CN101397569A - Method for improving tobacco virus resistance - Google Patents

Abstract

The invention discloses a method for improving the tobacco virus resistance, which comprises the steps: a PAP fusion gene which is composed of a DNA sequence for coding the tobacco pathogenesis protein-1a signal peptide and a DNA sequence for coding the pokeweed antiviral protein mature peptide (PAP) is artificially synthesized, the PAP fusion gene constructs a plant expression vector and is delivered to tobacco cells by agrobacterium mediation, resistant calli is screened in a screening culture medium and plant regeneration is induced, the transgenic plants are determined by the molecular biology method, and the transgenic plants which have strong virus resistance and normal growing development can be screened through the virus eliminating experiments. The invention can overcome the defect that the transgenic tobacco has PAP cytotoxicity and little-improved virus resistance after the tobacco is subjected to gene genetic transformation by the wild type pokeweed antiviral protein, and is beneficial to culturing the tobacco variety with broad-spectrum virus resistance.

Description

A kind of method that improves tobacco virus resistance

Technical field

The invention belongs to plant virus resistance breeding field, particularly, the present invention relates to a kind of method that improves tobacco virus resistance.

Background technology

Pokeweed antiviral protein (pokeweed antiviral protein, PAP) belong to type i strand ribosome inactivating protein (ribosome-inactiviting protein, RIP), RIP albumen is site-specific RNA N-Glycosylase, the N-glycosidic link of the special VITAMIN B4 of cracking eukaryote rrna 28S rRNA and prokaryotic organism rrna 23S rRNA Sarcin/ricin zone, rrna can not be combined with the eEF-2-GTP complex body, cause mRNA can not translate into protein, suppress virus in intracellular breeding.Irvin (1975,1980) at first from dyers' grapes (Phytolacca americana) blade, extract and obtain PAP, and research reported this protein broad-spectrum ntiviral characteristic, and (2002) such as (1994), Poyet etc. (1997) such as Lin etc. (1991), Poyet and Park have cloned PAP, PAP II, PAP-S and four kinds of cDNA of PAP-H respectively from dyers' grapes spring leaf, Xia Ye, seed and root.The former albumen of PAP by N end structure territory (22 amino acid), division center territory (262 amino acid) and C end structure territory (29 amino acid) form the PAP similar of other types, but each structural domain amino acid number and kind have than big-difference.The signal peptide effect is mainly played in N end structure territory, and PAP is positioned in the cell walls matrix; The division center territory is contained 4 at the conservative active catalytic residue of RIP camber, it is the active critical area of PAP, and C end structure territory, play the auxiliary localized effect of N end signal polypeptide cell, Ready etc. (1986) research is pointed out, PAP antivirus action mechanism belongs to " the local suicide " model, be that PAP and PAP II are positioned in the dyers' grapes cell walls matrix, during virus infection, PAP enters cell with pathogenic agent, and ribosomal deactivated with host cell causes susceptible necrocytosis, thereby the inhibition viral proliferation makes flanking cell not infected.This model obtains the support of Bonness etc. (1994) research, and Park etc. (2002) confirms that also PAP H is positioned at root-tip cells wall matrix.Studies show that: PAP has the antiviral function similar to RIP, can suppress animals and plants virus and phage activity, is the antiviral protein (Peumans etc., 2001) of wide spectrum.For example; PAP suppresses poliomyelitis virus (Polio virus); influenza virus (Influenza virus); hsv (Herpes simplex virus typeI); the mankind exempt from service defective virus (HIV-1) and phage MS2 activity; and various plants RNA viruses and dna virus such as cucumber mosaic virus (Cucumber mosaic virus; CMV); tobacco mosaic virus (TMV) (Tobacco mosaic virus; TMV); potato virus X (Potato virus X; PVX); marmor upsilon (Potato virus Y; PVY); cauliflower mosaic virus (Cauliflowermosaic virus; CaMV); african cassava mosaic virus (African cassavamosaic virus; ACMV); alfalfa mosaic virus (Alfalfa mosaic virus; AIMV); tobacco necrosis virus (Tobacconecrosis virus; TNV); bean mosaic virus 4 (Southernbean mosaic virus; SBMV) and Yuan mountain valley with clumps of trees and bamboo mosaic virus (Turnip mosaic virus, TuMV) etc.Therefore, PAP has great application prospect in field of medicaments, plant virus resistance gene engineering.

Plant (Lodge etc., 1993 of up to now, obtained tobacco, potato, the stem creeping bentgrass of crawling (Agrostis stolonifera), paddy rice and lily changeing the PAP gene; Tumer etc., 1997; Wang etc., 1998; Zhang Haiyan etc., 1998; Dai etc., 2003; Tabares etc., 2004; Wang Yanping etc., 2006; Wan Duorong etc., 2007).But PAP cytotoxicity symptom and the unfavorable result of virus resistance appear in transfer-gen plant in varying degrees, (1997) reports such as Lodge etc. (1993) and Tumer for example, and PAP transformation frequency very low (0.7%) maybe can not obtain transfer-gen plant; Even obtain changeing the plant of PAP gene, the cytotoxicity symptom that PAP such as that this plant then shows is sterile, dwarfing and withered spot produce; Virus inoculation 10 days, transgene tobacco does not wait from 33%～100% the infection rate of tobacco mosaic virus (TMV), and from 14%～86%, but the inoculation back is along with growth period prolongs transgenic Rhizoma Solani tuber osi to the infection rate of marmor upsilon, and infection rate increases.At reducing the PAP cytotoxicity and improving the transfer-gen plant virus resistance, in plant virus resistance research, utilize the technical characterstic of PAP gene to be:

(1) selects hypotoxic Pokeweed antiviral protein gene such as PAPII.Wang etc. (1998) use the common tobacco of PAPII gene transformation, and its transformation efficiency is 5%, and transfer-gen plant expression PAP II protein content is higher 10 times than PAP albumen at least, and cytotoxicity weakens.PAP II protein content reaches 150ng/mg albumen ability render transgenic tobacco leaf and the chlorosis toxicity symptom occurs, and it is normal that content is lower than the following transgene tobacco growth of 100ng/mg albumen.But, Dai etc. (2003) report, under the field planting condition, the transgenosis of expressing PAPII stem creeping bentgrass (Agrostis stolonifera) the plant mortality ratio of crawling is very high, and stronger PAP II cytotoxicity takes place in dead 11 strains of 18 strain transfer-gen plants.

(2) induce the PAP series jump.Tumer etc. (1997) induce the dna sequence dna base mutation in coding PAP maturation protein N end, C end and active centre, the result shows: the PAP sequence of C-terminal W237 nonsense mutation imports tobacco cell, transformation efficiency brings up to 11%, the transfer-gen plant growth is normal, has certain anti-PVX virus capable, but increase disease-resistant degree reduction along with infecting the back time; The change of N distal process still shows toxicity symptom, though the sequence of active centre base mutation imports tobacco cell, transfer-gen plant does not show the PAP cytotoxicity, its virus resistance forfeiture.Wan Duorong etc. (2007) induce PAP signal peptide amino acid mutation, and the PAP that signal peptide is modified imports tobacco cell, though the transfer-gen plant anti-virus ability increases but still performance PAP cytotoxicity symptom.

(3) utilize the damage inducible promoter, the render transgenic plant is expressed the PAP activity when being subjected to the damage of machinery or insect, avoids taking place under the normal growth condition PAP cytotoxicity.Zhang Haiyans etc. (1998) import swede type rape with the PAP cDNA that proteolytic enzyme suppresses sub-II promoters driven, and transfer-gen plant inoculation TuMV shows resistance in various degree after the virus, but the PAP cytotoxicity symptoms such as the withered spot of transfer-gen plant generation blade that have.

This shows that the existing technology of utilizing the PAP gene does not reach the purpose of not only eliminating the PAP cytotoxicity but also improving the transfer-gen plant virus resistance, has limited the PAP gene in the plant virus resistance gene application in engineering.

Summary of the invention

Technical problem to be solved by this invention is to overcome the existing deficiency of utilizing in the Pokeweed antiviral protein gene engineering, and the method that a kind of PAP of utilization fusion gene improves the transgene tobacco virus resistance proposed, promptly utilize tobacco disease engineered protein-1a signal peptide that the PAP maturation protein is navigated to function in the cell walls matrix, the dna sequence dna of encoding nicotiana course of disease albumen-1a signal peptide and the dna sequence dna of coding PAP maturation protein are merged synthetic PAP fusion gene.This fusion gene is imported tobacco cell and obtains transfer-gen plant, from transfer-gen plant, filter out virus resistance and by force, seldom or do not show the Cytotoxic tobacco plant of PAP.The present invention helps cultivating the tobacco bred of broad-spectrum antiviral.

Technical scheme provided by the invention is:

Utilize the dna sequence dna of encoding nicotiana pathogenesis-related proteins-1a signal peptide and the dna sequence dna synthetic PAP fusion gene of coding Pokeweed antiviral protein mature peptide, the sequence signature of this fusion gene is illustrated in the dna sequence dna shown in the sequence table.Wherein, 1～90bp is that (the GenBank accession number: D90196), 91～876bp is dna sequence dna (the GenBank accession number: X55383) of PAP maturation protein for the dna sequence dna of PR-1a signal peptide;

A kind of method that improves tobacco virus resistance may further comprise the steps:

(1) by reverse transcription and polymerase chain reaction (RT-PCR) amplification method, the cDNA sequence of clones coding tobacco disease engineered protein 1a gene (PR-1a) and coding Pokeweed antiviral protein gene (PAP);

(2) utilize gene splicing by overlap extension (gene splicing by overlapextension), with the dna sequence dna of the encoding nicotiana course of disease albumen 1a signal peptide of polymerase chain reaction (PCR) amplification and the dna sequence dna of coding Pokeweed antiviral protein mature peptide, synthetic PAP fusion gene, and clone PAP fusion gene;

(3) dna fragmentation with the PAP fusion gene is connected to binary vector plasmid pBI121, makes up the plant expression vector that carries the PAP fusion gene;

(4) utilize agrobacterium mediation method, the PAP fusion gene is imported tobacco cell, cultivate regeneration induction plant, Genetic identification transfer-gen plant by antibiotic-screening;

(5) mechanical process selects virus resistance by force, not show Cytotoxic transfer-gen plant tobacco mosaic virus (TMV) virus inoculation rotaring gene plant blades such as (TMV).

Embodiment

Embodiment one

1. the cDNA sequence of high-fidelity pcr amplification PAP and PR-1a

(1) the synthetic first chain cDNA

Infect the high potted plant tobacco bred K326 blade of 10～15cm with TMV, 4～6 susceptible plant leaf performance in week back manifest symptoms are got susceptible spire and are extracted mRNA; Spire in summer with the dyers' grapes full extension extracts mRNA.Specific practice is, respectively gets spire 0.1 gram, pulverizes in liquid nitrogen, uses the Pharmacia Quick Prep Micro mRNA Purification Kit of company, according to the test kit instruction manual, extracts and purified mRNA.Then, with the first chain cDNA synthetic agent box (Shen, Shanghai energy betting office), according to the test kit instruction manual, the synthetic first chain cDNA.

(2) pcr amplification PAP gene fragment

According to the PAP gene order of report such as Lin (1991) (GenBank accession number: X55383.1) design amplification PAP cDNA coding region sequence and carry BamH I and the sense primer and the antisense primer of Xam I restriction enzyme site, i.e. 5 '-GGATCCATGAAGTCGATGCTTGTGGT-3 ' and 5 '-CCCGGGTCAGAATCCTTCAAATAGAT-3 '.The high-fidelity pcr amplification carries out in 50 μ l systems, wherein contains pfu polysaccharase 2U, and the about 20ng of the dyers' grapes first chain cDNA, dNTP 200 μ mol/L, primer concentration respectively are 20pmol/L and 10 * pfu amplification buffer, 5 μ l.Amplification condition is: 94 ℃ of pre-sex change 4 minutes, and 94 ℃ of sex change are 1 minute then, 55 ℃ of annealing 1 minute, 72 ℃ were extended 2 minutes, and totally 30 circulations are at last 72 ℃ of insulations 10 minutes down.

(3) pcr amplification PR-1a gene fragment

According to the PR-1a gene order of Cornelissen (1986) report (GenBank accession number: D90196) design amplification PR-1a cDNA coding region sequence and carry BamH I and the sense primer and the antisense primer of Xam I restriction enzyme site, i.e. 5 '-GGATCCATGGGATTTGTTCTCTTT-3 ' and 5 '-CCCGGGTTAGTATGGACTTTCGCCTC-3 '.The first chain cDNA is a template with tobacco, pcr amplification PR-1a gene fragment, and amplification reaction system and condition are with this case step 1 (2).

2.PAP the clone of gene and PR-1a gene

Amplified production is electrophoresis in containing 1% agarose gel plate of ethidium bromide, TAE electrophoretic buffer, voltage 4V/cm.Behind the electrophoresis, under long wave ultraviolet light shines, cut the gel that only contains dna fragmentation fast.With 3S nucleic acid purification test kit (Shen energy betting office) purifying pcr amplified fragment.Use pGEM-T Easy test kit (Promega company), according to the test kit instruction manual, the dna fragmentation of amplification is connected to the pGEM-T carrier, transformed competence colibacillus coli strain JM109 cultivates on the LB substratum that contains X-gal 80mg/L, IPTG 100mg/L and penbritin 100mg/L.Select white colony, (Jin Dongyan, Li Mengfeng etc. translate: molecular cloning experiment guide, Science Press, 1992, P26) extraction plasmid with alkaline lysis.In 20 μ l reaction systems, add plasmid DNA 0.1 μ g, 10 * EcoR I enzyme buffer liquid, 2 μ l and EcoRI 10U, endonuclease reaction is 4 hours in 37 ℃ of waters bath with thermostatic control.Electrophoresis enzyme is cut product, identifies and carries the JM109 cell clone that contains the pGEM-T carrier that inserts target DNA fragment.After cloned genes carried out dna sequence dna order-checking (worker Bioisystech Co., Ltd is given birth in Shanghai), respectively with the NCBI gene pool in the sequence alignment of PAP gene and PR-1a gene, select to carry the JM109 cell clone that contains the correct pGEM-T plasmid of goal gene base sequence.

Embodiment two

Utilize the dna sequence dna of gene splicing by overlap extension composite coding PR-1a signal peptide and the synthetic fusion gene of dna sequence dna of coding PAP mature peptide.

(1) dna fragmentation of pcr amplification coding PR-1a signal peptide

Sense primer and antisense primer are respectively: 5 '-GGATCCATGGGATTTGTTCTCTTT-3 ' and 5 '-CCCGGGAACATTGTAGATGATTGTGGCACGGCAAGAGTGG-3 ', for touching plate, in 50 μ l systems, carry out the high-fidelity pcr amplification with the plasmid that carries PR-1a.Wherein, contain 10 * pfu amplification buffer, 5 μ l, plasmid DNA 20ng, dNTP 200 μ mol/L, primer 2 0pmol/L and pfu polysaccharase 2U.Amplification condition is: 94 ℃ of pre-sex change 4 minutes, and 94 ℃ of sex change are 1 minute then, 55 ℃ of annealing 1 minute, 72 ℃ were extended 2 minutes, carried out 30 circulations altogether, at last 72 ℃ of insulations 10 minutes down.The electrophoresis of pcr amplification product and purifying are with example one step 2.

(2) dna sequence dna of pcr amplification coding PAP maturation protein

Sense primer and antisense primer are respectively:

5 '-TCCCACTCTTGCCGTGCCACAATCATCTACAATGTT-3 ' and 5 '-CCCGGGTCAAGTTGTCTGACAGCTCCC for touching plate, carries out the high-fidelity pcr amplification with the plasmid that carries PAP in 50 μ l systems.Other steps are with this case step (1).

(3) pcr amplification PAP fusion gene

Sense primer and antisense primer are respectively: 5 '-GGATCCATGGGATTTGTTCTCTTT-3 ' and 5 '-CCCGGGTCAAGTTGTCTGACAGCTCCC, for touching plate, in 50 μ l systems, carry out the high-fidelity pcr amplification with the dna fragmentation of the dna fragmentation of the coding PR-1a signal peptide of purifying and coding PAP maturation protein.Other steps are with this case step (1).

(4) PAP fusion gene cloning

It is identical with example one step 2 that PAP fusion gene cloning and enzyme are cut authentication method.With the PAP fusion gene sequence after the order-checking,, select to carry the JM109 cell clone that contains the correct pGEM-T plasmid of target gene fragment base sequence with PR-1a signal peptide corresponding sequence in the NCBI gene pool and the comparison of PAP mature peptide corresponding sequence.

Embodiment three

1. make up the plant expression vector of PAP fusion gene

(1) PAP fusion gene dna fragmentation and the segmental purifying of line style binary vector plasmid pBI121

With BamH I and Xam I enzyme respectively double digestion contain the pGEM-T vector plasmid and the binary vector plasmid pBI121 of PAP fusion gene.Way is, adds plasmid DNA 0.5 μ g in 20 μ l reaction systems, 10 * BamH I reaction buffer, 2 μ l and BamH I 10U, and endonuclease reaction is 4 hours in 37 ℃ of waters bath with thermostatic control.Then, add dehydrated alcohol 40 μ l in reaction solution, mixing was placed 30 minutes down for-20 ℃, and per minute 12000 left the heart 10 minutes, abandoned solution, 17 μ l sterilized water dissolving DNAs precipitation.In this dna solution, add 10 * Xam I reaction buffer 2 μ l and Xam I 10U (10U/ μ l), endonuclease reaction is 4 hours in 37 ℃ of water-baths.Electrophoresis enzyme is cut product, cuts PAP fusion gene band and line style binary vector plasmid pBI121 band in the sepharose with scalpel, with 3S nucleic acid purification test kit (Shen can betting office) these two kinds of DNA bands of purifying respectively.

(2) the PAP fusion gene is connected with binary vector pBI121 plasmid and the clone

In 10 μ l ligation systems, add 2 * T ₄Dna ligase damping fluid 5 μ l, PAP fusion gene fragment 3 μ l, plasmid pBI121 1 μ l and T ₄Dna ligase 1 μ l, 4 ℃ of following connections spend the night.Mix with coli strain JM109 competent cell 50 μ l with connecting converted product 3 μ l, the coli strain JM109 cell of conversion processing is coated on the LB substratum that contains kantlex 100mg/L overnight incubation.Choose single bacterium colony, the alkaline lysis method of extracting plasmid, according to this case step 1 (1) method, with BamHI and Xam I enzyme double digestion, carry out electrophoresis then, the dna fragmentation of the PAP fusion gene that the double digestion product occurs in the sepharose behind the observation electrophoresis determines to carry the JM109 cell clone of the pBI121 plasmid that contains the PAP fusion gene with this.

(3) genetic transformation of Agrobacterium LBA4404

Mix with Agrobacterium LBA4404 competent cell with the pBI121 plasmid 0.1 μ g that carries the PAP fusion gene, freeze-thaw method transforms Agrobacterium.Agrobacterium after the conversion processing was cultivated 24～48 hours on the YEB substratum that contains Rifampin 80mg/L and kantlex 100mg/L, choose single bacterium colony, the alkaline lysis trace extracts plasmid, with BamH I and Xam I enzyme double digestion (method is with this case step 1 (1)), electrophoresis enzyme is cut product, observe the PAP fusion gene fragment that enzyme is cut product in the sepharose, identify the Agrobacterium bacterium colony that carries the pBI121 plasmid that contains the PAP fusion gene.

2. the genetic transformation of tobacco leaf

(1) cultivation of Agrobacterium

Incubated overnight Agrobacterium in the YEB substratum is got bacterium liquid 2ml, joins in the same substratum of 20ml, and concussion was cultivated about 6 hours under the condition that 28 ℃, per minute 180 change, and detecting bacterial concentration with spectrophotometer is OD ₆₀₀, be used for the genetic transformation of tobacco leaf cell at=0.2～0.5 o'clock.(2) tobacco leaf explant surface sterilization

Get the spire of full extension on the tobacco K326 kind 3 monthly age plant, clean with tap water and dry.On Bechtop, blade is put into aseptic beaker, with 70% alcohol-pickled blade 30 seconds, then with 0.1% mercury chloride to blade surface sterilization 6 minutes, sterile water wash 5 times stopped 5 minutes in sterilized water at every turn.In surface sterilization and cleaning process, need shake beaker off and on, make the blade surface sterilization and clean thoroughly.Then, aseptic blade is cut into 0.5～1.0cm ²Size is used to infect Agrobacterium.

(3) agroinfection tobacco leaf explant

The tobacco leaf explant is put into OD ₆₀₀In=0.2～0.5 the bacterium liquid, concussion was infected 10 minutes on the shaking table that per minute 80 changes.Then, in Bechtop, after absorbing bacterium liquid unnecessary on the leaf explant on the aseptic filter paper, blade inoculation is total to substratum (the fast quinoline 0.2mg/L+3% of the amino gland of MS minimum medium+naphthylacetic acid 0.02mg/L+6-benzyl sucrose+0.6% agar at MS, pH=5.8) on, under 26 ± 1 ℃, dark condition, cultivated altogether 3 days.

(4) screen resistant calli and induce plant regeneration

With the blade after cultivating altogether transfer to the MS screening culture medium (the fast quinoline 0.2mg/L+ of the amino gland of MS minimum medium+naphthylacetic acid 0.02mg/L+6-benzyl Pyocianil 100mg/L+ kantlex 80mg/L+3% sucrose+0.6% agar, pH=5.8) on.To cultivate leaf explant every 3～4 days transfers on the substratum of same new preparation.Culture condition is illumination 2000lx, 25 ℃ of temperature.After the cultivation through 3～4 weeks, indivedual positions form small callus from the leaf explant edge, cultivate through 1～2 week again, differentiate indefinite bud from callus.The aseptic seedling of kalamycin resistance grows into when growing about 2cm, aseptic seedling is cut off from basal part of stem, transfer to MS root media (the MS minimum medium of 1/2 macroelement concentration+naphthylacetic acid 0.01mg/L+ Pyocianil 100mg/L+ kantlex 80mg/L+1% sucrose+0.6% agar, pH=5.8) root induction in, culture condition is the same.After 10 days, produce many young roots from the aseptic seedling stem base portion.Root takes out the seedling of taking root after reaching 1～2cm length, carefully removes the agar of resistance shoot root system, is transplanted in the soil.The transplanting initial stage hides with plastics film, keeps relative humidity more than 90%, 20～25 ℃ of temperature.Transplanted seedling progressively reduces relative humidity, up to consistent with the relative humidity of physical environment after recovering growth.

3. the expression analysis of the evaluation of transfer-gen plant and PAP fusion gene

(1) pcr amplification is identified transfer-gen plant.

Get the plant spire 0.1g of antibiotics resistance, in liquid nitrogen, pulverize fast, change over to rapidly in the 1.5ml centrifuge tube of precooling, the 2 * cetyl trimethyl ammonia bromide (CTAB) that adds 65 ℃ of preheatings immediately extracts damping fluid 0.5ml, extracts 30 minutes in 65 ℃ of water-baths.Then, add the chloroform/primary isoamyl alcohol (24:1) of equivalent, put upside down extracting back and forth.Per minute 12000 left the heart 10 minutes.Get supernatant liquor 0.4ml, add the Virahol 0.8ml of-20 ℃ of precoolings, put upside down mixing back and forth, occur thread dna material in the solution.Choose thread dna material, after 20 minutes, dry a little with 76% ethanol (containing the 10mmol/L ammonium acetate) rinsing, with the dissolving of 0.5ml TE damping fluid.Identical (Nucleic Acids Res.18:6531-6535,1990) that (1990) such as the step that is further purified DNA and Williams are described.

Pcr amplified fragment with two kinds of dna sequence dnas is identified transfer-gen plant, a kind of is the dna fragmentation of npt II gene, its justice and antisense primer are respectively: 5 '-CCGACCTGTCCGGTGCCC-3 ' and 5 '-CCGCCACACCCAGCCGGCC-3 ', and the amplified production size is 495bp; Another kind is the 35S promoter fragment, and its justice and antisense primer are respectively: 5 '-CACAGATGGTTAGAGAGGCT-3 ' and 5 '-TTACGGCGAGTTCTGTTAGG-3 ', the amplified production size is 315bp.In 50 μ l pcr amplification systems, each adds the total DNA 100ng of blade, Tag archaeal dna polymerase 3U, and dNTP 200 μ mol/L, justice and antisense primer concentration respectively are 20 ρ mol/L, 10 * Tag DNA enzyme buffer liquid, 5 μ l, ultrapure water is supplied 50 μ l.Amplification condition is: 94 ℃ of pre-sex change 4 minutes, and 94 ℃ of sex change are 1 minute then, 55 ℃ of annealing 1 minute, 72 ℃ were extended 2 minutes, carried out 35 circulations altogether, last 72 ℃ of insulations 10 minutes.Simultaneously, be template with the plant expression carrier plasmid and the total DNA of non-transgenic plant leaf that carry the PAP fusion gene, respectively as the positive and negative control, under similarity condition, carry out pcr amplification.Amplified production is electrophoresis in containing 1.5% sepharose of ethidium bromide, TAE electrophoretic buffer, voltage 4V/cm.Observe electrophoresis result by the gel imaging instrument, the DNA band of 495bp and 315bp size appears in the PCR product of positive control and transfer-gen plant, and negative control does not have these DNA bands.

(2) expression of RT-PCR semi-quantitative analysis PAP fusion gene

Get on the transfer-gen plant unfolded spire 0.1g fully, in liquid nitrogen, fully pulverize, use plant RNA to extract test kit (Puli's Lay company, by specification experimentize operation) the total RNA of extraction transfer-gen plant mesophyll cell.Add DNase 2U in containing the 100 μ l systems of total RNA, 37 ℃ of temperature were bathed 10 minutes, eliminated the minim DNA among the RNA.Utilize the synthetic first chain cDNA of the first chain cDNA synthetic agent box (operation of test kit specification sheets is pressed by Shen, Shanghai energy lottery industry biotech company).

(the GenBank accession number: D63396) transcription product is confidential reference items, and the justice of its pcr amplification and antisense primer are respectively 5 '-TCACATCAACATTGTGGTCATTGGC-3 ' and 5 '-TTGATCTGGTCAAGAGCCTCAAG-3 ' with tobacco elongation factor-1 α (EF-1 α).The pcr amplification product size that obtains from the first chain cDNA is 654bp.Pcr amplification PAP fusion gene encoding mature protein sequence, designed sense primer and antisense primer are respectively 5 '-GGTTATTCTGATCCCTTTGA-3 ' and 5 '-TCTTACCCCATGTCTCTTGC-3 ', and the pcr amplification product size that obtains from the first chain cDNA is 409bp.In 50 μ l pcr amplification systems, add template cDNA 100ng, Tag archaeal dna polymerase 3U, dNTP 200 μ mol/L, justice and antisense primer concentration respectively are 20 ρ mol/L, 10 * Tag DNA enzyme buffer liquid, 5 μ l, ultrapure water is supplied 50 μ l.Amplification condition is, 94 ℃ of pre-sex change 4 minutes, and 30 seconds, 55 ℃ annealing of 94 ℃ of sex change were extended totally 30 circulations 45 seconds for 30 seconds, 72 ℃ then; Extended 7 minutes at 72 ℃ at last.Under the condition of the horizontal basically identical of pcr amplification product of each sample EF-1 α, according to the segmental electrophoresis observation result of pcr amplification PAP fusion gene in the different samples, determine whether the PAP fusion gene of transfer-gen plant expresses, and the power of PAP fusion gene expression.The transfer-gen plant that the PAP fusion gene is expressed is pressed the method detection virus resistance of this case step 4.

4. the virus resistance of transfer-gen plant

Get 4 ℃ of following kept dry, carry the tobacco leaf 20mg of TMV, place glass mortar, add phosphoric acid buffer (pH=7.0) 2ml of 0.1mol/L, grind to form homogenate, preparation TMV virus solution.Add a little 400 purpose quartz sand and viral liquid mixing.Tobacco transgenosis and non-transgenic plant with new growth 5～7 leaves in potted plant are material, dip in glass stick and get viral liquid, the blade surface that rubs gently, virus inoculation.It is the same to inoculate other viral methods simultaneously.Behind the virus inoculation 20 days, the susceptible symptom of routine observation record plant system, selection does not have or slightly shows virus stock togetherness such as TMV to dye symptom, and it is normal to grow, the transfer-gen plant that does not have the performance of PAP cytotoxicity, with its breeding material, be used to cultivate the tobacco of broad-spectrum antiviral as virus resistance.

Substratum that uses among the embodiment and damping fluid

(1) TAE electrode buffer (50 times of stock solutions of 100ml):

Tris, 24.2g; Glacial acetic acid, 5.7ml; EDTA, 10ml 0.5mol/L (pH8.0);

(2) LB substratum:

Trypsin, 5g/L; Yeast extract, 10g/L; NaCl, 10g/L; PH, 7.0;

(3) YEB liquid nutrient medium:

Peptone, 5g/L; Yeast extract, 10g/L; Extractum carnis, 5g/L; Sucrose, 5g/L; MgSO4,0.495g/L; PH, 7.0;

(4) MS minimum medium:

The macroelement composition:

KNO ₃，1900mg/L；NH ₄NO ₃，1650mg/L；CaCl ₂·2H ₂O，440mg/L；MgSO ₄·7H ₂O，370mg/L；KH ₂PO ₄，170mg/L；

The trace element composition:

FeSO ₄·7H ₂O，27.8mg/L；KI，0.83mg/L；H ₃BO ₃，6.2mg/L；MnSO ₄·4H ₂O，22.3mg/L；ZnSO ₄·7H ₂O，8.6mg/L；Na ₂MoO ₄·2H ₂O，0.25mg/L；CuSO ₄·5H ₂O，0.025mg/L；CoCl ₂·6H ₂O，0.025mg/L；

The organotrophy composition:

Inositol, 100mg/L; Nicotinic acid, 0.5mg/L; Pyridoxine hydrochloride, 0.5mg/L; Vitamin, 0.1mg/L; Glycine, 2.0mg/L;

(5) 2 * cetyl trimethyl ammonia bromides (CTAB) extract damping fluid:

Tris-HCl, 100mmol/L (pH8.0); CTAB, 2% (w/v); NaCl, 1.4mol/L; Beta-mercaptoethanol, 40mmol/L; EDTA, 20mmol/L;

(6) TE damping fluid:

Tris-HCl，10mmol/L(pH，7.4)；EDTA，1mmol/L；

Sequence table

＜110〉Beijing Normal University

＜120〉a kind of method that improves tobacco virus resistance

<130>1234

<160>2

<170>PatentIn?version?3.3

<210>1

<211>876

<212>DNA

＜213〉dyers' grapes (Phytolacca americana)

<220>

<221>CDS

<222>(1)..(876)

<220>

<221>sig_peptide

<222>(1)..(90)

<220>

<221>mat_peptide

<222>(91)..(876)

<400>1

<210>2

<211>292

<212>PRT

＜213〉dyers' grapes (Phytolacca americana)

<400>2

Claims (6)

1, a kind of PAP fusion gene of forming by the dna sequence dna of the dna sequence dna of encoding nicotiana course of disease albumen-1a signal peptide and the Pokeweed antiviral protein mature peptide of encoding, the aminoacid sequence shown in its code sequence tabulation SEQ NO:2.

2, fusion gene encoded polypeptides according to claim 1, it is by the dna sequence encoding shown in the sequence table SEQ NO:1.

3, the plant expression vector that contains the described fusion gene of claim 2.

4, plant expression vector according to claim 3, it contains the dna sequence dna of encoding nicotiana course of disease albumen-1a signal peptide and the fusion gene of the dna sequence dna composition of coding Pokeweed antiviral protein mature peptide.

5, the transgenic plant that contain the described fusion gene of claim 2.

6, plant according to claim 5 is common tobacco (Nicotiana tabacum).