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Acta Agron Sin ›› 2016, Vol. 42 ›› Issue (12): 1779-1786.doi: 10.3724/SP.J.1006.2016.01779

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Expression Analysis of a Functional Gene GbVWR Induced by Verticillium dahliae in Gossypium barbadense

ZHANG Li-Jia**,ZHANG Yan**,RONG Wei,YANG Jun,ZHANG Gui-Yin,WU Li-Qiang,LI Zhi-Kun,WU Jin-Hua,MA Zhi-Ying,WANG Xing-Fen*   

  1. North China Key Laboratory for Crop Germplasm Resources of Education Ministry / Key Laboratory for Crop Germplasm Resources of Hebei / Agricultural University of Hebei, Baoding 071001, China
  • Received:2016-03-14 Revised:2016-06-20 Online:2016-12-12 Published:2016-06-27
  • Contact: Wang Shengfen, E-mail: cotton@hebau.edu.cn E-mail:646560324@qq.com
  • Supported by:

    This study was supported by the Natural Science Foundation of Hebei Province (C2013204141), the Special Research Found for the Doctoral Program of Higher Education (20131302120002), and the Science and Technology Support Project of Hebei Province (14226308D).

Abstract:

Verticillium dahliae is a destructive, soil-borne fungal pathogen that causes severe losses in cotton yield and fiber quality. Mining functional genes related to resistance against V. dahliae will benefit efforts to genetically improve crop plants. In this study, we identified a gene that involved in cotton defense against V. dahliae based on screening the full-length cDNA library and suppression subtractive hybridization library (SSH) induced by V. dahliae in Gossypium barbadense and Gossypium. hirsutum, respectively. Sequence analysis indicated that there was no any annotation in NCBI database, and we named the sequence from G. barbadense as GbVWR. We characterized GbVWR gene and analyzed its expression. The full length cDNA of GbVWR was 520 bp including a 198 bp open reading frame (ORF), encoding 65 amino acid residues. Bioinformatic analyses suggested that GbVWR belonged to secretory protein and tis theoretical isoelectric point was 5.32. Using pET-32a(+) as a fused expression vector, a recombinant plasmid pET32a-GbVWR was constructed. The recombinant protein was induced in Escherichia coli BL21 (DE3) with 1.0 mmol L–1 IPTG then GbVWR could express about 7 kD protein in E. coli BL21 (DE3). In addition, diverse cis-acting promoter elements involved in fungal elicitor response, hormone response, wound-response, and flavonoid biosynthetic gene regulation were discovered in the promoter region of GbVWR. qPCR analysis showed that expression level of GbVWR was the highest in roots, and was significantly induced by V. dahliae. Besides, GbVWR could also be induced by SA, ET and GA treatments, respectively. In conclusion, GbVWR is a new functional gene, which involved in multiple signal pathways in cotton defense response to Verticillium wilt.

Key words: Gossypium barbadense, Verticillium wilt resistance, GbVWR, Clone, Gene expression

[1]Zhang J F, Fang H, Zhou H P, Sanogo S, Ma Z Y. Genetics, breeding, and marker-assisted selection for Verticillium wilt resistance in cotton. Crop Sci, 2014, 54: 1289–1303
[2]Blasingame D, Patel M V. Cotton disease loss estimate committee report. Proc, Beltwide Cotton Conf. National Cotton Council, Memphis, TN. 2011. pp 306–308
[3]Zhang J F, Sanogo S, Flynn R, Baral J B, Bajaj S, Hughs S E, Percy R G. Germplasm evaluation and transfer of Verticillium wilt resistance from Pima (Gossypium barbadense) to upland cotton (G. hirsutum). Euphytica, 2012, 187: 147–160
[4]徐理, 朱龙付, 张献龙. 棉花抗黄萎病机制研究进展. 作物学报, 2012, 38: 1553–1560
Xu L, Zhu L F, Zhang X L. Research on Resistance Mechanism of Cotton to Verticillium wilt. Acta Agron Sin, 2012, 38: 1553−1560 (in Chinese with English abstract)
[5]Sanogo S, Zhang J F. Resistance sources, resistance screening techniques and disease management for Fusarium wilt in cotton. Euphytica, 2016, 207: 255–271
[6]Wilhelm S, Sagen J E, Tietz H. Resistance to Verticillium wilt in cotton: sources, techniques of identification, inheritance trends, and the resistance potential of multilinecultivars. Phytopathology, 1974, 64: 924–931
[7]Bell A A. Verticillium wilt. In: Hillocks R J ed. Cotton Diseases. Wallingford, UK: CAB Intern, 1992. pp 87–126
[8]Bell AA. Verticillium wilt. in: Kirkpatrick T L, Rothrock C S, eds. Compendium of Cotton Diseases. Amer. St. Paul, MN: Phyto-pathol. Soc, 2001. pp 28–31
[9]Cai Y F, He X H, Mo J C, Sun Q, Yang J P, Liu J G. Molecular research and genetic engineering of resistance to Verticillium wilt in cotton. Afr J Biotechnol, 2009, 8: 7363–7372
[10]Wendel J F, Cronn R C. Polyploidy and the evolutionary history of cotton. Adv Agron, 2003, 78: 139–186
[11]Zhang J F, Sanogo S, Flynn R, Baral J B, Bajaj S, Hughs S E. Germplasm evaluation and transfer of Verticillium wilt resistance from Pima (Gossypium barbadense) to Upland cotton (G. hirsutum). Euphytica, 2012, 187: 147–160
[12]Zhou H P, Fang H, Sanogo S, Hughs S E, Jones D C, Zhang J F. Evaluation of Verticillium wilt resistance in commercial cultivars and advanced breeding lines of cotton. Euphytica, 2014, 196: 437–448
[13]Zhang J F, Percy R G, McCarty J J. Introgression genetics and breeding between Upland and Pima cotton-a review. Euphytica, 2014, 198: 1–12
[14]Zhang Y, Wang X F, Yang S, Chi J N, Zhang GY, Ma Z Y. Cloning and characterization of a Verticillium wilt resistance gene from Gossypium barbadense and functional analysis in Arabidopsis thaliana. Plant Cell Rep, 2011, 30: 2085–2096
[15]Zhang B L, Yang Y W, Chen T Z, Yu W G, Liu T L, Li H J, Fan X H, Ren Y Z, Shen D Y, Liu L, Duo D L, Chang Y H. Island cotton Gbve1 gene encoding a receptor-like protein confers resistance to both defoliating and non-defoliating isolates of Verticillium dahliae. PLoS One, 2012, 7(12): e51091
[16]Gao X Q, Wheeler T, Li Z H, Kenerley C M, He P, Shan L B. Silencing GhNDR1 and GhMKK2 compromises cotton resistance to Verticillium wilt. Plant J, 2011, 66: 293–305
[17]Zhang Y, Wang X F, Li Y Y, Wu L Z, Zhou H M, Zhang G Y, Ma Z Y. Ectopic expression of a novel Ser/Thr protein kinase from cotton (Gossypium barbadense), enhances resistance to Verticillium dahliae infection and oxidative stress in Arabidopsis. Plant Cell Rep, 2013, 32(11): 1703–1713
[18]Mo H J, Wang X F, Zhang Y, Zhang G Y, Zhang J F, Ma Z Y. Cotton polyamine oxidase is required for spermine and camalexin signalling in the defence response to Verticillium dahliae. Plant J, 2015, 83: 962–975
[19]杨君, 张艳, 王伟巧, 吴金华, 王国宁, 马峙英, 王省芬. 海岛棉GbHyPRP1克隆及其转基因拟南芥抗黄萎病验证. 植物资源学报, 2015, 16: 594–602
Yang J, Zhang Y, Wang W Q, Wu J H, Wang G N, Ma Z Y, Wang X F. Cloning of GbHyPRP1 from Gossypium barbadense and validation of Verticillium wilt resistance in transgenic Arabidopsis. J Plant Genet Resour, 2015, 16: 594–602 (in Chinese with English abstract)
[20]Xu L, Zhu L F, Tu L L, Yuan D J, Jin L, Long L, Zhang X L. Lignin metabolism has a central role in the resistance of cot-ton to the wilt fungus Verticillium dahliae as revealed by RNA-Seq-dependent transcriptional analysis and histochemistry. J Exp Bot, 2011, 62: 5607–5621
[21]Zhang Y, Wang X F, Ding Z G, Ma Q, Zhang G R, Zhang S L, Li Z K, Wu L Q, Zhang G Y, Ma Z Y. Transcriptome profiling of Gossypium barbadense inoculated with Verticillium dahliae provides a resource for cotton improvement. BMC Genom, 2013, 14(6): 1–18
[22]Wang K, Wang Z, Li F, Ye W, Wang J, Song G, Yue Z, Cong L, Shang H, Zhu S, Zou C, Li Q, Yuan Y, Lu C, Wei H, Gou C, Zheng Z, Yin Y, Zhang X, Liu K, Wang B, Song C, Shi N, Kohel R J, Percy RG, Yu J Z, Zhu YX, Wang J, Yu S. The draft genome of a diploid cotton Gossypium raimondii. Nat Genet, 2012, 44: 1098–1103
[23]Li F, Fan G, Wang K, Sun F, Yuan Y, Song G, Li Q, Ma Z, Lu C, Zou C, Chen W, Liang X, Shang H, Liu W, Shi C, Xiao G, Gou C, Ye W, Xu X, Zhang X, Wei H, Li Z, Zhang G, Wang J, Liu K, Kohel R J, Percy RG, Yu J Z, Zhu Y X, Wang J, Yu S. Genome sequence of the cultivated cotton Gossypium arboreum. Nat Genet, 2014, 46: 567–572
[24]Liu X, Zhao B, Zheng H J, et al. Gossypium barbadense genome sequence provides insight into the evolution of extra-long staple fiber and specialized metabolites. Sci Rep. 2015, 5: 14139
[25]Li F, Fan G, Lu C, Xiao G, Zou C, Kohel R J, Ma Z, Shang H, Ma X, Wu J, Liang X, Huang G, Percy R G, Liu K, Yang W, Chen W, Du X, Shi C, Yuan Y, Ye W, Liu X, Zhang X, Liu W, Wei H, Wei S, Huang G, Zhang X, Zhu S, Zhang H, Sun F, Wang X, Liang J, Wang J, He Q, Huang L, Wang J, Cui J, Song G, Wang K, Xu X, Yu JZ, Zhu Y, Yu S. Genome sequence of cultivated Upland cotton (Gossypium hirsutum TM-1) provides insights into genome evolution. Nat Biotechnol, 2015, 33: 524–530
[26]Zhang T, Hu Y, Jiang W, Fang L, Guan X, Chen J, Zhang J, Saski C A, Scheffler B E, Stelly D M, Hulse-Kemp A M, Wan Q, Liu B, Liu C, Wang S, Pan M, Wang Y, Wang D, Ye W, Chang L, Zhang W, Song Q, Kirkbride RC, Chen X, Dennis E, Llewellyn D J, Peterson D G, Thaxton P, Jones D C, Wang Q, Xu X, Zhang H, Wu H, Zhou L, Mei G, Chen S, Tian Y, Xiang D, Li X, Ding J, Zuo Q, Tao L, Liu Y, Li J, Lin Y, Hui Y, Cao Z, Cai C, Zhu X, Jiang Z, Zhou B, Guo W, Li R, Chen ZJ. Sequencing of allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement. Nat Biotechnol, 2015, 33: 531–537
[27]张纯颖, 王省芬, 张桂寅, 吴立强, 迟吉娜, 李志坤, 马峙英. 黄萎病菌诱导下陆地棉抗病品种SSH文库的EST分析. 棉花学报, 2010, 22: 17–22
EST analysis of suppression subtractive hybridization library from upland cotton resistant cultivar infection by Verticillium dahliae. Cotton Sci, 2010, 22: 17–22 (in Chinese with English abstract)
[28]王国宁, 赵贵元, 岳晓伟, 李志坤, 张艳, 张桂寅, 吴立强, 王省芬, 马峙英. 河北省棉花黄萎病菌致病性ISSR遗传分化. 棉花学报, 2012, 24: 348–357
Wang G N, Zhao G Y, Yue X W, Li Z K, Zhang Y, Wu L Q, Wang X F, Ma Z Y. Pathogenicity and ISSR genetic differentiation of Verticillium dahliae isolates from cotton growing areas of Hebei province. Cotton Sci, 2012, 24: 348–357 (in Chinese with English abstract)
[29]Zhao P, Zhao Y L, Jin Y, Zhang T, Guo H S. Colonization process of Arabidopsis thaliana roots by a green fluorescent protein-tagged isolate of Verticillium dahliae. Protein Cell, 2014, 5: 94–98
[30]彭姗, 吕学莲, 高峰, 李国英, 李晖. 一种新的棉花黄、枯萎病快速接种方法的研究. 棉花学报, 2008, 20: 174–178
Peng S, Lu X L, Gao F, Li G Y, Li H. Study on a new rapid inoculation method for Verticillium wilt and Fusarium wilt of cotton. Cotton Sci, 2008, 20: 174–178 (in Chinese with English abstract)
[31]Cheng C X, Jiao C, Singer S D, Gao M, Xu X Z, Zhou Y M, Li Z, Fei Z G, Wang Y J, Wang X P. Gibberellin-induced changes in the transcriptome of grapevine (Vitis labrusca × V. vinifera ) cv. Kyoho flowers. BMC Genom, 2015, 16(1): 1–16
[32]陈士林, 王春虎. 钙和赤霉素对棉花种子发芽力及活力的影响. 中国农学通报, 2004, 20(3): 112–113
Chen S L, Wang C H. Effects of Ca2+ and GA on the germination and the seed vigour in cotton. Chinese Agric Sci Bull, 2004, 20(3): 112–113 (in Chinese with English abstract)
[33]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCt method. Methods, 2001, 25: 402–408
[34]Bari R, Jones J D G. Role of plant hormones in plant defence responses. Plant Mol Biol, 2009 69: 473–488
[35]Clarke J D, Volko S M, Ledford H, Ausubel F M, Dong X. Roles of salicylic acid, jasmonic acid, and ethylene in cpr-induced resistance in Arabidopsis. Plant Cell, 2000, 12: 2175–2190

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