CN111393516A - Tobacco eISFiso 4E-T mutant and application thereof in cultivation of tobacco with virus resistance - Google Patents
Tobacco eISFiso 4E-T mutant and application thereof in cultivation of tobacco with virus resistance Download PDFInfo
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Abstract
A tobacco eISFiso 4E-T mutant and application thereof in cultivating antiviral tobacco. The amino acid sequence of the tobacco eILIO 4E-T mutant is shown as SEQ ID NO.2 or SEQ ID NO.3, and compared with the amino acid sequence of wild tobacco eILIO 4E-T shown as SEQ ID NO. 1, the amino acid sequence of the tobacco eILIO 4E-T mutant is mutated from A to T at the 76 th position or from G to D at the 98 th position. The eISFiso 4E-T mutant is mixed with the va locus or eIFiso4e-T resisting potato Y virusKOThe gene is polymerized to obtain the lasting resistance of tobacco to PVY. The eILSO 4E-T mutant provided by the invention has important significance for culturing persistent PVY-resistant tobacco.
Description
Technical Field
The invention belongs to the field of biotechnology, further belongs to the field of tobacco biotechnology breeding, and particularly relates to acquisition of a tobacco eEFiso 4E-T gene mutant and application of the tobacco disease-resistant breeding.
Background
Potato Virus Y (PVY) is a typical member of the genus Potyvirus of tobacco, and PVY mainly damages solanaceous crops such as Potato, tobacco, tomato, and pepper, and has been rising as a major disease on tobacco in recent years. PVY is non-sexually transmitted by aphids in the field. The aphid has short development period, strong fecundity and easy drug resistance, and the effect of controlling the disease by using chemical agents to control vector insects is limited, so planting disease-resistant varieties is the most fundamental, most economic and effective means for controlling PVY.
As an important economic crop and an important model crop for plant research, the tobacco has less research on persistent PVY resistance resource identification, disease resistance breeding and pathogen investigation identification. The va gene disease-resistant resources obtained by X-ray mutagenesis are widely applied to breeding tobacco disease-resistant varieties. Deletion of the tobacco eIF4E1-S gene (sometimes abbreviated as eIF4E-1, GenBank sequence accession KF155696) results in tobacco resistance to the Potato Virus Y (PVY) (Liu Yong et al, 2013; Julio et al, 2014), but there are few studies on persistent anti-PVY resource identification, disease-resistant breeding and pathogen investigation identification. No germplasm resource report of the common tobacco for enduring PVY resistance is found.
Under the ecological condition of farmland planted adjacently with the same crops, the control of PVY generation on one crop is not only beneficial to the disease control of the crop, but also beneficial to the disease control of other crops.
Disclosure of Invention
The first invention provides an eILESO 4E-T gene mutant. The second invention aims to provide application of the eILIO 4E-T gene mutant in breeding antiviral varieties.
The first invention of the present invention is realized by:
a tobacco eILIO 4E-T mutant is characterized in that the amino acid sequence of the tobacco eILIO 4E-T mutant is shown as SEQ ID NO.2 or SEQ ID NO.3, and compared with the amino acid sequence of wild type tobacco eFiso 4E-T shown as SEQ ID NO. 1, the amino acid sequence of the tobacco eILIO 4E-T mutant is mutated from A to T at the 76 th position or from G to D at the 98 th position.
The nucleotide sequence of the wild tobacco eILIO 4E-T is shown as SEQ ID NO. 4, and the nucleotide sequence of the tobacco eILIO 4E-T mutant is shown as SEQ ID NO. 5 or SEQ ID NO. 6.
The second object of the present invention is achieved by:
the application of the tobacco eISFiso 4E-T mutant in cultivating tobacco with virus disease resistance is disclosed, wherein the virus disease is potato virus Y.
Further, the application method is that eISFiso 4E-T mutation sites are introduced into target tobacco through chromosome segment introduction or gene introduction, so that tobacco containing an eISFiso 4E-T mutant is obtained, and the target tobacco is enabled to obtain potato Y virus resistance; the target tobacco is tobacco with eIF4E1-S gene deletion or eIF4E1-S gene knock-out; the target tobacco eIF4E1-S gene deletion nucleotide sequence is shown as SEQ ID NO. 7, and the target tobacco eIF4E1-S gene knockout amino acid sequence is shown as SEQ ID NO. 8.
Further, the method for introducing the eILIO 4E-T mutation site into the target tobacco through chromosome segment introduction comprises the steps of polymerizing an eILIO 4E-T mutant gene and a tobacco material with an eIF4E1-S gene knocked out through crossbreeding and protoplast fusion to obtain tobacco with virus disease resistance; the method for introducing the eILIO 4E-T mutation site into the target tobacco through gene introduction is characterized in that exogenous eILIO 4E-T mutant genes are introduced into the target tobacco to obtain the tobacco with virus disease resistance.
The target tobacco of the invention is any cultivated tobacco of plants of the genus Nicotiana, including flue-cured tobacco, burley tobacco, aromatic tobacco, cigar tobacco, sun-cured tobacco and yellow-flower tobacco.
The eISFiso 4E-T mutant has important application value, and is knocked out with eIF4E1-S (eIF4E1-S)KO) Or eIF4E1-S deletion (va) to obtain eifiso4E-tKOeif4e1-sKOOr eifiso4e-tKOva radicalDue to the combined tobacco plants. The tobacco plant can resist Potato Virus Y (PVY) for a long time, and can be used for breeding tobacco varieties with long-lasting resistance to PVY.
The tobacco eILIO 4E-T mutant is applied to cultivation of tobacco with virus resistance, a new durable PVY resistant tobacco variety, seeds and asexual propagules thereof can be obtained, and some genetic engineering products can be developed, including A, an expression cassette of the eILIO 4E-T mutant gene, a transgenic cell line, a recombinant bacterium and the like; b, an expression cassette comprising the eIF4E1-S gene knock out, a transgenic cell line, a recombinant bacterium and the like; c is the combination of A and B. The tobacco can obtain durable resistance to PVY by using the gene engineering product.
Drawings
FIG. 1 shows the structure of the eISFiso 4E-T gene;
FIG. 2 is a graph of the peaks of the genomic sequencing of the mutants with hybrid mutations of the eISHO 4E-T gene (T1);
FIG. 3 is a graph of the peaks from the genome sequencing of mutants homozygous for the eISFiso 4E-T gene (T2).
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting in any way, and any variations which are based on the teaching of the invention are intended to fall within the scope of the invention.
Unless otherwise specified, the following examples all employ conventional methods; unless otherwise specified, all test materials used were purchased from conventional biochemicals.
A tobacco eILIO 4E-T mutant is characterized in that the amino acid sequence of the tobacco eILIO 4E-T mutant is shown as SEQ ID NO.2 or SEQ ID NO.3, and compared with the amino acid sequence of wild type tobacco eFiso 4E-T shown as SEQ ID NO. 1, the amino acid sequence of the tobacco eFiso 4E-T mutant is mutated from A to T (short for A76T) at the 76 th position or from G to D (short for G98D) at the 98 th position.
The nucleotide sequence of the wild tobacco eILIO 4E-T is shown as SEQ ID NO. 4, and the nucleotide sequence of the tobacco eILIO 4E-T mutant is shown as SEQ ID NO. 5 or SEQ ID NO. 6.
The tobacco eISHO 4E-T mutant can be applied to cultivation of tobacco with virus disease resistance, and the virus disease is Potato Virus Y (PVY). The application method is to modify the endogenous eILIO 4E-T gene of the target tobacco, and carry out mutagenesis and screening on target tobacco cells, tissues, individuals or groups so that the coding gene of the target tobacco contains the tobacco eILIO 4E-T mutant. The specific technical means is to introduce an eISFiso 4E-T mutation site into target tobacco by chromosome segment introduction or gene introduction to obtain the tobacco containing the eISFiso 4E-T mutant, so that the target tobacco can obtain the durable resistance of the potato Y virus. The target tobacco is eIF4E1-S gene deletion (va genotype) or eIF4E1-S gene knockout (eIF4E1-S)KO) The tobacco of (2); the target tobacco eIF4E1-S gene deletion nucleotide sequence is shown as SEQ ID NO. 7, and the target tobacco eIF4E1-S gene knockout amino acid sequence is shown as SEQ ID NO. 8.
The method for introducing the eILSO 4E-T mutation site into the target tobacco through chromosome segment introduction is to knock out an eILSO 4E-T mutant gene (A76T or G98D) and an eIF4E1-S gene (eIF4E1-S) through crossbreeding and protoplast fusionKO) Polymerizing the tobacco material to obtain the tobacco with virus disease resistance. The method for introducing the eILIO 4E-T mutation site into the target tobacco through gene introduction is to introduce an eILIO 4E-T mutation gene (A76T or G98D) into the target tobacco to obtain the tobacco with virus disease resistance. The gene introduction comprises introduction (namely transgene) and direct introduction after foreign genes are introduced, and the most common method for transgene is an agrobacterium transformation method; the direct introduction method comprises transforming tobacco cells or tissues by using a conventional biological method such as microinjection, a pollen tube channel method, conductance, a gene gun and the like, and culturing the transformed tissues into plants.
The eILSO 4E-T gene mutant shown in SEQ ID NO.2 and SEQ ID NO.3 can be obtained by the method of the technicians in the field:
(1) the chemical mutagenesis can be achieved by chemical mutagenesis method, and can adopt mutagens such as EMS, nitrite and the like.
(2) The gene is obtained by a gene editing method which can be edited by methods such as CRISPR-Cas9, TA L EN, zinc finger protein and the like.
The specific application route of the eILIO 4E-T gene mutant is as follows:
step A: obtaining eILSO 4E-T gene mutation material;
and B: the eIF4E1-S gene knockout (eIF14E 1) is obtained by methods such as resource screening, gene editing, physical mutagenesis, chemical mutagenesis, germplasm resource screening, gene artificial synthesis, gene expression interference and the likeKO) Or eIF4E1-S gene deletion (va genotype);
and C: polymerizing the material obtained in the step A and the material obtained in the step B to obtain an eEFiso 4E-T mutant gene (eIFiso4e-T)KO) With eIF4E1-S knockout (eIF14E 1)KO) Or various combinations of eIF4E1-S gene deletion (va) polymerization.
The invention can obtain the target material of the step B on the basis of the material obtained in the step A; or on the basis of the material obtained in the step B, obtaining the target material in the step A; or respectively obtaining the target material of the step A and the target material of the step B. Then, the eISFiso 4E-T gene mutant (eIFiso4e-T) is obtained by means of crossbreeding, somatic cell hybridization and the likeKO) With eIF4E1-S knockout (eIF14E 1)KO) Or various combinations of eIF4E1-S gene deletion (va genotype) polymerization.
Further application routes of the invention are as follows:
(1) on the basis of eIF4E1-S normal function, eifiso-containing 4E-t is obtained by gene editing, chemical mutagenesis and physical mutagenesisKOA tobacco plant with a mutated gene; then, the mixture passes through eifiso4e-tKOPlant and eif4e1-sKOPlant or breeding means such as eIF4E1-S gene deletion (va genotype) hybridization, backcross and the like to obtain the plant containing eifiso4E-tKOeif4e1-sKOOr eifiso4e-tKOA tobacco plant of va gene having viral disease resistance.
(2) In the eIF4E1-S knockout (eIF4E1-S)KO) Or eIF4E1-S gene deletion (va genotype), gene editing, chemical mutagenesis and physical mutagenesis are carried out to obtain the gene containing eifiso4E-tKOeif4e1-sKOA tobacco plant of the gene having resistance to viral diseases.
The above eifiso4e-t having virus disease resistance was usedKOeif4e1-sKOTobacco material, or eifiso4e-tKOAnd (3) breeding an antiviral tobacco variety by the va tobacco material.
The target tobacco is any one of cultivated tobacco of tobacco plants, including flue-cured tobacco, burley tobacco, aromatic tobacco, cigar tobacco, sun-cured tobacco and yellow tobacco. Such as Nicotiana tabacum cv Yunyan87 (abbreviated as Yunyan87, genotype eI Fiso4E-T/eIF4E1-S, which is sensitive to PVY), safflower macrogol (genotype eI Fiso4E-T/eIF4E1-S, which is sensitive to PVY), 2-1398 (genotype eI Fiso4E-T/va, which is anti-PVY). The tobacco materials are all from the research institute of tobacco agricultural science in Yunnan province. The PVY virus is from the research institute of tobacco agricultural science in Yunnan province.
One implementation of the present invention is described in detail below with reference to Yunyan87 as an example.
eISIO 4E-T gene knockout EMS mutant material creation of persistent anti-PVY:
according to PVYvaBMechanism for completing infection cycle by using eISFiso 4E-T in transformation of va tobacco, and artificially creating PVY (human immunodeficiency virus) resistance through EMS (enhanced message service) mutagenesisvaBFirstly, an EMS mutant library (M2 generation) of a flue-cured tobacco main culture variety Yunyan87 is screened for an eISFiso 4E-T gene mutated single plant by a TI LL ING technology, 4 amino acid change (missense mutation) single plants are screened from 2000M 2 generation single plants, wherein M3 single plants with numbers of T1 and T2 verify base mutation of second exons G226A and G293A by sequencing, and respectively cause mutation of amino acids A76T (Alanine to Threonine) and G98D (Glycine to aspartic acid), and a seedling stage artificial inoculation test shows that M4 homozygous at a mutation site induces PVYvaB. Then, crossing with va tobacco, backcrossing and selfing to obtain BC1F2, artificially inoculating, and screening to obtain PVY-resistant tobaccovaBEifiso4e-tA76T、eifiso4e-tG98DWith 2 ditertiary lines of va (BC1F 3). The double-mutant strain with lasting PVY resistance is obtained by a non-transgenic technology and can be directly applied to the breeding of tobacco varieties.
1 materials and methods
1.1 Yunyan87 mutant and DNA library thereof
The Yunyan87 mutant and the DNA library thereof are prepared and stored by the research institute of tobacco agricultural science in Yunnan province. EMS mutagenesis is utilized to establish a mutant library of Yunyan87, and about 2200 parts of EMS 2 generation mutant plants are planted in a field. The genomic DNA was extracted from the collected leaf and the DNA concentration was diluted to 40 ng/. mu.l, and finally a DNA library containing 1842M 2 generation Yunyan87 mutant was established, each 8 DNA samples were mixed to form an 8-fold mixing pool and stored in a 96-well plate.
1.2 TI LL ING screening conditions
Designing specific primers according to eISFiso 4E-T gene CDS (GenBank MN897004) and genome sequence (GenBank L C217872.1) for PCR amplification, wherein YNot-F: 5 '-TTTATCCATACTTGGATGTTGTG; YNot-R: 5' -TGGGACAGATCATCAT TAAGAAAA, and the amplified product size is 710bp, and the amplified product size is 2 rd and 3 rd exon sequences, the PCR reaction system is configured as 10. about. buffer: 1.0. mu.l, dNTP (2.5mM) 0.8. mu.l, F-primer (10. mu.M) 0.16. mu.l, R-primer (10. mu.M) 0.16. mu.l, H2O: 6.78 μ l; template (20 ng/. mu.l): 1.0 μ l; total volume: 10 μ l. The amplification conditions were as follows: the reaction program is 95 ℃ for 3min, 94 ℃ for 30s, 62 ℃ for 30s, -1 ℃/cycle, 72 ℃ for 1min, 7 cycles; 30s at 94 ℃, 30s at 58 ℃, 1min at 72 ℃, 40 cycles, 5min at 72 ℃; 10min at 99 ℃; 20s at 70 ℃, 0.3 ℃/cycle, 70 cycles, and preservation at 4 ℃. The amplification products were analyzed by capillary electrophoresis.
1.3M 3 mutant screening
And (4) floating and seedling the selected M3 strains according to a conventional method, and performing pot culture on 64 strains of each strain when 5-6 leaves are obtained. Taking leaves of a single plant to extract DNA, and amplifying and sequencing. And (5) verifying the mutation site of the mutant individual. And (4) selecting homozygous mutant single plants and transplanting.
1.4 eifiso4e-tKOWith va polymerization
Selecting an M3 single plant, taking pollen, preparing F1 combination with Y87va, planting F1 self-crossing F2 seeds, floating and seedling F2 according to a conventional method, and potting 96 plants in each plant line when 5-6 leaves are planted. Post-survival inoculation of PVYvaBIsolates, investigated for morbidity every 7, 14 and 21d after inoculation, and for asymptomatic lines, individual plants were selected for transplant and continued until flowering was observed. SelectingThe branch young leaves with good growth vigor are detected by a double-antibody sandwich detection kit (Agdia product) to be PVY, and the single plant which is detected to be PVY negative by E L ISA is selfed to leave F3 seeds.
1.5 resistance analysis of disease-resistant mutant Material
F3 floating seedlings according to a conventional method, and when 5-6 leaves are planted, each strain pots 64 plants. Post-survival inoculation of PVYWTAnd (4) separating the plants, investigating the morbidity every 7 days after inoculation, continuously investigating until 70 days after inoculation, selecting single plants which have better growth vigor and have PVY negative branch young leaf E L ISA detection, and selfing and reserving seeds.
Respectively identifying the resistance of selected M4 strain and F1 hybridization combination in a plastic greenhouse, respectively inoculating PVY necrotic strain ZT-5 isolate in each strain potted plant when 5-6 leaves are planted, regularly investigating the incidence rate after inoculation, selecting young leaves of part of asymptomatic single plants for verifying the incidence condition, and adopting E L ISA to detect PVY, wherein the division standard of the resistance is that the incidence rate is 0-25.0 percent of High Resistance (HR), the incidence rate is 25.1-50.0 percent of Medium Resistance (MR), the incidence rate is 50.1-75.0 percent of susceptible disease (MS), and the incidence rate is 75.1-100 percent of High Sensitivity (HS).
2 results and analysis
2.1 eISFiso 4E-T Gene Structure analysis
The eISIO 4E-T gene CDS has overall length 588bp, comprises 5 exons and codes 195 amino acids (see figure 1). the research aims at 2 nd and 2 nd exon regions, and utilizes TI LL ING to screen mutants, YNot-F: 5 '-TTTATCCATACTTGGGATGTTGTG, YNot-R: 5' -TGGGACAGATCATCATTAAGAAAA, and 710bp of amplification product size is the 2 nd and 3 rd exon sequences.
2.2 eISFiso 4E-T Gene mutant TI LL ING screening
Collecting leaves to extract genome DNA, and establishing a DNA library of M2 generation Yunyan87 mutant. EMS mutant libraries of M2 generation Yunyan87 were screened using the Tilling screening system to obtain 18 base mutant strains in 1842 fractions of material. The mutant material genomic DNA was used as a template, and a PCR fragment obtained by amplification using the Ynisot F/R primer was ligated to pTOPO vector, followed by Sanger sequencing. Sequencing results indicated that there were 4 mutations that resulted in amino acid changes (missense mutations), 6 mutations that did not (synonymous mutations), and 8 mutations in the intron region (see Table 1). The influence of mutation sites on the Protein function is predicted by PROVEAN Protein online software, 2 mutations in 4 missense mutations have little influence (neutrality) on the Protein function, and 2 mutations cause the change of the physicochemical properties of amino acids and possibly change the Protein function (harmfulness) (see Table 2).
TABLE 1 eISFiso 4E-T gene mutation site screened by TI LL ING in Yunyan87 mutant library
| Number of the selection | Serial number | Type of base mutation | Mutated region | Type of amino acid mutation | |
| t1 | 1 | G-A HO | exon | Missense of the word | |
| 2 | G-A HO | exon | Synonymy | ||
| 3 | G-A HE | exon | Synonymy | ||
| 4 | G-A HE | exon | Synonymy | ||
| 5 | G-A HE | exon | Synonymy | ||
| t2 | 6 | G-A HE | exon | Missense of the word | |
| 7 | G-A HO | exon | Synonymy | ||
| 8 | G-A HE | exon | Synonymy | ||
| t3 | 9 | G-A HO | exon | Missense of the word | |
| 10 | G-A HE | intron | |||
| 11 | | intron | |||
| 12 | G-A HE | intron | |||
| 13 | G-A HE | intron | |||
| 14 | G-A HO | intron | |||
| t4 | 15 | G-A HE | exon | Missense of the word | |
| 16 | A-G HE | exon | Synonymy | ||
| 17 | G-A HO | intron | |||
| 18 | C-T HE | intron |
TABLE 2 functional PROVEN prediction of amino acid substitutions in eISFiso 4E-T missense mutants
(1) The threshold value is-2.5, the mutant score is less than or equal to-2.5 and is judged as harmful, and the mutant score is more than-2.5 and is judged as neutral "
Reference Choi Y, Sims GE, Murphy S, Miller JR, Chan AP (2012) Predicting the functional effects of Amino acids Substistions and indexes. P L oS ONE 7(10): e46688.
2.3 mutant Material mutation site validation
Planting M3 generation mutant material, extracting leaf genome DNA, sequencing to verify whether the M2 generation mutation can be inherited to the next generation, and screening homozygous mutant strains (see figure 2 and figure 3). Sequencing results show that T1 and T2 mutation sites are located at the second exons G226A and G293A base mutations of eILIO 4E-T gene, respectively result in mutation of A76T amino acid (Alanine to Threonine Threonine) and G98D amino acid (Glycine to aspartic acid) which can be stably inherited to M3 generation, and homozygous mutant individuals are selected to obtain M4 from inbreds through PCR product sequencing.
2.4M 4 Generation homozygous mutant resistance assay
Selecting 5M 4 generation homozygous mutant line tobacco seedlings from 3 missense mutants, potting, inoculating PVYvaBIsolate, after 21 and 28 days of inoculation, the single plants are close to 100% in disease occurrence, 5 single plants are positive to PVY virus through E L ISA detection, the disease occurrence rate of the control Yunyan87 reaches 100% (see table 3), 3 single plants are positive to PVY virus through E L ISA detection, symptom investigation and E L ISA detection results show that 3 missense mutants are sensitive to PVYvaBIsolate, eISHO 4E-T Gene missense mutation-induced PVY in Va backgroundvaB。eifiso4e-tKOResistance was verified against va polymerization.
TABLE 3 eISHO 4E-T Gene M4 mutant vs PVYvaBResistance of (2)
S means infection
2.5 polymerization of eifiso4e-tKOObtaining PVY from vavaBResistance to
eifiso4e-tKO(the genotype is abbreviated as isot) is hybridized with va (RY21-1-3) to obtain F1, and backcrossed with va to obtain BC1F 1. Performing PCR sequencing on BC1F1 tobacco seedlings, and screening to obtain tobacco seedlings containing eifiso4e-tKOAnd (4) selfing and harvesting single plants at loci to obtain BC1F 2. According to the Mendelian genetic model of 2 single genes, the proportion of vavassotiot genotypes in BC1F2 is theoretically 1/16. To reduce the workload, PVY was inoculated by inoculating BC1F2 seedlingsvaBScreening disease-resistant single plants (vavassotispot), verifying the disease-resistant single plants by symptom investigation and detection of PVY content of E L ISA, and verifying eifiso4E-t by PCR amplification sequencing of the disease-resistant single plantsKOMutating the locus, and marking and detecting to verify the va locus. Inoculation of PVYvaBIn the later 22 days, disease-resistant individuals which are in accordance with the theoretical prediction ratio exist in BC1F2 populations of t1 and t2, and the E L ISA is detected to be PVY negative (see tables 4 and 5), while the number of the disease-resistant individuals in the BC1F2 population of t3 is obviously lower than the theoretical prediction ratioA76TAnd eifiso4e-tG98DAfter polymerization with va, PVY is obtainedvaBAnd (5) resistance. Indicating the polymerization of eifiso4e-tKOAnd va persistent anti-PVY. Novel germplasm permanently resistant to PVY was created by EMS mutagenesis to knock out (knock out) eISIO 4E-T.
TABLE 4 polymerization of eifiso4e-tKOBC1F2 anti-PVY with vavaBIndividual plant screening and validation
TABLE 5 BC1F2 disease-free Individual E L ISA test PVY Virus concentration
Note: -represents negative, sample OD405 value/negative control OD value <1.5, + represents positive, sample OD405 value/negative control OD value > 1.5; double well loading (60min), 22 days after inoculation vaB
This example used TRIzol reagent (Invitrogen; Carlsbad, Calif.) to extract total RNA from tobacco leaves according to the manufacturer's protocol plasmid DNA extraction kit, agarose gel DNA recovery kit, DNA fragment purification kit were purchased from QIAGEN, Escherichia coli (Escherichia coli) DH5 α, restriction enzymes, reverse transcription kit, DNAmarker, PrimeSTAR GX L DNA Polymerase, T4 DNA Polymerase and T4 DNA ligase, spectinomycin were all purchased from Invitrogen, and Roche, RNA extraction kit Trizol was purchased from Invitrogen, Escherichia coli (Escherichia coli) DH5 α strain, Agrobacterium (Agrobacterium tumefaciens) EHA105, C58C1 strain were stored in this laboratory, cloning vector pMD18T was purchased from Dalibao.
In light of the detailed description of the invention, it is contemplated that numerous modifications and variations will readily occur to those skilled in the art in the practice of the invention, which modifications and variations are within the scope of the invention as claimed.
Sequence listing
<110> research institute of tobacco agricultural science in Yunnan province
<120> tobacco eILIO 4E-T mutant and application thereof in breeding tobacco with virus resistance
<160>8
<170>SIPOSequenceListing 1.0
<210>1
<211>195
<212>PRT
<213> amino acids of gene (eISFiso 4E-T)
<400>1
Met Ala Thr Glu Ala Pro Ile Glu Ala Thr Glu Val Leu Pro Ala Pro
1 5 10 15
Asp Thr Val Glu Lys Gln Pro His Lys Leu GluArg Arg Trp Thr Phe
20 25 30
Trp Phe Asp Lys Pro Lys Gln Gly Ala Val Trp Ala Ser Ala Leu Arg
35 40 45
Lys Ala Tyr Thr Phe Glu Thr Val Glu Glu Phe Trp Ser Leu Tyr Asp
50 55 60
Gln Ile Phe Lys Pro Ser Lys Leu Thr Ala Asn Ala Asp Phe His Leu
65 70 75 80
Phe Lys Ala Gly Ile Glu Pro Lys Trp Glu Asp Pro Glu Cys Ala Asn
85 90 95
Gly Gly Lys Trp Thr Val Thr Ser Ser Arg Lys Ala Asn Leu Glu Thr
100 105 110
Met Trp Leu Glu Thr Leu Met Ala Leu Val Gly Glu Gln Phe Asp Glu
115 120 125
Ser Glu Glu Ile Cys Gly Val Val Ala Ser Val Arg Arg Ser Gln Asp
130 135 140
Lys Leu Ser Leu Trp Thr Arg Thr Ala Ser Asn Glu Ala Ala Gln Met
145 150 155 160
Ser Ile Gly Arg Lys Trp Lys Glu Ile Ile Asp Ala Glu Lys Ile Ser
165 170 175
Tyr Ser Phe His Asp Asp Ser Lys Lys Glu Arg Ser Val Lys Ser Arg
180 185 190
Tyr Thr Val
195
<210>2
<211>195
<212>PRT
<213> mutant amino acids (eISFiso 4E-T)
<400>2
Met Ala Thr Glu Ala Pro Ile Glu Ala Thr Glu Val Leu Pro Ala Pro
1 5 10 15
Asp Thr Val Glu Lys Gln Pro His Lys Leu Glu Arg Arg Trp Thr Phe
20 25 30
Trp Phe Asp Lys Pro Lys Gln Gly Ala Val Trp Ala Ser Ala Leu Arg
35 40 45
Lys Ala Tyr Thr Phe Glu Thr Val Glu Glu Phe Trp Ser Leu Tyr Asp
50 55 60
Gln Ile Phe Lys Pro Ser Lys Leu Thr Ala Asn Thr Asp Phe His Leu
65 70 75 80
Phe Lys Ala Gly Ile Glu Pro Lys Trp Glu Asp Pro Glu Cys Ala Asn
85 90 95
Gly Gly Lys Trp Thr Val Thr Ser Ser Arg Lys Ala Asn Leu Glu Thr
100 105 110
Met Trp Leu Glu Thr Leu Met Ala Leu Val Gly Glu Gln Phe Asp Glu
115 120 125
Ser Glu Glu Ile Cys Gly Val Val Ala Ser Val Arg Arg Ser Gln Asp
130 135 140
Lys Leu Ser Leu Trp Thr Arg Thr Ala Ser Asn Glu Ala Ala Gln Met
145 150 155 160
Ser Ile Gly Arg Lys Trp Lys Glu Ile Ile Asp Ala Glu Lys Ile Ser
165 170 175
Tyr Ser Phe His Asp Asp Ser Lys Lys Glu Arg Ser Val Lys Ser Arg
180 185 190
Tyr Thr Val
195
<210>3
<211>195
<212>PRT
<213> mutant amino acids (eISFiso 4E-T)
<400>3
Met Ala Thr Glu Ala Pro Ile Glu Ala Thr Glu Val Leu Pro Ala Pro
1 5 10 15
Asp Thr Val Glu Lys Gln Pro His Lys Leu Glu Arg Arg Trp Thr Phe
20 25 30
Trp Phe Asp Lys Pro Lys Gln Gly Ala Val Trp Ala Ser Ala Leu Arg
35 40 45
Lys Ala Tyr Thr Phe Glu Thr Val Glu Glu Phe Trp SerLeu Tyr Asp
50 55 60
Gln Ile Phe Lys Pro Ser Lys Leu Thr Ala Asn Ala Asp Phe His Leu
65 70 75 80
Phe Lys Ala Gly Ile Glu Pro Lys Trp Glu Asp Pro Glu Cys Ala Asn
85 90 95
Gly Asp Lys Trp Thr Val Thr Ser Ser Arg Lys Ala Asn Leu Glu Thr
100 105 110
Met Trp Leu Glu Thr Leu Met Ala Leu Val Gly Glu Gln Phe Asp Glu
115 120 125
Ser Glu Glu Ile Cys Gly Val Val Ala Ser Val Arg Arg Ser Gln Asp
130 135 140
Lys Leu Ser Leu Trp Thr Arg Thr Ala Ser Asn Glu Ala Ala Gln Met
145 150 155 160
Ser Ile Gly Arg Lys Trp Lys Glu Ile Ile Asp Ala Glu Lys Ile Ser
165 170 175
Tyr Ser Phe His Asp Asp Ser Lys Lys Glu Arg Ser Val Lys Ser Arg
180 185 190
Tyr Thr Val
195
<210>4
<211>588
<212>DNA
<213> Gene nucleotides (eISFiso 4E-T)
<400>4
atggccactg aagcaccgat agaggcgacg gaggttctgc cggcgccgga tacggtggag 60
aagcagccgc ataagctaga gaggagatgg acattctggt tcgataagcc gaagcaaggc 120
gctgtttggg caagtgctct tcgaaaagcc tatactttcg aaactgttga ggaattctgg 180
agtttatatg atcagatatt caagcccagc aagttgactg ctaatgcgga ctttcatttg 240
ttcaaagctg ggattgagcc caaatgggaa gatcctgagt gtgccaatgg tggcaagtgg 300
actgtcacga gcagcagaaa ggctaatctt gagactatgt ggcttgaaac tctgatggca 360
ttggtgggtg agcaatttga tgaatcagaa gagatatgtg gagtggttgc cagtgttcgt 420
cggagtcagg ataaactttc cttgtggact aggactgcct ccaatgaagc agctcagatg 480
agcattggta ggaagtggaa ggagatcatc gatgctgaaa aaatatccta tagtttccat 540
gatgactcta aaaaggaaag gtcagttaag agtcgatata ctgtgtga 588
<210>5
<211>588
<212>DNA
<213> mutant nucleotide (eISFiso 4E-T)
<400>5
atggccactg aagcaccgat agaggcgacg gaggttctgc cggcgccgga tacggtggag 60
aagcagccgc ataagctaga gaggagatgg acattctggt tcgataagcc gaagcaaggc 120
gctgtttggg caagtgctct tcgaaaagcc tatactttcg aaactgttga ggaattctgg 180
agtttatatg atcagatatt caagcccagc aagttgactg ctaatacgga ctttcatttg 240
ttcaaagctg ggattgagcc caaatgggaa gatcctgagt gtgccaatgg tggcaagtgg 300
actgtcacga gcagcagaaa ggctaatctt gagactatgt ggcttgaaac tctgatggca 360
ttggtgggtg agcaatttga tgaatcagaa gagatatgtg gagtggttgc cagtgttcgt 420
cggagtcagg ataaactttc cttgtggact aggactgcct ccaatgaagc agctcagatg 480
agcattggta ggaagtggaa ggagatcatc gatgctgaaa aaatatccta tagtttccat 540
gatgactcta aaaaggaaag gtcagttaag agtcgatata ctgtgtga 588
<210>6
<211>588
<212>DNA
<213> mutant nucleotide (eISFiso 4E-T)
<400>6
atggccactg aagcaccgat agaggcgacg gaggttctgc cggcgccgga tacggtggag 60
aagcagccgc ataagctaga gaggagatgg acattctggt tcgataagcc gaagcaaggc 120
gctgtttggg caagtgctct tcgaaaagcc tatactttcg aaactgttga ggaattctgg 180
agtttatatg atcagatatt caagcccagc aagttgactg ctaatgcgga ctttcatttg 240
ttcaaagctg ggattgagcc caaatgggaa gatcctgagt gtgccaatgg tgacaagtgg 300
actgtcacga gcagcagaaa ggctaatctt gagactatgt ggcttgaaac tctgatggca 360
ttggtgggtg agcaatttga tgaatcagaa gagatatgtg gagtggttgc cagtgttcgt 420
cggagtcagg ataaactttc cttgtggact aggactgcct ccaatgaagc agctcagatg 480
agcattggta ggaagtggaa ggagatcatc gatgctgaaa aaatatccta tagtttccat 540
gatgactcta aaaaggaaag gtcagttaag agtcgatata ctgtgtga 588
<210>7
<211>660
<212>DNA
<213> nucleotide deletion in Gene (eIF4E1-S)
<400>7
atggcagagg aagctgagaa attgcgggta gatgaagtag aagtagtcga cgatggacct 60
gaagaaggag aaattgtgga tgaatctgat gatacggcgt cgtatttggg caaagaaatc 120
aaacctaagc atccattaga gaattcttgg actttttggt ttgataatcc tatggctaaa 180
tctagacaag ctgcttgggg cagttccctt cgcgaacttt acactttttc cactgtcgaa 240
gatttttggg gtgtttacaa taatatcaac cacccaagca agttagttgt gggagcagac 300
tttcattgtt ttaagcataa aattgagcca aagtgggaag atcctgtatg tgcgaatgga 360
gggaattgga caatgagctt tagtaagggt aaatctgata ccagctggct atacacgctg 420
ctggcaatga ttggacatca attcgatcat ggagaggaaa tttgtggagc agtagttagc 480
gtccgaaata agggggataa aatagcttta tggaccaaga atgctgcaaa tgaaacagct 540
caggttagca ttggtaagca atggaaggag tttctggatt acagcaactc gattggcttc 600
atatttcatg acgactcaat gaggctcggc agaggtgcca agaatcgtta tacagtatag 660
<210>8
<211>219
<212>PRT
<213> deletion of amino acids from Gene (eIF4E1-S)
<400>8
Met Ala Glu Glu Ala Glu Lys Leu Arg Val Asp Glu Val Glu Val Val
1 5 10 15
Asp Asp Gly Pro Glu Glu Gly Glu Ile Val Asp Glu Ser Asp Asp Thr
20 25 30
Ala Ser Tyr Leu Gly Lys Glu Ile Lys Pro Lys His Pro Leu Glu Asn
35 40 45
Ser Trp Thr Phe Trp Phe Asp Asn Pro Met Ala Lys Ser Arg Gln Ala
50 55 60
Ala Trp Gly Ser Ser Leu Arg Glu Leu Tyr Thr Phe Ser Thr Val Glu
65 70 75 80
Asp Phe Trp Gly Val Tyr Asn Asn Ile Asn His Pro Ser Lys Leu Val
85 90 95
Val Gly Ala Asp Phe His Cys Phe Lys His Lys Ile Glu Pro Lys Trp
100 105 110
Glu Asp Pro Val Cys Ala Asn Gly Gly Asn Trp Thr Met Ser Phe Ser
115 120 125
Lys Gly Lys Ser Asp Thr Ser Trp Leu Tyr Thr Leu Leu Ala Met Ile
130 135 140
Gly His Gln Phe Asp His Gly Glu Glu Ile Cys Gly Ala Val Val Ser
145 150 155 160
Val Arg Asn Lys Gly Asp Lys Ile Ala Leu Trp Thr Lys Asn Ala Ala
165 170 175
Asn Glu Thr Ala Gln Val Ser Ile Gly Lys Gln Trp Lys Glu Phe Leu
180 185 190
Asp Tyr Ser Asn Ser Ile Gly Phe Ile Phe His Asp Asp Ser Met Arg
195 200 205
Leu Gly Arg Gly Ala Lys Asn Arg Tyr Thr Val
210 215
Claims (6)
1. The tobacco eILIO 4E-T mutant is characterized in that the amino acid sequence of the tobacco eILIO 4E-T mutant is shown as SEQ ID NO.2 or SEQ ID NO.3, and compared with the amino acid sequence of wild type tobacco eILIO 4E-T shown as SEQ ID NO. 1, the amino acid sequence of the tobacco eILIO 4E-T mutant is mutated from A to T at the 76 th position or from G to D at the 98 th position.
2. The tobacco eIFiso4E-T mutant according to claim 1, wherein the nucleotide sequence of the wild type tobacco eIFiso4E-T is shown as SEQ ID No. 4 and the nucleotide sequence of the tobacco eIFiso4E-T mutant is shown as SEQ ID No. 5 or SEQ ID No. 6.
3. Use of the tobacco eIFiso4E-T mutant according to claim 1 or 2 for breeding tobacco with a viral disease, said viral disease being the potato virus Y.
4. The use of the tobacco eIFiso4E-T mutant according to claim 3 for breeding antiviral tobacco by introducing an eIFiso4E-T mutation site into a target tobacco by chromosome fragment introduction or gene introduction to obtain tobacco comprising the eIFiso4E-T mutant, thereby conferring potato Y virus resistance to the target tobacco; the target tobacco is tobacco with eIF4E1-S gene deletion or eIF4E1-S gene knockout; the target tobacco eIF4E1-S gene deletion nucleotide sequence is shown as SEQ ID NO. 7, and the amino acid sequence is shown as SEQ ID NO. 8.
5. The use of the tobacco eIFiso4E-T mutant according to claim 4 for breeding antiviral tobacco, wherein the method for introducing the eIFiso4E-T mutation site into the target tobacco through chromosome segment introduction comprises the steps of hybridizing breeding and protoplast fusion, and polymerizing the eIFiso4E-T mutant gene with the tobacco material with the eIF4E1-S gene knockout to obtain the antiviral tobacco; the method for introducing the eILIO 4E-T mutation site into the target tobacco through gene introduction is characterized in that the exogenous eILIO 4E-T mutant gene is introduced into the target tobacco to obtain the tobacco with virus disease resistance.
6. The use of the tobacco eIFiso4E-T mutant according to any of claims 3 to 5 for breeding antiviral tobacco, wherein the target tobacco is a plant of the nicotiana species, including any of flue-cured tobacco, burley tobacco, oriental tobacco, cigar tobacco, sun-cured tobacco, and yellow-wing tobacco.
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| CN109688806A (en) * | 2016-08-26 | 2019-04-26 | 日本烟草产业株式会社 | Virus resistance tobacco and preparation method thereof |
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