CN109610009A - A kind of tobacco disease resistance poison controlling gene screening technique and application - Google Patents
A kind of tobacco disease resistance poison controlling gene screening technique and application Download PDFInfo
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Abstract
The present invention provides a kind of tobacco disease resistance poison controlling gene screening technique and applications, this method is first using virus induced gene silencing VIGS carrier as purpose carrier, Tobacco cDNA library is constructed, obtains the library VIGS-cDNA that can be directly used for gene silencing screening analysis after carrying out homogenization processing；VIGS-cDNA Library plasmid is converted to Agrobacterium, Agrobacterium library is obtained；The coated plate culture of Agrobacterium library is obtained into single colonie, carries out VIGS silencing analysis respectively to each single colonie, screening obtains antiviral related bacterium colony；Sequencing analysis is carried out to the corresponding gene of antiviral correlation bacterium colony that screening obtains, and homology analysis and function prediction are carried out to the sequence of acquisition, obtains antiviral controlling gene.The method of the present invention is easy to operate, and time-consuming short without constructing transgenic plant or mutant, screening overall efficiency is high, especially suitable for the screening of mutational lethal gene, has a extensive future.
Description
Technical field
The invention belongs to the screening of plant disease-resistant controlling gene and applied technical fields, are related to a kind of tobacco disease resistance poison regulation base
Because of screening technique and application.
Background technique
Plant cDNA libraries refer to the set of all cDNA of plant species.These cDNA are usually inserted a load
Body, conversion are mixed into Escherichia coli and are saved.After the completion of building, for choosing use at any time.In addition, being used by selection different
Library construction carrier, different Functional Plant Genomics researchs can be carried out.Such as the text with plant expression vector construction
Library can be used for instantaneously being overexpressed analysis in plant, screen the controlling gene of specific objective function.Therefore, plant cDNA text
The utilization in library is the basic research means and scale selection of Functional Plant Genomics research, separation and clone's purpose tune
Control one of the main path of gene.The construction step of plant cDNA libraries mainly includes plant tissue Total RNAs extraction, and mRNA is pure
Change, reverse transcription, is connected into carrier, homogenization processing, the bacterium conversion of connection product, plasmid extraction, PCR and digestion inspection, capacity
With recombination fraction analysis etc..
Plant gene function is usually by comparing the phenotype in the case of the unconventional expression of analysis gene and normal expression
(Phenotype) or Function situation judges.The unconventional expression of gene includes the expression higher than normal level and is lower than
The expression two major classes of normal level.Expression higher than normal level is mainly overexpression/overexpression (Over-expression),
Mainly reached by way of driving destination gene expression connecting a strong promoter.It is main lower than the expression of normal level
Pass through RNA interference (RNA interfering, RNAi), Knockdown (Knock-down) and gene knockout (Knock-out) etc.
Mode reaches.Overexpression by construct and conversion one containing same sequence fragment opposite direction be inserted into an introne or its
Hairpin structure that sequence that it is not expressed is formed is realized, the result is that the reduction of destination gene expression.Knockdown can also lead to
Cross virus induced gene silencing (Virus-induced gene silencing, VIGS) Lai Shixian.Gene knockout (Knock-
Out) then by being inserted into the side such as a long non-plant sequence or excision target gene in target gene in the plant genome
Formula reaches, the result is that the nearly fully complete of destination gene expression inhibits.For individual gene, as long as building gene is super
Plant and/or overexpression plant, knock out mutants body are expressed, the phenotype and character and wild type/normal of these plant are compared
The difference of plant, the regulatory function for the gene pairs character that can have a definite purpose.But horizontal scale is learned for subgroup and group
Gene function analysis, then the overexpression library for needing to construct all genes, mutant library or the library VIGS can reach screening and divide
Purpose from target gene.
Due to the importing of foreign gene, cause consistent with the foreign gene and very high homology therewith plant endogenous in plant
The phenomenon that transcript accumulation of gene is suppressed referred to as posttranscriptional gene silencing (post-transcriptional gene
Silencing, PTGS)." virus induced gene silencing " (virus-induced gene silencing, VIGS) is plant
The middle widest PTGS phenomenon of research.VIGS phenomenon was a kind of natural mechanism that plant virus resistance infects originally.But in recent years,
It is developed to as a rapid gene Function Identification new technology.Plant target gene fragment need to be only inserted into viral vectors, building
At recombinant virus, can be used to inhibit the expression with the plant endogenous target gene of Insert Fragment very high homology after infecting plant,
To verify the gene function.By the research of several years, which, which has evolved into one, to study plant base with fast high-flux
Because of the mature technology of function, just had been more and more widely used in the research of Functional Plant Genomics.
It is existing at present to lose the method for (loss-of-function) research mainly including chemical mutation point for function
Analysis, transposons or the analysis of agrobatcerium T-DNA insertion mutation.These methods are in a small number of model plant gene functional research such as arabidopsis
In it is highly effective, but can not yet be applied on a large scale in other plant.In addition, the technology of inhibition destination gene expression further includes
Antisense RNA Technique, RNAi technology and chemicals induced gene silent technology.All these technologies are both needed to building transgenosis
Plant.VIGS technology has the following advantages that or feature compared with these existing technologies:
Firstly, VIGS analysis can be carried out in the case where full length sequence has not been obtained and does not even know sequence in advance.VIGS
Function Identification can be carried out by object of some segment using est sequence as object, mesh can also be carried out by object of cDNA library
Genescreen analysis.
Secondly, VIGS is easy to operate, it can quick obtaining phenotype.Function forfeiture table can be obtained in the plant present age using VIGS
Type, without obtaining mutant by Large-scale Screening.In addition, being directed into phenotype generation from VIGS only needs several weeks, therefore very fast
Speed.This is most one of clear advantage of VIGS.
Third, VIGS is without constructing transgenic plant.This obtains very difficult plant species for those transgenic plants
It is most important for class.
4th, VIGS can be used for being mutated lethal gene functional research.For mutational lethal gene, it is unable to get its mutation
Body.And VIGS can carry out the research of these gene functions in biggish seedling stage.
5th, VIGS can be used for the functional study of functional redundancy gene.Many plant genes exist in the form of gene family,
Gene members in one family often have similar function, therefore often to the mutation of single member gene in gene family
There is no apparent phenotype.VIGS can overcome this difficulty, need to only be cloned into most conservative region between gene family member
VIGS carrier, then carry out VIGS, that is, it can inhibit the expression of entire family gene, to observe phenotype.Certainly, if selection man
The most special sequence of race gene members carries out VIGS, then it can be observed that the loss of function phenotype of the individual gene.
Disease resistance of plant is inoculated with analysis by pathogen and carries out testing and evaluation.The inoculation method of different type pathogen is each
It is not identical.For fungi, can generate it is conidial, usually with conidial suspension spray inoculation plant.It does not generate mitogenetic
Spore, then with inoculated plants such as mycelia blocks.For oomycetes, generally first culture generates a large amount of sporangiums, and then Low- temperature culture makes
Release zoospore, finally with zoospore suspension inoculated plant.For bacterium, often pierced with thallus suspension liquid leaf-cutting, needle,
The methods inoculated plant such as injection, spraying.For virus, often with transcription product or extraction outside the virion of purification, prosthesis and mention
Pure virus is rubbed, is injected or by communication medias inoculated plants such as insects.For bacterium substance, be often inoculated with by vector or
Person's graft inoculation.For nematode, is often contacted in plant root basal part of stem or rhizosphere soil and be inoculated with a certain number of second instar larvaes
Plant.In most cases, it needs to keep higher relative humidity, suitable temperature and illumination condition after inoculation.By not after inoculation
Disease is observed continuously with time point, symptom, statistics disease incidence and morbidity severity occurs, calculates disease index, calculate bacterium amount or adopt
Pathogen biomass is detected with RT-PCR.By the comparative analysis with susceptible check plant incidence, hard objectives plant
Disease resistance.
Summary of the invention
The quick obtaining purpose virus tune that the object of the present invention is to provide a kind of without constructing transgenic plant or mutant
The method for controlling gene.
Tobacco disease resistance poison controlling gene screening technique provided by the invention, comprising the following steps:
(1) using VIGS carrier as purpose carrier, Tobacco cDNA library is constructed, obtains and can directly use after carrying out homogenization processing
In the library VIGS-cDNA of gene silencing screening analysis；
(2) cDNA library-VIGS is analyzed: the VIGS-cDNA Library plasmid that building is completed being converted to Agrobacterium, agriculture is obtained
The coated plate culture of Agrobacterium library is obtained single colonie, carries out VIGS silencing analysis respectively to each single colonie by bacillus library；
(3) screening of antiviral controlling gene: carrying out antiviral analysis to the VIGS silencing plant of each bacterium colony of cDNA library,
Screening obtains antiviral related bacterium colony；
(4) identification of antiviral controlling gene: the corresponding gene of antiviral correlation bacterium colony that screening obtains is sequenced
Analysis, and homology analysis and function prediction are carried out to the sequence of acquisition, obtain antiviral controlling gene.
In the above method of the invention, the method for step (1) building Tobacco cDNA library is:
1) tobacco total serum IgE is extracted, tobacco mRNA is obtained, building DSN uniforms cDNA primary libraries, by homogenization processing
CDNA is cloned into carrier by BP recombining reaction, then transformed competence colibacillus cell, is detected by coated plate and monoclonal, analyzes library
Capacity and homogenization effect；
2) carry out the building of VIGS secondary library: extraction step 1) obtained cDNA primary libraries plasmid, by obtain just
VIGS carrier is cloned by LR recombining reaction after grade Library plasmid dilution；It is detected by coated plate and monoclonal, analyzes library
Capacity and recombination fraction；
3) identification obtains the library tobacco VIGS-cDNA that capacity is big and recombination fraction is high.
Preferably, VIGS carrier is pTRVG, and the obtained library VIGS-cDNA is the library pTRVG-cDNA.
Tobacco disease resistance poison controlling gene screening technique step (2) of the invention carries out VIGS silencing to each single colonie respectively
The method of analysis are as follows:
1) the coated plate culture of part Agrobacterium bacterium solution is drawn, while cultivating the Agrobacterium library converted；Picking single colonie, connects
Kind is cultivated in antibiotic fluid nutrient medium, collects somatic cells, is resuspended to OD600Value is 2, after renewal cultivation, is pressed
1:1 (V:V) ratio mixes the VIGS-cDNA library transformation bacterium of the Agrobacterium converted and insertion target gene fragment；
2) 1- for the tobacco top full extension that agrobacterium liquid infiltration seedling age is 20-25d will the infiltration inoculation of plant: be mixed
2 blades, using the mixed bacteria liquid of the Agrobacterium of infiltration transformation and VIGS-eGFP Agrobacterium as control；
3) tobacco after infiltration inoculation is cultivated, target gene is allowed to carry out effective silencing.
One specific embodiment are as follows:
(1) acquisition of Agrobacterium single colonie: drawing the coated plate culture of part Agrobacterium bacterium solution, while cultivating TRVl Agrobacterium,
Picking single colonie；
(2) preparation of inoculum: picking them separately single colonie, is inoculated in the YEB Liquid Culture containing kanamycins and rifampin
It carries out shaking bacterium overnight incubation in a small amount in base, then carries out mass propgation；Somatic cells are collected, are resuspended with MMAi solution to OD600Value is
2；After renewal cultivation 2h, in 1:1 (V:V) ratio by TRV1 Agrobacterium and insertion target fragment pTRVG-cDNA library transformation bacterium
It is mixed；
(3) the infiltration inoculation of plant: using syringe infusion method, will mix the tobacco that agrobacterium liquid infiltration seedling age is 20-25d
1-2 blade of top full extension, using the mixed bacteria liquid of infiltrating T RV1 Agrobacterium and pTRVG-eGFP Agrobacterium as control；
(4) silencing culture: the tobacco after infiltration inoculation is placed in 21 DEG C, is cultivated under 16h/8h light dark cycles, allows purpose base
Because carrying out effective silencing.
The antiviral analysis method of tobacco disease resistance poison controlling gene screening technique step (3) provided by the invention are as follows: silencing
Processing carries out antiviral analysis after two weeks, to the VIGS silencing plant of each bacterium colony of cDNA library respectively, is inoculated with tobacco virus, inoculation
Afterwards, virus disease symptom is observed and recorded, the bacterium colony that silencing restrovirus Disease symptoms severity significantly changes is obtained, these
The corresponding gene of bacterium colony is the candidate gene of antiviral controlling gene.
Preferably, the virus is marmor upsilon (PVY) and tobacco mosaic virus (TMV) (CMV).
The antiviral related bacterium that tobacco disease resistance poison controlling gene screening technique step (4) provided by the invention obtains screening
It falls corresponding gene and carries out sequencing analysis, obtain the sequence of these genes, and tobacco and other plant bases are carried out to these sequences
Because of BLAST analysis, the conserved structure domain analysis of group database, predicted gene function, to obtain antiviral controlling gene.
The present invention provides application of the above-mentioned tobacco disease resistance poison controlling gene screening technique in tobacco virus prevention and treatment.
The present invention provides the tobacco disease resistance poison controlling gene screening techniques in preparing antiviral transgene tobacco
Using.
The present invention provides the tobacco disease resistance poison controlling gene screening techniques to obtain tobacco disease resistance poison controlling gene simultaneously
For the application in tobacco germplasm improvement.
The invention has the following advantages that the ingenious sieve for carrying out disease-resistant controlling gene using VIGS technology of (1) the method for the present invention
Choosing, without constructing mutant or transgenic plant, an analytical cycle only needs several weeks, very fast, obtains suitable for being difficult building
The screening that antiviral controlling gene is carried out in the species of transgenic plant is taken, it is easy to operate, it can quick obtaining purpose controlling gene.
(2) for the present invention in the horizontal screening for carrying out antiviral controlling gene of group, screening is comprehensively, high-efficient.The present invention uses library-
VIGS method, therefore be blind sieve at the genomic level, screening range is big, and candidate target is comprehensive, can be with Effective selection to newly
Antiviral controlling gene.(3) present invention is suitable for the screening of mutational lethal gene.For mutational lethal gene, it is unable to get
Its mutant, thus antiviral adjusting function analysis can not be carried out using mutant.And VIGS can in biggish seedling stage into
The research of these gene antiviral functions of row.Therefore, this method is suitable for the screening of mutational lethal gene, is to using mutant
Effective supplement of antiviral controlling gene screening technique is carried out, and is screened comprehensively, there is good market application prospect.
Specific embodiment
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case where spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art,
Biochemical reagents used in embodiment are commercially available.
The screening and identification of the antiviral controlling gene of 1 Ben Shi cigarette of embodiment
The sieve of the antiviral controlling gene of Ben Shi cigarette based on present embodiments providing a set of VIGS technology by cDNA library
Choosing and identification technology system.Key step includes:
(1) building in the library Ben Shi cigarette VIGS-cDNA
Using VIGS carrier pTRVG as purpose carrier, Ben Shi cigarette cDNA library is constructed, carries out homogenization processing, acquisition can be straight
Connect the library Ben Shi cigarette VIGS-cDNA that gene silencing screening analysis is carried out in Ben Shi cigarette.Specific step is as follows:
Ben Shi cigarette total serum IgE is extracted using TRIzol Reagent kit.It uses againMAG mRNA
Isolation Kit kit extracts Ben Shi cigarette mRNA.The structure of homogenization Uncut cDNA primary libraries is carried out according to the following steps
It builds:
Using CloneMiner II cDNA Library Construction Kit and Trimmer-2 cDNA
Normalization kit constructs DSN and uniforms cDNA primary libraries.First using mRNA as template, DSN-attB2-RT-primer
For primer, the first chain of cDNA is synthesized.Then the second chain of cDNA is synthesized.Again by cDNA and the DSN-attB1adapter phase for preparing
Even, the cDNA containing DSN-attB1 and DSN-attB2 connector is obtained.DSN solution is added, carries out cDNA hybridization reaction.With connector
In the PCR primer M1 that contains be primer, carry out PCR amplification, obtain DSN and uniform cDNA.By the cDNA of homogenization processing
It is cloned into pDONR222 carrier by BP recombining reaction, then converts Escherichia coli.It is detected by coated plate and monoclonal, analysis text
The capacity and homogenization effect in library.
Then, the building of pTRVG-DEST secondary library is carried out according to the following steps:
UsingHiPure Plasmid Filter Midiprep Kit extracts primary libraries plasmid.Will
To primary libraries plasmid be diluted to 300ng/uL, pTRVG carrier is cloned by LR recombining reaction.Pass through coated plate and monoclonal
Detection, analyzes the capacity and recombination fraction in library.
Identification obtains the library Ben Shi cigarette pTRVG-cDNA that capacity is big and recombination fraction is high.The present invention constructs the Ben Shi cigarette obtained
The library capacity in the library VIGS-cDNA is 4.5 × 106Number is always cloned up to 1.3 × 10 in cfu/mL, library7Cfu can be used for purpose base
The VIGS of cause is screened.
(2) Ben Shi cigarette cDNA library-VIGS is analyzed
The Ben Shi cigarette VIGS-cDNA Library plasmid that building is completed is obtained by electroporated to agrobacterium strains GV3101
Obtain Agrobacterium library.Library coated plate culture is obtained into single colonie, carries out VIGS silencing analysis respectively to each single colonie.Specific step
It is rapid:
The coated plate culture of part bacterium solution is drawn, while cultivating TRVl Agrobacterium, picks them separately single colonie, is inoculated in 1ml containing card
In the YEB fluid nutrient medium of that mycin (Kan, 50 μ g/ml) and rifampin (Rif, 50 μ g/ml), 28 DEG C, 200rpm was cultivated
Night.It draws the 100 cultured bacterium solutions of μ l to be seeded in new 100ml YEBi solution, 28 DEG C, 200rpm culture, until bacterium solution OD600
For 0.8-1.2.8000rpm is centrifuged 10min, collects somatic cells, is then resuspended with MMAi solution to OD600=2 (VMMAi=VBacterium solution
×OD600/2).The bacterium solution of resuspension is in 28 DEG C, 60rpm renewal cultivation 2h.After renewal cultivation is good, in 1:1 (V:V) ratio by TRV1
It is mixed with the pTRVG-cDNA library transformation bacterium of insertion target fragment.Using syringe infusion method, by mixing agrobacterium liquid leaching
Moisten 1-2 blade of the Ben Shi cigarette top full extension that seedling age is 20-25d, with infiltrating T RV1 Agrobacterium and pTRVG-eGFP agriculture
The mixed bacteria liquid of bacillus is as control.Treated that Ben Shi cigarette is placed in 21 DEG C by VIGS, cultivates under 16/8 light dark cycles, allows purpose
Gene carries out effective silencing.
(3) screening of the antiviral controlling gene of Ben Shi cigarette
Above-mentioned silencing processing after two weeks, carries out antiviral analysis to the VIGS silencing plant of each bacterium colony of cDNA library respectively,
It is inoculated with marmor upsilon (PVY) and tobacco mosaic virus (TMV) (CMV) respectively.Acquisition shows the leaf of obvious PVY and CMV symptom respectively
Piece extracts virus with PBS, and the supernatant crude extract after centrifugation is for being inoculated with.Using two, juice frictional inoculation top tender leaf, every leaf
200 μ L virus crude extracts.2 weeks after inoculation, observation and record virus disease symptom.Obtain silencing restrovirus Disease symptoms severity
The bacterium colony significantly changed.The corresponding gene of these bacterium colonies is the candidate gene of anti-PVY and CMV virus regulatory gene.
The present invention passes through to 907 library Ben Shi cigarette pTRVG-cDNA bacterium colony genes in Ben Shi cigarette plant a more than 3600
VIGS analysis obtains symptom and disease severity significantly change after being inoculated with PVY after gene silencing gene 42, is inoculated with CMV
Significantly change gene 31 of symptom and disease severity afterwards.
(4) identification of the antiviral controlling gene of Ben Shi cigarette
Sequencing analysis is carried out to the corresponding gene of antiviral correlation bacterium colony that screening obtains, obtains the sequence of these genes,
And these sequences are carried out with BLAST analysis, the conserved structure domain analysis of Ben Shi cigarette and other Plant Genome databases, predict base
Because of function, to obtain antiviral controlling gene.
The present invention corresponds to gene to 73 bacterium colonies that above-mentioned screening obtains and is sequenced, further to the sequence of acquisition into
Row homology analysis and function prediction, identification obtains the anti-PVY controlling gene of potential Ben Shi cigarette 10 respectively and anti-CMV regulates and controls base
Because of 10.It discloses proteins ubiquitin and proteasome degradation, transcription regulatory factor and one group of information conduct factors may be anti-
It plays an important role in PVY virus, and apoptosis (PCD), the protein film of DAD1 and cathepsin B proteases mediate
Transhipment and endocytosis and subcellular localization variation may play an important role in anti-CMV virus.
Claims (10)
1. a kind of tobacco disease resistance poison controlling gene screening technique, which comprises the following steps:
(1) using VIGS carrier as purpose carrier, Tobacco cDNA library is constructed, acquisition can be directly used for base after carrying out homogenization processing
Because of the library VIGS-cDNA of silencing screening analysis；
(2) cDNA library-VIGS is analyzed: the VIGS-cDNA Library plasmid that building is completed being converted to Agrobacterium, Agrobacterium is obtained
The coated plate culture of Agrobacterium library is obtained single colonie, carries out VIGS silencing analysis respectively to each single colonie by library；
(3) antiviral analysis, screening the screening of antiviral controlling gene: are carried out to the VIGS silencing plant of each bacterium colony of cDNA library
Obtain antiviral related bacterium colony；
(4) identification of antiviral controlling gene: carrying out sequencing analysis to the corresponding gene of antiviral correlation bacterium colony that screening obtains,
And homology analysis and function prediction are carried out to the sequence of acquisition, obtain antiviral controlling gene.
2. tobacco disease resistance poison controlling gene screening technique as described in claim 1, which is characterized in that step (1) constructs tobacco
The method of cDNA library is:
1) tobacco total serum IgE is extracted, tobacco mRNA is obtained, building DSN uniforms cDNA primary libraries, by the cDNA of homogenization processing
It is cloned into carrier by BP recombining reaction, then transformed competence colibacillus cell, is detected by coated plate and monoclonal, analyze the appearance in library
Amount and homogenization effect；
2) building of VIGS secondary library: extraction step 1 is carried out) obtained cDNA primary libraries plasmid, the primary text that will be obtained
VIGS carrier is cloned by LR recombining reaction after the dilution of library plasmid；It is detected by coated plate and monoclonal, analyzes the capacity in library
And recombination fraction；
3) identification obtains the library tobacco VIGS-cDNA that capacity is big and recombination fraction is high.
3. tobacco disease resistance poison controlling gene screening technique as claimed in claim 2, which is characterized in that VIGS carrier is pTRVG,
The obtained library VIGS-cDNA is the library pTRVG-cDNA.
4. tobacco disease resistance poison controlling gene screening technique as described in claim 1, which is characterized in that step (2) is to each list
The method that bacterium colony carries out VIGS silencing analysis respectively are as follows:
1) the coated plate culture of part Agrobacterium bacterium solution is drawn, while cultivating the Agrobacterium library converted；Picking single colonie, is inoculated in
It is cultivated in antibiotic fluid nutrient medium, collects somatic cells, be resuspended to OD600Value is 2, after renewal cultivation, by 1:1
(V:V) ratio mixes the VIGS-cDNA library transformation bacterium of the Agrobacterium converted and insertion target gene fragment；
2) the infiltration inoculation of plant: will mix the blade for the tobacco top full extension that agrobacterium liquid infiltration seedling age is 20-25d,
Using the mixed bacteria liquid of the Agrobacterium of infiltration transformation and VIGS-eGFP Agrobacterium as control；
3) tobacco after infiltration inoculation is cultivated, target gene is allowed to carry out effective silencing.
5. the tobacco disease resistance poison controlling gene screening technique as described in claim 1-4 is any, which is characterized in that step (3)
Antiviral analysis method are as follows: silencing processing after two weeks, carries out the VIGS silencing plant of each bacterium colony of cDNA library antiviral respectively
Analysis is inoculated with tobacco virus, and after inoculation, observation and record virus disease symptom obtain silencing restrovirus Disease symptoms severity
The bacterium colony significantly changed, the corresponding gene of these bacterium colonies are the candidate gene of antiviral controlling gene.
6. tobacco disease resistance poison controlling gene screening technique as claimed in claim 5, which is characterized in that the virus is potato
Y virus and tobacco mosaic virus (TMV).
7. the tobacco disease resistance poison controlling gene screening technique as described in claim 1-4 is any, which is characterized in that step (4) is right
The corresponding gene of antiviral correlation bacterium colony that screening obtains carries out sequencing analysis, obtains the sequence of these genes, and to these sequences
BLAST analysis, the conserved structure domain analysis of column progress tobacco and other Plant Genome databases, predicted gene function, thus
Obtain antiviral controlling gene.
8. tobacco disease resistance poison controlling gene screening technique as claimed in claim 1 to 7 answering in tobacco virus prevention and treatment
With.
9. tobacco disease resistance poison controlling gene screening technique as claimed in claim 1 to 7 is in preparing antiviral transgene tobacco
Application.
10. tobacco disease resistance poison controlling gene screening technique as claimed in claim 1 to 7 is obtaining tobacco disease resistance poison regulation base
Cause and the application being used in tobacco germplasm improvement.
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| CN201811340502.3A CN109610009A (en) | 2018-11-12 | 2018-11-12 | A kind of tobacco disease resistance poison controlling gene screening technique and application |
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| CN114480486A (en) * | 2022-01-20 | 2022-05-13 | 西北农林科技大学 | Screening method and application of plant antiviral RNA silencing related transcription factors |
| CN117511989A (en) * | 2023-11-08 | 2024-02-06 | 安徽农业大学 | Method for screening functional genes of plasmodiophora |
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