Systemic Agrobacterium tumefaciens–mediated transfection of viral replicons for ef?cient transient expression in plants
Sylvestre Marillonnet 1,2,Carola Thoeringer 1,2,Romy Kandzia 1,Victor Klimyuk 1&Yuri Gleba 1
Plant biotechnology relies on two approaches for delivery and expression of heterologous genes in plants:stable genetic
transformation and transient expression using viral vectors.Although much faster,the transient route is limited by low infectivity of viral vectors carrying average-sized or large genes.We have developed constructs for the ef?cient delivery of RNA viral vectors as DNA precursors and show here that Agrobacterium–mediated delivery of these constructs results in gene ampli?cation in all mature leaves of a plant simultaneously (systemic transfection).This process,called ‘magnifection’,can be performed on a large scale and with different plant species.This technology combines advantages of three biological systems (the transfection ef?ciency of A.tumefaciens ,the high expression yield obtained with viral vectors,and the post-translational capabilities of a plant),does not require genetic modi?cation of plants and is faster than other existing methods.
Viral vectors designed for expression of recombinant proteins in plants hold great promise because of high absolute and relative yields,and because of the speed provided by transient expression.Most of the results of practical interest achieved so far have been obtained with vectors built on the backbones of plus-sense RNA viruses such as tobacco mosaic virus (TMV)or potato virus X 1–4.
We have recently shown that TMV-based vectors can be delivered to plant tissues using A.tumefaciens 5(agroinfection).However,one step of this process,namely the formation of active replicons from the primary nuclear transcript,is inef?cient.In a standard leaf transfec-
tion experiment,this inef?ciency is masked by the subsequent ability of the replicons to move to neighboring cells by cell-to-cell movement.Here we show that this bottleneck can be fully remedied by incorpora-tion of silent nucleotide substitutions into the vector and by addition of multiple introns.We demonstrate that such modi?cations provide for ef?cient processing of the DNA information into active replicons in almost all cells (as high as 94%)of Nicotiana benthamiana ,an up to 1,000-fold improvement over nonoptimized TMV-based vectors,and an even higher improvement (4106-fold)in Nicotiana tabacum (tobacco).Finally,we show that the resulting vectors allow the development of a fully scalable and versatile whole-plant transfection protocol,that we term magnifection,for production of heterologous proteins in plants.
RESULTS
Viral replication following agroin?ltration of TMV-based vectors Agroin?ltration of a TMV-based viral vector containing the gene encoding green ?uorescent protein (GFP)(pICH16707,Fig.1a )into N.benthamiana leaves leads to the formation of foci of GFP
?uorescence 3d post-in?ltration (d.p.i.)(shown in ref.5and in Supplementary Fig.1online).T o quantify the proportion of cells initiating viral replication,a 489-bp deletion was made within the movement protein (MP)coding sequence,resulting in construct pICH14833(Fig.1a ).Replicons derived from this construct cannot move from cell-to-cell but are able to replicate autonomously within each infected cell.Three days after agroin?ltration of pICH14833in N.benthamiana leaf (OD 600of the A.tumefaciens in in?ltration solution was 0.7),a small number of cells expressing GFP appeared (see Supplementary Fig.1online),and the same pattern was still visible 2weeks after in?ltration.By counting protoplasts prepared from the in?ltrated area (Figs.1and 2),we found that 0.6–1.6%of cells initiated viral replication.
There are several reasons why RNA viral vectors might have dif?culties starting the replication cycle.First,RNA viruses,such as TMV ,replicate in the cytoplasm and never enter the nucleus,and have therefore evolved in an environment where they are not exposed to the nuclear pre-mRNA processing machinery.As a result,pre-mRNA transcripts made in the nucleus from viral constructs may not be re-cognized and processed properly.Second,viral vector constructs encode very large transcripts (B 7.6kb for the primary transcript of a viral vector containing a GFP gene),a size much larger than the average size (1–2kb)of plant genes.Moreover,in nature,large eukaryotic genes often contain numerous introns that facilitate processing and export of the pre-mRNAs from the nucleus 6.We therefore hypothesized that modi?cations of the constructs that would increase the ef?ciency of processing and export of primary transcripts from the nucleus to the cytoplasm could lead to an increase in the number of cells that would initiate viral replication.Two types of modi?cations were made:
Published online 8May 2005;doi:10.1038/nbt1094
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Genetics,Biozentrum Halle,Weinbergweg 22,D-06120Halle (Saale),Germany.2These authors contributed equally to this work.Correspondence and requests for materials should be addressed to Y.G.(gleba@icongenetics.de).A R T I C L E S
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(i)removal of sequence features that might be improperly recognized by the RNA processing machinery (such as cryptic splice sites and thymine-rich intron-like sequences),and (ii)addition of introns.Removal of putative intron-like features
T o identify sequence features that might induce abnormal RNA processing events,we analyzed the sequence of pICH14833using the NetgeneII program (http://www.cbs.dtu.dk/services/NetGene2/(ref.7))with parameters set for Arabidopsis thaliana sequences.We noticed several intron-like sequence features consisting of putative cryptic splice sites and several thymine-rich sequences (see Supple-mentary Fig.2online).
We ?rst removed some of the putative cryptic splice sites by using PCR with primers designed to introduce silent nucleotide substitu-tions.However,the resulting constructs (pICH15011and pICH17266)were not signi?cantly more ef?cient than the initial construct,pICH14833(Fig.1and Supplementary Table 1online).Then,we mutagenized a 0.6-kb thymine-rich region located at the beginning of the RdRp coding sequence by introducing 54silent nucleotide sub-stitutions (two substitutions being the two splice site mutations also present in pICH15011)to increase its GC content.The resulting clone,pICH15466,worked substantially better than the unmodi?ed clone,with 13%of cells in the in?ltrated area initiating viral replication (Fig.1)compared to 1.6%for pICH14833.Another potentially problematic region corresponds to a 220-bp thymine-rich sequence at the 3￠end of the RdRp coding sequence,a region that contains the MP subgenomic promoter (see Supplementary Fig.2online).Forty-three silent nucleotide substitutions were introduced in this area (leaving all other regions unmodi?ed).With the resulting construct,pICH15900,53%of cells from the in?ltrated area expressed GFP (Fig.2b ,Fig.2e in?ltration 4).In a separate assay,we measured the amount of GFP ?uorescence in the in?ltrated area,and found an increase from 3.3?uorescence units (for pICH14833)to 53.3,a relative increase consistent with the increase in the number of GFP-expressing protoplasts.A construct similar to pICH15900but with no deletion in the MP ,pICH16989,gave similar rates of initiation of replication,indicating that the improvement was due to modi?cation of the codon usage and not to the deletion in the MP (Fig.1).
T o test whether these modi?cations had an effect on viral replica-tion,pICH15466and pICH15900were in?ltrated into the leaves of transgenic N.benthamiana plants expressing MP (plants transformed with pICH10745).The size of the GFP-expressing foci and the intensity of GFP ?uorescence appeared similar for both constructs and pICH14833(Fig.2f ),showing that modi?cation of the RNA sequence did not signi?cantly affect the replication or cell-to-cell movement abilities of the viral vector.
pICH15025 pICH15034pICH16877
pICH15488pICH15755pICH15477 pICH17200 pICH15922 pICH15499pICH16433 pICH14030 pICH15041pICH16100pICH16191
pICH16200 pICH15860 pICH16141pICH17494 (pICH16707)
pICH18535
(pICH18000)pICH18722
(pICH18711)
pICH17466 (pICH16424)
pICH17474(pICH17272)
pICH18523(pICH17282) pICH15466 pICH15900pICH17266 pICH15011 pICH14833 pICH16989pICH5661
(pICH15662)
pICH17144pICH18000pICH17272a
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Figure 1Constructs maps and quanti?cation
of the ef?ciency of initiation of replication.(a )Schematic representation of the constructs.The MP is shown as a gray box between the TVCV RNA-dependent RNA polymerase (RdRp)and the GFP coding sequence.Deletions of 489and 575nt are labeled d1and d2,respectively.Introns are shown as narrow white boxes,and the designation of the insertion sites (numbered 1–23,position given in the methods section)are indicated by a number above the introns.1a and 1b refer to the insertion of two different introns at the same
position (position 1).Vertical black lines show the position of mutated putative splice sites.Two mutagenized regions containing 54and 43silent nucleotide substitutions are shown as a gray box underlined with a black line,and as a dotted box,respectively.A frameshift in the MP is shown as an X at the beginning of the MP .Construct numbers written in italics under construct names correspond to versions of the constructs not containing a frameshift in the MP .Act2,A.thaliana ACT2promoter;N,cr-TMV 3￠untranslated region;T,Nos terminator.
(b )Quanti?cation of the ef?ciency of initiation of replication of viral constructs measured by counting the proportion of protoplasts expressing GFP in in?ltrated areas (gray columns,expressed as a percentage of all protoplasts)or by
measuring GFP ?uorescence with a luminescence spectrometer (black columns,expressed in
?uorescence units,all values were multiplied by 0.7in order to be visualized on the same scale as the protoplast counts).Error bars indicate standard deviation.For protoplasts counts,two samples of 400–500protoplasts were counted from each protoplast preparation.For GFP
?uorescence,values were determined from three samples taken from different in?ltrated leaves.
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Addition of introns
Next,introns were added at up to 19positions within the RdRp and the MP sequences.When introns were inserted into the MP coding sequence,the MP was made nonfunctional by a frameshift rather than by a deletion.The presence of most introns increased the ef?ciency of initiation of viral replication,although some introns were not as ef?-cient as others (Fig.1b ).Insertion of additional introns usually further increased the ef?ciency of initiation of viral replication.For example,with pICH18535,which contains 12introns,92%(90–94%)of protoplasts from the in?ltrated area expressed GFP .With the same construct (but with a functional MP),pICH18000,93%(90–96%)of protoplasts expressed GFP .This suggests that with constructs with more than 12introns,initiation of viral replication starts in virtually every N.benthamiana cell,at least when in?ltration solution containing agrobacteria at an OD 600of 0.7is used.Also,at the protein level,expression of GFP was similar in 16-intron constructs with or without a functional MP (pICH18711-described below,and pICH18722,Fig.2g ).
We also tested the effect of the presence of introns within the gene of interest.Four introns were inserted within the GFP coding sequence,resulting in construct pICH17144.This construct worked better than the same construct without introns,with 9.5%of cells in the in?ltrated area expressing GFP .
Viral replication of fully-optimized viral vectors
We then tested the performance of pICH18711,a fully optimized construct containing most of the modi?cations described above (the ?rst mutagenized region at the beginning of the RdRp and 16introns in the RdRp and MP coding sequences),but also containing a functional MP (Fig.1a ).A dilution series of A.tumefaciens in in?ltration solution was in?ltrated into N.benthamiana and tobacco leaves (Fig.3a ,b ).Whereas no GFP-expressing sector could be detected with the original construct in N.benthamiana at dilutions higher than 10à3,GFP-expressing foci were obtained until the 10à6dilution with pICH18711.In?ltration of a 10à4dilution for pICH18711gave a similar number of GFP-expressing foci as the 10à1dilution for the nonoptimized construct,pICH16707,indicating
a 1,000-fold increase in the ef?ciency of initiation of viral replication.The number of bacteria present in each dilution was estimated by plating an aliquot of the in?ltration solution and counting the number of colonies for the 10à5and 10à6dilutions.This showed that approximately eight A.tumefaciens cells are required per
GFP-expressing event in N.benthamiana ,whereas 5,700agrobacteria are required for the unmodi?ed control construct.This represents a 712-fold improvement,in accordance with the 1,000-fold increase cited above.For tobacco,23agrobacteria were required per GFP-expressing foci.In contrast,a nonoptimized construct did not work properly in tobacco,even at high bacterial concentration:at the 10à1and 10à2dilutions,only a few individual cells in the in?ltrated area expressed GFP ,but replicons were unable to move outside of these few initial cells.Optimized viral vectors were also tested in N.excelsior,and the same level of improvement was seen as in N.benthamiana .
Characterization of the optimized viral vectors
We replaced the relatively small GFP coding sequence (0.7kb)in pICH16707and pICH18711by the larger coding sequence (1.8kb)of the b -glucuronidase gene (GUS),resulting in constructs pICH18841(0introns)and pICH18851(16introns),respectively.With pICH18841,fewer replication foci were observed in in?ltrated areas than when using a construct expressing GFP (not shown),suggesting that longer genes may negatively affect the frequency of initiation of viral replication.By using the intron-optimized viral vector pICH18851,the entire in?ltrated area was expressing GUS.
A time-course experiment was carried out to measure the time required to obtain maximal gene expression after in?ltration with either GFP -or GUS -containing constructs (Fig.3c ,d ).A 10à1dilution
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Figure 2Performance of different synthetic vectors.(a –d )Protoplasts
prepared from N.benthamiana leaves with pICH14833(a ),pICH15900(b ),pICH18722(c )and pICH18711(d ),photographed under blue light.Scale bar,100m m.(e )N.benthamiana leaves were in?ltrated with pICH14833(1),pICH15011(2),pICH15466(3),pICH15900(4),pICH15477(5),pICH15034(6),pICH16433(7),pICH16141(8),pICH17466(9),
pICH17144(10),pICH18722(11),pICH18711(12)and photographed under UV light at 7d.p.i.In?ltrations 13–15were similar to in?ltrations 10–12except that pICH10745was coin?ltrated to provide transient MP expression.In?ltrations in a –e were performed with A.tumefaciens in in?ltration solution at an OD 600of 0.7.(f )Leaf of a transgenic N.benthamiana plant (pICH10745)in?ltrated with pICH14833(1),
pICH15466(2)and pICH15900(3).A.tumefaciens in?ltration solutions were diluted to between 10à3to 10à4relative to the overnight-grown
A.tumefaciens culture in order to obtain separate GFP foci.(g )Coomassie-stained SDS protein gel loaded with crude extracts prepared from
N.benthamiana nonin?ltrated leaf tissue (u)or from the in?ltrated areas shown in e (numbering is as in panel e ).Molecular weights (kDa)are shown on the left.The arrow indicates GFP .
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of the A.tumefaciens in?ltration solution (OD 600?0.35)was used for this experiment.For both GFP and GUS,levels of expression increased faster and reached higher levels with intron-containing constructs.As a control for transient expression using nonreplicating constructs,a time course was performed for GFP expressed from the CaMV 35S promoter (pICH5290)in the absence or presence of the suppressor of silencing p19to enhance expression 8(Fig.3c ).In both cases,GFP expression levels were lower than with viral vectors.
Transfection of whole plants:‘magnifection’
Since TMV-based vectors that lack a coat protein gene cannot move systemically,production in entire plants requires inoculating all leaves of a plant.However,this process is inef?cient with unimproved viral vectors owing to low infectivity of the vectors.Having improved viral vectors in hand,we attempted to inoculate entire plants using a variety of treatments,including immersing or spraying entire plants,applying (or not)a vacuum treatment,in?ltrating whole plants or detached leaves,wilting plants/leaves before treatments,using detergents and solubilizers.The best and most reliable results were obtained by simply immersing all aerial parts of an entire plant into a bacterial suspension
and applying a weak vacuum (0.5–1bar)for 1–2min,followed by a gentle (o 1min)gradual return to atmospheric pressure.This simple procedure,which is similar to a protocol commonly used for transient
expression in detached leaves 9,leads to in?ltration of A.tumefaciens suspension into the intercellular space of all mature leaves of tobacco or N.benthamiana plants.The treated plants are then simply returned to the greenhouse (under standard conditions)where they fully recover.This procedure leads to GFP expression in all leaves,with the exception of the young nonexpanded leaves of the apex (Fig.4).The in?ltration procedure was tested on plants and seedlings of different ages,ranging from 2weeks old to ?owering age (6–8weeks depending on growth conditions).High levels of GFP expression were obtained in the mature leaves of all plants,but a higher ratio of expressing to nonexpressing tissue was obtained for larger plants (43weeks old).High levels of GFP expression were also obtained with plants in?ltrated at ?owering stage,although at later stages,older leaves showed reduced expression.
We also tested bacterial suspensions that were diluted 10à1to 10à6relative to a saturated overnight bacterial culture (OD 600of the 10à1dilution was 0.35or B 1.8?108colony forming units per ml).In?ltrations were performed with N.benthamiana and N.tabacum plants.For both species,the 10à3dilution provided the highest yield,indicating that the 10à1and 10à2dilutions are somewhat inhibitory or toxic to plant cells (Fig.5).For the 10à3or lower dilutions,development of the infection was delayed,indicating that the primary infection by agrobacteria occurred in a minority of cells only,and that
c d.p.i.
d.p.i.Figure 3Ef?ciency of ‘agrodelivery’and of gene expression of the ?nal,fully optimized construct.(a )Tobacco leaf in?ltrated with dilution series of
A.tumefaciens in in?ltration solution for constructs pICH16707(lower half of the leaf)and pICH18711(upper half).Dilutions labeled –1to –6correspond to 10à1to 10à6dilutions of the A.tumefaciens relative to the starting overnight culture (OD 600of the 10à1dilution was 0.35).The picture was taken under UV light at 9d.p.i.(b )Same as in a but in N.benthamiana.The picture was taken at 5d.p.i.(c )Time course showing the level of GFP ?uorescence in N.benthamiana leaf from 2or 3to 10d.p.i.All samples from individual curves were harvested from the same leaf.Leaves A and B were in?ltrated with pICH18711,leaves C and D with pICH16707,leaf E with pICH5290(35S-GFP )and leaf F with pICH5290+pICH6692(35S-p19);A.tumefaciens concentration was at 0.35under OD 600for all in?ltrations.f.u.,?uorescence units.(d )As in c but with GUS ?uorescence by in?ltration of pICH18851(16introns)in leaves A and B and pICH18841(0intron)in leaves C and D.
Figure 4Expression of GFP in N.benthamiana plants and Beta vulgaris .Whole plants were vacuum-in?ltrated with pICH18711and viewed under UV light.(a –d )N.benthamiana plants 4d after in?ltration with in?ltration solution containing agrobacteria diluted 10à1(d ),10à2(c ),10à3(b )or 10à4(a )relative to the overnight saturated A.tumefaciens culture (OD 600?0.35of the 10à1dilution).(e )N.benthamiana plants of various ages (17–35d after sowing)were
in?ltrated with a 10à1diluted in?ltration solution and photographed 4d later.(f )same plant as shown in e )(in?ltrated 28d after sowing),but pictured 6d.p.i.(g ,h )Beta vulgaris var.conditiva vacuum-in?ltrated with pICH18711(A.tumefaciens OD 600?0.35),photographed under normal (g )or UV light (h )10d.p.i.
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(virally controlled)cell-to-cell spread of the replicons was then required to complete transfection of all leaf tissue.
The absolute protein yield seen in our experiments was as high as 4g of recombinant protein per kg of fresh leaf biomass in N.benthamiana and up to 2.5g/kg in tobacco (N.tabacum),and the relative yield as high as 25%and 40%of total soluble protein in both species,respectively (Fig.5).Assuming such protein yield,and based on realistic yields of 100tons of plant leaf biomass per hectare of a greenhouse per year,a 1ha facility should be capable of producing 280–400kg of recombinant protein per year.Our measurements show that in?ltration consumes 1–1.5liters of bacterial suspension per kg of plant leaf biomass,indicating that,at the optimal 10à3dilution,1liter of overnight A.tumefaciens culture is suf?cient to treat 700–1,000kg of N.benthamiana biomass and to produce 2.5–4.0kg of recombinant protein.
Analysis of magnifection in different plant species
Whole-plant in?ltration was tested on over 50dicotyledonous plant species belonging to eight plant families.Good expression was found in seven species (petunia,cucumber,sun?ower,red beets,spinach,
Chenopodium capitatum and Tetragonia expansa ),representing ?ve plant families (expression in red beets shown in Figure 4).Some expression was also detected in six other species,including A.thaliana ,Brassica spp.and Lepidium sativum .Since the leaves of some species supporting the transfection,including red beets,spinach,Chenopo-dium or Tetragonia,can be used as uncooked food,the technology proposed here can in principle be used for manufacturing edible/topical vaccines or for production of minimally processed functional foods/feeds.
DISCUSSION
The ?rst demonstration of A.tumefaciens –mediated infection of a plant with a TMV-based vector was reported in 1993(ref.10).The authors quanti?ed the effectiveness of the process and concluded that for a wild-type TMV virus (the U1strain),transfection is very inef?cient,requiring B 108bacteria for one successful infection event in a tobacco plant.Since agroinfection by DNA viruses is generally much more effective (103–105bacteria per event),the authors concluded that the low ef?ciency for RNA viruses is a result of either viral RNA degradation in the nucleus,premature termination of transcription,or low in vivo rates of transcription or poor transcript transport to the cytoplasm.The results of our study both support those conclusions and provide effective remedies,resulting in a process that requires 20bacteria to generate one transfection event
in a tobacco plant,an up to 107-fold improvement over the original process.Several investigators had previously modi?ed the cDNA of RNA viruses by introduction of introns,but mainly with the goal of eliminating the toxicity caused by viral sequences in bacteria 11–13.The general principle of modifying a DNA copy of an RNA virus-derived replicon for increased infectivity is most likely also applicable to other cytoplasmic plant and animal RNA viruses other than the crucifer-speci?c TMV strain used in our experi-ments 14,15(see also ref.16).
Having a more infectious viral vector allowed us to develop magnifection,an ef?cient whole-plant in?ltration protocol.This straightforward protocol requires,in addition to well-established industrial upstream (plant cultivation)and downstream (protein extraction and puri?cation)components,a simple technology block that contains an apparatus for vacuum-in?ltration of batches of plants and a chamber/greenhouse for subsequent short-term incubation,as well as a small bacterial fermenter 17.Such a block would of course require certain safety locks so as to prevent the release of agrobacteria into the open environment and to protect the operating personnel.The magnifection process relies on vectors that do not express a coat protein.Although such vectors cannot move systemically,the combination with magnifection provides a solution that has many advantages over existing systems:(i)the vectors are less affected by transgene size since they do not have to move systemically;(ii)simultaneous infection of multiple cells in multiple leaves means that expression is more synchronous and faster;(iii)a larger propor-tion of the host plant is infected by the virus than with traditional vectors (which move systemically primarily to young emerging host tissue);(iv)all metabolic resources of the cell are directed toward synthesis of the protein and not wasted to make large amounts of coat protein;(v)replicon RNAs are not packaged into viral particles and therefore cannot be transferred to secondary untransfected plants or generate wild-type virus.
The magnifection process described here is safe,since,in addition to the lack of of viral particle formation,the high yield achievable allows for full containment of the process,thus avoiding open ?eld cultiva-tion.We have also shown that vectors lacking MP are fully comple-mented by host plants engineered to express this viral protein,and therefore using such vectors would lead to an even safer process (unpublished data).Finally,A.tumefaciens is a ubiquitous soil bacter-ium that,for industrial-scale applications,can be genetically rendered unable to survive in the natural environment 18or to transfer Ti plasmid via conjugation to other bacteria.Undesired DNA transfer can also be limited by deleting from the bacterial genome some
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Figure 5Time course of GFP expression in N.benthamiana and tobacco.(a )GFP in N.benthamiana plants in?ltrated with pICH18711with in?ltration solution diluted 10à1to 10à5relative to the overnight saturated A.tumefaciens culture,2–12d.p.i.(b)Coomassie-stained SDS gels loaded with crude protein extracts from N.benthamiana plants in?ltrated with the 10à1and 10à3diluted in?ltration solutions.m,molecular weight markers (94,67,43,30,20and 14kDa);u,unin?ltrated plant;s,GFP standard.(c )As in a ,but with tobacco.(d )As in b ,but with tobacco.
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essential functions necessary for T-DNA transfer (from bacteria to plant host)and engineering the plant host to provide those in trans 19,20,or by mixing two agrobacteria that require intercellular complementa-tion for the transfer to occur (ref.21and unpublished data).METHODS
Constructs.pICH16707is a GFP-expressing TMV-based viral vector derived from pICH4351(ref.5),but differs from this construct by lack of a LoxP recombination site and by a different vector backbone.pICH15011and pICH17266were made by mutating two putative splice sites (CG/G T GA to CG/G A GA,position 829relative to GenBank accession no.BRU03387,and GCA G /GA to GCA A /GA,position 1,459)or four putative sites (AA/G TAC to AA/A TAC,position 4,201;GC A G/CC to GC C G/CC,position 4411;AA/G TAT to AA/A TAT,position 4,570;AT A G/TC to AT C G/TC position 4,884),respec-tively.Several derivatives were also made from pICH16707by introducing 54and 43silent nucleotide substitutions in areas extending from nt 827to 1,462and 4,655to 4,871,respectively (numbering relative to GenBank accession no.BRU03387,Fig.1a ).The mutated areas were synthesized by PCR using overlapping oligonucleotides containing the desired modi?cations.Introns ranging in size from 91to 443nt were ampli?ed from A.thaliana genomic DNA by PCR.Sites for intron insertion in the viral sequence were selected that either matched the consensus AG/GT or that could be mutated with silent nucleotide substitutions to match the consensus.Nineteen different positions (shown in Fig.1a )were selected (position given relative to turnip vein-clearing virus (TVCV)sequence,GenBank accession no.BRU03387):1,nt 209;2,nt 423;3,nt 828;4,nt 1,169;5,nt 1,378;6,nt 1,622;7,nt 1,844;8,nt 2,228;9,nt 2,589;10,nt 2,944;11,nt 3,143;12,nt 3,381;13,nt 3,672;14,nt 3,850;15,nt 4,299;16,nt 4,497;17,nt 5,099;18,nt 5,287;19,nt 5,444.Four insertion sites were also selected in GFP (positions given relative to the start of the ORF:20,nt 155;21,nt 275;22,nt 383;23,nt 490).
The TVCV MP coding sequence was ampli?ed by PCR from cloned TVCV cDNA (GenBank accession no.BRU03387,bp 4,802–5,628)and subcloned in pICBV10(a pBIN19-derived binary vector)under control of the 35S promoter,resulting in plasmid pICH10745.pICH6692contains the suppressor of silenc-ing p19ampli?ed from tomato bushy stunt virus (TBSV)cDNA using primers 5￠-TTCCATGGAACGAGCTATACAAGGAAACG-3￠and 5￠-CGGGATCCTTAC TCGCTTTCTTCTTCGAAGGT-3￠and cloned under control of a 1.3kb 35S promoter fragment in pICBV10.pICH5290contains the gene encoding GFP 22under control of a 1.3-kb 35S promoter fragment in pICBV1(a pBIN19-derived binary vector).
In?ltration of plants and protoplast isolation.In?ltrations of individual leaf sectors were performed as described 5.Agrobacteria were resuspended in in?ltration solution at various dilutions relative to the overnight saturated A.tumefaciens culture,from a 5-fold dilution (OD 600?0.7)to a 10à6dilution (see main text and ?gure legends).
For inoculation of entire plants,Agrobacteria were inoculated to 300ml of Luria-Bertani medium containing 50m g/ml rifampicin and 50m g/ml kanamycin (selection for the binary vector)and grown at saturation.The bacteria were pelleted at 4,800g for 10min and resuspended in 3liters of in?ltration buffer (10mM 2-[N -morpholino]ethanesulfonic acid (MES)pH 5.5,10mM MgSO 4)in order to get a 10à1dilution relative to the saturated A.tumefaciens culture or in a larger volume for higher dilutions.A beaker containing the in?ltration solution was placed in a vacuum chamber (30-cm diameter),with the aerial parts of a plant dipped into the solution.A vacuum was applied for 2min using a Type PM 16763-860.3pump from KNF Neuberger,with pressure ranging from 0.5to 0.9bar.The plants were returned to the greenhouse under standard conditions.
The protoplast isolation procedure is also as previously described 5.Quanti?cation of GFP and GUS.GFP quanti?cation was performed by spectro?uorometry as previously described 5.Absolute GFP protein
concentration was determined by comparing values of the protein extracts to a standard curve made with recombinant GFP (rGFP from Roche,concentra-tion 1mg/ml).The GUS assay were performed using 50-mg leaf tissue samples according to the protocol described 23.
Note:Supplementary information is available on the Nature Biotechnology website.
ACKNOWLEDGMENTS
We thank Robert Erwin and Yuri Dorokhov for useful discussions.
COMPETING INTERESTS STATEMENT
The authors declare competing ?nancial interests (see the Nature Biotechnology website for details).
Received 14December 2004;accepted 15March 2005
Published online at https://www.doczj.com/doc/1c9172582.html,/naturebiotechnology/
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A R T I C L E S
?2005 N a t u r e P u b l i s h i n g G r o u p h t t p ://w w w .n a t u r e .c o m /n a t u r e b i o t e c h n o l o g y
员工质量意识培训 一、对品质的一些认识： 品质是检验出来的 错！品质是做出来的而不是检验出来的。检验一般是抽检，而抽检具一定的风险性；产品是由作业员一个一个的做出来的，他们对每一个产品不但进行生产，还要进行判定。 品质是很抽象的东西，需要很高深的知识才可以掌握 错！品质随时随地可见，如生活质量（吃、穿、住、用），工作质量（效果、速度、方法），产品质量（尺寸、性能、外观）等。它们可用（好、差；合格、不合格等）来形容。 就产品来说，对现场作业员，产品尺寸、性能可能不大好掌握和控制，需要专检采用一些检测手段进行管制。但外观这一品质特性，很直观，浅而易见，作业员应该是可以掌握和控制的。即使是新进员工，只要经过一段时间的熟悉和了解，也就可以了。每一个产品都是作业员自己做出来的，可以这么说，在外观检测方面，作业员比检验员还有经验，还要懂得多。 品质就在我们的身边，就在我们生活当中，工作当中，并在我们做的每一件事情里面得到体现，在我们做的每一个产品里得到体现。 99%良品率意味着公司品质水平已经很高了 错！提高质量目标或者达到100%的质量目标就是降低成本，增加利润。 任何一个公司追求的质量目标都应该是100%或者说是零缺陷。如果达不到，就是我们还做得不够，还做得不好。那就应该采取措施进行改善，努力的去提高，去完善。 通过所有人都做好每件事，做好每一个产品，100%和零缺陷的质量目标是能够达到的。 顾客的要求和期望，是要100%满意，你达不到100%，我就不放心，或者我就不要你的东西。 试想，如果我们去商场买东西，不管是电视机，还是微波炉，或是其它什么东西，如果卖东西的人给你说，我们的产品只有80%好，即使说有99%好，你想你会放心买吗？ 这一点小问题，没关系的 错！比如说，做一个产品，我的上一道工序有一点小问题，没关系，到我这，也出现一点小问题，也没关系，到下一道，又有一点小问题，还是没关系，最后下来，这产品存在着很多的小问题，也就不再是小问题，它就成了大问题。 一个问题的解决，首先是要寻找问题的原因，问题的原因往往是很多，因为存在着很多小问题，那问题的解决就得从这些小问题的解决开始，一个一个突破，最终得到完全解决。 所以，小问题不容忽视。在日常工作当中，一旦发生问题，即使是小问题，我们都应当立即将其消除。绝不忽略和拖延！如果我们的每一件事都做好，每个小问题都得以消除，那就不会产生什么大问题和长期问题，我们的工作我们的产品就能做好。 许多人总是认为工作中缺陷是不可能避免的，也习惯接受缺陷并容许其不断发生。但我们在个人生活中，却常常会坚持零缺陷的标准。我们会对饭店上菜的片刻延误而喋喋不休，会对汽车的误点而
烟草遗传转化实验文件管理序列号：[K8UY-K9IO69-O6M243-OL889-F88688]
实验八植物细胞悬浮培养 实验目的：学习和掌握植物细胞悬浮培养的操作技术与方法。 实验器材： 超净工作台、恒温培养室、高压灭菌器、冰箱、恒温培养箱、培养瓶、250 mL 、500 mL三角瓶、镊子、酒精灯等。 配置MS液体培养基（2，4-D 2 mg/L+1%甘露醇 +3% 蔗糖，pH 5.8）分装于250ml的三角瓶中，每瓶50ml。 实验材料：烟草叶片愈伤组织。 实验方法： 1.70%乙醇净化工作台并擦洗干净，将所用的材料、工具、培养基等放入 工作台。打开紫外灯和风机，15分钟后关闭紫外灯开始方可操作。 2. 在超净台上用无菌镊子夹取出生长旺盛的松软愈伤组织，放入150ml 三角瓶中并轻轻夹碎,每个三角瓶加入培养基20-30ml，每瓶接种1-2g愈伤组织，按愈伤组织与液体培养基1∶10的比例，以保证最初培养物中有足够量的细胞。 3.接种后的三角瓶用天平称取重量并记载，然后置于摇床上，在转速 100-120rpm，25℃下培养以及散射光条件下，进行振荡悬浮培养。4.每周更换新鲜液体培养基两次，每次更换1/3。每次更换新鲜培养基时 称取重量。 5. 每个人接种悬浮培养细胞1瓶，观察并记录细胞生长情况。 6. 培养7天后，制作细胞生长曲线：为了解县浮培养细胞的生长动态，可用以下方法绘制生长曲线图： 鲜重法：在转代培养的不同时间，取一定体积的悬浮培养物，离心收集后，称量细胞的鲜重，以鲜重为纵座标，培养时间为横座标，绘制 鲜重增长曲线。 烟草遗传转化实验 实验目的
第一章概述 第一节烟叶分级的重要性 一、烟叶分级的目的 对烟叶内在质量的优劣进行等级划分，充分发挥农业资源，达到烟叶以质论价，优质优价。 二、以外观质量因素（外观质量）评价内在质量 三、烟叶分级的重要性 1、有利于合理利用国家资源，物尽其用，充分发挥资源的经济效益。 2、满足卷烟工业的需要。 3、有利于贯彻以质论价，优质优价的价格政策。 4、有利于促进烟叶生产的发展，提高烟农的经济收入。 5、有利于商业经营和对外贸易。 6、合理分级，有利于烟叶副产品的开发利用。 第二节国标的作用及组成 一、国标的作用 1、质量导向作用。 2、对烟草农业生产（栽培）起引导作用。即市场需要什么样的烟叶就生产什么。 3、对卷烟制品质量起规范作用。即卷烟原料对卷烟制品起规范作用。 4、协调国家、烟草企业、烟农三者利益。国家得到税收，企业得到利润，烟农等到收益，要通过落实国标来实现。 5、连接国际市场的纽带作用。与国际接轨，利于创汇。 二、国标的组成 1、文字标准：即国标对每一个等级所做的文字规定。每一个等级包涵了级别代号和相应的分级因素。文字标准是国标的核心，实物样品制订以文字标准为依据。 2、实物样品：即按国标的文字标准制作的实物样品。 3、名词术语：即国标中所作用的概念、术语等。 4、分组：分组分级的前奏，是烟叶分级过程中的重要环节。 5、分级因素及档次：界定烟叶等级的重要依据。 6、验收规格：国标中对水分、纯度允差、砂土率、杂色、微带青、青黄、扎把等指标的规定。 7、检验方法：样品抽取，水分、砂土率等的检验方法。 8、烟叶包装、运输、保管：国标中对烟叶包装、运输、保管的具体规定。 第三节烤烟分级标准的发展历史 三、现行烤烟国标的研究制订过程 ?（一）我国15级标准与国外的差距 ?改革开放以后，为实现与国际接轨，对比了当时的标准与国外先进标准，主要的差距有： ?1、部位分得过粗; ?2、对上等烟颜色、光泽要求偏严; ?3、没按颜色、性质、用途分组; ?4、不利于扩大出口; ?5、分级少，等级数目少，级差大，价差也大，收购时等级矛盾突出，难保收购秩序。
本氏烟草（N. benthamian）瞬时表达及相关实验方法： 一、农杆菌介导的烟草瞬时转化： A、实验步骤： 1、根据实验需要，将所要表达的基因克隆到含有不同标签的双元载体中，并转化农杆菌。 2、将新活化的农杆菌单克隆接种到含有相应抗生素的YEP中，28℃，200rpm过夜。 *估算时间，防止农杆菌液浓度超过1OD，否则会影响转化效率。 3、当菌液OD值介于0.6~1.0之间时，1000g，5min离心收集农杆菌。 4、用2ml Induction medium（without AS）轻柔重悬农杆菌，然后再次离心收集菌液。 5、重复步骤4。 6、所得沉淀用1ml Induction medium 重悬。 7、室温放置1~4小时 8、测OD值，根据实验需要，配置侵染液（组合详见下文）。 9、用不加针头的注射器将侵染液注射进6~8周大的本氏烟草叶片中。 *使用注射器时注意安全，防止针头扎到手，使用完的注射器要把针头套套上再扔，或者将针头放到注射器里面，避免伤害他人；注射时应戴乳胶手套并在每次注射完成后清洗手套，防止交叉污染。B、试剂： Induction medium： MES-KOH PH 5.7 10mM MgCl210mM AS 200uM 推荐提前配制母液 1M MES-KOH PH5.7 过滤灭菌，4℃保存，用时稀释100倍。 1M MgCl2 过滤灭菌，4℃保存，用时稀释100倍。 0.2M AS 溶于DMSO 有机溶剂专用滤膜过滤灭菌，分装（避免反复冻融），-20℃。用高压灭菌的超纯水稀释。 C、关于表达时间： 烟草瞬时表达系统中蛋白的表达可以维持比较长的时间，一般注射24小时之后到一周之内都会有表达。严格来讲需要摸索每个蛋白的最佳表达时段，但一般注射后48小时至72小时不同蛋白表达量都比较可观，不要错过。 D、关于侵染液浓度： 推荐每个菌株的浓度在0.1~0.2之间。过高的农杆菌浓度会引起叶片萎蔫甚至枯萎。
烟草叶片的组织培养 一、实验原理与实验步骤 在植物组织培养中，主要目标是诱导愈伤组织形成和形态发生，使一个离体的细胞、一块组织或一个器官的细胞，通过脱分化形成愈伤组织，并由愈伤组织再分化形成植物体。从一块外植体形成典型的愈伤组织，大致要经历三个时期：起动期、分裂期和形成期。 植物材料：烟草植株 药品：（2,4-D、NAA、6-BA浓度均可）、1mol/L NaOH、1mol/L HCL 蒸馏水、70%酒精0.01%升汞 仪器：玻璃杯、玻璃棒、pH试纸(5.5-9.0) 、1瓶无菌水、1个无菌烧杯、1包无菌滤纸、 1个无菌白瓷板、培养瓶若干移液器、微波炉、灭菌器、超净工作台、酒精灯、解剖刀、镊子 二、实验方法与步骤
注意： 1、大量元素按照使用时高10倍的数值称取，分别将各种化合物称量后，除CaCl2·2H2O单独配制外，其余化合物混合在500ml烧杯中加适量蒸馏水溶解，用玻璃棒搅拌促溶，倒入1000ml容量瓶中用蒸馏水定容至刻度，置小口瓶中保存，贴上标签注明化合物名称（或编号），浓缩倍数，配制日期和配制者姓名，CaCl2·2H2O配制同上置于另一小口瓶中。 2、微量元素母液的配制 按要求浓缩100倍的数值称取，分别将各种化合物称量除铁盐（FeSO4·7H2O 和Na2-EDTA.2H2O）作为一组单独配制外，其余化合物可混合置于烧杯内加少量蒸馏水溶解后，定容在1000ml容量瓶中，置小口瓶中保存，贴上标签 3、铁盐配制将FeSO4·7H2O和Na2-EDTA.2H2O分别溶于450ml蒸馏水中，加热，（很重要）不断搅拌，溶解后，两液混合，调PH至5.5加水定容至1000ml，置于小口瓶中，贴上标签。 4、有机物母液配制，按母液要求浓缩50倍，除蔗糖按3%单独临时称量外，其余分别称量后，溶解，定容在500ml容量瓶中，置于小口瓶中保存，贴上标签。5．母液最好在2~4℃的冰箱中贮存，特别是有机类物质，贮存时间不宜过长，无机盐母液最好在一个月内用完，如发现有霉菌和沉淀产生，就不能再使用。（1）制备母液和营养培养基时，所用蒸馏水或无离子水必须符合标准要求，化学药品必须是高纯度的（分析纯）。 （2）称量药物采用高灵敏度的天平，每种药品专用一药匙。 生长调节剂母液配制： 为了操作方便，节约时间，生长调节剂也可如同配制母液一样，先配成原液，这样配制培养基时只要稍加计算，按需要量取即可。 不同药品在配制时若不溶于水，可用少量不同的溶剂先溶解，萘乙酸（NAA），吲哚乙酸（IAA）,赤霉素（GA3），2，4-D等生长素和玉米素（ZT）可先用少量95%酒精溶解，然后加水，如溶解不完全再加热。激动素（KT）和6-苄基嘌呤（BA）可溶于少量1mol/L的盐酸中，叶酸需用少量稀氨水溶解。 称取50mg生长调节物质，溶解后，在100ml的容量瓶中定容，配制的母液每毫升则含有生长调节物质0.5mg，配制后一般要求在低温（0~4℃）保存，配制培养基时如每升（1000ml）需添加的生长调节剂物质为0.5mg时，则取1ml母液即可。 三、培养基配制和灭菌 本次实验使用 （1）愈伤组织诱导及其幼芽分化培养基:MS+2,4-D 0.5 mg/L +6-BA 1.0 mg/L；（2）幼芽增殖培养基：MS+6-BA 1.0 mg/L +NAA 0.2 mg/L； （3）生根培养基：MS+NAA 0.2 mg/L。 注意事项：上述培养基均在MS固体培养基溶化后降低到50左右后，加入相应激素所得， MS固体培养基的配置过程在此不作过多赘述
提升烟叶质量之我见 随着我国加入WTO，国家关于农村经济结构的调整战略的实施，给当前烟叶生产带来了机遇和挑战。县级公司烟叶生产当前面临的共性矛盾和问题，一是烟叶生产基础仍然比较薄弱，抵御自然和市场风险能力低。二是烟叶质量既面临国内结构性矛盾的压力，又面临入世后的冲击，结构调整的工作显得十分突出。三是由于受地理、环境的制约，不同程度地存在着生产规模小，市场空间窄的问题。那么，如何正视困难，知难而进呢？ 国际上的市场竞争日趋激烈，而市场竞争的焦点就是质量和价格。我们要在不违背“游戏规则”的前提下，守住国门，保住市场，扩大出口，单靠政策调控是远远不够的。我们必须紧跟时代步伐，围绕“控制总量、提高质量、优化布局、优化结构”的烟叶工作重点，依靠科技进步，加速推进“良种化、区域化、规范化”生产技术措施，大力普及实用技术，使我们的烟叶质量尽快赶上国际先进水平。 一、加强组织领导，各方齐抓共管 烤烟生产和收购是一项系统工程，它政策性强，涉及面广，仅靠一个或几个部门是难以抓好的，必须加强领导，加强配合。各级政府要加大烟叶市场整治力度，严厉打击烟贩的非法活动，努力维护烟叶市场的正常秩序。从烟草部门来说，各级领导要把烟叶工作作为一项战略任务，继续加强组织领导，一把手要亲自
抓烟叶，要主动向当地政府汇报情况，请示工作，争取当地政府的重视和支持；此外，还要加强与农业、工商、公安、银行等有关部门的联系，取得大家的支持，及时安排落实烟叶生产、收购所需的物资和资金。烟草公司要切实做好思想政治工作，加强和充实烟叶力量。烟叶工作要上去，各级干部要下去，烟草公司都要安排干部和科技人员深入第一线办点，以点带面。公司机关和领导干部要带头转变作风，坚持为人民服务的宗旨，帮助基层排忧解难，为基层搞好服务工作，并及时发现和总结推广典型经验，把烟叶工作做得更扎实，更有成效。 二、调整烟叶生产布局，优化烟叶资源配置 目前，一些地区尤其在山区县烟叶种植仍然比较分散，必须尽快做到三个转移：非适应区向适应区转移，分散区向集中区转移，不规范种植区向规范化种植区转移。 三、打造一支过硬的专业技术队伍 能否快速提高烟叶品质，技术队伍是关键。现阶段烟草技术队伍中仍然存在着一些共性问题和不良倾向：一是部分技术员工作的计划性差，思路不清，工作效率低。二是部分技术员文化程度低，工作责任心不强。三是部分技术员虽然身在农村，但缺乏农村工作经验，不能和烟农打成一片。四是部分技术员知识更新速度慢，上进心不强，不学无术者依然存在。五是少部分技术员不能正确处理权力和义务的关系。上述问题和倾向的存在，严重制约着烟叶生产的健康发展。因此，我们必须加大教育培训力度，
Systemic Agrobacterium tumefaciens–mediated transfection of viral replicons for ef?cient transient expression in plants Sylvestre Marillonnet 1,2,Carola Thoeringer 1,2,Romy Kandzia 1,Victor Klimyuk 1&Yuri Gleba 1 Plant biotechnology relies on two approaches for delivery and expression of heterologous genes in plants:stable genetic transformation and transient expression using viral vectors.Although much faster,the transient route is limited by low infectivity of viral vectors carrying average-sized or large genes.We have developed constructs for the ef?cient delivery of RNA viral vectors as DNA precursors and show here that Agrobacterium–mediated delivery of these constructs results in gene ampli?cation in all mature leaves of a plant simultaneously (systemic transfection).This process,called ‘magnifection’,can be performed on a large scale and with different plant species.This technology combines advantages of three biological systems (the transfection ef?ciency of A.tumefaciens ,the high expression yield obtained with viral vectors,and the post-translational capabilities of a plant),does not require genetic modi?cation of plants and is faster than other existing methods. Viral vectors designed for expression of recombinant proteins in plants hold great promise because of high absolute and relative yields,and because of the speed provided by transient expression.Most of the results of practical interest achieved so far have been obtained with vectors built on the backbones of plus-sense RNA viruses such as tobacco mosaic virus (TMV)or potato virus X 1–4. We have recently shown that TMV-based vectors can be delivered to plant tissues using A.tumefaciens 5(agroinfection).However,one step of this process,namely the formation of active replicons from the primary nuclear transcript,is inef?cient.In a standard leaf transfec- tion experiment,this inef?ciency is masked by the subsequent ability of the replicons to move to neighboring cells by cell-to-cell movement.Here we show that this bottleneck can be fully remedied by incorpora-tion of silent nucleotide substitutions into the vector and by addition of multiple introns.We demonstrate that such modi?cations provide for ef?cient processing of the DNA information into active replicons in almost all cells (as high as 94%)of Nicotiana benthamiana ,an up to 1,000-fold improvement over nonoptimized TMV-based vectors,and an even higher improvement (4106-fold)in Nicotiana tabacum (tobacco).Finally,we show that the resulting vectors allow the development of a fully scalable and versatile whole-plant transfection protocol,that we term magnifection,for production of heterologous proteins in plants. RESULTS Viral replication following agroin?ltration of TMV-based vectors Agroin?ltration of a TMV-based viral vector containing the gene encoding green ?uorescent protein (GFP)(pICH16707,Fig.1a )into N.benthamiana leaves leads to the formation of foci of GFP ?uorescence 3d post-in?ltration (d.p.i.)(shown in ref.5and in Supplementary Fig.1online).T o quantify the proportion of cells initiating viral replication,a 489-bp deletion was made within the movement protein (MP)coding sequence,resulting in construct pICH14833(Fig.1a ).Replicons derived from this construct cannot move from cell-to-cell but are able to replicate autonomously within each infected cell.Three days after agroin?ltration of pICH14833in N.benthamiana leaf (OD 600of the A.tumefaciens in in?ltration solution was 0.7),a small number of cells expressing GFP appeared (see Supplementary Fig.1online),and the same pattern was still visible 2weeks after in?ltration.By counting protoplasts prepared from the in?ltrated area (Figs.1and 2),we found that 0.6–1.6%of cells initiated viral replication. There are several reasons why RNA viral vectors might have dif?culties starting the replication cycle.First,RNA viruses,such as TMV ,replicate in the cytoplasm and never enter the nucleus,and have therefore evolved in an environment where they are not exposed to the nuclear pre-mRNA processing machinery.As a result,pre-mRNA transcripts made in the nucleus from viral constructs may not be re-cognized and processed properly.Second,viral vector constructs encode very large transcripts (B 7.6kb for the primary transcript of a viral vector containing a GFP gene),a size much larger than the average size (1–2kb)of plant genes.Moreover,in nature,large eukaryotic genes often contain numerous introns that facilitate processing and export of the pre-mRNAs from the nucleus 6.We therefore hypothesized that modi?cations of the constructs that would increase the ef?ciency of processing and export of primary transcripts from the nucleus to the cytoplasm could lead to an increase in the number of cells that would initiate viral replication.Two types of modi?cations were made: Published online 8May 2005;doi:10.1038/nbt1094 1Icon Genetics,Biozentrum Halle,Weinbergweg 22,D-06120Halle (Saale),Germany.2These authors contributed equally to this work.Correspondence and requests for materials should be addressed to Y.G.(gleba@icongenetics.de).A R T I C L E S ?2005 N a t u r e P u b l i s h i n g G r o u p h t t p ://w w w .n a t u r e .c o m /n a t u r e b i o t e c h n o l o g y
农杆菌介导的瞬时表达系统 一．溶液配制 乙酰丁香酮AS 0.1M，取0.101gAS溶于5mLDMSO中，在工作台上用灭过菌的0.45μM滤膜过滤,分装入无菌小管，-20℃冰冻保存； MES 1M 调pH=5.6，过滤除菌； MgCl2 1M 过滤后灭菌； Kan 终浓度100mg/ml，过滤除菌。 (AS ，MES均购自泰安齐旺试剂公司) YEB过夜培养液(30ml) MES AS Kan 10mM 20μM 50μg/ mL 300μl 6μl 15μl 悬浮培养液(50ml) MES 10mM 500μl AS 200μM 100μl MgCl2 10mM 500μl YEB培养基(pH 7.2) 酵母膏 牛肉膏 蛋白胨 蔗糖 MgSO4·7H2O 1g/L 5g/L 5g/L 5g/L 493mg/L 二．操作步骤 1. 挑起单个农杆菌菌落，接种3mL YEB培养基（含抗生素）在200r/min，28℃摇床培养过夜，储存时间比较长的菌液可多活化两次； 2. 接种2mL过夜培养的50mL的YEB液体培养基（含抗生素，10mM MES和20μM乙酰丁香酮），28℃摇床培养2-4h； 3. 将上述过夜培养菌液在4℃，8000g下离心6min，收集菌体； 4. 用渗透培养液重新悬浮沉淀的农杆菌细胞。调节浓度OD600至0.5-1.0（一般需要100-150mL渗透培养液），置室温培养至少3小时，无需振荡； 5. 对需要渗透处理的烟草植株无一定大小要求，一般4-5叶期，已经生长一个月左右的本生烟比较合适。一般渗透处理下部2-3个比较大的叶片。用针头在需要渗透处理的叶片背面非常细微地扎一细小的浅微孔，然后用3mL的无针头的注射器吸取2-3mL悬浮有农杆菌的渗透培养液，从针孔处将培养液轻微的注入叶片内。注意用一手指从叶片下面拖住叶片，将注射器平平地堵住针孔，不让液体从叶片边缘挤压出来。注射区域接近叶片边缘即可或根据自己的实验目的决定； 6. 将处理过的烟草放回温室，照常管理。一般沉默发生在2-5天内； 7. 在暗室里，利用长波紫外灯（MODEL SB-100P/F,365nm）观察渗透处理过的叶片，并拍照记录叶片侵染区绿色荧光表达的变化过程。
品质学习资料 1、质量意识 质量意识是一个企业从领导决策层到每一个员工对质量和质量工作的认识和理解， 这对质量意识和质量行为起着极其重要的影响和制约作用。质量意识体现在每一位员工的岗位工作中，也集中体现在企业最高决策层的岗位工作中，是一种自觉地去保证企业所生产顾客需求产品的硬件、软件和流程性材料产品质量、工作质量和服务质量的意志力。企业以质量求生存，求发展，质量意识则是企业生存和发展的思想基础。质量意识是通过企业质量管理、质量教育和质量责任等来建立和影响的，并且通过质量激励机制使之自我调节而一步步地、缓慢地形成起来的质量意识。 2、品质意识 品质意识就是指人们在生产经营中，对品质（包括产品品质、工作品质）以及与之相关的各种活动的客观及主观的看法和态度，也就是通常所说的对提高产品品质的认识程度和重视程度，以及对提高产品品质的决心和愿望。而人的行动受大脑意识支配，有什么样的意识，就会产生什么样的行动，一个有错误品质意识的人，很难想像他能做出什么好的产品。只有当一个人的思想意识上升到一定的高度，再加上相应的工作技能，他才会做出好的产
品。一位哲学家曾经说过：“高度”很重要，它反映出一个人的修养和内涵，一个做过大事或见过大事的人，再想平庸都难。可想而知，品质意识对产品品质的重要作用。 3、如何提高质量意识 很多时候我们都提到要提高质量意识，但究竟什么是质量意识，如何提高质量意识？不知道有多少人仔细研究过，又深入地思考过？质量意识是摸不着看不到的东西，是人们对质量的一种想法态度，一种心理，一种潜在的行为指导规范。既然质量意识是一个看不见摸不着的东西，那如何提高质量意识就更是难上加难了。 多数人的说法，提高质量意识就是要培训，培训能提高质量意识吗？能提高多少啊？有没有仔细考虑分析过？质量意识通过培训能够提高，例如经过1小时的培训，人们对质量都有了理解，但只是短时间的认识，过几天后就逐步淡忘了。质量意识的培训只能让人们对质量有个初步了解，一般不会将质量的想法、心理带入到工作中，形成有益的质量活动。 举例，一次生产了一批产品，由于交货要求急，生产中发现产品中有一个尺寸有部分产品尺寸超差，一个孔稍微小一点，但由于出货紧就这样出货了。结果全部被退货，直接损失近10万，还差点丢了这个客户，退货回来之后对全体员工进行了教育，说明了事情的经过及问题的严重性，从此以后只要有问题员工都会反映出来，领导对于出现的一些问题也很重视，从此以后产品质量不断的提高了。
0 前言 一、首件应符合的条件 二、如何做好首件确认 三、首件确认的时机 四、制程中如何处理品质控制 五、品质异常的处理 六、重工、重检的处理 七、注塑件常见外观不良 0 前言 公司主要生产的产品是呼吸类（属于急救类）医疗器械产品，目前主要有面罩类、管路类（氧气类）和电刀类产品。公司的规模发展有，在美国、加拿大有销售公司，在台北、台南有研发和生产基地，在厦门有崇仁医疗生产公司和崇宜医疗销售公司，全球约有40~50%呼吸类医疗器械产品是我们公司生产的。 我们生产的产品主要是为生命垂危的人、患者、为健康生活的人提供帮助。我们从事的是一种非常令人自豪的行业，因为我们从事生产的产品是为急需帮助的人提供服务，为人类生命的延续提供机会。为此，我们必须对产品品质负极大的责任。 我们对产品的品质负责，如我们所使用的原料有毒、生产的产品有毛屑、杂质、油污等让患者吸入肺中，导致产生医疗事故的话，公司将会面临：承担医疗责任及患者的赔偿、产品的招回（可能被强制销毁）、药监局网上通报、产品重新认证等巨大的经济损失，严重的可能使公司失去市场的业务、倒闭。 综上所述，公司追求品质零缺陷，产品质量从源头做起，从我做起。 一、首件应符合的条件 首件应符合如下要求： 1、符合SIP规定 a、外观要求： 主要由作业员自检、班组长确认；QC检验； TP0001的规定； 以往产品的品质履历记录。 b、尺寸要求：主要由QC负责检测； c、功能要求：主要由QC负责检测。 说明：首件是开机生产的第一模产品，而是生产 稳定时，自检确认合格时生产的第5~10模中任一 模产品，包含所有模穴；作业员在生产时可以问 品管员此产品应注意哪些品质事项、品质重点是 什么、要检查哪些项目，品管员有义务告之。 2、符合SOP的规定 a、确认符合注塑成型条件表，确认此表已得生产主 管的认可； b、包装方式的确认：是否有包装SOP或包装方式得 到工程的认可、产品成型后，如何包装，这样包 装是否会影响产品的变形，折皱等 3、符合样品的要求
烟草行业质量管理解决方案 我国烟草产量和消费量都居世界首位，烟草行业是国内最大的行业之一。随着市场经济体制的逐步建立和中国”入世”后自由贸易规则的普遍推行，国家的反垄断指向不断增强，烟草专卖体制将会受到来自各方面的日益加大的压力和挑战，中国的烟草行业正面临着重新洗牌的格局。如何解决国外卷烟大量涌入我国市场后对原有的卷烟市场的挤压和冲击，实现从”烟草大国”向”烟草强国”转变是摆在企业面前的重要课题。将过去以传统手段为主的烟草管理模式逐步转变为以信息化手段为主的现代先进管理模式，正是提高企业效益和整体竞争力的重要措施。 方案背景： 我国烟草企业普遍具有较好的信息化基础，并且重视质量文化的建设，但是质量管理水平仍然停留在传统的检验控制阶段。近年来，通过质量管理体系认证制度的推广，我国烟草行业质量管理水平有了长足的进步，但同时也还存在着一些不足之处。主要包括： 1、产品生命周期全过程质量信息采集缺乏有效的收集和管理系统，致使大量数据流失，管理分散，统计、分析和查询困难 2、检验标准等产品质量基础资料的管理不规范，检验标准管理分散，更改和换版周期长，查询不方便。更改的不准确和不及时经常会造成不合格品的产生，建立产品的基础资料库势在必行 3、关键质量控制过程的运作不规范。企业生产过程的检验过程、合格判定过程、不合格品审理过程和市场反馈的质量问题的处理过程等大都由人工通过纸质表单传递的方式实现，出现质量问题后的无法有效追溯 4、缺乏生产制造过程质量控制的科学途径。目前许多烟厂仍处在以检验把关为主的质量控制阶段。生产制造过程中的质量控制是依靠工艺方法和操作者的经验来控制，易出现大量的不合格品，给企业造成了巨大的经济损失 5、质量信息难以实现快速查询和追溯 6、质量系统与ERP等其它信息系统的数据难以共享 7、质量诊断与协同监控问题不及时，不能实现质量预防 北京北科汇智软件技术有限公司充分研究中国烟草行业的质量管理现状和信息化需求，以质量为优化目标，结合现代质量管理思想和信息技术，为企业构建了全面、系统的质量管理信息化解决方案——烟草行业质量管理信息系统（QIS）。 北科汇智烟草行业QIS产品功能设计遵循ISO 9000标准，结合北科汇智专家多年来在烟草行业的质量管理研究和国内先进质量管理技术的研究，依托公司强大的技术研发实力，打造的一体化的企业质量管理综合集成平台。 系统构架如下图示： 方案优势： 1、以质量为核心优化企业管理流程 通过信息系统整合企业质量管理资源，优化企业质量管理流程，是现代系统理论、控制理论与质量思想在信息化时代的突出表现。北科汇智QIS以质量为优化目标，整合系统资源，尤其是质量信息资源，突破传统质量管理模式下的信息手工传递和人工处理，提高流程的运行效率，使科学、规范、严格的流程控制实现成为可能。 2、将预防和过程控制思想与企业实践相结合
烟草农杆菌转化实验 实验配方：1L RMOP: 20×大量元素50ml 200×微量元素5ml 200×有机5ml 200×铁盐5ml 3%蔗糖30g 0.8%琼脂（国产）4g/500ml 灭菌后，每500ml培养基加入: 0.1mg/L NAA 1mg/ml NAA 50μL 1mg/L 6-BA 1mg/ml6-BA 500μL YEB: 液体培养基：1L 酵母提取物1g 牛肉膏5g 蛋白胨5g 蔗糖5g MgSO4?7H2O 0.5g pH7.0 高压灭菌。 固体培养基： 每升YEB 液体培养基加15g 琼脂粉，高压灭菌。 卡那霉素（Kan）储液：100mg/ml 利福平（Rif）储液：50mg/ml YEB 固体培养基平板: 灭菌后的YEB 固体培养基待其温度降至50℃时加入卡那霉素（Kan）和利福平（Rif），至终浓度为50mg/l，混匀后立即倒入培养皿，凝固后4℃倒置保存。 一．农杆菌感受态细胞的制备 （1）划线活化农杆菌，挑取农杆菌单菌落于3ml的YEB液体培养基（含Rif 50mg/l）中，28℃振荡培养过夜； （2）取过夜培养菌液1ml接种于50ml YEB（Rif 50mg/l）液体培养基中，28℃振荡培养至OD600为0.5； （3）取2ml菌液，13000rpm，离心30sec, 弃上清； （4）加入10ml 0.1M CaCl2，使农杆菌细胞充分悬浮，冰浴30min；
（5）13000rpm，离心30sec，弃上清，置于冰上，加入2.5ml预冷的0.1M CaCl2，充分悬浮细胞，分装在1.5ml EP管中，液氮中速冻1min，置-80℃冰箱保存备用。二．质粒DNA导入农杆菌 ①电转法： 1.取农杆菌感受态在冰上冻融； 2.加2μL质粒DNA于100μL感受态细胞中，用枪头轻轻搅拌混匀； 3.然后再将其转入电转杯中（不要产生气泡），在2500V高压下电击； 4.取出电转杯，加入500μL预冷的YEB培养基（不含抗生素），轻轻吹打混匀，吸出菌液转入1.5ml离心管中，28℃，200rpm振荡培养5h； 5.取30-40μL菌液涂在含相应抗生素（50mg/l Kan和50mg/l Rif）的YEB平板上，28℃倒置培养1.5-2天； 6.挑选单菌落PCR检测，将阳性菌落保存。 ②冻融法： 1、在200μl感受态细胞中加入2－6μl质粒DNA，冰浴5min，液氮中速冻5min； 2、迅速转入37℃水浴中，热激5min； 3、加入1ml YEB（不含抗生素）液体培养基，28℃慢速振荡培养2－4小时； 4、3000rpm离心4min，去一部分上清，留取200μl菌液涂布于含有50mg/l Kan 和50mg/l Rif的YEB平板； 5、放置约0.5h，待水份干后，28℃培养约24小时至长出菌落。 6、挑选单菌落PCR检测，将阳性菌落保存。 三．烟草遗传转化实验 ①准备农杆菌 （1）将农杆菌在平板上划线，从平板上挑取单菌落，接种到20mL附加相应抗生素（kan 50mg/L, Rif 50mg/L）的YEB液体培养基中，在恒温摇床上，于28℃下以180r/min培养至OD600为0.6~0.8（约需17h）。 （2）将OD600为0.6~0.8的菌液，按1%~2%的比例，转入新配制的无抗生素（含Rif）的YEB液体培养基中，可同时加入100~500μmol/L的乙酰丁香酮。在上述相同的条件下再培养6h左右，待OD600为0.2~0.5时即可用于转化。
龙源期刊网 https://www.doczj.com/doc/1c9172582.html, 农杆菌介导的基因瞬时表达技术及其应用 作者：宋建刘仲齐 来源：《天津农业科学》2008年第01期 摘要：主要介绍了农杆菌介导的基因瞬时表达方法的原理、技术、影响因素及其在外源基因表达分析、启动子分析、基因沉默及防卫反应等方面的应用。 关键词：农杆菌；植物；基因瞬时表达 中图分类号：Q789文献标识码：A文章编号：1006—6500(2008)01—0020—03 把外源基因导入受体植物内，是研究基因功能和获得遗传修饰有机体的主要手段。农杆菌介导法是目前最常用的遗传转化方法，当农杆菌感染植物受伤组织后，质粒上的目标基因可以进入植物细胞内并整合到植物染色体中，这种转化细胞经过诱导分化，再生成为转基因植株。通常大多数植物的遗传转化和再生效率低下，费时且费用昂贵。即使对于转化程序大大简化的植物，例如拟南芥，仍然需要花费数月的时间来产生适合分析的转基因植株。农杆菌介导的瞬时表达提供了一种快速分析基因型功能的方法，该方法是Rossi等在1993年创建的。他们将带有重组质粒的农杆菌，经诱导后通过抽真空渗透入植物叶片进而渗透入植物细胞，通过目的基因瞬时表达来检测植物中农杆菌介导的T-DNA转移的效率。随后人们又采用针管注射活体植株叶片，来进行农杆菌介导的基因瞬时表达检测。近几年该项技术不断完善、发展，已被广泛用于外源基因表达分析、无毒基因与抗性基因的相互作用、基因沉默、启动子分析等许多植物分子生物学领域。 1主要原理 农杆菌介导的瞬时表达是将目的基因插入共整合载体或双元载体，转化根癌农杆菌，后者经酚类化合物诱导处理后，通过真空渗透或针管注射入植物叶片组织中，农杆菌在叶片内与植物细胞紧密接触。诱导处理在转录水平激活Vir区基因，真空渗透或注射使得农杆菌与植株叶片细胞接触，从而实现了T-DNA转移进入植物细胞核。大部分T-DNA并未整合入植物基因组而是暂时存在于核内并在植物细胞转录、翻译成分的协助下瞬时表达T-DNA基因，通常在数小时后即可检测到外源基因的表达，并在1～2d内达到最高值。而少量整合进植物染色体的 T-DNA在瞬时表达中不起作用或极为微弱。
目录 实验一烟草形态与类型的观察与识别 (2) 实验二育苗技术观察 (5) 实验三烤烟烟苗素质考察 (10) 实验四主要栽培技术与烤烟生长分析 (11) 实验五打顶时期与留叶数对叶片生长的影响观察 (12) 实验六烤烟叶片经济性状考察 (13)
实验一烟草形态与类型的观察与识别 一、目的 通过观察烤烟根、茎、叶等器官构造与形态，建立对烟草初步的感性认识；观察掌握不同类型烟草的形态特征并能识别。 二、内容 观察烤烟植株形态及根、茎、叶等器官特征，观察烤烟、白肋烟、香料烟、晒烟、晾烟、黄花烟六种类型的外观特征，并能描述。 1、烤烟 烤烟亦称火管烤烟，源于美国的弗吉尼亚州，具有特殊的形态特征，因而也被称为弗吉尼亚型。烤烟的主要特征是植株高大，叶片分布较疏而均匀。一般株高120-150cm，单株着叶20-30片，叶片厚苤适中，中部的质量最佳。栽培上不宜施用过多的氮素肥料。叶片自下而上成熟，分次采收最初的调制方法也是晾晒。后来（1869）年改用火管烘烤。目前是在烤房内调制，烤后呈金黄色。其化学成分的特点是含糖量较高，蛋白质含量较低，烟碱含量中等。烤烟是我国也是世界上栽培面积最大的烟草类型，是卷烟工业的主要原料，也被供作斗烟。世界上生产烤烟的国家主要有中国、美国、印度，其次是巴西、津巴布韦、泰国、加拿大、日本等。我国烤烟种植面积和总产量都居世界第一位。重点产区有河南、山东、云南、贵州、黑龙江、湖南、湖北、陕西、安徽等省，四川、广东、福建、辽宁、江西、广西、吉林等省（区）也有较大面积的栽培。 2、晒烟 晒烟的烟叶利用阳光调制，主要有晒红烟与晒黄烟。一般晒黄烟外观特征和所含化学成分与烤烟相近，而晒红烟则同烤烟差别较大。晒红烟的叶片一般较少，叶肉较厚，分次采收或一次采收，晒制后多呈深褐色或褐色，以上部叶片质量最好。烟叶一般含糖量较低，蛋白质和烟碱含量较高，烟味浓，劲头大。晒烟主要用于斗烟、水烟和卷烟，也作为雪茄芯叶、束叶和鼻烟、嚼烟的原料。此外，有些晒烟还可以加工成杀虫剂。世界上生产晒烟的主要国家是中国和印度。我国各省都有晒烟种植，但分布零散，比较集中的有四川、广东、贵州、湖南、湖北、云南、吉林、山东、陕西等省。 3、晾烟 晾烟有浅色晾烟（白肋、马里兰）和深色晾烟之别，都是在阴凉通风场所晾制而成。而其中的白肋烟、马里兰烟和雪茄包叶烟因别具一格，均已自成一类。但在我国，除将白肋烟单独作为一个烟草类型外，其余所有的晾制烟草，包括雪茄包叶烟、马里兰烟和其他传统晾烟，均归属于晾烟类型。 4、白肋烟 白肋烟是马里兰深色晒烟品种的一个突变种。1864年美国俄亥俄州布朗县的一个农场在马里兰阔叶烟苗床里初次发现了奶黄色的突变烟株，后经专门种植