 mutant FT536(Shim and Woloshuk,2001).Geneticin-resistant transformants were isolated and con?rmed by PCR for the trans-forming CID1gene.Unlike the original D fcc1mutant,the D fcc1/ CID1transformant grew as fast as the wild-type strain on V8agar plates(Fig.7A).It also produced similar amount of conidia as the wild-type strain(Fig.7B).While the D fcc1mutant produced80%less FB1fumonisin than the wild-type strain,the D fcc1/CID1trans-formant was normal in FB1production(Fig.7C),indicating that when expressed in F.verticillioides,the CID1gene fully comple-mented the D fcc1mutant.It is likely that these two genes are func-tionally conserved between F.graminearum and F.verticillioides, two closely related species that produce different mycotoxins and cause different diseases.
4.Discussion
FCC1and CID1are homologous to the S.cerevisiae UME3gene, which also is known as SSN8or SRB11.Ume3and its cyclin-dependent kinase(Cdk)Ume5are components of the RNA poly-merase II holoenzyme and are required for the repression of genes
Table3
Effect of different pH on conidiation and DON production.
Strain pH Conidiation*(5?104conidia/ml)DON(ppm)
PH-1302950±58
5112.0±0.91276±34
7138.0±0.71585±67
9100.0±0.31124±23
D cid130856±24
Mutant528.0±1.5356±31
756.0±1.2418±16
949.0±0.6165±13
*Means and standard errors were calculated from three independent replicates.
Table4
Primers used in this study.
Primers Sequence(50–30)*
CID1-F1GCTTTAACTGCCGCGAGGACCC
CID1-R2GACCTCCACTAGCTCCAGCCAAGCCGTACTGTTCGAGCAGATTGCCTG CID1-F3ATAGAGTAGATGCCGACCGGCCATACGAGATACGAGACATGAAG CID1-R4GCTTGGAGGCCAACTTTTCTGTTAC
CID1-F5CGCCGAACGAATCCGTATCTCG
CID1-R6CACTTATCAAGCCCTCGCGCC
HYR GTATTGACCGATTCCTTGCGGTCCGAA
YGF GATGTAGGAGGGCGTGGATATGTCCT
HYGF GGCTTGGCTGGAGCTAGTGGAGGTCAA
HYGR CGGTCGGCATCTACTCTAT
CID1-CF ATATGGATCCCTGCTTCCGTGTTAGTTGTCGTTGACAC
CID1-CR ATATGCGGCCGCTCGCTCTGTCTCGGCATAAACTGA
TRI5QF TGAGGGATGTTGGATTGAGCAGTAC
TRI5QR TGCTTCCGCTCATCAAACAGGT
PKS12QF TATCAAGAACCGGCAACTGCAACA
PKS12QR TACTGCTTCGGCTGAGTGGTTTG
TubQF GTCAGTGCGGTAACCAAATCGT
TubQR
CTCAGAGGTGCCGTTGTAAACACC
TRI101QF ATACCCTATGGCGATGTTTGACGAGAA
TRI101QR CTGTCCGTTGACAGTGAGGATGA
PKS10F TGACCAAGCAGGCCATCGAATCT
PKS10R CTTGAGGCCTAAATCCCTTAGGCC
*Nucleotide sequences from the hygromycin resistance gene were https://www.doczj.com/doc/439986144.html,plementation of the F.verticillioides D fcc1mutant with the CID1gene.
(A)Colonies of the wild-type,D fcc1(FT536),and D fcc1/CID1(CID-com)strains grown on V8agar plates.Typical colonies were photographed after incubating for 7days.(B)The wild-type and D fcc1/CID1complemented strains,but not the D fcc1 mutant,formed abundant conidia in liquid cultures.(C)Fumonisin FB1production in cracked corn cultures of the wild-type,FT536,and CID-com strains.Mean and standard error were calculated from three independent experiments.
X.Zhou et al./Fungal Genetics and Biology47(2010)143–151149
involved in stress responses,meiosis,and other metabolic or developmental processes(Ansari et al.,2005;Cohen et al.,2003; Cooper and Strich,2002).This cyclin-Cdk kinase can modify the general transcriptional machinery by phosphorylation of the car-boxy-terminal domain of RNA polymerase II.Deletion of UME3 is not lethal but results in reduced glycogen accumulation and in-creased sensitivities to various stresses,such as alkaline pH and heavy metals(Cohen et al.,2003;Cooper et al.,1997).The ume3 mutant is reduced in diploid?lamentous growth,blocked in hap-loid invasive growth,and has increased telomere length and re-duced sporulation(Cooper and Strich,2002).In addition,it produces small buds with nuclei(budding growth defects)on sporulation medium.
Like UME3in yeast,CID1and FCC1are not essential genes.How-ever,the growth rate of the D fcc1and D cid1mutants was reduced although no obvious changes in conidium germination and hyphal branching were observed in the D cid1mutant.Because Cid1p is likely a component of the RNA polymerase II holoenzyme,deletion of CID1may result in a general reduction in transcription.How-ever,the expression of PKS12,TRI5,and PKS1in the D cid1mutant was increased,decreased,and unaltered,respectively.Therefore, the effect of D cid1mutation on transcription must be speci?c for different genes.Deletion of CID1may result in the derepression of genes that are normally repressed.Abnormal or unbalanced transcription of different genes may disturb normal physiology and reduce fungal growth.
In yeast,the Ume3-Ume5kinase system is required for entry into and execution of meiosis(Cooper and Strich,2002).Ume3is required for the normal exit from the mitotic cell cycle prior to meiotic induction and is degraded before meiosis I.The cid1mu-tant was female sterile but it could function as the male.We were able to isolate normal D cid1ascospore progeny from a D cid1?CID1cross.Because the D cid1mutant is female sterile,it is impossible to test the fertility of the D cid1?D cid1cross.There-fore,it remains possible that CID1is important for meiosis in F. graminearum.Loss of female fertility in the D cid1mutant may be related to a possible role of CID1in switching from asexual to sex-ual reproduction.However,many mutants deleted for genes of unrelated functions are known to be female sterile,such as the mgv1,gpmk1,and ftl1mutants in F.graminearum(Jenczmionka and Schafer,2005;Urban et al.,2003;Hou et al.,2002).
Our data indicate that the D cid1mutant produced a very low amount of DON on wheat kernels and it was reduced signi?cantly in TRI5expression.In F.verticilloides,the D fcc1mutant also was reduced in FB1fumonisin production when cultured on cracked corn kernels.However,unlike the D cid1mutant with detectable TRI5expression,the expression of FUM5was undetectable in the fcc1mutant(Shim and Woloshuk,2001).In the D cid1mutant, the TRI101gene also had a reduced expression level.TRI101is not located in the same TRI cluster with TRI5and its expression is not regulated by the pathway-speci?c transcription factor TRI6. Therefore,the effect of CID1deletion on trichothecene synthesis must be at a global or semi-global level.Nevertheless,it is unli-kely that deletion of CID1has a universal effect on secondary metabolism because the D cid1mutant had increased expression of PKS12and pigmentation.Various secondary metabolites may have different biological functions in F.graminearum and are reg-ulated by different mechanisms.
Like the D fcc1mutant,the D cid1mutant was reduced in conidiation.The FCC1orthologs may have a conserved role in?la-mentous fungi to positively regulate asexual reproduction.Inter-estingly,conidia formed by the D cid1mutant had fewer septa and cell numbers.Conidium germination also was delayed in the D cid1mutant.These defects were not described for the D fcc1mu-tant.However,it is worth noting here that unlike most Fusarium species,F.graminearum does not form microconidia.Conidiation was assayed with macroconidia in F.graminearum.For F.verticillio-ides,microconidia are formed more abundantly than macroco-nidia.It was microconidia that were examined in this study and the original characterization of the D fcc1mutant(Shim and Wolo-shuk,2001).
In F.verticillioides,acidic pH could suppress the defects of the D fcc1mutant in fumonisin production and conidiation(Shim and Woloshuk,2001).In contrast,acidic pH had a negative impact on the production of macroconida in both the wild-type and D cid1 mutant strains in F.graminearum.No conidia were produced when cultured at pH3(Table3).Therefore,the effect of acidic pH on con-idiation must be different between these two Fusarium species.In F.graminearum,low pH increases the expression of TRI5and DON production in axenic cultures(Gardiner et al.,2009b).The D cid1 mutant produced more DON at pH3than at pH5or pH7(Table3), indicating that it still responded to acidic pH.However,in compar-ison with the wild-type strain,DON production at lower pH was still signi?cantly reduced in the D cid1mutant.Therefore,although acidic pH had a stimulatory effect on DON production,it could not suppress the defect caused by the deletion of CID1.The relationship between CID1and pH regulation on DON synthesis in F.graminea-rum must be different from that of FCC1and acidic pH on fumon-isin synthesis in F.verticillioides.
Although its homologs are well conserved in?lamentous asco-mycetes,the CID1gene is the?rst one to be shown to be important for plant infection.The D cid1mutant was signi?cantly reduced in virulence on?owering wheat heads(Fig.3A).While the30%reduc-tion in growth rate may contribute to the defects in plant infection, other important pathogenicity factors are probably affected in the mutant because its disease index was reduced more than16-fold. The D cid1mutant had a disease index score of0.5(nearly non-pathogenic).In stalk rot assays,it had a75%reduction in virulence (Fig.4),which also was much greater than its reduction in growth rate.However,the D cid1mutant appeared to be more virulent on corn stalks than on wheat heads.One possible reason is that corn pith mainly consists of dead plant tissues.The mutant may face more effective defense responses on?owering wheat heads than inside corn stalks.In addition,DON is an important virulence factor in F.graminearum and DON production was signi?cantly reduced in the D cid1mutant.In F.verticillioides,the D fcc1mutant was not examined for its defects in plant infection.While it is possible that the D fcc1mutant also is reduced in virulence,fumonisin is not an important virulence factor in F.verticillioides.
In yeast,Ume5is the Cdk associated with Ume3.The Ume3-Ume5kinase can bind to and phosphorylate various transcriptional regulators,including Gal4,Gcn4,Ste12,Msn2,Sip4,and Med2,that are involved in different metabolic and developmental processes (Ansari et al.,2005;Hallberg et al.,2004;Larschan and Winston, 2005).In F.verticillioides,Fck1is the Cdk that is homologous to Ume5and directly interacts with Fcc1(Bluhm and Woloshuk, 2006).The F.graminearum genome has the orthologous gene (FGSG_04484)of UME5and FCK1.It is tempting to speculate that the normal function of Cid1and the corresponding CDK is to re-press the expression of stage-speci?c regulatory genes that nega-tively control sexual reproduction,conidiation,plant infection, and mycotoxin production.Deletion of CID1may result in the im-proper expression of these negative regulators and the phenotypes observed in the D cid1mutant.Although conidiation and myco-toxin production may be co-regulated,it is unlikely that loss of fe-male fertility and reduced virulence are controlled by the same regulatory mechanism.Therefore,multiple negative regulators may be regulated by CID1in F.graminearum.The F.graminearum genome contains homologs of many yeast transcription factors regulated by Ume3-Ume5,including Gal4,Gcn4,Ste12,and Msn4.It will be important to characterize their functions and reg-ulation by CID1in F.graminearum.
150X.Zhou et al./Fungal Genetics and Biology47(2010)143–151
Acknowledgments
We thank Dr.Charles Woloshuk for providing the D fcc1mutant and Dr.Yanhong Dong for DON and ergosterol measurements.We also thank https://www.doczj.com/doc/439986144.html,rry Dunkle and Charles Woloshuk for critical reading of this manuscript.This work was supported by the departmental REU program to CH and Grants to JX from the US Wheat and Barley Scab Initiative and the National Research Initia-tive of the USDA CSREES(#2003-35319-13829and#2007-35319-102681).
References
Ansari,A.Z.et al.,2005.Transcriptional activating regions target attached substrates to a cyclin-dependent https://www.doczj.com/doc/439986144.html,A102,2346–2349. Bai,G.H.et al.,2002.Deoxynivalenol-nonproducing Fusarium graminearum causes initial infection,but does not cause disease spread in wheat spikes.
Mycopathologia153,91–98.
Bai,G.H.,Shaner,G.,2004.Management and resistance in wheat and barley to Fusarium head blight.Annu.Rev.Phytopathol.42,135–161.
Bluhm,B.H.,Woloshuk,C.P.,2005.Amylopectin induces fumonisin B-1production by Fusarium verticillioides during colonization of maize kernels.Mol.Plant–Microbe Interact.18,1333–1339.
Bluhm,B.H.,Woloshuk,C.P.,2006.Fck1,a C-type cyclin-dependent kinase,interacts with Fcc1to regulate development and secondary metabolism in Fusarium verticillioides.Fungal Genet.Biol.43,146–154.
Bluhm, B.H.et al.,2007.RAS2regulates growth and pathogenesis in Fusarium graminearum.Mol.Plant–Microbe Interact.20,627–636.
Catlett,N.L.et al.,2003.Split-marker recombination for ef?cient targeted deletion of fungal genes.Fungal Genet.Newslett.50,9–11.
Cohen,T.J.et al.,2003.Ask10p mediates the oxidative stress-induced destruction of the Saccharomyces cerevisiae C-type cyclin Ume3p/Srb11p.Eukaryot.Cell2, 962–970.
Cooper,K.F.et al.,1997.Stress and developmental regulation of the yeast C-type cyclin Ume3p(Srb11p/Ssn8p).EMBO J.16,4665–4675.
Cooper,K.F.,Strich,R.,2002.Saccharomyces cerevisiae C-type cyclin Ume3p/Srb11p is required for ef?cient induction and execution of meiotic development.
Eukaryot.Cell1,66–74.
Desjardins, A.E.et al.,2000.Analysis of aberrant virulence of Gibberella zeae following transformation-mediated complementation of a trichothecene-de?cient(Tri5)mutant.Microbiology146,2059–2068.
Desjardins, A.E.et al.,1996.Reduced virulence of trichothecene-nonproducing mutants of Gibberella zeae in wheat?eld tests.Mol.Plant–Microbe Interact.9, 775–781.
Flaherty,J.E.,Woloshuk, C.P.,2004.Regulation of fumonisin biosynthesis in Fusarium verticillioides by a zinc binuclear cluster-type gene,ZFR1.Appl.
Environ.Microbiol.70,2653–2659.
Gardiner,D.M.,Kazan,K.,Manners,J.M.,2009a.Nutrient pro?ling reveals potent inducers of trichothecene biosynthesis in Fusarium graminearum.Fungal Genet.
Biol.8,604–613.
Gardiner,D.M.,Osborne,S.,Kazan,K.,Manners,J.M.,2009b.Low pH regulates the production of deoxynivalenol by Fusarium graminearum.Microbiology155, 3149–3156.
Gale,L.R.et al.,2002.Population analysis of Fusarium graminearum from wheat ?elds in eastern China.Phytopathology92,1315–1322.
Goswami,R.S.,Kistler,H.C.,2004.Heading for disaster:Fusarium graminearum on cereal crops.Mol.Plant Pathol.5,515–525.
Goswami,R.S.,Kistler,H.C.,2005.Pathogenicity and in planta mycotoxin accumulation among members of the Fusarium graminearum species complex on wheat and rice.Phytopathology95,1397–1404.
Hallberg,M.et al.,2004.Site-speci?c Srb10-dependent phosphorylation of the yeast mediator subunit Med2regulates gene expression from the2-mu m plasmid.
https://www.doczj.com/doc/439986144.html,A101,3370–3375.
Han,Y.K.et al.,2007.A novel F-box protein involved in sexual development and pathogenesis in Gibberella zeae.Mol.Microbiol.63,768–779.
Harris,L.J.et al.,1999.Possible role of trichothecene mycotoxins in virulence of Fusarium graminearum on maize.Plant Dis.83,954–960.
Hohn,T.M.et al.,1999.Characterization of a transcriptional activator controlling trichothecene toxin biosynthesis.Fungal Genet.Biol.26,224–235.Hou,Z.M.et al.,2002.A mitogen-activated protein kinase gene(MGV1)in Fusarium graminearum is required for female fertility,heterokaryon formation,and plant infection.Mol.Plant–Microbe Interact.15,1119–1127.
Jenczmionka,N.J.et al.,2003.Mating,conidiation and pathogenicity of Fusarium graminearum,the main causal agent of the head-blight disease of wheat,are regulated by the MAP kinase gpmk1.Curr.Genet.43,87–95.
Jenczmionka,N.J.,Schafer,W.,2005.The Gpmk1MAP kinase of Fusarium graminearum regulates the induction of speci?c secreted enzymes.Curr.
Genet.47,29–36.
Kang,Z.,Buchenauer,H.,1999.Immunocytochemical localization of Fusarium toxins in infected wheat spikes by Fusarium culmorum.Physiol.Mol.Plant Pathol.55,275–288.
Kim,J.E.et al.,2005.Putative polyketide synthase and laccase genes for biosynthesis of aurofusarin in Gibberella zeae.Appl.Environ.Microbiol.71,1701–1708. Kimura,M.et al.,2003.The trichothecene biosynthesis gene cluster of Fusarium graminearum F15contains a limited number of essential pathway genes and expressed non-essential genes.FEBS Lett.539,105–110.
Kimura,M.et al.,2007.Molecular and genetic studies of Fusarium trichothecene biosynthesis:pathways,genes,and evolution.Biosci.Biotech.Biochem.71, 2105–2123.
Kuchin,S.et al.,1995.Cyclin-dependent protein-kinase and cyclin homologs Ssn3 and Ssn8contribute to transcriptional control in yeast.Proc.Natl.Acad.Sci.92, 4006–4010.
Larschan,E.,Winston,F.,2005.The Saccharomyces cerevisiae Srb8–Srb11complex functions with the SAGA complex during Gal4-activated transcription.Mol.Cell.
Biol.25,114–123.
Lee,B.N.et al.,2005.Functional analysis of all nonribosomal peptide synthetases in Cochliobolus heterostrophus reveals a factor,NPS6,involved in virulence and resistance to oxidative stress.Eukaryot.Cell4,545–555.
Lee,T.et al.,2002.Tri13and Tri7determine deoxynivalenol-and nivalenol-producing chemotypes of Gibberella zeae.Appl.Environ.Microbiol.68,2148–2154.
Lu,S.W.et al.,2003.A novel class of gene controlling virulence in plant pathogenic ascomycete https://www.doczj.com/doc/439986144.html,A100,5980–5985.
Malz,S.et al.,2005.Identi?cation of a gene cluster responsible for the biosynthesis of aurofusarin in the Fusarium graminearum species complex.Fungal Genet.
Biol.42,420–433.
McMullen,M.et al.,1997.Scab of wheat and barley:a re-emerging disease of devastating impact.Plant Dis.81,1340–1348.
Pirttila,A.M.et al.,2004.Expression pro?le analysis of wild-type and fcc1mutant strains of Fusarium verticillioides during fumonisin biosynthesis.Fungal Genet.
Biol.41,647–656.
Pritsch,C.et al.,2000.Fungal development and induction of defense response genes during early infection of wheat spikes by Fusarium graminearum.Mol.Plant–Microbe Interact.13,159–169.
Proctor,R.H.et al.,1995.Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene.Mol.Plant–Microbe Interact.8,593–601.
Sambrook,J.,Russell,D.,2001.Molecular Cloning–A Laboratory Manual.Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY.
Seong,K.et al.,2005.Random insertional mutagenesis identi?es genes associated with virulence in the wheat scab fungus Fusarium graminearum.
Phytopathology95,744–750.
Seong,K.et al.,2006.Cryptic promoter activity in the coding region of the HMG-CoA rediactase gene in Fusarium graminearum.Fungal Genet.Biol.43,34–41. Seong,K.Y.et al.,2009.Global gene regulation by Fusarium transcription factors Tri6and Tri10reveals adaptations for toxin biosynthesis.Mol.Microbiol.72, 354–367.
Shim,W.B.et al.,2006.FSR1is essential for virulence and female fertility in Fusarium verticillioides and F.Graminearum.Mol.Plant–Microbe Interact.19, 725–733.
Shim,W.B.,Woloshuk,C.P.,2001.Regulation of fumonisin B-1biosynthesis and conidiation in Fusarium verticillioides by a cyclin-like(C-type)gene,FCC1.Appl.
Environ.Microbiol.67,1607–1612.
Tag,A.G.et al.,2001.A novel regulatory gene,Tri10,controls trichothecene toxin production and gene expression.Appl.Environ.Microbiol.67,5294–5302. Trail,F.et al.,2005.Ejection mechanics and trajectory of the ascospores of Gibberella zeae(anamorph Fuarium graminearum).Fungal Genet.Biol.42,528–533. Trail, F.et al.,2003.Analysis of expressed sequence tags from Gibberella zeae (anamorph Fusarium graminearum).Fungal Genet.Biol.38,187–197.
Urban,M.et al.,2003.The Fusarium graminearum MAP1gene is essential for pathogenicity and development of perithecia.Mol.Plant Pathol.4,347–359. Yu,H.Y.et al.,2008.Functional analyses of heterotrimeric G protein G alpha and G beta subunits in Gibberella zeae.Microbiology154,392–401.
X.Zhou et al./Fungal Genetics and Biology47(2010)143–151151
分子生物学 1.ORF 答:ORF是open reading frame的缩写，即开放阅读框架。在DNA链上，由蛋白质合成的起始密码开始，到终止密码为止的一个连续编码列，叫做一个开放阅读框架。 2.结构基因 答：结构基因（structural genes）可被转录形成mRNA,并翻译成多肽链，构成各种结构蛋白质或催化各种生化反应的酶和激素等。 3.断裂基因 答：基因是核酸分子中贮存遗传信息的遗传单位，一个基因不仅仅包括编码蛋白质或 RNA 的核酸序列，还包括保证转录所必需的调控序列、位于编码区 5 ' 端与 3 ' 端的非编码序列和内含子。真核生物的结构基因，由若干个编码区和非编码区互相间隔开但又连续镶嵌而成，去除非编码区再连接后，可翻译出由连续氨基酸组成的完整蛋白质，这些基因称为断裂基因（split gene）。 4.选择性剪接 答：选择性剪接（也叫可变剪接）是指从一个mRNA前体中通过不同的剪接方式（选择不同的剪接位点组合）产生不同的mRNA剪接异构体的过程，而最终的蛋白产物会表现出不同或者是相互拮抗的功能和结构特性，或者，在相同的细胞中由于表达水平的不同而导致不同的表型。 5.C值 答：基因组的大小通常以其DNA的含量来表示，我们把一种生物体单倍体基因组DNA的总量成为C值（C value）。 6.生物大分子 答：生物大分子指的是作为生物体内主要活性成分的各种分子量达到上万或更多的有机分子。常见的生物大分子包括蛋白质、核酸、脂类、糖类。 7.酚抽提法 答：酚抽提法最初于1976年由Stafford及其同事提出，通过改良，以含EDTA、SDS及无DNA酶的RNA酶裂解缓冲液破碎细胞，经蛋白酶K处理后，用pH8.0的Tris饱和酚抽提DNA，重复抽提至一定纯度后，根据不同需要进行透析或沉淀处理获得所需的DNA样品。 8.凝胶过滤层析 答：凝胶过滤层析也称分子排阻层析或分子筛层析，利用凝胶分子筛对大小、形状不同的分子进行层析分离，是根据分子大小分离蛋白质混合物最有效的方法之一。 9.多重PCR 答：多重PCR技术是在一个反应体系中加入多对引物，同时扩增出多个核酸片段，由于每对引物扩增的片段长度不同，可用琼脂糖凝胶电泳或毛细管电泳等技术加以鉴别。 10.荧光域值 答：荧光阈值是在荧光扩增曲线上人为设定的一个值，它可以设定在荧光信号指数扩增阶段任意位置上，一般荧光阈值的设置是基线荧光信号的标准偏差的10倍。 11.退火 答：温度突然降至37-58℃时，变性的DNA单链在碱基互补的基础上重新形成氢
现代分子生物学 复习提纲 第一章绪论 第一节分子生物学的基本含义及主要研究内容 1 分子生物学Molecular Biology的基本含义 ?广义的分子生物学：以核酸和蛋白质等生物大分子的结构及其在遗传信息和细胞信息传递中的作用为研究 对象，从分子水平阐明生命现象和生物学规律。 ?狭义的分子生物学：偏重于核酸（基因）的分子生物学，主要研究基因或DNA的复制、转录、表达和调控 等过程，也涉及与这些过程相关的蛋白质和酶的结构与功能的研究。 1.1 分子生物学的三大原则 1) 构成生物大分子的单体是相同的 2) 生物遗传信息表达的中心法则相同 3) 生物大分子单体的排列（核苷酸、氨基酸）的不同 1.3 分子生物学的研究内容 ●DNA重组技术（基因工程） ●基因的表达调控 ●生物大分子的结构和功能研究(结构分子生物学） ●基因组、功能基因组与生物信息学研究 第二节分子生物学发展简史 1 准备和酝酿阶段 ?时间：19世纪后期到20世纪50年代初。 ?确定了生物遗传的物质基础是DNA。 DNA是遗传物质的证明实验一：肺炎双球菌转化实验 DNA是遗传物质的证明实验二：噬菌体感染大肠杆菌实验 RNA也是重要的遗传物质-----烟草花叶病毒的感染和繁殖过程 2 建立和发展阶段 ?1953年Watson和Crick的DNA双螺旋结构模型作为现代分子生物学诞生的里程碑。 ?主要进展包括： ?遗传信息传递中心法则的建立 3 发展阶段 ?基因工程技术作为新的里程碑，标志着人类深入认识生命本质并能动改造生命的新时期开始。 ? 第三节分子生物学与其他学科的关系 思考 ?证明DNA是遗传物质的实验有哪些？ ?分子生物学的主要研究内容。 ?列举5～10位获诺贝尔奖的科学家，简要说明其贡献。
分子生物学试题及答案 一、名词解释 1．cDNA与cccDNA：cDNA是由mRNA通过反转录酶合成的双链DNA；cccDNA是游离于染色体之外的质粒双链闭合环形DNA。 2．标准折叠单位：蛋白质二级结构单元α－螺旋与β－折叠通过各种连接多肽可以组成特殊几何排列的结构块，此种确定的折叠类型通常称为超二级结构。几乎所有的三级结构都可以用这些折叠类型，乃至他们的组合型来予以描述，因此又将其称为标准折叠单位。 3．CAP：环腺苷酸（cAMP）受体蛋白CRP（cAMP receptor protein ），cAMP与CRP结合后所形成的复合物称激活蛋白CAP（cAMP activated protein ） 4．回文序列：DNA片段上的一段所具有的反向互补序列，常是限制性酶切位点。 5．micRNA：互补干扰RNA或称反义RNA，与mRNA序列互补，可抑制mRNA的翻译。 6．核酶：具有催化活性的RNA，在RNA的剪接加工过程中起到自我催化的作用。 7．模体：蛋白质分子空间结构中存在着某些立体形状和拓扑结构颇为类似的局部区域 8．信号肽：在蛋白质合成过程中N端有15~36个氨基酸残基的肽段，引导蛋白质的跨膜。 9．弱化子：在操纵区与结构基因之间的一段可以终止转录作用的核苷酸序列。 10．魔斑：当细菌生长过程中，遇到氨基酸全面缺乏时，细菌将会产生一个应急反应，停止全部基因的表达。产生这一应急反应的信号是鸟苷四磷酸(ppGpp)和鸟苷五磷酸（pppGpp）。PpGpp与pppGpp的作用不只是一个或几个操纵子，而是影响一大批，所以称他们是超级调控子或称为魔斑。 11．上游启动子元件：是指对启动子的活性起到一种调节作用的DNA序列，-10区的TATA、-35区的TGACA 及增强子，弱化子等。 12．DNA探针：是带有标记的一段已知序列DNA，用以检测未知序列、筛选目的基因等方面广泛应用。13．SD序列：是核糖体与mRNA结合序列，对翻译起到调控作用。 14．单克隆抗体：只针对单一抗原决定簇起作用的抗体。 15．考斯质粒：是经过人工构建的一种外源DNA载体，保留噬菌体两端的COS区，与质粒连接构成。16．蓝-白斑筛选：含LacZ基因（编码β半乳糖苷酶）该酶能分解生色底物X-gal(5-溴-4-氯-3-吲哚-β-D-半乳糖苷)产生蓝色，从而使菌株变蓝。当外源DNA插入后，LacZ基因不能表达，菌株呈白色，以此来筛选重组细菌。称之为蓝-白斑筛选。 17．顺式作用元件：在DNA中一段特殊的碱基序列，对基因的表达起到调控作用的基因元件。18．Klenow酶：DNA聚合酶I大片段，只是从DNA聚合酶I全酶中去除了5’→3’外切酶活性 19．锚定PCR：用于扩增已知一端序列的目的DNA。在未知序列一端加上一段多聚dG的尾巴，然后分别用多聚dC和已知的序列作为引物进行PCR扩增。 20．融合蛋白：真核蛋白的基因与外源基因连接，同时表达翻译出的原基因蛋白与外源蛋白结合在一起所组成的蛋白质。 二、填空 1． DNA的物理图谱是DNA分子的（限制性内切酶酶解）片段的排列顺序。 2． RNA酶的剪切分为（自体催化）、（异体催化）两种类型。 3．原核生物中有三种起始因子分别是（IF-1）、（IF-2）和（IF-3）。 4．蛋白质的跨膜需要（信号肽）的引导，蛋白伴侣的作用是（辅助肽链折叠成天然构象的蛋白质）。5．启动子中的元件通常可以分为两种：（核心启动子元件）和（上游启动子元件）。 6．分子生物学的研究内容主要包含（结构分子生物学）、（基因表达与调控）、（DNA重组技术）三部分。7．证明DNA是遗传物质的两个关键性实验是（肺炎球菌感染小鼠）、（ T2噬菌体感染大肠杆菌）这两个实验中主要的论点证据是：（生物体吸收的外源DNA改变了其遗传潜能）。 8．hnRNA与mRNA之间的差别主要有两点：（hnRNA在转变为mRNA的过程中经过剪接，）、 （mRNA的5′末端被加上一个m7pGppp帽子，在mRNA3′末端多了一个多聚腺苷酸(polyA)尾巴）。 9．蛋白质多亚基形式的优点是（亚基对DNA的利用来说是一种经济的方法）、（可以减少蛋白质合成过程中随机的错误对蛋白质活性的影响）、（活性能够非常有效和迅速地被打开和被关闭）。 10．蛋白质折叠机制首先成核理论的主要内容包括（成核）、（结构充实）、（最后重排）。 11．半乳糖对细菌有双重作用；一方面（可以作为碳源供细胞生长）；另一方面（它又是细胞壁的成分）。所以需要一个不依赖于cAMP—CRP的启动子S2进行本底水平的永久型合成；同时需要一个依赖于cAMP—CRP的启动子S1对高水平合成进行调节。有G时转录从（ S2）开始，无G时转录从（ S1）开
Northern blot：是DNA/RNA的杂交，它是一项用于检测特异性RNA的技术，RNA混合物首先按照它们的大小和相对分子量通过变性琼脂糖凝胶电泳加以分离，凝胶分离后的RNA 通过southern印迹转移到尼龙膜或硝酸纤维素膜上，再与标记的探针进行杂交反应，通过杂交结果分析可以对转录表达进行定量或定性。它是研究基因表达的有效手段。与Southern blot 相比，它的条件更严格些，特别是RNA容易降解，前期制备和转膜要防止Rnase的污染。实验步骤：1.用具的准备2．用RNAZaP去除用具表面的RNase酶污染3．制胶4. RNA样品的制备5.电泳6.转膜7.探针的制备8.探针的纯化及比活性测定9．预杂交10．探针变性11．杂交12．洗膜13．曝光14．去除膜上的探针15．杂交结果 半定量PCR要求比普通PCR更严格一些，另外往往通过转膜后的同位素杂交检测或凝胶成像后的灰度测定比较样品间的差异。 半定量RT-PCR一般是在没有条件做实时PCR 的情况下使用，用于测定体内目的基因的表达增加减少与否，即通过目的基因跑出来的电泳带与管家基因（如β-actin）的电泳带的相对含量比较，观测目的基因表达增减，另外还要做一个β-actin的内参照对照。 实时荧光定量PCR技术，是指在PCR反应体系中加入荧光基团，利用荧光信号积累实时监测整个PCR进程，最后通过标准曲线对未知模板进行定量分析的方法。 1．实时荧光定量PCR无需内标 2．内标对实时荧光定量PCR的影响 Sybr green（荧光染料掺入法）和Taqman probe（探针法） 检测两种蛋白质相互作用方法 1共纯化、共沉淀，在不同基质上进行色谱层析 2蛋白质亲和色谱基本原理是将一种蛋白质固定于某种基质上（如Sepharose），当细胞抽提液经过改基质时，可与改固定蛋白相互作用的配体蛋白被吸附，而没有吸附的非目标蛋白则随洗脱液流出。被吸附的蛋白可以通过改变洗脱液或者洗脱条件而回收下来。 3免疫共沉淀免疫共沉淀是以抗体和抗原之间的专一性作用为基础的用于研究蛋白质相互作用的经典方法。改法的优点是蛋白处于天然状态，蛋白的相互作用可以在天然状态下进行，可以避免认为影响；可以分离得到天然状态下相互作用的蛋白复合体。缺点：免疫共沉淀同样不能保证沉淀的蛋白复合物时候为直接相互作用的两种蛋白。另外灵敏度不如亲和色谱高4 Far-Western 又叫做亲和印记。将PAGE胶上分离好的凡百样品转移到硝酸纤维膜上，然后检测哪种蛋白能与标记了同位素的诱饵蛋白发生作用，最后显影。缺点是转膜前需要将蛋白复性。 1.酵母双杂交 2.GSTpull-down实验 3.免疫共沉淀 4.蛋白质细胞内定位 RACE是基于PCR技术基础上由已知的一段cDNA片段，通过往两端延伸扩增从而获得完整的3'端和5'端的方法 1.此方法是通过PCR技术实现的，无须建立cDNA文库，可以在很短的时间内获得有 利用价值的信息 2.节约了实验所花费的经费和时间。 3.只要引物设计正确，在初级产物的基础上可以获得大量的感兴趣基因的全长 基因特异性引物（GSPs）应该是： 23－28nt 50－70％GC Tm值≥65度，Tm值≥70度可以获得好的结果 注意事项 1.cDNA的合成起始于polyA+RNA。如果使用其它的基因组DNA或总RNA，背景会很高
【期末复习总结】基础分子生物学 基础分子生物学 第一章 1. DNA的发现 Avery的肺炎双球菌转化实验 Hershey和Chase的噬菌体侵染细菌试验 2. 基因工程操作的工具 限制性内切酶。DNA连接酶。运载体。 3. 原核生物的基因组和染色体结构都比较简单，转录和翻译在同一时间和空间内发生，基因表达的调控主要发生在转录水平。 真核生物转录和翻译过程在时间和空间上都被分隔开，且在转录和翻译后都有复杂的信息加工过程，其基因表达的调控可以发生在各种不同的水平上。其基因表达调控主要表现在信号传导研究、转录因子研究及RNA 剪辑3个方面。 弟一早 1. 原核细胞染色体： 一般只有一条大染色体且大都带有单拷贝基因，除少数基因外（如rRNA基因）。整个染色体DNA儿乎全部由功能基因和调控序列所 组成。 几乎每个基因序列都与它所编码蛋白质序列呈线性对应关系。
2. 真核生物 真核生物染色体中相对分子质量一般大大超过原核生物，并结合有大 量的蛋白质DNA具体组成成分为：组蛋白、非组蛋白、DNAo 其蛋白质与相应DNA的质量之比约为2:lo 5. 组蛋白 组蛋白是染色体的结构蛋白，其与DNA组成核小体。根据其凝胶电泳 性质可将其分为HL H2A、H2B、H3及H4。 6. 组蛋白的特性： 进化上极端保守性。其中H3、H4最保守，H1较不保守。 无组织特异性. 肽链上氨基酸分布的不对称性. 组蛋白的修饰作用。包括甲基化、乙基化、磷酸化及ADP核糖基化等。 富含赖氨酸的组蛋白H5. 7. 非组蛋白 色体上除了存在大约与DNA等量的组蛋白以外，还存在大量的非组蛋白。 组蛋白的量大约是组蛋白的60%?70%,非组蛋白的组织专一性和种属专一性。 组蛋白包括酶类、骨架蛋白、核孔复合物蛋白以及肌动蛋白、肌球蛋白等。它们也可能是染色质的组成成分。 类常见的非组蛋白： HMC蛋白。一般认为可能与DNA的超螺旋结构有关。
1.定义重组DNA技术 将不同的DNA片段按照人们的设计定向连接起来，然后在特定的受体细胞中与载体同时复制并得到表达，产生影响受体细胞的新的遗传性状。 2.说出分子生物学的主要研究内容 1.DNA重组技术 2.基因表达研究调控 3.生物大分子的结构功能研究 4.基因组、功能基因组与生物信息学研究 3.简述DNA的一、二、三级结构 一级：4种核苷酸的连接及排列顺序，表示了该DNA分子的化学成分 二级：2条多核苷酸连反向平行盘绕所形成的双螺旋结构 三级：DNA双螺旋进一步扭曲盘绕所形成的特定的空间结构 4.原核生物DNA具有哪些不同于真核生物DNA的特征？ ①DNA双螺旋是由2条互相平行的脱氧核苷酸长链盘绕而成，多核苷酸的方向由核苷酸间的磷酸二酯键的走向决定，一条是5---3，另一条是3---5②DNA双螺旋中脱氧核糖和磷酸交替连接，排在外侧构成基本骨架，碱基排在内侧③两条链上的碱基通过氢键相结合，形成碱基对 5.DNA双螺旋结构模型是由谁提出的？沃森和克里克 6.DNA以何种方式进行复制，如何保证DNA复制的准确性？ 线性DNA的双链复制：将线性复制子转变为环状或者多聚分子，在DNA末端形成发卡式结构，使分子没有游离末端，在某种蛋白质的介入下在真正的末端上启动复制。环状DNA 复制：θ型、滚环型、D型 ①以亲代DNA分子为模板进行半保留复制，复制时严格按照碱基配对原则 ②DNA聚合酶I 非主要聚合酶，可确保DNA合成的准确性
③DNA修复系统：错配修复、切除修复、重组修复、DNA直接修复、SOS系统 7.简述原核生物DNA复制特点 只有一个复制起点，复制起始点上可以连续开始新的DNA复制，变现为虽只有一个复制单元，但可以有多个复制叉 8.真核生物DNA的复制在哪些水平上受到调控？ 细胞生活周期水平调控；染色体水平调控；复制子水平调控 9.细胞通过哪几种修复系统对DNA损伤进行修复？ 错配修复，恢复错配；切除修复，切除突变的碱基和核苷酸片段；重组修复，复制后的修复；DNA直接修复，修复嘧啶二聚体；SOS系统，DNA的修复，导致变异 10.什么是转座子？分为哪些种类？ 是存在于染色体DNA上可自主复制和移动的基本单位。可分为插入序列和复合型转座子11.什么是编码链？什么是模板链？ 与mRNA序列相同的那条DNA链称为编码链，另一条根据碱基互补配对原则指导mRNA 合成DNA链称为模板链 12.简述RNA的种类及其生物学作用 mRNA：编码了一个或多个多肽链序列。 tRNA：把mRNA上的遗传信息变为多肽中的氨基酸信息。 rRNA：是核糖体中的主要成分。 hnRNA：由DNA转录生成的原始转录产物。 snRNA：核小RNA，在前体mRNA加工中，参与去除内含子。 snoRNA：核仁小RNA，主要参与rRNA及其它RNA的修饰、加工、成熟等过程。scRNA：细胞质小RNA在蛋白质合成过程起作用。
分子生物学 第一章绪论 分子生物学研究内容有哪些方面？ 1、结构分子生物学； 2、基因表达的调节与控制； 3、DNA重组技术及其应用； 4、结构基因组学、功能基因组学、生物信息学、系统生物学 第二章DNA and Chromosome 1、DNA的变性：在某些理化因素作用下，DNA双链解开成两条单链的过程。 2、DNA复性：变性DNA在适当条件下，分开的两条单链分子按照碱基互补原则重新恢复天然的双螺旋构象的现象。 3、Tm(熔链温度)：DNA加热变性时，紫外吸收达到最大值的一半时的温度，即DNA分子内50%的双链结构被解开成单链分子时的温度） 4、退火：热变性的DNA经缓慢冷却后即可复性，称为退火 5、假基因：基因组中存在的一段与正常基因非常相似但不能表达的DNA序列。以Ψ来表示。 6、C值矛盾或C值悖论:C值的大小与生物的复杂度和进化的地位并不一致，称为C值矛盾或C值悖论(C-Value Paradox)。 7、转座：可移动因子介导的遗传物质的重排现象。 8、转座子：染色体、质粒或噬菌体上可以转移位置的遗传成分 9、DNA二级结构的特点：1）DNA分子是由两条相互平行的脱氧核苷酸长链盘绕而成；2）DNA分子中的脱氧核苷酸和磷酸交替连接，排在外侧，构成基本骨架，碱基排列在外侧；3）DNA分子表面有大沟和小沟；4）两条链间存在碱基互补，通过氢键连系，且A=T、G ≡ C（碱基互补原则）；5）螺旋的螺距为3.4nm，直径为2nm，相邻两个碱基对之间的垂直距离为0.34nm，每圈螺旋包含10个碱基对；6）碱基平面与螺旋纵轴接近垂直，糖环平面接近平行 10、真核生物基因组结构：编码蛋白质或RNA的编码序列和非编码序列，包括编码区两侧的调控序列和编码序列间的间隔序列。 特点：1）真核基因组结构庞大哺乳类生物大于2X109bp；2）单顺反子（单顺反子：一个基因单独转录，一个基因一条mRNA，翻译成一条多肽链；）3）基因不连续性断裂基因（interrupted gene）、内含子(intron)、外显子(exon)；4）非编码区较多，多于编码序列(9:1) 5）含有大量重复序列 11、Histon(组蛋白)特点：极端保守性、无组织特异性、氨基酸分布的不对称性、可修饰作用、富含Lys的H5 12、核小体组成：由组蛋白和200bp DNA组成 13、转座的机制：转座时发生的插入作用有一个普遍的特征，那就是受体分子中有一段很短的被称为靶序列的DNA会被复制，使插入的转座子位于两个重复的靶序列之间。 复制型转座：整个转座子被复制，所移动和转位的仅为原转座子的拷贝。 非复制型转座：原始转座子作为一个可移动的实体直接被移位。 第三章DNA Replication and repair 1、半保留复制：DNA生物合成时，母链DNA解开为两股单链，各自作为模板(template)按碱
分子生物学 1．DNA的一级结构：指DNA分子中核苷酸的排列顺序。 2．DNA的二级结构：指两条DNA单链形成的双螺旋结构、三股螺旋结构以及四股螺旋结构。3．DNA的三级结构：双链DNA进一步扭曲盘旋形成的超螺旋结构。 4．DNA的甲基化：DNA的一级结构中，有一些碱基可以通过加上一个甲基而被修饰，称为DNA的甲基化。甲基化修饰在原核生物DNA中多为对一些酶切位点的修饰，其作用是对自身DNA产生保护作用。真核生物中的DNA甲基化则在基因表达调控中有重要作用。真核生物DNA中，几乎所有的甲基化都发生于二核苷酸序列5’-CG-3’的C上，即5’-mCG-3’. 5．CG岛：基因组DNA中大部分CG二核苷酸是高度甲基化的,但有些成簇的、稳定的非甲基化的CG小片段,称为CG岛,存在于整个基因组中。“CG”岛特点是G+C含量高以及大部分CG二核苷酸缺乏甲基化。 6．DNA双螺旋结构模型要点： （1）DNA是反向平行的互补双链结构。 （2）DNA双链是右手螺旋结构。螺旋每旋转一周包含了10对碱基，螺距为3.4nm. DNA 双链说形成的螺旋直径为2 nm。每个碱基旋转角度为36度。DNA双螺旋分子表面 存在一个大沟和一个小沟，目前认为这些沟状结构与蛋白质和DNA间的识别有关。（3）疏水力和氢键维系DNA双螺旋结构的稳定。DNA双链结构的稳定横向依靠两条链互补碱基间的氢键维系，纵向则靠碱基平面间的疏水性堆积力维持。 7．核小体的组成： 染色质的基本组成单位被称为核小体，由DNA和5种组蛋白H1,H2A,H2B,H3和H4共同构成。各两分子的H2A,H2B,H3和H4共同构成八聚体的核心组蛋白，DNA双螺旋缠绕在这一核心上形成核小体的核心颗粒。核小体的核心颗粒之间再由DNA和组蛋白H1构成的连接区连接起来形成串珠样结构。 8．顺反子（Cistron）：由结构基因转录生成的RNA序列亦称为顺反子。 9．单顺反子（monocistron）：真核生物的一个结构基因与相应的调控区组成一个完整的基因，即一个表达单位，转录物为一个单顺反子。从一条mRNA只能翻译出一条多肽链。10．多顺反子(polycistron): 原核生物具有操纵子结构，几个结构基因转录在一条mRNA 链上，因而转录物为多顺反子。每个顺反子分别翻译出各自的蛋白质。 11．原核生物mRNA结构的特点: (1) 原核生物mRNA往往是多顺反子的，即每分子mRNA带有几种蛋白质的遗传信息。 （2）mRNA 5‘端无帽子结构，3‘端无多聚A尾。 （3）mRNA一般没有修饰碱基。 12．真核生物mRNA结构的特点： （1）5‘端有帽子结构。即7－甲基鸟嘌呤－三磷酸鸟苷m7GpppN。 （2）3‘端大多数带有多聚腺苷酸尾巴。 （3）分子中可能有修饰碱基，主要有甲基化。 （4）分子中有编码区和非编码区。 14．tRNA的结构特点 （1）tRNA是单链小分子。 （2）tRNA含有很多稀有碱基。 （3）tRNA的5‘端总是磷酸化，5’末端核苷酸往往是pG. （4）tRNA的3‘端是CCA－OH序列。是氨基酸的结合部位。 （5）tRNA的二级结构形状类似于三叶草，含二氢尿嘧啶环（D环）、T环和反密码子环。
分子生物学实验基础知识 分子生物学是在生物化学基础上发展起来的，以研究核酸和蛋白质结构、功能等生命本质的学科，在核酸、蛋白质分子水平研究发病、诊断、治疗和预后的机制。其中基因工程（基因技术，基因重组）是目前分子生物学研究热点，这些技术可以改造或扩增基因和基因产物，使微量的研究对象达到分析水平，是研究基因调控和表达的方法，也是分子水平研究疾病发生机制、基因诊断和基因治疗的方法。转化（trans formation）、转染、转导、转位等是自然界基因重组存在的方式，也是人工基因重组常采用的手段。基因重组的目的之一是基因克隆（gene clone），基因克隆可理解为以一分子基因为模板扩增得到的与模板分子结构完全相同的基因。使需要分析研究的微量、混杂的目的基因易于纯化，得以增量，便于分析。 外来基因引起细胞生物性状改变的过程叫转化（transformation），以噬菌体把外源基因导入细菌的过程叫转染（transfection）。利用载体（噬菌体或病毒）把遗传物质从一种宿主传给另一种宿主的过程叫转导（transduction）。一个或一组基因从一处转移到基因组另一处的过程叫转位（transposition），这些游动的基因叫转位子。 一、基因工程的常用工具 （一）载体 载体（Vector）是把外源DNA（目的基因）导入宿主细胞，使之传代、扩增、表达的工具。载体有质粒（plasmid）、噬菌体、单链丝状噬菌体和粘性末端质粒（粘粒）、病毒等。载体具有能自我复制；有可选择的，便于筛选、鉴定的遗传标记；有供外源DNA插入的位点；本身体积小等特征。 质粒存在于多种细菌，是染色体（核）以外的独立遗传因子，由双链环状DNA组成，几乎完全裸露，很少有蛋白质结合。质粒有严紧型和松弛型之分。严紧型由DNA多聚酶Ⅲ复制，一个细胞可复制1-5个质粒。而松弛型由DNA多聚酶Ⅰ复制，一个细胞可复制30-50个质粒，如果用氯霉素可阻止蛋白质合成，使质粒有效利用原料，复制更多的质粒。质粒经过改造品种繁多，常用的有pBR322、pUC系列等。这些质粒都含有多个基本基因，如复制起动区（复制原点Ori），便于复制扩增；抗抗生素标记（抗氨芐青霉素Ap r、抗四环素Tc r等）或大肠埃希菌部分乳糖操纵子（E.coli LacZ）等，便于基因重组体的筛选；基因发动子（乳糖操纵子Lac、色氨酸操纵子Trp等）和转录终止序列，便于插入的外源基因转录、翻译表达。质粒上还有许多限制性内切酶的切点，即基因插入位点，又叫基因重组位点，基因克隆位点。 常用噬菌体载体有单链噬菌体M13系统；双链噬菌体系统。噬菌体应和相应的宿主细胞配合使用。以上载体各有特点，便于选择，灵活应用。 （二）工具酶
《分子生物学》复习题 1、染色体：是指在细胞分裂期出现的一种能被碱性染料强烈染色，并具有一定 形态、结构特征的物体。携带很多基因的分离单位。只有在细胞分裂中才可见的形态单位。 2、染色质：是指细胞周期间期细胞核内由DNA、组蛋白、非组蛋白和少量RNA 组成的复合结构，因其易被碱性染料染色而得名。 3、核小体：染色质的基本结构亚基，由约200 bp的DNA和组蛋白八聚体所组 成 4、C值谬误：一个有机体的C值与它的编码能力缺乏相关性称为C值矛盾 5、半保留复制：由亲代DNA生成子代DNA时，每个新形成的子代DNA中， 一条链来自6、亲代DNA，而另一条链则是新合成的，这种复制方式称半保留复制 6、DNA重组技术又称基因工程，目的是将不同的DNA片段（如某个基因或基 因的一部分）按照人们的设计定向连接起来，在特定的受体细胞中与载体同时复制并得到表达，产生影响受体细胞的新的遗传性状。 7、半不连续复制：DNA复制时其中一条子链的合成是连续的，而另一条子链的 合成是不连续的，故称半不连续复制。 8、引发酶：此酶以DNA为模板合成一段RNA,这段RNA作为合成DNA的引 物（Primer)。实质是以DNA为模板的RNA聚合酶。 9、转坐子：存在与染色体DNA上可自主复制和位移的基本单位。 10、多顺反子：一种能作为两种或多种多肽链翻译模板的信使RNA，由DNA 链上的邻近顺反子所界定。 11、基因：产生一条多肽链或功能RNA所必需的全部核甘酸序列。 12、启动子：指能被RNA聚合酶识别、结合并启动基因转录的一段DNA序列。 13、增强子：能强化转录起始的序列 14、全酶：含有表达其基础酶活力所必需的5个亚基的酶蛋白复合物，拥有σ因子。 （即核心酶+σ因子） 15、核心酶：仅含有表达其基础酶活力所必需亚基的酶蛋白复合物，没有σ因子。 16、核酶：是一类具有催化功能的RNA分子 17、三元复合物：开放复合物与最初的两个NTP相结合，并在这两个核苷酸之间形成磷酸二酯键后，转变成包括RNA聚合酶，DNA和新生的RNA的三元复合物。 18、SD序列：mRNA中用于结合原核生物核糖体的序列。30S亚基通过其
基础分子生物学 三、选择题 1、RNA 合成的底物是------ ---------。 A dATP, dTTP , dGTP , d CTP BATP, TTP , GTP , CTP C ATP ,GTP, CTP,UTP D 、GTP, CTP,UTP，TTP 2．模板DNA的碱基序列是3′—TGCAGT—5′，其转录出RNA碱基序列是：A．5′—AGGUCA—3′ B．5′—ACGUCA—3′ C．5′—UCGUCU—3′ D．5′—ACGTCA—3′ E．5′—ACGUGT—3′ 3、转录终止必需。 A、终止子 B、ρ因子 C、DNA和RNA的弱相互作用 D上述三种 4、在转录的终止过程中，有时依赖于蛋白辅因子才能实现终止作用，这种蛋白辅因子称为---- -----。 A σ因子 B ρ因子 C θ因子 D IF因子 5．识别RNA转转录终止的因子是： A．α因子 B．β因子 C．σ因子 D．ρ因子 E．γ因子 6．DNA复制和转录过程有许多异同点,下列DNA复制和转录的描述中错误的是： A．在体内以一条DNA链为模板转录，而以两条DNA链为模板复制 B．在这两个过程中合成方向都为5′→3′ C．复制的产物通常情况下大于转录的产物 D．两过程均需RNA引物 E．DNA聚合酶和RNA聚合酶都需要Mg2+ 7、核基因mRNA 的内元拼接点序列为。 A、AG……GU B、GA……UG C、GU……AG D、UG……GA 8、真核生物mRNA分子转录后必须经过加工，切除---------，将分隔开的编码序列连接在一起，使其成为蛋白质翻译的模板，这个过程叫做RNA的拼接。 A 外显子 B 启动子 C 起始因子 D 内含子 9、在真核生物RNA polⅡ的羧基端含有一段7个氨基酸的序列，这个7肽序列为Tyr-Ser-Pro-Thr-Ser-Pro-Ser ,被称作。 A C末端结构域 B 帽子结构 C Poly(A)尾巴 D 终止子 10．真核生物RNA的拼接需要多种snRNP的协助，其中能识别左端（5’）拼接点共有序列的snRNP 是： A．U1 snRNP B．U2 snRNP C．U5 snRNP E．U2 snRNP+ U5 snRNP 四、是非题 1、所有的启动子都位于转录起始位点的上游。（ X ） 2、RNA分子也能像蛋白酶一样，以其分子的空间构型产生链的断裂和和合成所必须的微环境。（对） 3、真核生物的mRNA中的poly A 尾巴是由DNA编码，经过转录形成的。（ X ） 4、在大肠杆菌RNA聚合酶中，β亚基的主要功能是识别启动子。（ X ） 5、所有起催化作用的酶都是蛋白质。（ X ） 五、问答题
肿瘤分子生物学讲义 第一节概述 (1) 第二节肿瘤的发生机制 (4) 第三节癌基因及其致癌的分子机制 (5) 第四节抑癌基因及其抑癌的分子机制 (9) 第五节肿瘤转移相关基因 (11) 第六节肿瘤的预防和治疗 (13) 第一节概述 一、肿瘤及肿瘤分子生物学的概念 肿瘤（tumor）是一类疾病的总称，它们的基本特征是细胞增殖与凋亡失控，扩张性增生形成新生物。肿瘤可分为良性肿瘤（benign tumor）和恶性肿瘤（malignant tumor）。 良性肿瘤生长缓慢，虽可增长至相当大的体积，但仍保留正常细胞的某些特性，通常在瘤体外有完整的包膜，手术切除后患者预后良好。绝大多数良性肿瘤基本上是无害的，不引起或很少引起宿主损伤。不过有极少数良性肿瘤因其靠近生命中枢或能合成大量生物活性物质也可能杀伤宿主。例如，脑膜上生长缓慢的良性肿瘤通过压迫使得生命中枢萎缩破坏，最终导致宿主死亡；胰岛细胞良性肿瘤可以分泌大量胰岛素而引起体内胰岛素过量，导致低血糖和死亡。 恶性肿瘤统称为癌症（cancer），它不同于良性肿瘤的最重要的特性是能侵袭周围组织，疾病晚期癌细胞发生远端转移，破坏受侵袭的脏器，最终使机体衰亡，但如能在侵袭转移前切除癌瘤，一般预后明显改善。由于技术水平的限制，目前临床诊断的癌症患者多处于中晚期。加上不良生活方式如吸烟、过度饮酒、不合理饮食习惯，以及环境污染增加等因素，在刚过去的20世纪，世界各国许多常见癌症的发病率在总体上呈上升趋势，或维持在高水平，在我国的情况亦大致如此。目前除几种较少见的癌症如妇科的宫颈癌、绒癌等的死亡率有明显下降外，多数常见恶性肿瘤死亡率还处于令人忧心的高位态势下。有研究者预测，在21世纪癌症仍将是危害人类健康的主要疾病之一，故应引起预防、临床和基础研究者的高度关注。 恶性肿瘤几乎在所有类型的细胞中均可发生。根据组织学来源，癌症的起源可分为三种：癌（carcinoma）起源于上皮细胞，大部分成人癌症属此类；淋巴瘤起源于脾和淋巴结等的淋巴细胞；肉瘤（sarcoma）起源于间叶组织如结缔组织、骨和肌肉等。以上在各种实质性组织、脏器中发生的癌症属实体肿瘤（solid tumor）。白血病起源于骨髓造血细胞，恶性细胞存在于流动的血液中，属液体肿瘤（liquid tumor）。 肿瘤分子生物学，就是用分子生物学的理论和技术来研究肿瘤的一门科学，是医学和生物学的一门交叉学科 二、肿瘤的生物学特征 1、癌症是体细胞遗传病 就本质而论，癌症是一种遗传学疾病或体细胞遗传学疾病，可简称为遗传病。在癌细胞中发生的遗传学变异有：基因内的碱基替代、缺失、插入和基因扩增等，以及染色体的数量和结构的改变，如非整倍体、易位等；表遗传学改变有：DNA甲基化型式改变、组蛋白修饰和染色质改型等。这些改变引起了肿瘤抑制基因灭活和原癌基因的活化，它们所产生的恶性表型通过有丝分裂能在细胞世代间传递。上述过程均发生在体细胞，这是占全部癌症中绝大多数的、散发性癌症的发生模式。遗传性癌综合征不同于其他一些遗传病，它遗传的仅是癌易感性，还需要体细胞的多次击中才能产生恶性表型。 基因组内存在两类癌相关基因：一类基因直接调控细胞增殖与凋亡、运动与黏着，以及细胞基质的改型等，并参与细胞的信号转导，结果得以维持正常组织细胞的自稳性。当这些基因缺陷造成上述过程失衡，随着细胞各种恶性特征的积累，最终癌症发生。这些基因包括癌基因、肿瘤抑制基因中把关基因（gatekeeper gene）；另一类基因并不直接调控细胞的增殖和凋亡，而是影响第一类癌相关基因的突变速率的管护基因（caretaker gene），这包括各类DNA修复基因，还包括代谢酶多态性在内的一组修饰基因（modifier gene）。 2、癌细胞的恶性生物学特征
分子生物学作业 第一次 1、Promoter：（启动子）一段位于结构基因5…端上游、能活化RNA聚合酶的DNA序列，是RNA聚合酶的结合区，其结构直接关系转录的特异性与效率。 2、Cis-acting element：（顺式作用元件）影响自身基因表达活性的非编码DNA序列，组成基因转录的调控区包括：启动子、增强子、沉默子等 一、简述基因转录的基本特征。（作业）P35 二、简述蛋白质生物合成的延长过程。P58 肽链的延伸由于核糖体沿mRNA5 ′端向3′端移动，开始了从N端向C端的多肽合成。 起始复合物，延伸AA-tRNA，延伸因子，GTP,Mg 2+，肽基转移酶 每加一个氨基酸完成一个循环，包括: 进位：后续AA-tRNA与核糖体A位点的结合 起始复合物形成以后，第二个AA-tRNA在EF-Tu作用下，结合到核糖体A位上。 通过延伸因子EF-Ts再生GTP,形成EF-Tu?GTP复合物，参与下一轮循环。 需要消耗GTP，并需EF-Tu、EF-Ts两种延伸因子。 转位：P位tRNA的AA转给A位的tRNA,生成肽键； 移位：tRNA和mRNA相对核糖体的移动； 核糖体向mRNA3’端方向移动一个密码子，二肽酰－tRNA2进入P位，去氨酰-tRNA 被挤入E位，空出A位给下一个氨酰-tRNA。移位需EF-G并消耗GTP。 三、真核细胞mRNA分子的加工过程有哪些？P40 1、5’端加帽 加帽指在mRNA前体刚转录出来或转录尚未完成时，mRNA前体5’端在鸟苷酸转移酶催化下加G,然后在甲基转移酶的作用下进行甲基化。 帽子的类型 0号帽子(cap1) 1号帽子(cap1) 2号帽子(cap2) 2、3’端的产生和多聚腺苷酸花 除组蛋白基因外，真核生物mRNA的3?末端都有poly(A)序列，其长度因mRNA种类不同而变化，一般为40～200个A 。 大部分真核mRNA有poly(A)尾巴，1/3没有。 带有poly(A)的mRNA称为poly(A)+， 不带poly(A)的mRNA称为poly(A)－。 加尾信号： 3?末端转录终止位点上游15～30bp处的一段保守序列AAUAAA。 过程： ①内切酶切开mRNA3?端的特定部位； ②多聚A合成酶催化加poly(A)。 3、RNA的剪接
分子生物学实验基础 分子生物学是在生物化学基础上发展起来的，以研究核酸和蛋白质结构、功能等生命本质的学科，在核酸、蛋白质分子水平研究发病、诊断、治疗和预后的机制。其中基因工程（基因技术，基因重组）是目前分子生物学研究热点，这些技术可以改造或扩增基因和基因产物，使微量的研究对象达到分析水平，是研究基因调控和表达的方法，也是分子水平研究疾病发生机制、基因诊断和基因治疗的方法。转化（transforma tion）、转染、转导、转位等是自然界基因重组存在的方式，也是人工基因重组常采用的手段。基因重组的目的之一是基因克隆（gene clone），基因克隆可理解为以一分子基因为模板扩增得到的与模板分子结构完全相同的基因。使需要分析研究的微量、混杂的目的基因易于纯化，得以增量，便于分析。 外来基因引起细胞生物性状改变的过程叫转化（transformation），以噬菌体把外源基因导入细菌的过程叫转染（transfection）。利用载体（噬菌体或病毒）把遗传物质从一种宿主传给另一种宿主的过程叫转导（transduction）。一个或一组基因从一处转移到基因组另一处的过程叫转位（transposition），这些游动的基因叫转位子。 一、基因工程的常用工具 （一）载体 载体（Vector）是把外源DNA（目的基因）导入宿主细胞，使之传代、扩增、表达的工具。载体有质粒（p lasmid）、噬菌体、单链丝状噬菌体和粘性末端质粒（粘粒）、病毒等。载体具有能自我复制；有可选择的，便于筛选、鉴定的遗传标记；有供外源DNA插入的位点；本身体积小等特征。 质粒存在于多种细菌，是染色体（核）以外的独立遗传因子，由双链环状DNA组成，几乎完全裸露，很少有蛋白质结合。质粒有严紧型和松弛型之分。严紧型由DNA多聚酶Ⅲ复制，一个细胞可复制1-5个质粒。而松弛型由DNA多聚酶Ⅰ复制，一个细胞可复制30-50个质粒，如果用氯霉素可阻止蛋白质合成，使质粒有效利用原料，复制更多的质粒。质粒经过改造品种繁多，常用的有pBR322、pUC系列等。这些质粒都含有多个基本基因，如复制起动区（复制原点Ori），便于复制扩增；抗抗生素标记（抗氨芐青霉素Apr、抗四环素Tcr等）或大肠埃希菌部分乳糖操纵子（E.coli LacZ）等，便于基因重组体的筛选；基因发动子（乳糖操纵子Lac、色氨酸操纵子Trp等）和转录终止序列，便于插入的外源基因转录、翻译表达。质粒上还有许多限制性内切酶的切点，即基因插入位点，又叫基因重组位点，基因克隆位点。 常用噬菌体载体有单链噬菌体M13系统；双链噬菌体系统。噬菌体应和相应的宿主细胞配合使用。以上载体各有特点，便于选择，灵活应用。
分子生物学知识点Last revision on 21 December 2020
一、名词解释： 1. 基因：基因是位于染色体上的遗传基本单位，是负载特定遗传信息的DNA片段，编码具有生物功能的产物包括RNA和多肽链。 2. 基因表达：即基因负载遗传信息转变生成具有生物学功能产物的过程，包括基因的激活、转录、翻译以及相关的加工修饰等多个步骤或过程。 3.管家基因：在一个生物个体的几乎所有组织细胞中和所有时间段都持续表达的基因，其表达水平变化很小且较少受环境变化的影响。如GAPDH、β-肌动蛋白基因。 4. 启动子：是指位于基因转录起始位点上游、能够与RNA聚合酶和其他转录因子结合并进而调节其下游目的基因转录起始和转录效率的一段DNA片段。 5.操纵子：是原核生物基因表达的协调控制单位，包括有结构基因、启动序列、操纵序列等。如：乳糖操纵子、色氨酸操纵子等。 6.反式作用因子：指由其他基因表达产生的、能与顺式作用元件直接或间接作用而参与调节靶基因转录的蛋白因子（转录因子）。 7.顺式作用元件：即位于基因附近或内部的能够调节基因自身表达的特定DNA序列。是转录因子的结合位点，通过与转录因子的结合而实现对真核基因转录的精确调控。 8. Ct值：即循环阈值（cycle threshold，Ct），是指在PCR扩增过程中，扩增产物的荧光信号达到设定的荧光阈值所经历的循环数。（它与PCR扩增的起始模板量存在线性对数关系，由此可以对扩增样品中的目的基因的模板量进行准确的绝对和（或）相对定量。） 9.核酸分子杂交：是指核酸分子在变性后再复性的过程中，来源不同但互不配对的核酸单链（包括DNA和DNA，DNA和RNA，RNA和RNA）相互结合形成杂合双链的特
分子生物学总结完整版 1、结构分子生物学； 2、基因表达的调节与控制； 3、DNA重组技术及其应用； 4、结构基因组学、功能基因组学、生物信息学、系统生物学 第二章DNA and Chromosome 1、DNA的变性：在某些理化因素作用下，DNA双链解开成两条单链的过程。 2、 DNA复性：变性DNA在适当条件下，分开的两条单链分子按照碱基互补原则重新恢复天然的双螺旋构象的现象。 3、 Tm(熔链温度)： DNA加热变性时，紫外吸收达到最大值的一半时的温度，即DNA分子内50%的双链结构被解开成单链分子时的温度） 4、退火：热变性的DNA经缓慢冷却后即可复性，称为退火 5、假基因：基因组中存在的一段与正常基因非常相似但不能表达的DNA序列。以Ψ来表示。 6、 C值矛盾或C值悖论:C值的大小与生物的复杂度和进化的地位并不一致，称为C值矛盾或C值悖论(C-Value Paradox)。 7、转座：可移动因子介导的遗传物质的重排现象。 8、转座子：染色体、质粒或噬菌体上可以转移位置的遗传成分
9、 DNA二级结构的特点：1）DNA分子是由两条相互平行的脱氧核苷酸长链盘绕而成；2）DNA分子中的脱氧核苷酸和磷酸交替连接，排在外侧，构成基本骨架，碱基排列在外侧；3）DNA分子表面有大沟和小沟；4）两条链间存在碱基互补，通过氢键连系，且A=T、G ≡ C（碱基互补原则）；5）螺旋的螺距为 3、4nm，直径为2nm，相邻两个碱基对之间的垂直距离为0、34nm，每圈螺旋包含10个碱基对；6）碱基平面与螺旋纵轴接近垂直，糖环平面接近平行 10、真核生物基因组结构：编码蛋白质或RNA的编码序列和非编码序列，包括编码区两侧的调控序列和编码序列间的间隔序列。特点：1）真核基因组结构庞大哺乳类生物大于2X109bp；2）单顺反子（单顺反子：一个基因单独转录，一个基因一条mRNA，翻译成一条多肽链；）3）基因不连续性断裂基因（interrupted gene）、内含子(intron)、外显子(exon)；4）非编码区较多，多于编码序列(9:1) 5）含有大量重复序列1 1、Histon(组蛋白)特点：极端保守性、无组织特异性、氨基酸分布的不对称性、可修饰作用、富含Lys的H5 12、核小体组成： 由组蛋白和200bp DNA组成 13、转座的机制：转座时发生的插入作用有一个普遍的特征，那就是受体分子中有一段很短的被称为靶序列的DNA会被复