CN107699572A - A kind of identification and application of tomato SolyWRKY54 transcription factors regulation and control tomato yellow leaf curl virus - Google Patents

A kind of identification and application of tomato SolyWRKY54 transcription factors regulation and control tomato yellow leaf curl virus Download PDF

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CN107699572A
CN107699572A CN201610628562.XA CN201610628562A CN107699572A CN 107699572 A CN107699572 A CN 107699572A CN 201610628562 A CN201610628562 A CN 201610628562A CN 107699572 A CN107699572 A CN 107699572A
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tomato
solywrky54
leaf curl
yellow leaf
curl virus
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CN107699572B (en
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熊爱生
黄莹
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Nanjing Agricultural University
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Abstract

The present invention is entitled ' a kind of identification and application of tomato SolyWRKY54 transcription factors regulation and control tomato yellow leaf curl virus '.The present invention identifies the WRKY transcription factor gene SolyWRKY54 of a response tomato yellow leaf curl virus, the Group III subtribe that the albumen of the gene code belongs in WRKY transcription factor families from tomato.Semiquantitive PCR result shows that viral accumulation is higher than disease-resistant variety ' Zhejiang miscellaneous 301 ' in susceptible variety ' golden canopy 1 '.In disease-resistant tomato variety, ' in Zhejiang miscellaneous 301 ', the expression quantity of SolyWRKY54 genes reduces quantitative fluorescent PCR analysis shows.Gene Silencing experiment further demonstrates negative regulation function of the SolyWRKY54 genes during tomato yellow leaf curl virus is responded.The SolyWRKY54 transcription factor genes that the present invention identifies are advantageous to the resistance mechanism of analysis and research tomato response tomato yellow leaf curl virus, the cultivation available for disease-resistant tomato.

Description

A kind of identification of tomato SolyWRKY54 transcription factors regulation and control tomato yellow leaf curl virus And application
Technical field
The invention belongs to plant genetic engineering field, a WRKY Group III transcription factor being related in tomato SolyWRKY54 functional study and application.SolyWRKY54 genes respond tomato yellow leaf curl virus in different tomato varieties Pattern respond tomato yellow leaf curl virus for further further investigation WRKY transcription factors and provide foundation.
Background technology
Tomato (Solanum lycopersicum L.), also known as tomato, it is western originating from South America, belong to annual Herbaceous plant.Tomato is widely used in various scientific researches, such as breeding, Fruit development, degeneration-resistant defence regulation (Cuartero et al., J Exp Bot 2006,57：1045-1058；Kennedy et al., Annu Rev Entomology 2003,48：51-72；Klee et al., Annu Rev Genet 2011,45：41-59；Schauer et Al., Plant Cell 2008,20：509-523).
Tomato yellow leaf curl virus (tomato yellow leafcurly virus, TYLCV) is a kind of Genome Size For 2.7~2.8kb single stranded circle DNA virus (Regenmortel et al., Arch Virol 2000,145：2227- 2232).TYLCV mainly infects dicotyledon, such as tomato, tobacco, cotton (Jones et al., Eur J by Bemisia tabaci Plant Pathol 2003,109：195-219).Plant infection TYLCV symptoms are obvious, can cause whole yellowing leaf, Atrophy, fold, fructescence extend, and have a strong impact on yield and quality (Glick et al., Plant Protect Sci 2009,45：81-97).
WRKY transcription factors because WRKY domains (WRKYGQK) are contained at its N- end and name (Eulgem et al., Trends Plant Sci 2000,5：199-206).WRKY transcription factors are identified in various plants, such as Chinese cabbage, are intended (Tang et al., the Plant Mol Biol Rep 2014,32 such as southern mustard, tea tree, rice, willow：781-795；Wu et Al., Mol Genet Genomics 2016,291：255-269；Wang et al., Biol Direct 2015,10：1- 27).Compared with I, II class subtribe, WRKY Group III transcription factor C-terminals contain C2HC(C-X7-C-X23-H-X1- C) zinc finger knot Structure.The expression of WRKY Group III transcription factor genes is influenceed by various environmental conditions, such as pathogen, environment stress, aging (Mao et al., Plant Cell Physiol 2007,48：833-842；Zhou et al., Plant Physiol Bioch 2015,96：311-320；Besseau et al., J Exp Bot 2012,63：2667-2679).
The content of the invention
Present invention obtains a kind of tomato SolyWRKY54 transcription factor genes for responding tomato yellow leaf curl virus.Tomato The functional study and application of SolyWRKY54 genes, the defense mechanism that TYLCV is responded for further further investigation tomato provide reason By basis.
Brief description of the drawings
The phylogenetic analysis of Fig. 1 tomato WRKY families, square frame mark for tomato SolyWRKY54 transcription factors.
Fig. 2 difference resistant tomato kinds TYLCV virus accumulation assays.
Fig. 3 tomato SolyWRKY54 genes respond TYLCV expression analysis in different tomato varieties.
Fig. 4 Gene Silencings verify tomato SolyWRKY54 gene functions.
Embodiment
With reference to specific embodiment, the invention will be further described, and specific experiment step is as follows：
1. the clone of tomato transcription factor gene SolyWRKY54 genes
Utilize RNA simple Total RNA Kit (Tiangeng biochemical technology Co., Ltd, Beijing) and Prime Script ' RNA of the miscellaneous 301 ' blade in Zhejiang is simultaneously inverted to RT reagent Kit (precious bioengineering Co., Ltd, Dalian) extractions tomato cDNA.So that ' the miscellaneous 301 ' cDNA in Zhejiang is (positive according to special primer as template：5 '-ATGGATTGTGGATTCAATTATGAAT-3 ', Reverse 5 '-TTATCTGAAAAAATCAGAGAAATTTG-3 ') expanded.PCR reaction conditions are：94 DEG C of pre-degeneration 5min；94 DEG C denaturation 30s, 54 DEG C annealing 30s, 72 DEG C extension 1min 10s, totally 35 circulation；72 DEG C of extension 10min.Purpose band is used The agarose gel electrophoresis of quality volume fraction 1.2% is reclaimed, and connection pMD18-T carriers (precious bioengineering Co., Ltd, greatly Even), bacillus coli DH 5 alpha is converted, extraction plasmid picking positive colony after PCR is detected send Nanjing Jin Sirui biotechnologies limited Company is sequenced.
2. different tomato variety TYLCV viral levels measure
According to TYLCV genome sequences (NCBI accession number：AM282874.1), design a pair of detection primers and be used for sxemiquantitative PCR, it is positive：5 '-ATGTCGAAGCGACCAGGCGATATAAT-3 ', reverse 5 '-TTAATTTGATATTGAATCATAGAAAT- 3’.' Zhejiang miscellaneous 301 ' (vegetables institute of academy of agricultural sciences of Zhejiang Province, Hangzhou) and the sense of extraction control and infection TYLCV 7d disease-resistant tomato variety Sick tomato variety ' golden canopy 1 ' (Seed Co., Ltd. of roc of money of Xi'an, Xi'an) leaf DNA.Tomato Tubulin genes are as reference Gene, detect different tomato variety TYLCV viral levels (Chen et al., PLoS One 2013,8：e80816).
3. real-time quantitative PCR reacts
The Bemisia tabaci for carrying TYLCV is raised in insect prevention greenhouse.By long ' Zhejiang miscellaneous 301 ' and ' golden canopy 1 ' to 2 leaf states Tomato seedling, which is put in insect prevention greenhouse, infects TYLCV 2,4,6,8,10d, takes blade sample to be used to extract RNA.Quantitative fluorescent PCR Completed using SYBR Premix Ex Taq kit (precious bioengineering Co., Ltd, Dalian) by operating instruction.According to SolyWRKY54 gene orders design specific primer and carry out quantitative fluorescent PCR.Primer：5’- CCACAGAAACATAAACACAAGAAAC-3 ', reverse 5 '-AGGAGAGACAAAAGATGGTGAATAA-3 '.
4. Gene Silencing and TYLCV assays
By the SolyWRKY54 genetic fragments expanded be building up to pTRV2 carriers (Liu et al., Plant J2002, 30：415-429).By recombinant plasmid pTRV2-SolyWRKY54, pTRV2-PDS (phytoene dehydrogenase) and pTRV1, PTRV2 plasmids import Agrobacterium GV3101 (Beijing day bounties Gene Tech. Company Limited, Beijing).Bacterium solution is injected into ' Zhejiang is miscellaneous In 301 ' cotyledons, after 7d, control group (pTRV1+pTRV2) and treatment group tomato leaf is taken to freeze in liquid nitrogen and be stored in -80 DEG C of ice In case.By the tomato seedling inoculation TYLCV viruses after silence, sampling extraction DNA, detects viral level after one week.Design is special Primer, TYLCV expressing viral detection primers are detected, it is positive：5 '-AGTACCGCAACTGTGAAGAATGATT-3 ', reverse 5 '- CATACTTGGCTGCCTCCTGATGAT-3’。
5. result of the test：
1) after being inoculated with TYLCV 7d, viral level is higher than disease-resistant variety ' Zhejiang miscellaneous 301 ' in susceptible variety ' golden canopy 1 ' blade (Fig. 1).
2) the tomato SolyWRKY54 transcription factors that the present invention analyzes and identifies belong to WRKY Group IIIs subtribe (Fig. 2).
3) fluorescent quantitative PCR result shows tomato SolyWRKY54 response TYLCV infection.In disease-resistant variety ' Zhejiang miscellaneous 301 ' In, SolyWRKY54 expression quantity reduces.In susceptible variety ' golden canopy 1 ', SolyWRKY54 expression, which is presented, first rises what is declined afterwards Trend, when handling 4d, expression quantity reaches peak value, and expression quantity adds 1.7 times (Fig. 3).
4) Gene Silencing shows, tomato SolyWRKY54 negative regulations TYLCV infection.Compared with control group, SolyWRKY54 expression quantity after silence reduces by 50%, and viral level is then 10% (Fig. 4) of control group.

Claims (4)

  1. A kind of 1. WRKY Group III transcription factor genes SolyWRKY54 obtained in tomato.
  2. 2. the nucleotide sequence of the tomato WRKY Group III transcription factor genes SolyWRKY54 described in claim 1.
  3. 3. the functional study of the tomato WRKY Group III transcription factor genes SolyWRKY54 described in claim 1；
    1) method for using semiquantitive PCR, shows that viral level is higher than disease-resistant tomato variety in susceptible tomato variety ' golden canopy 1 ' ' Zhejiang miscellaneous 301 '；
    2) fluorescence quantifying PCR method is used, it was demonstrated that tomato WRKY Group III transcription factor gene SolyWRKY54 are in different tomatoes The pattern that tomato yellow leaf curl virus is responded in kind is different；
    3) tomato SolyWRKY54 gene response tomato yellow leaf curl virus is further verified by Gene Silencing Function.
  4. 4. the application of the tomato WRKY Group III transcription factor genes SolyWRKY54 described in claim 1.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109456979A (en) * 2018-11-05 2019-03-12 浙江大学 Regulatable tomato genic male sterile line and its creation method and application are created using SlPIF3 gene
CN112430261A (en) * 2020-11-24 2021-03-02 上海交通大学 WRKY32 regulation and control YFT1 expression to influence tomato fruit color and application thereof in tomato quality improvement
CN113061614A (en) * 2021-03-30 2021-07-02 四川大学 Application of tomato SlWRKY35 gene in improving content of carotenoid compounds or/and chlorophyll in tomatoes
CN113151305A (en) * 2021-06-01 2021-07-23 安徽农业大学 Tea tree WRKY29 gene and application thereof in improving cold resistance of plants
CN115960916A (en) * 2022-10-13 2023-04-14 安徽农业大学 Tea tree WRKY transcription factor gene and cold-resistant application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101173002A (en) * 2007-10-29 2008-05-07 中国科学院遗传与发育生物学研究所 Plants stress tolerance correlation transcription factor GmWRKY54, encoding gene and application thereof
CN102344487A (en) * 2011-10-18 2012-02-08 中国科学院植物研究所 Plant resistance-related protein ATWRKY46 as well as encoding gene and application thereof
CN102816773A (en) * 2012-08-30 2012-12-12 中山大学 Application of OsWRKY28 transcription factor gene of rice in improvement of plant disease resistance
CN105543274A (en) * 2016-01-19 2016-05-04 浙江省农业科学院 Application of solanum lycopersicum SlWRKY81 gene

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101173002A (en) * 2007-10-29 2008-05-07 中国科学院遗传与发育生物学研究所 Plants stress tolerance correlation transcription factor GmWRKY54, encoding gene and application thereof
CN102344487A (en) * 2011-10-18 2012-02-08 中国科学院植物研究所 Plant resistance-related protein ATWRKY46 as well as encoding gene and application thereof
CN102816773A (en) * 2012-08-30 2012-12-12 中山大学 Application of OsWRKY28 transcription factor gene of rice in improvement of plant disease resistance
CN105543274A (en) * 2016-01-19 2016-05-04 浙江省农业科学院 Application of solanum lycopersicum SlWRKY81 gene

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GENBANK: "NCBI Reference Sequence: XM_004245482.2", 《GENBANK》 *
TIANZI CHEN等: "Comparative Transcriptome Profiling of a Resistant vs. Susceptible Tomato (Solanum Lycopersicum) Cultivar in Response to Infection by Tomato Yellow Leaf Curl Virus", 《PLOS ONE》 *
YING HUANG等: "Members of WRKY Group III Transcription Factors Are Important in TYLCV Defense Signaling Pathway in Tomato (Solanum Lycopersicum)", 《BMC GENOMICS》 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109456979A (en) * 2018-11-05 2019-03-12 浙江大学 Regulatable tomato genic male sterile line and its creation method and application are created using SlPIF3 gene
CN112430261A (en) * 2020-11-24 2021-03-02 上海交通大学 WRKY32 regulation and control YFT1 expression to influence tomato fruit color and application thereof in tomato quality improvement
CN112430261B (en) * 2020-11-24 2022-06-28 上海交通大学 WRKY32 regulation and control YFT1 expression to influence tomato fruit color and application thereof in tomato quality improvement
CN113061614A (en) * 2021-03-30 2021-07-02 四川大学 Application of tomato SlWRKY35 gene in improving content of carotenoid compounds or/and chlorophyll in tomatoes
CN113061614B (en) * 2021-03-30 2023-04-28 四川大学 Application of tomato SlWRKY35 gene in improving carotenoid compound or/and chlorophyll content of tomatoes
CN113151305A (en) * 2021-06-01 2021-07-23 安徽农业大学 Tea tree WRKY29 gene and application thereof in improving cold resistance of plants
CN115960916A (en) * 2022-10-13 2023-04-14 安徽农业大学 Tea tree WRKY transcription factor gene and cold-resistant application thereof

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