Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (21): 4065-4073.doi: 10.3864/j.issn.0578-1752.2016.21.001

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

Subcellular Localization and Verticillium Wilt Resistance Analysis of Cotton GbRvd in Overexpressed Tobacco

YANG Zhan-wu, YANG Jun, ZHANG Yan, WU Jin-hua, LI Zhi-kun, WANG Xing-fen, WU Li-qiang, ZHANG Gui-yin, MA Zhi-ying   

  1. College of Agronomy, Hebei Agricultural University/North China Key Laboratory for Crop Germplasm Resources of Ministry of Education, Baoding 071001, Hebei
  • Received:2016-04-29 Online:2016-11-01 Published:2016-11-01

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Abstract: 【Objective】In order to resolve the mechanism of GbRvd-mediated resistance to Verticillium wilt in cotton, subcellular localization and resistance of GbRvd-overexpressed tobacco were studied.【Method】Expression vector pJCV52 was modified by inserting gfp gene into SpeⅠenzyme site and the resulted vector was named as GPJCV52 which can be used for subcellular localization. Gateway-based constructs, made for subcellular localization and overexpression of GbRvd, were transformed into Agrobacterium strain GV3101 for tobacco transformation. Subcellular localization of GbRvd was predicted and observed by bioinformatics analysis and Agrobacterium-mediated transient expression in tobacco leaf, respectively. Transformation of tobacco was made by Agrobacterium-mediated method, and final transgenic tobacco plants were obtained through tissue culture. PCR and semi-quantitative RT-PCR were used for screening positive transgenic plants and detecting gene expression levels, respectively. Verticillium dahliae conidial suspension was prepared and incubated with T3 generation of transgenic tobacco strains through the method of soil drench. The disease severity for plants was graded from 0 to 4 and then the disease index was calculated. 【Result】ProtComp, an online analysis software, predicted that GbRvd is an extracellular (secreted) protein. Transmembrane prediction showed that GbRvd contains three transmembrane domains. On-line analysis software WoLF PSORT prediction results showed that GbRvd localization in the cell membrane, endoplasmic reticulum and chloroplast scores were 7, 3 and 1, respectively, indicating that GbRvd mainly existed in the plant cell membrane. In order to further confirm the location of GbRvd in the cell, expression of GbRvd fused with GFP was examined. Fluorescence signal of control GFP protein in tobacco cells was observed in the nucleus, cytoplasm and cell membrane, and fluorescence signals of GbRvd fused with GFP were mainly detected in the plasma membrane and cytoplasm of tobacco epidermal cells. The transgenic tobacco plants were produced by Agrobacterium-mediated infection and tissue culture method. PCR detection results showed that the expected size of DNA band could be amplified in positive transgenic tobacco plants, and that could not appear in wild type plants. By semi-quantitative RT-PCR expected band could also be detected in positive transgenic tobacco plants, which indicates that GbRvd successfully integrated to the tobacco genome and can be normally transcribed. Three independent T3 transgenic tobacco lines were selected to analyze Verticillium wilt resistance. The results showed that the disease index of transgenic lines was significantly lower than that of wild-type, indicating that overexpression of GbRvd can effectively improve plant resistance to Verticillium wilt. 【Conclusion】GbRvd mainly exist in plant cell plasma and membrane, and its overexpression can significantly increase the resistance of tobacco to Verticillium wilt.

Key words: cotton, GbRvd, subcellular location, transgenic tobacco, Verticillium wilt

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