棉花GbRvd的亚细胞定位及其超表达烟草抗病性分析

doi:10.3864/j.issn.0578-1752.2016.21.001

摘要/Abstract

摘要： 【目的】研究棉花NB-LRR家族基因GbRvd的亚细胞定位及超表达烟草的黄萎病抗性，为解析GbRvd介导的植物抗黄萎病机制提供依据。【方法】对真核表达载体pJCV52进行改造，在SpeⅠ酶切位点插入gfp，使其兼具亚细胞定位的功能，命名为GPJCV52。应用Gateway技术构建GbRvd的亚细胞定位和超表达载体，并分别转化农杆菌GV3101。利用生物信息学和农杆菌介导的烟草瞬时表达技术对GbRvd分别进行亚细胞定位预测与观察。采用农杆菌介导法转化烟草，并通过组织培养技术获得转基因植株。运用PCR对超表达GbRvd的烟草进行阳性植株筛选，且利用半定量RT-PCR对阳性转基因植株进行表达检测。制备棉花黄萎病菌孢子悬浮液，通过灌根法对T3代转基因烟草株系进行接种，利用5级标准统计发病情况并计算病情指数。【结果】利用在线分析软件ProtComp预测GbRvd为一种胞外（分泌）蛋白；GbRvd的跨膜预测结果显示其包含3个跨膜域；在线分析软件WoLF PSORT预测结果显示GbRvd在细胞膜、内质网及叶绿体的预测分值分别为7、3和1，表明GbRvd主要存在于植物细胞膜上。利用农杆菌介导的烟草瞬时表达技术进一步对GbRvd的亚细胞定位进行观察，结果显示在单独表达GFP蛋白的烟草细胞中，荧光信号在细胞核、细胞质和细胞膜均有分布；而GbRvd与GFP融合表达后的荧光信号主要分布于烟草表皮细胞质和细胞膜。综合生物信息学分析和亚细胞定位观察结果，表明细胞膜和细胞质是GbRvd的主要分布位置。利用农杆菌介导和组织培养获得了转基因烟草植株，通过对转基因烟草植株的PCR检测显示，阳性转基因烟草植株能够通过PCR扩增出与预期大小一致的目的条带，而野生型植株未出现相应条带；阳性转基因烟草植株半定量RT-PCR同样能够检测出与预期大小一致的目的条带，由此表明GbRvd成功整合于烟草基因组并能够进行正常转录。对3个T3代超表达烟草株系的黄萎病抗性进行分析，结果显示超表达GbRvd的烟草植株病情指数显著低于野生型对照植株，表明过表达GbRvd可有效提高植株对黄萎病的抗性。【结论】GbRvd主要存在于植物细胞质和细胞膜，其超表达可显著提高烟草对黄萎病的抗性。

关键词: 棉花, GbRvd, 亚细胞定位, 转基因烟草, 黄萎病

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

杨占武，杨君，张艳，吴金华，李志坤，王省芬，吴立强，张桂寅，马峙英. 棉花GbRvd的亚细胞定位及其超表达烟草抗病性分析[J]. 中国农业科学, 2016, 49(21): 4065-4073.

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. Subcellular Localization and Verticillium Wilt Resistance Analysis of Cotton GbRvd in Overexpressed Tobacco[J]. Scientia Agricultura Sinica, 2016, 49(21): 4065-4073.

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