海岛棉水杨酸途径脂肪酶基因GbEDS1抗黄萎病功能及分子机制

Breeding resistant variety is an efficient strategy to control cotton disease.It is important for cotton breeding community to ascertain the molecular mechanism against Verticillium wilt. A gene homologous to EDS1 (Enhanced Disease Susceptibility1) was isolated from full-length cDNA library of G. barbadense induced by V.dahliae. The marked Verticillium wilt resistance was observed in transgenic Arabidopsis. In this study, the molecular mechanism analysis of GbEDS1 will be carried out. The cotton expression pattern of the EDS1 will be conducted by using Real-time PCR when induced by V. dahliae pathogen and signal molecules, respectively. Virus-induced gene silencing (VIGS) expression vector will be employed to transform cotton and silence endogenous EDS1 gene of G. barbadense, and to mine cotton metabolic pathways that the EDS1 participates in by detecting expression of some marker genes, such as PR1a and PR5a in SA, PR4 in MeJA, EREBP in ET and CAT1, GST, APX in ROS pathways. Furthermore, we will construct GbEDS1-pBI121 overexpressing vector and transform susceptible upland cotton for analyzing the resistance of transgenic plants under V. dahliae stress and investigate the effects of GbEDS1 overexpression to ethylene(ET), salicylic acid(SA) and hydrogen peroxide(H2O2). These will be of significance to understanding the molecular mechanism of cotton Verticillium wilt resistance, as well as to providing important scientific reference to cotton breeding.

抗病育种是防治棉花重要病害-黄萎病的经济有效措施，研究抗黄萎病分子机制对棉花抗病育种具有重要意义。项目组从黄萎病菌诱导的海岛棉全长cDNA文库分离了Enhanced Disease Susceptibility 1 （EDS1）基因，初步研究表明转基因拟南芥对黄萎病具有很好的抗性。本研究拟对GbEDS1进行抗黄萎病作用机制解析。研究棉花分别受黄萎病菌和信号分子处理后基因的表达；构建VIGS表达载体并转化棉花，反向快速验证基因功能，并进一步研究该基因对不同抗病途径重要基因（水杨酸：PR1a、PR5a；茉莉酸：PR4；乙烯：EREBP； ROS：CAT1、GST、APX）的影响；构建真核过表达载体转化感病陆地棉，研究转基因材料黄萎病菌胁迫下基因表达和抗性反应；分析GbEDS1基因超表达对抗病内源性激发因子乙烯、水杨酸和H2O2表达量的影响，探明基因的抗病作用及机制，为抗黄萎病育种提供理论依据。

棉花黄萎病是毁灭性的、对棉花产量影响最大的病害之一，抗黄萎病遗传改良和抗病机制一直是棉花遗传育种领域的重大科学问题。本研究基于项目组前期克隆的海岛棉GbEDS1基因及其在拟南芥上的初步功能鉴定结果，在棉花和拟南芥上从细胞学、生理生化及分子生物学等方面深入研究GbEDS1基因的抗病分子机制。通过比较不同棉属EDS1基因及来自不同物种的EDS1基因发现棉花EDS1基因具有很强的保守性。qPCR结果显示，GbEDS1基因受黄萎病菌诱导表达，且在抗病海岛棉中具有最高的表达量；同时，GbEDS1基因参与SA等信号转导途径。转基因拟南芥抗病性的提高与植株体内增加的SA含量、H2O2水平和抗氧化系统有关，GbEDS1介导的抗病反应需要SA通路中NDR1和BAK1的参与。构建了GbEDS1植物超表达载体，利用花粉管通道法转化感病陆地棉品种，获得了T2代转基因株系。抗病性鉴定表明GbEDS1基因可以提高陆地棉的黄萎病抗性。利用VIGS技术抑制內源GbEDS1基因的转录水平获得基因沉默棉花，沉默植株表型出抗病性显著下降，SA含量和H2O2水平降低，且SA信号通路中NDR1和BAK1基因的表达显著受到抑制。此外，还克隆了上述NDR1基因及海岛棉中的一个新的抗病相关基因VWR，并研究了这两个基因的表达与基因功能，明确该基因具有抗黄萎病功能。上述研究结果丰富了棉花抗黄萎病分子机制，对深入了解棉花与黄萎病菌互作过程的基因表达与调控机制具有重要意义。