CN113416736B - Artificial improved mutant of disease-resistant gene Sw-5b and application thereof - Google Patents

Artificial improved mutant of disease-resistant gene Sw-5b and application thereof Download PDF

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CN113416736B
CN113416736B CN202110544417.4A CN202110544417A CN113416736B CN 113416736 B CN113416736 B CN 113416736B CN 202110544417 A CN202110544417 A CN 202110544417A CN 113416736 B CN113416736 B CN 113416736B
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陶小荣
黄海宁
李佳
黄申
戴婧
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Nanjing Agricultural University
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Abstract

The mutant is characterized in that 33 th leucine is mutated into proline, 319 th lysine is mutated into glutamic acid, 927 th arginine is mutated into alanine or glutamine, the rest amino acid sites are kept unchanged, and three amino acid sites are simultaneously mutated. Sw-5b obtained by the invention L33P/K319E/R927A And Sw-5b L33P/K319E/R927Q Two mutants of TSWV wild type and two virus field variant TSWV C118Y 、TSWV T120N All have disease resistance, and provide effective disease-resistant material for the prevention and control of TSWV field resistance breakthrough variant strains.

Description

Artificial improved mutant of disease-resistant gene Sw-5b and application thereof
Technical Field
The invention belongs to the field of plant genetic engineering, and particularly relates to an artificially improved mutant of a disease-resistant gene Sw-5b and application thereof.
Background
TSWV is a representative species of the order Bunyaviridae, family Lycopersicon esculentum Betularensis Virus, genus Lycopersicon esculentum Betul. TSWV has wide infection range, and can infect more than 800 plants of 80 different families. TSWV causes black spots on tomato leaves, and also causes dead leaf tips, unilateral growth and developmental retardation. TSWV may cause failure of tomato to seed early in infestation; in the late stage of infestation, concentric spots appear on the fruit.
At present, the most effective means for preventing and treating TSWV is to select and breed disease-resistant varieties. Disease-resistant gene from Peru tomatoSw- 5bHas good disease resistance to TSWV. In addition to the TSWV,Sw-5balso has good antiviral effect on American viruses such as tomato chlorosis virus and impatiens necrotic spot virus in the genus of positive tomato spotted wilt virus.
Is carried along withSw-5bThe disease-resistant variety of the disease-resistant gene is popularized and used for a long time, and the gradual generation in the field can overcomeSw-5bMediated disease resistanceThe TSWV variant strain of (1) (hereinafter referred to as a resistant mutant virus strain). In 2003, resistance-breaking virus strains were first discovered in the Spanish field, and subsequently emerged in Italy, California, USA, and so on. Of these, the incidence of TSWV resistant breakthrough strains in California in the United states was about 50-80% in 2016, causing severe economic losses. Researches of Carmelo Lopez and the like in 2011 find that TSWV resistant burst strains are mainly divided into two types, one type is that cysteine at position 118 of TSWV mobile protein NSm is mutated into tyrosine, namely NSm C118Y (ii) a Another is the mutation of threonine at position 120 of the TSWV movement protein NSm to aspartic acid, namely NSm T120N . Currently, there is no effective control means for these two resistance breakthrough virus strains. Because the germplasm resources and varieties of the crops for resisting the tomato spotted wilt virus are deficient, the disease-resistant genes are screened by manual methodSw-5bIt is very important to improve and design to obtain new disease-resistant gene resources.
Combined with the current disease-resistant geneSw-5bThe research on the identification mechanism of TSWV NSm and the artificial improvement design method of disease-resistant gene is advanced bySw-5bAnd carrying out accurate artificial molecular design and improvement to endow the disease-resistant gene with new disease resistance to field variation strains of the tomato spotted wilt virus.
Disclosure of Invention
The technical problem to be solved is as follows: aiming at the technical problems, the invention provides an artificial improved mutant of a disease-resistant gene Sw-5b and application thereof. Two Sw-5b mutants (Sw-5 b) L33P/K319E/R927A And Sw-5b L33P/K319E/R927Q ) For TSWV wild type and two virus field variation strains TSWV C118Y 、TSWV T120N All have disease resistance.
The technical scheme is as follows: the disease-resistant gene Sw-5b artificially improves mutants, 33 th leucine is mutated into proline, 319 th lysine is mutated into glutamic acid, 927 th arginine is mutated into alanine or glutamine, the rest amino acid sites are kept unchanged, and three amino acid sites are simultaneously mutated.
One of the artificial improved mutants of the disease-resistant gene Sw-5b has an amino acid sequence shown in SEQ ID NO. 1.
The second of the artificial improved mutants of the disease-resistant gene Sw-5b has an amino acid sequence shown in SEQ ID NO. 2.
A coding gene encoding the mutant.
A vector containing the above-mentioned coding gene.
Recombinant bacteria or recombinant cells containing the vector.
The artificial improved mutant of the disease-resistant gene Sw-5b is used for cultivating anti-TSWV wild type and anti-TSWV C118Y And TSWV T120N Application in plants.
The plant is tomato.
Has the advantages that: the invention obtains Sw-5b L33P/K319E/R927A And Sw-5b L33P/K319E/R927Q Two mutants of TSWV wild type and two virus field variant TSWV C118Y 、TSWV T120N All have disease resistance. The invention provides an effective disease-resistant material for the prevention and control of TSWV field resistance breakthrough variant strains.
Drawings
FIG. 1 is a pair Sw-5b R927A Performing random mutation on the SD functional domain;
FIG. 2 shows Sw-5b L33P/K319E/R927A And Sw-5b L33P/K319E/R927Q And NSm WT 、NSm C118Y 、NSm T120N (ii) an HR symptom map of transient infiltration of burley tobacco leaves;
FIG. 3 shows Sw-5b L33P/K319E/R927A And Sw-5b L33P/K319E/R927Q Transgenic Benyan to TSWV wild type and two virus field variation strains TSWV C118Y 、TSWV T120N The disease resistance is good;
FIG. 4 shows the result of detecting the accumulation of TSWV in the systemic leaves of the plant of FIG. 3.
FIG. 5 shows two Sw-5b mutants (Sw-5 b) L33P/K319E/R927A And Sw-5b L33P/K319E/R927Q ) Schematic amino acid sequence.
Detailed Description
The following examples are presented to enable one of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. It is within the scope of the present invention to modify or substitute the strategy, method, procedure or conditions of the present invention without departing from the spirit and substance of the present invention.
Example 1: to Sw-5b R927A Random mutagenesis of the SD Domain
About Sw-5b R927A Construction of mutant pools and Sw-5b R927A Strategy for mutant selection (FIG. 1), briefly, a large number of base-mutated Sw-5b SD PCR products were amplified by the Mn-dITP PCR method, as follows: PCR amplification used a universal primer pair (forward primer: 5'-ATGGCTGAAAATGAAATTGAGGAAATGTTA-3', reverse primer: 5'-GGGGAACTGAGATGGAGTAAATTTTAAGGA-3'). In the first round of PCR amplification, PCR reaction (50. mu.L): 10 XPCR buffer 5. mu.L, MnCl 2 （1 mM）2 μL，MgCl 2 4. mu.L (25 mM), 1. mu.L of dNTP (10 mM), 1. mu.L of each primer (10. mu.M), 4. mu.L of DNA template (25 ng), 1. mu.L of Taq DNA polymerase (5U), and 31. mu.L of double distilled water; the PCR amplification conditions were: pre-denaturation at 94 deg.C for 3 min, denaturation at 94 deg.C for 1min, annealing at 51 deg.C for 1min, extension at 72 deg.C for 1min15s, and circulation for 20 times, and extension at 72 deg.C for 10 min. In the second round of PCR amplification, PCR reaction (50. mu.L): 10 XPCR buffer 5. mu.L, dITP (10 mM) 2. mu.L, MgCl 2 4. mu.L (25 mM), 1. mu.L of dNTP (10 mM), 1. mu.L of each primer (10. mu.M), 2. mu.L of DNA template (first round PCR product), 1. mu.L of Taq DNA polymerase (5U), and 33. mu.L of double distilled water; the PCR amplification conditions were: pre-denaturation at 94 deg.C for 3 min, denaturation at 94 deg.C for 1min, annealing at 51 deg.C for 1min, extension at 72 deg.C for 1min15s, and circulation for 30 times, and extension at 70 deg.C for 10 min. Simultaneous amplification of p2300-Sw-5b R927A The specific operation of the product of the Δ SD PCR is as follows: the PCR amplification uses a universal primer pair (forward primer: 5'-TCCTTAAAATTTACTCCATCTCAGTTCCCC-3', reverse primer: 5 ' -TAACATTTCCTCAATTT)
CATTTTCAGCCAT-3'). PCR reaction (50. mu.L): 2 × 25 μ L of Phanta Max buffer, 1 μ L of dNTP Mix (10 mM), 2 μ L of each primer (10 μ M), 2 μ L of DNA template, 1 μ L of Phanta Max Super-Fidelity DNA Polymerase, and 17 μ L of double distilled water; the PCR amplification conditions were: pre-denaturation at 95 deg.C for 3 min, denaturation at 95 deg.C for 15s, annealing at 58 deg.C for 15s, and extension at 72 deg.C for 6min30sCirculating for 35 times, and extending for 10 min at 72 ℃. . The recovered Sw-5b SD PCR product was combined with the recovered p2300-Sw-5b R927A Homologous recombination of the Δ SD PCR products, homologous recombination system (20 μ L): 5 × CE II Buffer 4. mu.L, linearized cloning vector (p 2300-Sw-5 b) R927A Δ SD PCR) 240 ng, insert amplification product (Sw-5 b SD) 48 ng, Exnase II 2 μ L, double distilled water to make up to 20 μ L. And (3) placing the configured homologous recombination system at 37 ℃ for reaction for 30 min. After the reaction was completed, the reaction tube was immediately placed in an ice-water bath to cool for 5 min. Then the homologous recombination products are transformed by electric shock to obtain a large amount of chimeric Sw-5b R927A And (3) mutants.
Example 2: screening can simultaneously identify TSWV NSm WT 、NSm C118Y 、NSm T120N Sw-5b of R927A Mutants
To screen for NSm capable of interacting with TSWV WT 、NSm C118Y Or NSm T120N Sw-5b for HR production R927A Mutant, inventive pairs 2036 Sw-5b R927A The mutants were verified by picking chimeric Sw-5b R927A The mutant was cultured in 3-4 mL of LB liquid medium (1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 1% (w/v) NaCl at 28 ℃ in a constant temperature incubator at 220 rpm for 24 hours. Placing the bacterial liquid in a 2 mL centrifuge tube, and centrifuging at 6000 rpm for 10 min; the supernatant was discarded, and Agrobacterium treatment was used ((10 mM MES pH 5.6, 10 mM MgCl) 2 And 150 mM acetosyringone) suspended precipitate, OD 600 Adjusting to 0.5. Sw-5b R927A Mutants and NSm WT 、NSm C118Y Or NSm T120N Isovoluminal mixing and co-infiltrating Benshi tobacco leaf, screening and NSm WT 、NSm C118Y Or NSm T120N Sw-5b all capable of producing HR reactions R927A Mutants, eventually screened for two simultaneously with NSm WT 、NSm C118Y Or NSm T120N (iii) Sw-5b producing HR response without immune self-activation R927A And (3) mutants. Sequencing results show that the two mutants are mutated from leucine (L) to proline (P) at the position 33 of the SD functional domain, namely L33P; mutation of lysine (K) at position 319 to glutamic acid (E), namely K319E(ii) a No other functional domains have base mutations. By means of constructing a plant expression vector in a site-specific mutagenesis mode, instantly infiltrating Nicotiana benthamiana leaves by agrobacterium and the like, it is found that Sw-5b and NSm can only be subjected to mutation at three amino acid sites (33 th leucine is mutated into proline, 319 th lysine is mutated into glutamic acid, 927 th arginine is mutated into alanine) simultaneously WT 、NSm C118Y Or NSm T120N An HR reaction occurs. In addition, Sw-5b 33 leucine to proline, 319 lysine to glutamic acid, 927 arginine to glutamine, and NSm WT 、NSm C118Y Or NSm T120N An HR response was generated (FIG. 2) and no immune self-activation was observed.
Example 3: sw-5b L33P/K319E/R927A And Sw-5b L33P/K319E/R927Q Systemic disease resistance of transgenic Oryza glutinosa to TSWV wild type and resistance-breakthrough type strains
Infiltrating TSWV wild type and resistant outbreak type virus infectious clones (rTSWV respectively) on Sw-5b transgenic Benzense tobacco leaf slices WT 、rTSWV C118Y 、rTSWV T120N ) After 10-15 days, the symptoms of the leaves of the Nicotiana benthamiana were observed and the amount of accumulated viral proteins was measured. The results show that rTSWV WT 、rTSWV C118Y And rTSWV T120N Typical symptoms of leaf rolling following viral infection were observed on leaves of the wild type P.benthamiana system and accumulation of viral protein expression could be detected (FIGS. 3 and 4). rTSWV on transgenic Sw-5b Benzen tobacco C118Y And rTSWV T120N Can infect systemic leaves, causing symptoms of leaf rolling and wilting; rTSWV WT Systemic leaves were not infected, symptoms caused by TSWV were not observed on systemic leaves, and expression accumulation of viral proteins was not detected (fig. 3 and 4). In the transgene Sw-5b L33P/K319E/R927A And Sw-5b L33P/K319E/R927Q On Bunshi tobacco, rTSWV WT 、rTSWV C118Y And rTSWV T120N None of the systemic leaves could be infected, no symptoms such as leaf rolling and wilting could be observed on the systemic leaves, and no expression and accumulation of viral proteins could be detected (fig. 3 and 4). In summary, Sw-5b L33P/K319E/R927A And Sw-5b L33P /K319E/R927Q For rTSWV WT 、rTSWV C118Y And rTSWV T120N Has systemic disease resistance.
Sequence listing
<110> Nanjing university of agriculture
Artificial improved mutant of <120> disease-resistant gene Sw-5b and application thereof
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Lys Ser Gly Gly Asp Leu Asp Trp Leu Asp Ile Leu Arg Ile Glu Glu
20 25 30
Pro Glu Met Val Leu Arg Val Phe Arg Thr Phe Thr Lys Tyr Asn Asp
35 40 45
Val Leu Leu Pro Asp Ser Leu Val Glu Leu Thr Lys Arg Ala Lys Leu
50 55 60
Ile Gly Glu Ile Leu His Arg Leu Phe Gly Arg Ile Pro His Lys Cys
65 70 75 80
Lys Thr Asn Leu Asn Leu Glu Arg Leu Glu Ser His Leu Leu Glu Phe
85 90 95
Phe Gln Gly Asn Thr Ala Ser Leu Ser His Asn Tyr Glu Leu Asn Asn
100 105 110
Phe Asp Leu Ser Lys Tyr Met Asp Cys Leu Glu Asn Phe Leu Asn Asp
115 120 125
Val Leu Met Met Phe Leu Gln Lys Asp Arg Phe Phe His Ser Arg Glu
130 135 140
Gln Leu Ala Lys His Arg Ser Ile Lys Glu Leu Lys Ile Val Gln Lys
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Lys Ile Arg Phe Leu Lys Tyr Ile Tyr Ala Thr Glu Ile Asn Gly Tyr
165 170 175
Val Asp Tyr Glu Lys Gln Glu Cys Leu Glu Asn Arg Ile Gln Phe Met
180 185 190
Thr Asn Thr Val Gly Gln Tyr Cys Leu Ala Val Leu Asp Tyr Val Thr
195 200 205
Glu Gly Lys Leu Asn Glu Glu Asn Asp Asn Phe Ser Lys Pro Pro Tyr
210 215 220
Leu Leu Ser Leu Ile Val Leu Val Glu Leu Glu Met Lys Lys Ile Phe
225 230 235 240
His Gly Glu Val Lys Ala Ser Lys Phe Thr Gln Ser Lys Thr Phe Lys
245 250 255
Asp Lys Lys Leu Pro Lys Gly Phe Ser His His Leu His Asn Leu Leu
260 265 270
Met Tyr Leu Arg Asn Lys Lys Leu Glu Asn Phe Pro Asn Asn Ile Ala
275 280 285
Ala Gln Asn Ile Asp Val Ala Ile Glu Phe Leu Leu Val Phe Leu Asp
290 295 300
Ala Asp Val Ser Asn His Val Ile Asn Gly Asn Trp Leu Lys Glu Val
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Leu Leu Lys Val Gly Ala Ile Ala Gly Asp Ile Leu Tyr Val Ile Gln
325 330 335
Lys Leu Leu Pro Arg Ser Ile Asn Lys Asp Glu Thr Ser Asn Ile Ser
340 345 350
Leu Cys Ser Ile Gln Ile Leu Glu Lys Thr Lys Asp Leu Lys Ala Gln
355 360 365
Val Glu Thr Tyr Tyr Lys Ser Leu Lys Phe Thr Pro Ser Gln Phe Pro
370 375 380
Thr Phe Gly Gly Leu Ser Phe Leu Asn Ser Leu Leu Arg Lys Leu Asn
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Leu Gly Asn Leu Glu Lys Glu Leu Ser Ser Leu Thr Ser Ile Leu Glu
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Ile Pro Lys Asp Leu Gln Arg Arg Thr Ile Asn Leu Ser Tyr Glu Ala
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Glu Val Ala Ile Asp Ser Ile Leu Ala Gln Tyr Asn Ala Phe Leu His
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Ile Phe Cys Ser Leu Pro Thr Ile Val Lys Glu Ile Lys Gln Ile Asn
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Ala Glu Val Thr Glu Met Trp Ser Ala Asp Ile Pro Leu Asn Pro His
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Tyr Val Ala Ala Pro Leu Lys His Leu Pro Asp Arg His Ser Asn Leu
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Val Thr Asp Glu Glu Val Val Gly Phe Glu Asn Lys Ala Glu Glu Leu
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Ile Asp Tyr Leu Ile Arg Gly Thr Asn Glu Leu Asp Val Val Pro Ile
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Val Gly Met Gly Gly Gln Gly Lys Thr Thr Ile Ala Arg Lys Leu Tyr
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Asn Asn Asp Ile Ile Val Ser Arg Phe Asp Val Arg Ala Trp Cys Ile
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Ile Ser Gln Thr Tyr Asn Arg Arg Glu Leu Leu Gln Asp Ile Phe Ser
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Gln Val Thr Gly Ser Asp Asp Asn Gly Ala Thr Val Asp Val Leu Ala
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Asp Met Leu Arg Arg Lys Leu Met Gly Lys Arg Tyr Leu Ile Val Leu
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Asp Asp Met Trp Asp Cys Met Val Trp Asp Asp Leu Arg Leu Ser Phe
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Pro Asp Asp Gly Ile Arg Ser Arg Ile Val Val Thr Thr Arg Leu Glu
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Glu Val Gly Lys Gln Val Lys Tyr His Thr Asp Pro Tyr Ser Leu Pro
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Phe Leu Thr Thr Glu Glu Ser Cys Gln Leu Leu Gln Lys Lys Val Phe
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Gln Lys Glu Asp Cys Pro Pro Glu Leu Gln Asp Val Ser Gln Ala Val
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Ile Ile Lys Lys Arg Lys Met Glu Glu Ser Trp Trp Asn Glu Val Lys
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Asp Ala Leu Phe Asp Tyr Leu Asp Ser Glu Phe Glu Glu Tyr Ser Leu
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Ala Thr Met Gln Leu Ser Phe Asp Asn Leu Pro His Cys Leu Lys Pro
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Cys Leu Leu Tyr Met Gly Met Phe Ser Glu Asp Ala Arg Ile Pro Ala
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Ser Thr Leu Ile Ser Leu Trp Ile Ala Glu Gly Phe Val Glu Asn Thr
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Glu Ser Gly Arg Leu Met Glu Glu Glu Ala Glu Gly Tyr Leu Met Asp
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Leu Ile Ser Ser Asn Leu Val Met Leu Ser Lys Arg Thr Tyr Lys Gly
835 840 845
Arg Val Lys Tyr Cys Gln Val His Asp Val Val His His Phe Cys Leu
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Glu Lys Ser Arg Glu Ala Lys Phe Met Leu Ala Val Lys Gly Gln Tyr
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Ile His Phe Gln Pro Ser Asp Trp Lys Gly Thr Arg Val Ser Phe Ser
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Phe Ser Glu Glu Leu Ser Lys Phe Ala Ser Leu Val Ser Lys Thr Gln
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Lys Pro Phe His Gln His Leu Arg Ser Leu Ile Thr Thr Asn Ala Ala
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Lys Ser Ile Asn Asp Ile Phe Ser Cys Gln Ile Ser Glu Leu Arg Leu
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Leu Lys Val Leu Asp Leu Ser Ser Tyr Ile Val Glu Phe Leu Ser Leu
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Ala Thr Phe Lys Pro Leu Asn Gln Leu Lys Tyr Leu Ala Val Gln Ala
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Phe Glu Phe Tyr Phe Asp Pro Gly Ser His Leu Pro His Ile Glu Thr
980 985 990
Phe Ile Val Met Asn Leu Pro Tyr Tyr Asp Ile Leu Leu Pro Val Ser
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Phe Trp Glu Met Lys Lys Leu Arg His Ala His Phe Gly Lys Ala Glu
1010 1015 1020
Phe Asp Lys Gln Gly Leu Ser Glu Gly Ser Ser Lys Leu Glu Asn Leu
1025 1030 1035 1040
Arg Ile Leu Lys Asn Ile Val Gly Phe Asp Arg Val Asp Val Leu Ser
1045 1050 1055
Arg Arg Cys Pro Asn Leu Gln Gln Leu Gln Ile Thr Tyr Phe Gly Asn
1060 1065 1070
Asn Glu Glu Pro Phe Cys Pro Lys Leu Glu Asn Leu Thr Gln Leu Gln
1075 1080 1085
Gln Leu Gln Leu Ser Phe Ala Arg Pro Arg Thr Leu Ser Gly Leu Gln
1090 1095 1100
Leu Pro Ser Asn Leu Asn Lys Leu Val Leu Glu Gly Ile His Ile Gly
1105 1110 1115 1120
Cys Val Ile Pro Phe Ile Ala Gly Leu Pro Ser Leu Glu Tyr Leu Gln
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Leu His Asp Val Cys Phe Pro Gln Ser Glu Glu Trp Cys Leu Gly Asp
1140 1145 1150
Ile Thr Phe His Lys Leu Lys Leu Leu Lys Leu Val Lys Leu Asn Ile
1155 1160 1165
Ser Arg Trp Asp Val Ser Glu Glu Ser Phe Pro Leu Leu Glu Thr Leu
1170 1175 1180
Val Ile Lys Lys Cys Ile Asp Leu Glu Glu Ile Pro Leu Ser Phe Ala
1185 1190 1195 1200
Asp Ile Pro Thr Leu Glu Gln Ile Lys Leu Ile Gly Ser Trp Lys Val
1205 1210 1215
Ser Leu Glu Asp Ser Ala Val Arg Met Lys Glu Glu Ile Lys Asp Thr
1220 1225 1230
Glu Gly Cys Asp Arg Leu His Leu Val Lys Gln Arg Ser Asp
1235 1240 1245
<210> 2
<211> 1246
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Ala Glu Asn Glu Ile Glu Glu Met Leu Glu His Leu Arg Arg Ile
1 5 10 15
Lys Ser Gly Gly Asp Leu Asp Trp Leu Asp Ile Leu Arg Ile Glu Glu
20 25 30
Pro Glu Met Val Leu Arg Val Phe Arg Thr Phe Thr Lys Tyr Asn Asp
35 40 45
Val Leu Leu Pro Asp Ser Leu Val Glu Leu Thr Lys Arg Ala Lys Leu
50 55 60
Ile Gly Glu Ile Leu His Arg Leu Phe Gly Arg Ile Pro His Lys Cys
65 70 75 80
Lys Thr Asn Leu Asn Leu Glu Arg Leu Glu Ser His Leu Leu Glu Phe
85 90 95
Phe Gln Gly Asn Thr Ala Ser Leu Ser His Asn Tyr Glu Leu Asn Asn
100 105 110
Phe Asp Leu Ser Lys Tyr Met Asp Cys Leu Glu Asn Phe Leu Asn Asp
115 120 125
Val Leu Met Met Phe Leu Gln Lys Asp Arg Phe Phe His Ser Arg Glu
130 135 140
Gln Leu Ala Lys His Arg Ser Ile Lys Glu Leu Lys Ile Val Gln Lys
145 150 155 160
Lys Ile Arg Phe Leu Lys Tyr Ile Tyr Ala Thr Glu Ile Asn Gly Tyr
165 170 175
Val Asp Tyr Glu Lys Gln Glu Cys Leu Glu Asn Arg Ile Gln Phe Met
180 185 190
Thr Asn Thr Val Gly Gln Tyr Cys Leu Ala Val Leu Asp Tyr Val Thr
195 200 205
Glu Gly Lys Leu Asn Glu Glu Asn Asp Asn Phe Ser Lys Pro Pro Tyr
210 215 220
Leu Leu Ser Leu Ile Val Leu Val Glu Leu Glu Met Lys Lys Ile Phe
225 230 235 240
His Gly Glu Val Lys Ala Ser Lys Phe Thr Gln Ser Lys Thr Phe Lys
245 250 255
Asp Lys Lys Leu Pro Lys Gly Phe Ser His His Leu His Asn Leu Leu
260 265 270
Met Tyr Leu Arg Asn Lys Lys Leu Glu Asn Phe Pro Asn Asn Ile Ala
275 280 285
Ala Gln Asn Ile Asp Val Ala Ile Glu Phe Leu Leu Val Phe Leu Asp
290 295 300
Ala Asp Val Ser Asn His Val Ile Asn Gly Asn Trp Leu Lys Glu Val
305 310 315 320
Leu Leu Lys Val Gly Ala Ile Ala Gly Asp Ile Leu Tyr Val Ile Gln
325 330 335
Lys Leu Leu Pro Arg Ser Ile Asn Lys Asp Glu Thr Ser Asn Ile Ser
340 345 350
Leu Cys Ser Ile Gln Ile Leu Glu Lys Thr Lys Asp Leu Lys Ala Gln
355 360 365
Val Glu Thr Tyr Tyr Lys Ser Leu Lys Phe Thr Pro Ser Gln Phe Pro
370 375 380
Thr Phe Gly Gly Leu Ser Phe Leu Asn Ser Leu Leu Arg Lys Leu Asn
385 390 395 400
Glu Met Ser Thr Ser Lys Ser Gly Leu Gly Phe Leu Met Lys Pro Leu
405 410 415
Leu Gly Asn Leu Glu Lys Glu Leu Ser Ser Leu Thr Ser Ile Leu Glu
420 425 430
Lys Glu Leu Ser Ser Ile Phe Arg Asp Val Val His His Glu His Asn
435 440 445
Ile Pro Lys Asp Leu Gln Arg Arg Thr Ile Asn Leu Ser Tyr Glu Ala
450 455 460
Glu Val Ala Ile Asp Ser Ile Leu Ala Gln Tyr Asn Ala Phe Leu His
465 470 475 480
Ile Phe Cys Ser Leu Pro Thr Ile Val Lys Glu Ile Lys Gln Ile Asn
485 490 495
Ala Glu Val Thr Glu Met Trp Ser Ala Asp Ile Pro Leu Asn Pro His
500 505 510
Tyr Val Ala Ala Pro Leu Lys His Leu Pro Asp Arg His Ser Asn Leu
515 520 525
Val Thr Asp Glu Glu Val Val Gly Phe Glu Asn Lys Ala Glu Glu Leu
530 535 540
Ile Asp Tyr Leu Ile Arg Gly Thr Asn Glu Leu Asp Val Val Pro Ile
545 550 555 560
Val Gly Met Gly Gly Gln Gly Lys Thr Thr Ile Ala Arg Lys Leu Tyr
565 570 575
Asn Asn Asp Ile Ile Val Ser Arg Phe Asp Val Arg Ala Trp Cys Ile
580 585 590
Ile Ser Gln Thr Tyr Asn Arg Arg Glu Leu Leu Gln Asp Ile Phe Ser
595 600 605
Gln Val Thr Gly Ser Asp Asp Asn Gly Ala Thr Val Asp Val Leu Ala
610 615 620
Asp Met Leu Arg Arg Lys Leu Met Gly Lys Arg Tyr Leu Ile Val Leu
625 630 635 640
Asp Asp Met Trp Asp Cys Met Val Trp Asp Asp Leu Arg Leu Ser Phe
645 650 655
Pro Asp Asp Gly Ile Arg Ser Arg Ile Val Val Thr Thr Arg Leu Glu
660 665 670
Glu Val Gly Lys Gln Val Lys Tyr His Thr Asp Pro Tyr Ser Leu Pro
675 680 685
Phe Leu Thr Thr Glu Glu Ser Cys Gln Leu Leu Gln Lys Lys Val Phe
690 695 700
Gln Lys Glu Asp Cys Pro Pro Glu Leu Gln Asp Val Ser Gln Ala Val
705 710 715 720
Ala Glu Lys Cys Lys Gly Leu Pro Leu Val Val Val Leu Val Ala Gly
725 730 735
Ile Ile Lys Lys Arg Lys Met Glu Glu Ser Trp Trp Asn Glu Val Lys
740 745 750
Asp Ala Leu Phe Asp Tyr Leu Asp Ser Glu Phe Glu Glu Tyr Ser Leu
755 760 765
Ala Thr Met Gln Leu Ser Phe Asp Asn Leu Pro His Cys Leu Lys Pro
770 775 780
Cys Leu Leu Tyr Met Gly Met Phe Ser Glu Asp Ala Arg Ile Pro Ala
785 790 795 800
Ser Thr Leu Ile Ser Leu Trp Ile Ala Glu Gly Phe Val Glu Asn Thr
805 810 815
Glu Ser Gly Arg Leu Met Glu Glu Glu Ala Glu Gly Tyr Leu Met Asp
820 825 830
Leu Ile Ser Ser Asn Leu Val Met Leu Ser Lys Arg Thr Tyr Lys Gly
835 840 845
Arg Val Lys Tyr Cys Gln Val His Asp Val Val His His Phe Cys Leu
850 855 860
Glu Lys Ser Arg Glu Ala Lys Phe Met Leu Ala Val Lys Gly Gln Tyr
865 870 875 880
Ile His Phe Gln Pro Ser Asp Trp Lys Gly Thr Arg Val Ser Phe Ser
885 890 895
Phe Ser Glu Glu Leu Ser Lys Phe Ala Ser Leu Val Ser Lys Thr Gln
900 905 910
Lys Pro Phe His Gln His Leu Arg Ser Leu Ile Thr Thr Asn Gln Ala
915 920 925
Lys Ser Ile Asn Asp Ile Phe Ser Cys Gln Ile Ser Glu Leu Arg Leu
930 935 940
Leu Lys Val Leu Asp Leu Ser Ser Tyr Ile Val Glu Phe Leu Ser Leu
945 950 955 960
Ala Thr Phe Lys Pro Leu Asn Gln Leu Lys Tyr Leu Ala Val Gln Ala
965 970 975
Phe Glu Phe Tyr Phe Asp Pro Gly Ser His Leu Pro His Ile Glu Thr
980 985 990
Phe Ile Val Met Asn Leu Pro Tyr Tyr Asp Ile Leu Leu Pro Val Ser
995 1000 1005
Phe Trp Glu Met Lys Lys Leu Arg His Ala His Phe Gly Lys Ala Glu
1010 1015 1020
Phe Asp Lys Gln Gly Leu Ser Glu Gly Ser Ser Lys Leu Glu Asn Leu
1025 1030 1035 1040
Arg Ile Leu Lys Asn Ile Val Gly Phe Asp Arg Val Asp Val Leu Ser
1045 1050 1055
Arg Arg Cys Pro Asn Leu Gln Gln Leu Gln Ile Thr Tyr Phe Gly Asn
1060 1065 1070
Asn Glu Glu Pro Phe Cys Pro Lys Leu Glu Asn Leu Thr Gln Leu Gln
1075 1080 1085
Gln Leu Gln Leu Ser Phe Ala Arg Pro Arg Thr Leu Ser Gly Leu Gln
1090 1095 1100
Leu Pro Ser Asn Leu Asn Lys Leu Val Leu Glu Gly Ile His Ile Gly
1105 1110 1115 1120
Cys Val Ile Pro Phe Ile Ala Gly Leu Pro Ser Leu Glu Tyr Leu Gln
1125 1130 1135
Leu His Asp Val Cys Phe Pro Gln Ser Glu Glu Trp Cys Leu Gly Asp
1140 1145 1150
Ile Thr Phe His Lys Leu Lys Leu Leu Lys Leu Val Lys Leu Asn Ile
1155 1160 1165
Ser Arg Trp Asp Val Ser Glu Glu Ser Phe Pro Leu Leu Glu Thr Leu
1170 1175 1180
Val Ile Lys Lys Cys Ile Asp Leu Glu Glu Ile Pro Leu Ser Phe Ala
1185 1190 1195 1200
Asp Ile Pro Thr Leu Glu Gln Ile Lys Leu Ile Gly Ser Trp Lys Val
1205 1210 1215
Ser Leu Glu Asp Ser Ala Val Arg Met Lys Glu Glu Ile Lys Asp Thr
1220 1225 1230
Glu Gly Cys Asp Arg Leu His Leu Val Lys Gln Arg Ser Asp
1235 1240 1245
<210> 3
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atggctgaaa atgaaattga ggaaatgtta 30
<210> 4
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggggaactga gatggagtaa attttaagga 30
<210> 5
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tccttaaaat ttactccatc tcagttcccc 30
<210> 6
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
taacatttcc tcaatttcat tttcagccat 30

Claims (6)

1. The artificial improved mutant of the disease-resistant gene Sw-5b is characterized in that 33 th leucine is mutated into proline, 319 th lysine is mutated into glutamic acid, 927 th arginine is mutated into alanine or glutamine, the rest amino acid sites are kept unchanged, and three amino acid sites are simultaneously mutated, wherein the amino acid sequence of the artificial improved mutant of the disease-resistant gene Sw-5b is shown as SEQ ID No.1 or SEQ ID No. 2.
2. A coding gene encoding the mutant of claim 1.
3. A vector comprising the gene encoding the gene of claim 2.
4. A recombinant bacterium or a recombinant cell comprising the vector of claim 3.
5. Use of the artificially improved mutant of the disease-resistant gene Sw-5b as claimed in claim 1 in culturing TSWV-resistant wild type TSWV C118Y And TSWV T120N Application in plants.
6. Use according to claim 5, wherein the plant is tomato.
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CN113416736B true CN113416736B (en) 2022-08-05

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160319300A1 (en) * 2013-12-20 2016-11-03 ISI Sementi s.p.a. Isolated nucleotide sequence from solanum lycopersicum for improved resistance to tomato spotted wilt virus, tswv.

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