黄萎病抗性基因bs2在棉花中的功能解析

Cotton is a pillar industry of our country, which occupies an importantly strategic position in China's cotton industry . In recent years, verticillium wilt, as one of the most important diseases of cotton, is more frequencies occurrence and more harmful in the main region of cotton production, which should be seriously affecting the stable development of cotton production. Exploring of disease resistance genes is only effective method for improving resistance to verticillium wilt in upland cotton. Our team isolated a new protein BS2 from bacillus subtitle B111 resisting deciduous verticillium wilt fungus. By spectrometry sequencing and validation of antibacterial activity, BS2 was indentified as a new antifungal protein. Therefore, we cloned the full-length gene of BS2 protein, bs2. This project expected to transgenic bs2 gene into cotton for enhancing resistance to Verticillium wilt disease by nanocarriers and Agrobacterium tumefaciens mediated method. In this project, we should (1) dissect molecular mechanism of bs2 by analyzing mRNA/protein expression at different periods in root/stem/leaf, subcellular localization and yeast two-hybrid methods; (2) dissect resistant mechanism of bs2 at physiological and biochemical level by comparing the difference of some compounds and enzymes between trensgenic plants and negative controls; and at tissue and cellular level by comparing the difference of tissue structure and cellular size using microsection and electron microscopy; (3) identify verticillium wilt disease-resistance and genetic stability in multi-plot disease areas for several years.After carrying out this project, the bs2 function and resistant mechanism to verticillium wilt disease should be distected, which could provide some instructions for disease-resistant breeding of cotton. And this project should provide new resistant gene and develop new materials for cotton disease-resistant breeding.

棉花在我国的农业产业中占有重要的战略地位。近年来，黄萎病在主产棉区的发生和危害呈显著加重趋势，已严重影响棉花产业的稳定发展。发掘内、外源的抗病基因，是提高陆地棉黄萎病抗性的唯一有效途径。本研究从枯草芽孢杆菌B111分离出一个新型抗菌蛋白BS2，经验证：该蛋白高效抑制落叶型黄萎病菌的生长。因此，克隆了该蛋白的基因bs2。本研究拟构建过表达载体和维管束特异表达载体，转化棉花，通过该基因在棉花体内的时空表达分析、亚细胞定位，结合酵母双杂交、RNA-senquence等方法，深入研究该抗病基因bs2抗棉花黄萎病的分子机理；此外，通过病圃鉴定，分析转基因植株与对照（受体）在组织结构、生理生化指标以及抗病性上的变化，解析bs2基因对棉花黄萎病抗性的生理机制。通过本项目的实施，一方面为陆地棉黄萎病抗性提供可靠的基因资源和重要的育种材料，另一方面对深入解析棉花抗黄萎病的机理研究具有重要意义。

棉花在我国的农业产业中占有重要的战略地位。近年来，黄萎病在主产棉区的发生和危害呈显著加重趋势，已严重影响棉花产业的稳定发展。发掘内、外源的抗病基因，是提高陆地棉黄萎病抗性的唯一有效途径。本研究从枯草芽孢杆菌B111分离出一个新型抗菌蛋白BS2，经验证：该蛋白高效抑制落叶型黄萎病菌的生长。因此，克隆了该蛋白的基因bs2。项目实施期内，由于本研究前期获得的棉花转化体平板对峙试验发现所有检测样品均未出现抑菌圈，这与T0代结果不相符，因此，重新构建了植物表达载体vsp1-bs2-Red-Kan-GUS和35S-bs2-Red-Kan-GUS转化烟草、棉花工作同时正在进行，由于其转化时期较长，2021年冬拿到了阳性植株，计划后续进行更进一步的功能分析。转录组分析发现一些GO的富集分类与细胞壁的组成发生很大变化，其中包括细胞碳水化合物生物合成过程，植物型细胞壁生物发生和多糖生物合成。细胞壁的主要成分是糖，这些富集途径模式可能是细胞壁防御机制的一部分。通过WGCNA筛选出的一个基因GH_D11G0391，基因敲除后节间缩短，真叶增多，抗病性提高，后期将进一步转化至棉花植株中，做进一步的研究。本项目在实施期内发表文章1篇，申请专利1项（实审），培养研究生2名。