WO2020052415A1 - 隐性抗烟草脉带花叶病毒的烟草eIFiso4E-S基因及其应用 - Google Patents

隐性抗烟草脉带花叶病毒的烟草eIFiso4E-S基因及其应用 Download PDF

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WO2020052415A1
WO2020052415A1 PCT/CN2019/101922 CN2019101922W WO2020052415A1 WO 2020052415 A1 WO2020052415 A1 WO 2020052415A1 CN 2019101922 W CN2019101922 W CN 2019101922W WO 2020052415 A1 WO2020052415 A1 WO 2020052415A1
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tobacco
eifiso4e
gene
tvbmv
mosaic virus
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PCT/CN2019/101922
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English (en)
French (fr)
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刘勇
黄昌军
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云南省烟草农业科学研究院
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Publication of WO2020052415A1 publication Critical patent/WO2020052415A1/zh
Priority to US17/191,643 priority Critical patent/US20210189419A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
    • A01H1/126—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance for virus resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
    • A01H6/823—Nicotiana, e.g. tobacco
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance

Definitions

  • the invention belongs to the field of biotechnology, further belongs to the field of tobacco biotechnology breeding, and particularly relates to a tobacco eIFiso4E-S gene of a recessive tobacco vein mosaic virus, obtaining the gene knockout (Knockout, KO) mutant and tobacco resistance Disease breeding applications.
  • TVBMV Tobacco vein band mosaic virus
  • Potyvirus tobacco potato Y virus genus
  • TVBMV mainly harms solanaceous crops such as potato, tobacco, tomato and pepper.
  • TVBMV has risen to tobacco Major diseases.
  • TVBMV is transmitted non-sexually by aphids in the field. Because aphids have a short development cycle, strong fertility, and are prone to drug resistance, the use of chemical agents to control vector insects to control the disease has limited effectiveness. Therefore, planting TVBMV disease-resistant varieties is the most fundamental and cost-effective way to prevent and control TVBMV.
  • Tobacco as an important cash crop and an important model crop for plant research, has few studies on the identification of PVY-resistant resources, disease-resistant breeding, and pathogen investigation and identification. Among them, va gene resistance resources obtained by X-ray mutagenesis have been widely used in breeding tobacco resistance varieties. Tobacco eIF4E1 gene (some documents abbreviated as eIF4E-1, GenBank sequence accession number KF155696) caused tobacco to develop resistance to Potato virus Y (PVY) (Liu Yong et al., 2013; Julio et al., 2014), There are few studies on the identification of anti-TVBMV resources, resistance breeding and pathogen investigation and identification. There were no reports of germplasm resources of common tobacco against TVBMV.
  • eIF4E1 gene (some documents abbreviated as eIF4E-1, GenBank sequence accession number KF155696) caused tobacco to develop resistance to Potato virus Y (PVY) (Liu Yong et al., 2013; Julio
  • the first object of the present invention is to provide a recessive anti-TVBMV eIFiso4E-S gene.
  • the second object of the present invention is to provide an amino acid sequence of a recessive anti-TVBMV eIFiso4E-S gene.
  • the third object of the present invention is to provide an application of the recessive anti-TVBMV eIFiso4E-S gene, that is, to provide the eIFiso4E-S gene knockout (Knock out, represented by eifiso4e-s KO ) in the selection and breeding of TVBMV-resistant tobacco. application.
  • the fourth object of the present invention is to provide a tobacco variety obtained from the application of the TVBMV-resistant eifiso4e-s KO gene, a seed thereof, and an asexual propagule.
  • the fifth object of the present invention is to provide a Knockout expression cassette of the recessive anti-TVBMV eIFiso4E-S gene.
  • the first object of the present invention is achieved as follows: The base sequence of the eIFiso4E-S gene of the recessive anti-TVBMV is shown in SEQ ID No.1.
  • the second object of the present invention is achieved in such a way that the amino acid sequence of the polypeptide encoded by the eIFiso4E-S gene of the recessive anti-TVBMV is shown in SEQ ID No. 2.
  • the purpose of the third invention of the present invention is achieved as follows: the application of the eIFiso4E-S gene of recessive anti-tobacco tobacco vein mosaic virus, which is characterized by introduction of chromosome fragments, gene introduction, gene editing, gene silencing or physicochemical mutagenesis To obtain tobacco with eIFiso4E-S gene knockout (eifiso4e-s KO ), the target tobacco can obtain TVBMV resistance.
  • the purpose of the fourth invention of the present invention is as follows: the application of the eIFiso4E-S gene of the recessive anti-tobacco tobacco vein mosaic virus is characterized in that the tobacco containing eifiso4e-s KO is obtained by introducing a chromosome fragment.
  • the step is: firstly, The chromosomal fragments of eifiso4e-s KO were transferred to target tobacco through cross breeding and protoplast fusion to obtain TVBMV-resistant tobacco.
  • the application of the eIFiso4E-S gene of the recessive anti-tobacco vein mosaic virus is characterized in that the step of introducing tobacco containing the eifiso4e-s KO gene through gene introduction is: introducing an exogenous eifiso4e-s KO gene into a target tobacco, TVBMV resistant tobacco was obtained.
  • the application of the recessive anti-tobacco tobacco mosaic virus eIFiso4E-S gene is characterized in that the step of obtaining tobacco containing eifiso4e-s KO through gene editing is as follows: the eIFiso4E-S gene homologue present in the target tobacco is passed through Deletion, addition, and / or replacement of a specific nucleotide position of the specific nucleotide site renders the sequence shown in SEQ ID No. 1 nonfunctional and obtains TVBMV-resistant tobacco.
  • the application of the eIFiso4E-S gene of recessive anti-tobacco tobacco vein mosaic virus is characterized in that it is transformed into tobacco containing eifiso4e-s KO through physical or chemical mutagenesis.
  • the step is: physical or chemical mutagenesis to make the target tobacco SEQ ID
  • the sequence shown in No. 1 loses function, and TVBMV-resistant tobacco is obtained.
  • the application of the eIFiso4E-S gene of recessive resistance to tobacco tobacco vein mosaic virus is characterized in that the target tobacco is va, or eIF4E1 gene knockout (eif4E1 KO ) tobacco.
  • the fifth object of the present invention is achieved by: Knockout expression cassette of eIFiso4E-S gene of recessive resistance to tobacco tobacco vein mosaic virus.
  • the recessive anti-TVBMV eIFiso4E-S gene has important application value.
  • the eIFiso4E-S knockout can be obtained through gene editing, physical mutagenesis, chemical mutagenesis, germplasm resource screening, gene artificial synthesis, gene expression interference and other methods. (eifiso4e-s KO ).
  • eIF4E1 knockout (eif4e1 KO) or eIF4E1 modified (eif4e1 LOF) of the material is polymerized to afford containing eifiso4e-s KO eif4e1 KO, eifiso4e -s LOF eif4e1 KO, eifiso4e-s KO eif4e1 LOF, eifiso4e-s LOF eif4e1 LOF isogenic Combined tobacco plants.
  • the above tobacco plants are resistant to TVBMV and can be used for breeding tobacco varieties that are resistant to TVBMV.
  • the base sequence of the eIFiso4E-S gene of a recessive anti-TVBMV according to the present invention is shown in SEQ ID No. 1.
  • SEQ ID No. 1 The nucleotide sequence information shown in SEQ ID No. 1 provided by the present invention, those skilled in the art can easily obtain the gene by the following methods: (1) obtained by searching through a genomic database; (2) using SEQ ID NO. 1 Obtained by screening tobacco genomic library or cDNA library for probes; (3) Designing oligonucleotide primers based on the sequence information of SEQ ID NO.1, and using PCR amplification method to obtain from tobacco genome and cDNA; (4) using chemical The gene was obtained synthetically. (5) Obtained by mutating one or several base pairs of codons and / or mutating one or several base pairs.
  • a knockout mutant of the nucleotide sequence information gene shown in SEQ ID No. 1 or a mutant inactive with TVBMV and VPg interaction function by the following methods: (1) by a gene editing method Obtained, gene editing methods can be edited using CRISPR-Cas9, TALEN, zinc finger protein and other methods. (2) Obtained through chemical mutagenesis. Chemical mutagenesis can use mutagen such as EMS and nitrite. (3) Obtained through physical mutagenesis methods. The physical mutagenesis methods can use radiation mutations such as gamma rays, X-rays, fast neutrons, and heavy ions.
  • Artificially generated variants also include polynucleotides of synthetic origin, such as variant polynucleotides generated using site-directed mutagenesis but still sharing significant sequence identity with the naturally occurring sequences disclosed herein, and thus obtain TVBMV strain resistance. Generally, these variants have a sequence identity rate of more than 90% with the sequence shown in SEQ ID NO.1.
  • Polynucleotide variants can also be evaluated by comparing the sequence shown in SEQ ID NO. 2 with the amino acid sequence of the polypeptide encoded by the variant.
  • the sequence identity rate between any two polypeptides can be calculated using sequence alignment programs and parameters to compare the percent sequence identity shared by the two polypeptides. In general, the sequence identity between the two encoded polypeptides should be above 90%.
  • sequence identity rate can be calculated using molecular biology methods such as MEGA, BLAST.
  • amino acid sequence of the polypeptide encoded by the eIFiso4E-S gene of a recessive anti-TVBMV according to the present invention is shown in SEQ ID No. 2.
  • the application route of the recessive anti-TVBMV eIFiso4E-S gene according to the present invention is: Step A. Through gene editing, physical mutagenesis, chemical mutagenesis, germplasm resource screening, gene synthesis, gene expression interference and other methods To obtain eIFiso4E-S knockout (eifiso4e-s KO ) material. Step B. Obtain eIF4E1 knockout (eif14e1 KO ) material through methods such as gene editing, physical mutagenesis, chemical mutagenesis, germplasm resource screening, gene synthesis, and gene expression interference. Step C.
  • step A Polymerize the material obtained in step A and the material obtained in step B to obtain various combinations of polymerization of eIFiso4E-S knockout (eifiso4e-s KO ) and eIF14E1 knockout (eif4e1 KO ).
  • a preferred application approach is: (1) obtaining the target material of step B on the basis of the material obtained in step A; or obtaining the target material of step A on the basis of the material obtained in step B above. (2) Obtain the target material of step A and B of the target material respectively, and then obtain the eIFiso4E-S knockout (eifiso4e-s KO ) and eIF14E1 knockout (eif4e1 KO ) by cross breeding, somatic cell hybridization, and other methods. Various combinations of polymerization.
  • a further preferred application route is: (1) on the basis of eIF4E1 knockout (eif4e1 KO ), through gene editing, chemical mutagenesis, and physical mutagenesis to become tobacco plants containing the eifiso4e-s KO eif4e1 KO gene; ( 2) On the basis of the eIF4E1 knockout (eif4e1 KO ), determine the specific amino acids of eIFiso4E-S that interact with specific amino acids of TVBMV's VPg, and use biotechnology, including gene editing, introduction of synthetic genes, and selection of mutants.
  • a specific amino acid of eIFiso4E-S which can interact with a specific amino acid of TVBMV's VPg is converted into an amino acid that cannot interact with each other, and a tobacco plant containing the eifiso4e-s KO eif4e1 KO gene is obtained.
  • a germplasm resource containing the eifiso4e-s KO gene is selected from Nicotiana plants; the germplasm resource includes wild tobacco species, cultivars, and hybrids of wild species and cultivars.
  • tobacco plants containing the eifiso4e-s KO eif4e1 KO gene were obtained by breeding means such as crossing and backcrossing of eifiso4e-s KO germplasm resources with eif4e1 KO plants.
  • breeding means such as crossing and backcrossing of eifiso4e-s KO germplasm resources with eif4e1 KO plants.
  • eIF4E1 On the basis of normal function of eIF4E1, it can be transformed into tobacco plants containing eifiso4e-s KO gene through gene editing, chemical mutagenesis, and physical mutagenesis; then, cross and backcross the eif4e1 KO plant with eifiso4e-s KO plant, etc. Breeding means to obtain tobacco plants containing the eifiso4e-s KO eif4e1 KO gene.
  • eIF4E1 Based on the normal function of eIF4E1, use biotechnology, including gene editing, mutant screening, to convert specific amino acids of eIFiso4E-S that can interact with specific amino acids of TVBMV's VPg into non-interactive amino acids, and then Tobacco plants containing the eifiso4e-s KO eif4e1 KO gene were obtained by breeding means such as crossing and backcrossing of the eifiso4e-s KO plant with the eif4e1 KO plant.
  • breeding means such as crossing and backcrossing of the eifiso4e-s KO plant with the eif4e1 KO plant.
  • the TVBMV-resistant eifiso4e-s KO eif4e1 KO tobacco material or the eifiso4e-s KO va tobacco material is used to breed TVBMV-resistant tobacco varieties.
  • the gene introduction of the present invention is to introduce an exogenous eifiso4e-s KO gene into a target tobacco, and includes introducing the exogenous gene after introduction (ie, transgene) and direct introduction.
  • the most commonly used method for transgene is Agrobacterium transformation method; direct introduction Methods include conventional biological methods such as microinjection, pollen tube channel method, conductance, and gene gun to transform tobacco cells or tissues, and cultivate the transformed tissues into plants.
  • the gene editing of the present invention is a technology developed in recent years that can complete precise modification of the genome. It can complete InDel mutation, knock-in, simultaneous multi-site mutation and deletion of small fragments, etc., and can perform precise at the genome level. Gene editing. By editing the eIFiso4E-S gene, the eIFiso4E-S polypeptide loses its function of interacting with TVBMV and VPg, so that the edited material has the potential to resist TVBMV.
  • the present invention proposes the following hypothesis: TVBMV interacts with two members of the tobacco eIF4E family to complete the infection cycle, and knocking out both members at the same time is expected to obtain TVBMV resistance.
  • the preferred solution is: knock out another unknown member in common tobacco (va) lacking the eIF4E1 gene, and hope to obtain TVBMV resistance breeding material.
  • using the eifiso4e-s KO / eif4e1 or eifiso4e-s KO / va double mutation material of the present invention can obtain anti-TVBMV gene resources and disease-resistant materials. It has important reference for TVBMV breeding of tobacco, potato, pepper and tomato in solanaceae.
  • a new TVBMV resistant tobacco variety, a seed thereof, and an asexual propagule can be obtained.
  • some genetic engineering products can also be developed, including A: Knockout expression cassette of the eIFiso4E-S gene, transgenic cell lines and recombinant bacteria.
  • C A combination of A and B.
  • D The eIFiso4E-S gene and the eIF4E1 gene are simultaneously Knockout expression cassettes, transgenic cell lines, recombinant bacteria, and the like.
  • the above-mentioned genetically engineered products are used to make tobacco resistant to TVBMV.
  • Gene knockout refers to the use of genetic manipulation techniques to make one or more genes of an organism inactive. Gene knockout methods include homologous recombination and site-specific nucleases. Site-specific nuclease technologies include Zinc-finger nucleases (ZFNs) and transcription-activator-like effector nucleases (TALENs). Regularly clustered spaced short palindromic nucleases (Clustered, regularly spaced, short spaced, short, Palindromic repeats, CRISPR). Gene knockouts can result in loss-of-function (KO) genes.
  • ZFNs Zinc-finger nucleases
  • TALENs transcription-activator-like effector nucleases
  • Regularly clustered spaced short palindromic nucleases Clustered, regularly spaced, short spaced, short, Palindromic repeats, CRISPR.
  • chromosome fragments usually by systematic backcrossing and selfing, and by means of molecular marker-assisted selection, fragments of donor parents are introduced into recurrent parents.
  • Gene introduction is the introduction of foreign genes into the target tobacco, including the introduction of foreign genes after introduction (ie, transgenes) and direct introduction.
  • the most commonly used method of transgene is Agrobacterium transformation method; direct introduction methods include microinjection, pollen Conventional biological methods such as tube channel method, conductance, gene gun and the like are used to transform tobacco cells or tissues, and the transformed tissues are cultivated into plants.
  • Gene editing is a technology developed in recent years that can complete precise modification of the genome. It can complete the site-specific InDel mutation, knock-in, simultaneous multi-site mutation, and deletion of small fragments, etc., and can perform precise genes at the genome level. edit.
  • the most commonly used methods for gene editing include zinc finger nucleases, transcription activator-like effector nucleases, and regular clustered spaced short palindrome repeat nuclease technology (Gaj, 2013).
  • Gene silencing A phenomenon in which a foreign gene exists in an organism and is not lost or damaged, but the gene is not expressed or the expression level is extremely low. Gene silencing is divided into transcription-level silencing (TGS) and post-transcriptional silencing (PTGS). TGS refers to the gene silencing in the nucleus of RNA, which leads to gene silencing. PTGS refers to the stable transcription of genes in the nucleus, but no corresponding mRNA exists in the cytoplasm.
  • TGS transcription-level silencing
  • PTGS post-transcriptional silencing
  • Methods for gene silencing include, but are not limited to, sense / co-suppression, antisense suppression, double-stranded RNA (dsRNA) interference, hairpin RNA interference and intron-containing hairpin RNA interference, amplicon-mediated interference , Ribozymes and small interfering RNAs or microRNAs.
  • dsRNA double-stranded RNA
  • amplicon-mediated interference Ribozymes and small interfering RNAs or microRNAs.
  • Physical and chemical mutagenesis refers to the use of physical or chemical factors to make plant genes mutate.
  • Physical mutagens mainly include ultraviolet, X-ray, gamma-ray, fast neutron, laser, microwave, ion beam and so on.
  • Chemical mutagens are mainly known as alkylating agents, base analogs, hydroxylamines, acridine pigments, nitrous acid, sodium azide, and the like.
  • Alkylating agents include, but are not limited to, alkyl sulfonates and alkyl sulfates. Representative agents are ethyl methanesulfonate (EMS) and diethyl sulfate (DES). 2.
  • Nitrosyl alkyl compounds representing The agents are nitrosoethylurea (NEH), N-nitroso-N-ethylureane (NEU); 3. Ethylamine and ethylene oxide, the representative agent is ethyleneimine (EI); 4. Mustard gas, nitrogen mustard, sulfur mustard.
  • test materials used are all purchased from conventional biochemical reagent companies.
  • Tobacco materials are Nicotiana tabacum cun Yunyan87 (referred to as Yunyan 87, genotype eIFiso4E-S / eIF4E1, sense TVBMV), safflower gold (genotype eIFiso4E-S / eIF4E1, sense TVBMV), 2-1398 (genotype eIFiso4E- S / va, anti-PVY) are from Yunnan Academy of Tobacco Agricultural Sciences.
  • Yunyan 87 genotype eIFiso4E-S / eIF4E1, sense TVBMV
  • 2-1398 genotype eIFiso4E- S / va, anti-PVY
  • TVBMV virus comes from Yunnan Academy of Tobacco Agricultural Sciences.
  • Example 1 Sequence analysis of eIFiso4E gene in common tobacco
  • Ruffel et al. Ruffel S, et al. Simultaneous mutations, translation, initialization, factors, eIF4E, and eIF (iso) 4E, are required), prevent, prevent, prevent, and prevent, and reduce the number of viruses. Infection of the pipeline is based on J.Gen. Virol. 87: 2089.
  • isoS-F (5’-ATGGCCACTGAAGCACCGATAGAG-3 ’)
  • PCR product cloning and sequencing The PCR products recovered by the gel were constructed on a cloning and sequencing vector, and 30 positive clones were selected and sent to Thermo Fisher Scientific (Guangzhou) for sequencing.
  • the sequence of the obtained clone sequence and the sequence of eIFiso4E-S, and the identity of the nucleotide sequence of eIFiso4E-S to 99% or more is the DNA sequence of the eIFiso4E-S gene, as shown in SEQ ID No. 1 of the Sequence Listing .
  • Example 3 Polypeptide sequence encoded by the eIFiso4E-S gene
  • the amino acid sequence of the polypeptide encoded by the eIFiso4E-S gene was deduced using molecular biology software MEGA6 as shown in SEQ ID No. 2.
  • RNA-guided gene editing in plants using CRISPR-Cas system is described in the document “Xie, K. and Y. Yang (2013).” RNA-guided gene editing in plants using CRISPR-Cas system. “Mol Plant 6 : 1975-1983. "
  • the target site of the present invention is designed on the first exon of the eIFiso4E-S gene.
  • PAM NVG or CCN, the form of the gene sequence is 5'-NNNNNNNNNNNNNNNNNNNGG-3 'or 5'-CCNNNNNNNNNNNNNNNNNNNNN-3'
  • Oligo synthesis and annealing of gRNA With gRNA target sites as templates, primers oligo are designed according to the following format.
  • the present invention selects two target sites for the first exon of the eIFiso4E-S gene.
  • the primer sequences are designed as follows, where F and R represent forward and reverse primers, respectively:
  • Primer annealing The synthesized pair of complementary DNAs are annealed to form dsDNA.
  • the annealing system is as follows:
  • the annealing procedure is: 95 ° C 5mim, 90 ° C 1mim, 80 ° C 1mim, 70 ° C 1mim, 60 ° C 1mim, 50 ° C 1mim, 40 ° C 1mim, 30 ° C 1mim, 20 ° C 1mim, 10 ° C 1mim.
  • the ligation product was transformed into E. coli and colony PCR was performed to identify positive clones.
  • the colony PCR detection forward primer was: OsU3 5'F 5'-aaggaatctttaaacatacgaacag-3 'reverse primer was the reverse sequence of sgRNA (gRNA1R or gRNA2R).
  • the amplified positive clones were shaken and sequenced to analyze whether the gRNA was correct.
  • the sequencing primer was OsU3 5'F.
  • Example 5 Plant transformation of gene knockout vectors and detection of eIFiso4E-S gene knockout plants
  • the correct plasmid will be ligated and transformed into Agrobacterium GV3101 by electric shock.
  • Agrobacterium-mediated transformation of tobacco callus to obtain transgenic plants The wild-type tobacco Yunyan 87 and tobacco 2-1398 containing homozygous va gene loci were used as materials to induce callus, and Agrobacterium-mediated tobacco transformation experiments were performed.
  • Agrobacterium GV3101 was used for infection and transformation. After screening for hygromycin resistance, resistant callus differentiated and regenerated to obtain transgenic positive strains.
  • Detection of eIFiso4E-T gene mutants in transgenic tobacco Design primers for the target gene detection. According to the target gene, primers are designed upstream and downstream of the target site sequence. The primer sequences are:
  • EditestF 5’-caattccattacgcctctccgttcgct-3 ’
  • EditestR 5’-ggaacaaaatccgaatttatcaataact-3 ’
  • Genomic DNA was extracted from the obtained transgenic positive plants for PCR reaction. PCR products were used for sequencing.
  • the sequencing company was Thermo Fisher Scientific (Guangzhou) and the sequencing primer sequence was EditestF.
  • Example 6 Polymerized eIFiso4E-S KO and va have TVBMV resistance (g1-1C,)
  • a CRISPR-Cas9 gene editing vector was designed for exIF1 of eIFiso4E-S (see Example 4).
  • the CRISPR-Cas9 gene editing vector of eIFiso4E-S was used to transform va tobacco 2-1398.
  • the T0 generation seedlings were amplified and sequenced by PCR, and the eIFiso4E-S hybrid edited single strain 2-1398g1-1C, and the sequence of the eIFiso4E-S hybrid mutation of 2-1298g1-1C were selected as shown in SEQ ID No. 1
  • the 11 bits increase by an A or decrease by an A.
  • the T1 generation of eifiso4e-s KO / va tobacco was obtained by self-seeding, and the T1 plants were cultivated by conventional methods.
  • leaf DNA was extracted to screen eIFiso4E-S biallelic or homozygous editor and eIFiso4E-T was a wild-type single plant for resistance identification.
  • TVBMV disease leaf sap was inoculated 40 times. Take 2-1398 as a control. The incidence of TVBMV was investigated at 14 days, 21 days, and 28 days after vaccination.
  • PCR primers were designed based on the TVBMV VPg sequence. The size of the amplified product was 765 bp, and the annealing temperature was 60 ° C.
  • a chromosome fragment containing the Knockout allele (eifiso4e-s KO ) of the eIFiso4E-S gene shown in SEQ ID NO. 1 in Nicotiana plants is transferred to the target tobacco by conventional breeding methods.
  • the target tobacco is va or eif4E1 KO (the same applies hereinafter).
  • germplasm resources containing eifiso4e-s KO gene were screened from Nicotiana plants.
  • Germplasm resources include wild tobacco species, hybrids and cultivars of wild species and cultivated tobacco.
  • Non-transgenic tobacco materials with improved TVBMV resistance were obtained by using conventional cross breeding or protoplast fusion or chromosome fragment introduction techniques to introduce chromosomal fragments containing the eifiso4e-s KO gene into target tobacco. Through breeding methods such as crossbreeding and backcrossing, the tobacco material with improved resistance is bred into a commercial variety, and the resistance of the tobacco variety to TVBMV is improved.
  • Example 7 Application of gene editing and breeding of eIFiso4E-S gene
  • the eIFiso4E-S gene in the target tobacco was modified by biotechnology such as gene editing, so that it lost the biological function of interaction with TVBMV VPg, and TVBMV-resistant tobacco was obtained.
  • the target tobacco is va, or eif4E1 KO .

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Abstract

一种烟草脉带花叶病毒(Tobacco vein banding mosaic virus，TVBMV)隐性抗性基因(recessive resistance gene)eIFiso4E-S的分离克隆与育种应用。所述烟草隐性抗TVBMV基因eIFiso4E-S的核苷酸序列如SEQ ID NO.1所示，其编码多肽的氨基酸如SEQ ID NO.2所示。将烟草中该基因敲除(Knockout,KO)可获得eIFiso4E-S KO材料。将eIFiso4E-S KO与抗马铃薯Y病毒(PVY)的va或eIF4E1 KO基因聚合，可获得烟草对TVBMV和PVY的抗性。

Description

隐性抗烟草脉带花叶病毒的烟草eIFiso4E-S基因及其应用 技术领域
本发明属于生物技术领域，进一步属于烟草生物技术育种领域，具体涉及一种隐性烟草脉带花叶病毒的烟草eIFiso4E-S基因、该基因敲除(Knockout,KO)突变体获得及其烟草抗病育种应用。
背景技术
烟草脉带花叶病毒(Tobacco vein banding mosaic virus，TVBMV)是烟草马铃薯Y病毒属(Potyvirus)的典型成员，TVBMV主要危害马铃薯、烟草、番茄和辣椒等茄科作物，近年来TVBMV已经上升为烟草上的主要病害。TVBMV在田间由蚜虫非性传播。由于蚜虫发育周期短、繁殖力强和易产生抗药性，利用化学药剂防治媒介昆虫来治理该病害效果有限，因此，种植TVBMV抗病品种是防控TVBMV最根本、最经济有效的手段。
烟草作为重要的经济作物和植物研究重要的模式作物，有关抗PVY资源鉴定、抗病育种和病原调查鉴定研究较少。其中通过X-射线诱变获得的va基因抗病资源，已广泛应用于选育烟草抗病品种。烟草eIF4E1基因(有的文献缩写为eIF4E-1，GenBank序列登录号KF155696)缺失导致烟草产生针对马铃薯Y病毒(Potato virus Y,PVY)的抗性(刘勇等，2013；Julio等，2014)，有关抗TVBMV资源鉴定、抗病育种和病原调查鉴定研究较少。未见普通烟草抗TVBMV的种质资源报道。
在同科作物相邻种植的农田生态下，控制一种作物上TVBMV的发生不仅有利于该作物的病害防治，也有利于其他作物的病害治理。
发明内容
本发明第一发明目的是提供一种隐性抗TVBMV的eIFiso4E-S基因。第二发明目的在于提供一种隐性抗TVBMV的eIFiso4E-S基因的氨基酸序列。第三发明目的是提供一种隐性抗TVBMV的eIFiso4E-S基因的应用，即提供一种所述eIFiso4E-S基因敲除(Knock out,以eifiso4e-s KO表示)在选育抗TVBMV烟草的应用。第四发明目的是提供一种根据所述抗TVBMV的eifiso4e-s KO基因的应用所得到的烟草品种、及其种子和无性繁殖体。第五发明目的是提供一种所述隐性抗TVBMV的eIFiso4E-S基因的Knockout表达盒。
本发明第一发明目的是这样实现的：所述隐性抗TVBMV的eIFiso4E-S基因的碱基序列如SEQ ID No.1所示。
本发明第二发明目的是这样实现的：所述隐性抗TVBMV的eIFiso4E-S基因编码的多肽的氨基酸序列如SEQ ID No.2所示。
本发明第三发明目的是这样实现的：隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因的应用，其特征在于通过染色体片段导入、基因导入、基因编辑、基因沉默或物理化学诱变方式获得eIFiso4E-S基因敲除(eifiso4e-s KO)的烟草，从而使目标烟草获得TVBMV抗性。
本发明第四发明目的是这样实现的：隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因的应用，其特征在于通过染色体片段导入得到包含eifiso4e-s KO的烟草步骤为：先将含有eifiso4e-s KO的染色体片段通 过杂交育种、原生质体融合转育至目标烟草中，得到抗TVBMV的烟草。
隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因的应用，其特征在于通过基因导入得到包含eifiso4e-s KO基因的烟草步骤为：将外源的eifiso4e-s KO基因导入目标烟草中，得到抗TVBMV的烟草。
隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因的应用，其特征在于通过基因编辑得到包含eifiso4e-s KO的烟草步骤为：将目标烟草中存在的eIFiso4E-S基因同源体，通过对其特定核苷酸位点的缺失、增加和/替换，使SEQ ID No.1所示序列失去功能，得到抗TVBMV的烟草。
隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因的应用，其特征在于通过物理或化学诱变得到包含eifiso4e-s KO的烟草步骤为：通过物理或化学诱变，使目标烟草SEQ ID No.1所示序列失去功能，得到抗TVBMV的烟草。
隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因的应用，其特征在于目标烟草为va、或eIF4E1基因敲除(eif4E1 KO)的烟草。
隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因的应用所得到的烟草品种、及其种子和无性繁殖体。
本发明第五发明目的是这样实现的：的隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因的Knockout表达盒。
本发明隐性抗TVBMV的eIFiso4E-S基因具有重要的应用价值，通过基因编辑、物理诱变、化学诱变、种质资源筛选、基因人工合成、 基因表达干涉等方式，获得eIFiso4E-S敲除(eifiso4e-s KO)的材料。与eIF4E1敲除(eif4e1 KO)或eIF4E1修饰(eif4e1 LOF)的材料聚合，得到含eifiso4e-s KOeif4e1 KO，eifiso4e-s LOFeif4e1 KO，eifiso4e-s KOeif4e1 LOF，eifiso4e-s LOFeif4e1 LOF等基因组合的烟草植株。上述烟草植株抗TVBMV，可用于选育对TVBMV具有抗性的烟草品种。
本发明所述一种隐性抗TVBMV的eIFiso4E-S基因，其碱基序列如SEQ ID No.1所示。根据本发明提供的SEQ ID NO.1所示的核苷酸序列信息，本领域技术人员可以通过以下方法容易地获得该基因：(1)通过基因组数据库检索获得；(2)以SEQ ID NO.1为探针筛选烟草基因组文库或cDNA文库获得；(3)根据SEQ ID NO.1序列信息设计寡核苷酸引物，用PCR扩增方法从烟草的基因组和cDNA中获得；(4)用化学合成的方法获得该基因。(5)通过将缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的突变得到。
本领域技术人员可以通过以下方法容易地获得SEQ ID NO.1所示的核苷酸序列信息基因的敲除突变体或与TVBMV VPg互作功能失活的突变体：(1)通过基因编辑方法获得，基因编辑方法可采用CRISPR-Cas9、TALEN、锌指蛋白等方法编辑。(2)通过化学诱变方法获得，化学诱变可采用EMS、亚硝酸盐等诱变剂。(3)通过物理诱变方法获得，物理诱变方法可采用伽马射线、X-射线、快中子、重离子等辐射诱变。(4)在自然界可能存在与本发明SEQ ID NO.1所示的多核苷酸基因功能敲除的突变体。与SEQ ID NO.1所示的序列相比，天然变体内一个或多个位置处删除和/或添加和/或取代一种或多种核 苷酸，导致TVBMV的VPg不能与其互作，不能支持TVBMV完成侵染循环。天然产生的变体可通过用熟知的分子生物学技术鉴定，例如用聚合酶链式反应(PCR)和本领域已知杂交技术。人为产生的变体还包括合成来源的多核苷酸，如用定点诱变生成但仍与本文公开的天然产生序列共有显著序列相同性的变体多核苷酸，且因此获得TVBMV株系抗性。通常，这些变体与SEQ ID NO.1所示的序列具有90％以上的序列一致率。
多核苷酸变体还可通过比较SEQ ID NO.2所示的序列与变体所编码的多肽的氨基酸序列评估。任何两种多肽之间的序列一致率可用序列比对程序和参数计算，比较两种多肽共有的序列相同性百分比。一般来说，两种编码的多肽之间的序列一致率应在90％以上。
所述序列一致率可采用MEGA,BLAST等分子生物学方法计算。
本发明所述一种隐性抗TVBMV的eIFiso4E-S基因编码的多肽，其氨基酸序列如SEQ ID No.2所示。
采用组合方式：
本发明所述的一种隐性抗TVBMV的eIFiso4E-S基因的应用途径为：A步骤.通过基因编辑、物理诱变、化学诱变、种质资源筛选、基因人工合成、基因表达干涉等方法，获得eIFiso4E-S敲除(eifiso4e-s KO)的材料。B步骤.通过基因编辑、物理诱变、化学诱变、种质资源筛选、基因人工合成、基因表达干涉等方法，获得eIF4E1敲除(eif14e1 KO)的材料。C步骤.将上述A步骤获得的材料与上述B步骤获得的材料聚合，获得eIFiso4E-S敲除(eifiso4e-s KO)与eIF14E1 敲除(eif4e1 KO)的聚合的各种组合。
优选的应用途径为：(1)在上述A步骤获得的材料的基础上，获得上述B步骤的目标材料；或者在上述B步骤获得的材料的基础上，获得上述A步骤的目标材料。(2)分别获得上述A步骤的目标材料、B步骤的目标材料，然后利用杂交育种、体细胞杂交等方式获得获得eIFiso4E-S敲除(eifiso4e-s KO)与eIF14E1敲除(eif4e1 KO)的聚合的各种组合。
进一步优选的应用途径(步骤)为：(1)在eIF4E1敲除(eif4e1 KO)的基础上，通过基因编辑、化学诱变、物理诱变得到含eifiso4e-s KOeif4e1 KO基因的烟草植株；(2)在eIF4E1敲除(eif4e1 KO)的基础上，确定与TVBMV的VPg的特定氨基酸互作的eIFiso4E-S的特定氨基酸，利用生物技术，包括基因编辑、导入人工合成基因、突变体筛选，将能与TVBMV的VPg的特定氨基酸互作的eIFiso4E-S的特定氨基酸转换为不能互作的氨基酸，获得含eifiso4e-s KOeif4e1 KO基因的烟草植株。(3)从烟草属植物中筛选获得包含eifiso4e-s KO基因的种质资源；所述的种质资源包含烟草野生种、栽培种、以及野生种与栽培种的杂交种。然后，通过eifiso4e-s KO种质资源植株与eif4e1 KO植株杂交、回交等育种手段，获得含eifiso4e-s KOeif4e1 KO基因的烟草植株。(4)在eIF4E1功能正常的基础上，通过基因编辑、化学诱变、物理诱变得到含eifiso4e-s KO基因的烟草植株；然后，通过eifiso4e-s KO植株与eif4e1 KO植株杂交、回交等育种手段，获得含eifiso4e-s KOeif4e1 KO基因的烟草植株。(5)在eIF4E1功能正常的基础 上，利用生物技术，包括基因编辑、突变体筛选，将能与TVBMV的VPg的特定氨基酸互作的eIFiso4E-S的特定氨基酸转换为不能互作的氨基酸，然后，通过eifiso4e-s KO植株与eif4e1 KO植株杂交、回交等育种手段，获得含eifiso4e-s KOeif4e1 KO基因的烟草植株。利用上述具有TVBMV抗性的eifiso4e-s KOeif4e1 KO烟草材料、或者eifiso4e-s KOva烟草材料选育抗TVBMV的烟草品种。
本发明基因导入是将外源的eifiso4e-s KO基因导入目标烟草中，包括将外源基因转入后导入(即转基因)和直接导入，转基因最常用的方法为农杆菌转化法；直接导入的方法包括显微注射、花粉管通道法、电导、基因枪等常规生物学方法转化烟草细胞或组织，并将转化的组织培育成植株。
本发明基因编辑是近年来发展起来的可以对基因组完成精确修饰的一种技术，可完成基因定点InDel突变、敲入、多位点同时突变和小片段的删除等，可在基因组水平上进行精确的基因编辑。通过对eIFiso4E-S基因进行编辑，使eIFiso4E-S多肽失去与TVBMV VPg互作的功能，从而使被编辑材料获得具有抗TVBMV的潜力。
本发明提出如下假说：TVBMV与烟草eIF4E家族两个成员互作完成侵染循环，同时敲除两个成员有望获得TVBMV抗性。优选的方案为：在缺失eIF4E1基因的普通烟草(va)中敲除另一个未知成员，有望获得TVBMV抗性的育种材料。比如，利用本发明的eifiso4e-s KO/eif4e1或eifiso4e-s KO/va双突变材料可获得抗TVBMV基因资源和抗病材料。对茄科作物烟草、马铃薯、辣椒、番茄抗TVBMV育种具有 重要的借鉴作用。
根据所述获得对辣椒脉斑驳病毒抗性的烟草植株的方法，可以得到新的抗TVBMV烟草品种、及其种子和无性繁殖体。另外，还可以开发一些基因工程产品，包括A:所述eIFiso4E-S基因Knockout的表达盒，转基因细胞系和重组菌等。B:包括所述eIF4E1基因Knockout的表达盒，转基因细胞系和重组菌等。C:A和B的组合。D:所述eIFiso4E-S基因和eIF4E1基因同时Knockout的表达盒，转基因细胞系和重组菌等。利用上述基因工程产品使烟草获得对TVBMV的抗性。
定义：基因敲除：gene knockout(简写:KO)，指利用遗传操作技术使生物体的一个或多个基因失去功能。基因敲除的方法有同源重组(homologous recombination)和位点特异性核酸酶技术(site-specific nucleases)。位点特异性核酸酶技术包括锌指核酸酶(Zinc-finger nucleases，ZFN)、类转录激活因子效应物核酸酶(Transcription activator-like effector nucleases，TALENs))。规律成簇的间隔短回文重复核酸酶技术(Clustered regularly interspaced short palindromic repeats，CRISPR)。基因敲除可产生基因失去功能突变体(Loss-of-function,KO)。
染色体片段导入：通常通过系统回交和自交，并借助于分子标记辅助选择使供体亲本的片段导入到轮回亲本中。
基因导入是将外源基因导入目的烟草中，包括将外源基因转入后导入(即转基因)和直接导入，转基因最常用的方法为农杆菌转化法； 直接导入的方法包括显微注射、花粉管通道法、电导、基因枪等常规生物学方法转化烟草细胞或组织，并将转化的组织培育成植株。
基因编辑是近年来发展起来的可以对基因组完成精确修饰的一种技术，可完成基因定点InDel突变、敲入、多位点同时突变和小片段的删除等，可在基因组水平上进行精确的基因编辑。基因编辑最常用的方法括锌指核酸酶、类转录激活因子效应物核酸酶、规律成簇的间隔短回文重复核酸酶技术(Gaj，2013)。
基因沉默(gene silencing)：外源基因存在于生物体内，并未丢失或损伤，但该基因不表达或表达量极低的现象。基因沉默分为转录水平的沉默(TGS)和转录后水平的沉默(PTGS)。TGS是指基因在细胞核内RNA合成受到了阻止导致基因沉默，PTGS是指基因在细胞核中能够稳定的转录，但在细胞质中无对应的mRNA存在。基因沉默所述方法包括但不限于有义抑制/共抑制、反义抑制、双链RNA(dsRNA)干扰、发夹RNA干扰和含内含子的发夹RNA干扰、扩增子介导的干扰、核酶和小干扰RNA或微小RNA。
物理化学诱变：指利用物理因素或化学因素使植物基因产生变异。物理诱变剂主要有紫外线，X-射线，γ-射线，快中子，激光，微波，离子束等。化学诱变剂主要有已知的有烷化剂、碱基类似物(base analog)、羟胺(hydroxylamine)、吖啶色素、亚硝酸、叠氮化钠等。烷化剂包括但不限于：烷基磺酸盐和烷基硫酸盐，代表药剂为甲基磺酸乙酯(EMS)、硫酸二乙酯(DES)；2.亚硝基烷基化合物，代表药剂为亚硝基乙基脲(NEH)、N-亚硝基-N-乙基脲烷(NEU)；3.次乙 胺和环氧乙烷类，代表药剂为乙烯亚胺(EI)；4.芥子气类，氮芥类、硫芥类。
具体实施方式
下面对本发明作进一步说明，但不以任何方式对本发明加以限制，基于本发明教导所作的任何变换，均落入本发明保护范围。
下面结合实施例进行进一步的说明和验证。
如无特殊说明，下述各实施例使用的均为常规方法；如无特殊说明，所用的试验材料均为自常规生化试剂公司购买得到的。
烟草材料为Nicotiana tabacum cv Yunyan87(简称云烟87，基因型eIFiso4E-S/eIF4E1，感TVBMV)，红花大金元(基因型eIFiso4E-S/eIF4E1，感TVBMV)，2-1398(基因型eIFiso4E-S/va，抗PVY)均来自云南省烟草农业科学研究院。TVBMV病毒来自云南省烟草农业科学研究院。
使用TRIzol试剂(Invitrogen；Carlsbad，CA)按照厂商的方案从烟草叶片中提取总RNA。质粒DNA提取试剂盒、琼脂糖凝胶DNA回收试剂盒、DNA片段纯化试剂盒购自QIAGEN公司。大肠杆菌(Escherichia coli)DH 5α；限制性内切酶、反转录试剂盒、DNA Marker、PrimeSTAR GXL DNA Polymerase、T4DNA聚合酶及T4DNA连接酶、壮观霉素均购自大连宝生物公司和Roche公司。RNA提取试剂盒Trizol购自Invitrogen公司，大肠杆菌(Escherichia coli)DH5α菌株、农杆菌(Agrobacterium tumefaciens)EHA105、C58C1菌株由本实验室保存。克隆载体pMD18T购自大连宝生物公司。
实施例1：普通烟草eIFiso4E基因序列分析
根据Ruffel等文献(Ruffel S,et al.Simultaneous mutations in translation initiation factors eIF4E and eIF(iso)4E are required to prevent pepper veinal mottle virus infection of pepper.J Gen Virol 87:2089-2098[J].Journal of General Virology,2006,87(Pt 7):2089-2098.)，以辣椒eIFiso4E基因(Genebank：DQ022080)序列为参照，在Genbank数据库中，通过Blastn获得普通烟草(N.tabacum)eIFiso4E的同源序列，将普通烟草中的eIFiso4E与祖先种林烟草(N.sylvestris)和绒毛状烟草(N.tomentosiformis)的eIFiso4E核苷酸序列一致率高的eIFiso4E基因分别命名为eIFiso4E-S、eIFiso4E-T。针对eIFiso4E-S设计特异引物：
isoS-F:(5’-ATGGCCACTGAAGCACCGATAGAG-3’)
isoS-R:(5’-TCACACAGTATATCGACTCT-3’)
实施例2：eIFiso4E-S基因克隆与序列分析
(1)烟草总RNA的提取：取普通烟草红花大金元新鲜嫩叶，采用Trizol试剂(Invitrogen)提取叶片组织的总RNA。以Oligo dT-Adapter作为反转录引物，通过RT-PCR扩增普通烟草的cDNA。
(2)eIFiso4E-S基因的克隆：以cDNA为模板，用引物isoS-F和引物isoS-R进行PCR扩增。PCR的反应体系总体积为50μL，其中100ng/μL cDNA样品4.0μL，5×PCR buffer 10.0μL，dNTPs(2.5mmol/L each)4μL，10μmol/L的引物isoS-F和isoS-R各2.0μL，PrimeSTAR GXL DNA Polymerase 1μL，ddH 2O 27μL。所用试剂购自宝生物公司。PCR的反应条件为：98℃ 2min，98℃ 10s，60℃ 15s，68℃ 1min，35个循环，68℃ 10min。
(3)PCR产物回收与纯化：将PCR产物在1.5％的琼脂糖凝胶 上电泳，电泳缓冲液为1×TAE，当电泳指示剂溴酚蓝在120V，60min条件下迁移至足够分离DNA片段时，取下凝胶，用凝胶图像分析系统记录结果。在紫外灯下割下DNA片段凝胶。用胶回收试剂盒(QIAGEN公司出品)回收DNA。
(4)PCR产物克隆测序：胶回收的PCR产物，构建到克隆测序载体上，挑选30个阳性克隆，送赛默飞世尔科技公司(广州)测序。将得到的克隆序列和eIFiso4E-S的序列比对，与eIFiso4E-S核苷酸序列一致率达到99％以上的序列为eIFiso4E-S基因的DNA序列，如序列表的SEQ ID No.1所示。
实施例3：eIFiso4E-S基因所编码的多肽序列
根据eIFiso4E-S基因的核苷酸序列，采用分子生物学软件MEGA6推导出eIFiso4E-S基因所编码的多肽的氨基酸序列如SEQ ID No.2所示。
实施例4：烟草eIFiso4E-S基因敲除载体的构建
用于本发明基因敲除表达载体构建的质粒pRGEB31在文献“Xie,K.and Y.Yang(2013)."RNA-guided genome editing in plants using a CRISPR-Cas system."Mol Plant 6(6):1975-1983.”中公开过；
根据CRISPR/Cas9技术设计靶位点的原则，本发明靶位点设计在eIFiso4E-S基因第一个外显子上。寻找靶位点时，首先从第一个外显子序列上找到PAM(NGG或CCN，在基因序列上的形式为5’-NNNNNNNNNNNNNNNNNNNNNGG-3’或5’-CCNNNNNNNNNNNNNNNNNNN NN-3’)位点，gRNA的oligo合成与退火：以gRNA靶位点为模板，按照如下格式设计引物oligo，为确保基因敲除事件效率，本发明针对eIFiso4E-S基因第一个外显子选择了两个靶位点，设计引物序列如下，其中F和R分别代表正、反向引物：
gRNA1F 5’-GGCAcgcctctatcggtgcttcag-3’
gRNA1R 5’-AAACctgaagcaccgatagaggcg-3’
gRNA2F 5’-GGCActagagaggagatggacattc-3’
gRNA2R 5’-AAACgaatgtccatctcctctctag-3’
引物退火：合成的一对互补DNA oligo退火形成dsDNA，退火体系如下：
Figure PCTCN2019101922-appb-000001
退火程序为:95℃ 5mim，90℃ 1mim，80℃ 1mim，70℃ 1mim，60℃ 1mim，50℃ 1mim，40℃ 1mim，30℃ 1mim，20℃ 1mim，10℃ 1mim。
酶切pRGEB31质粒体系
Figure PCTCN2019101922-appb-000002
酶切后回收大片段的酶切产物。
pRGEB31质粒回收的片段与退火后形成的dsDNA的连接体系：
Figure PCTCN2019101922-appb-000003
连接产物转化大肠杆菌并进行菌落PCR鉴定阳性克隆，菌落PCR 的检测正向引物为：OsU3 5'F 5’-aaggaatctttaaacatacgaacag-3’反向引物为退火合成sgRNA的反向序列gRNA1R或gRNA2R)
将扩增得到的阳性克隆摇菌并进行测序，分析gRNA是否正确。测序引物为OsU3 5'F。
实施例5：基因敲除载体的植物转化和eIFiso4E-S基因敲除植株检测
将连接正确的质粒，电击转化农杆菌GV3101。农杆菌介导转化烟草愈伤组织获得转基因植株。分别以野生型烟草云烟87和含有纯合va基因位点的烟草2-1398为材料诱导愈伤组织，进行农杆菌介导的烟草转化实验。以GV3101农杆菌进行侵染转化，经过潮霉素抗性筛选，抗性愈伤组织分化再生获得转基因阳性株系。
转基因烟草中eIFiso4E-T基因突变体的检测：设计目的基因检测引物。根据目的基因在靶位点序列上游和下游分别设计引物，引物序列分别为：
EditestF：5’-caattccattacgcctctccgttcgct-3’
EditestR：5’-ggaacaaaatccgaatttatcaataact-3’
将获得的转基因阳性植株提取基因组DNA进行PCR反应。利用PCR产物进行测序，测序公司为赛默飞世尔科技公司(广州)，测序引物序为EditestF。
实施例6：聚合eIFiso4E-S KO和va具有TVBMV抗性(g1-1C，)
针对eIFiso4E-S的exon1设计CRISPR-Cas9基因编辑载体(见实施例4)，利用eIFiso4E-S的CRISPR-Cas9基因编辑载体，转化va烟草2-1398。对T0代苗通过PCR扩增测序，筛选出eIFiso4E-S杂合编辑的单株2-1398g1-1C，2-1298g1-1C的eIFiso4E-S杂合突变的序列如SEQ ID No.1所示第11位增加一个A或者减少一个A。自交收 种获得eifiso4e-s KO/va烟草T1代种子，常规方法育苗盆栽T1植株。4-5片叶时，提取叶片DNA筛选出eIFiso4E-S双等位或纯合编辑且eIFiso4E-T为野生型的单株用于抗性鉴定。接种TVBMV病叶汁液40倍。以2-1398为对照。接种后14d、21d、28d调查TVBMV发病情况。结果表明(表4)，接种后14天对照2-1398(基因型va)的发病率为100％，接种后14天至接种后35天eifiso4e-s KO/va的发病率为0，表明eifiso4e-s KO/va具有抗TVBMV的功能。
表4eifiso4e-s KO/va对TVBMV的抗性
Figure PCTCN2019101922-appb-000004
接种后第32天，采集单株叶片，提取总RNA，采用OligoT引物反转录获得cDNA。根据TVBMV的VPg序列设计PCR引物，扩增产物大小为765bp，退火温度60℃。
TVBMVVPg_F：5’-AACTCAAGAGTCGTTGGAACA-3’
TVBMVVPgR：5’-CAAGCAAGCATATACACTTAGC-3’
通过PCR扩增检测。结果表明，2-1398g1-1C接种TVBMV后的12个样品无目标扩增条带，而2-1398接种TVBMV后有目标扩增条带。表明2-1398g1-1C抗TVBMV，而对照2-1398感TVBMV。
实施例5：eIFiso4E-S等位基因eifiso4e-s KO的育种应用
将烟草属植物中包含SEQ ID NO.1所示eIFiso4E-S基因Knockout等位基因(eifiso4e-s KO)的染色体片段，通过常规育种手段转育至目标烟草中。目标烟草为va、或eif4E1 KO的烟草(下同)。通过利用eifiso4e-s KO的功能分子标记或连锁的分子标记，或者人工接种TVBMV方法，从烟草属植物中筛选获得包含eifiso4e-s KO基因种质资 源。种质资源包含烟草野生种、野生种与栽培烟草的杂交种和栽培种。利用常规杂交育种或者原生质体融合或者染色体片段导入等技术手段，将种质资源中包含eifiso4e-s KO基因的染色体片段导入目标烟草，获得TVBMV抗性提高的非转基因烟草材料。通过杂交和回交等育种手段，将该抗性提高的烟草材料育成商业品种，改良烟草品种对TVBMV的抗性。
实施例7：eIFiso4E-S基因的基因编辑育种应用
将目标烟草中eIFiso4E-S基因通过基因编辑等生物技术修饰，使其失去与TVBMV VPg互作的生物学功能，得到抗TVBMV的烟草。目标烟草为va、或eif4E1 KO的烟草。
考虑到本发明前面的详细描述，预期在本发明的实施中本领域技术人员可进行很多改进和变化。因此，这些改进和变化都包括在本发明权利要求的范围内。

Claims (10)

  1. 一种隐性抗烟草脉带花叶病毒的烟草eIFiso4E-S基因，其特征在于其碱基序列如SEQ ID No.1所示。
  2. 根据权利要求1所述的隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因编码的多肽，其特征在于其氨基酸序列如SEQ ID No.2所示。
  3. 隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因的应用，其特征在于通过染色体片段导入、基因导入、基因编辑、基因沉默或物理化学诱变方式获得eIFiso4E-S基因敲除(eifiso4e-s KO)的烟草，从而使目标烟草获得TVBMV抗性。
  4. 根据权利要求3所述的隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因的应用，其特征在于通过染色体片段导入得到包含eifiso4e-s KO的烟草步骤为：先将含有eifiso4e-s KO的染色体片段通过杂交育种、原生质体融合转育至目标烟草中，得到抗TVBMV的烟草。
  5. 根据权利要求3所述的隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因的应用，其特征在于通过基因导入得到包含eifiso4e-s KO基因的烟草步骤为：将外源的eifiso4e-s KO基因导入目标烟草中，得到抗TVBMV的烟草。
  6. 根据权利要求3所述的隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因的应用，其特征在于通过基因编辑得到包含eifiso4e-s KO的烟草步骤为：将目标烟草中存在的eIFiso4E-S基因同源体，通过对其特定核苷酸位点的缺失、增加和/替换，使SEQ ID No.1所示序列失去功能，得到抗TVBMV的烟草。
  7. 根据权利要求3所述的隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因的应用，其特征在于通过物理或化学诱变得到包含eifiso4e-s KO的烟草步骤为：通过物理或化学诱变，使目标烟草SEQ ID No.1所示序列失去功能，得到抗TVBMV的烟草。
  8. 根据权利要求3～8任一所述的隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因的应用，其特征在于目标烟草为va、或eIF4E1基因敲除(eif4E1 KO)的烟草。
  9. 根据权利要求3～8任一所述的隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因的应用所得到的烟草品种、及其种子和无性繁殖体。
  10. 一种含有根据权利要求1所述的隐性抗烟草烟草脉带花叶病毒的eIFiso4E-S基因的Knockout表达盒。
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