利用农杆菌介导法快速鉴定小麦病程相关蛋白基因TaPR1的功能  

Rapid identification of the function of wheat pathogenesis-related gene TaPR1 by Agrobacterium-mediated method

作　　者：梁芳 刘亦菲 崔钟池 王海燕[1] 刘大群[1] LIANG Fang;LIU Yifei;CUI Zhongchi;WANG Haiyan;LIU Daqun(College of Plant Protection, Hebei Agricultural University /Biological Control Center of Plant Diseases and PlantPests of Hebei province, National Engineering Research Center for Agriculture in Northern mountainous Areas,Baoding 071000, China)

机构地区：[1]河北农业大学植物保护学院/河北省农作物病虫害生物防治工程技术研究中心/国家北方山区农业工程技术研究中心,河北保定071000

出　　处：《河北农业大学学报》2019年第2期12-17,共6页Journal of Hebei Agricultural University

基　　金：国家自然科学基金(31501623);河北省高等学校科学技术研究项目(QN2015171)

摘　　要：本研究拟采用农杆菌瞬时表达体系,在小麦叶片中快速表达目的基因,以鉴定其在小麦抗叶锈病防御反应中的功能。在前期研究基础上,将已克隆获得的小麦病程相关蛋白基因TaPR1构建到小麦过表达载体pLGY-02,转化农杆菌菌株GV3101。然后以感病小麦品种‘金禾9123’为试材,将携带pLGY-02-TaPR1重组质粒的农杆菌GV3101注射到小麦叶片中,以空载体pLGY-02为对照。3 d后接种叶锈菌,14 d后观察小麦叶片表型。同时,在接菌后24、36、48 h时分别采集小麦叶片材料,利用DAB组织化学染色法,监测接种叶锈菌后叶片中H_2O_2的变化,并通过组织病理学显微观察,快速验证TaPR1在小麦与锈菌互作过程中的功能。结果发现,注射重组质粒pLGY-02-TaPR1的叶片发病程度、叶片孢子堆数均轻于、少于对照组叶片,且发病滞后;DAB组织化学染色结果表明,TaPR1表达量增加使得H_2O_2的清除在一定程度上受到抑制,小麦叶片中H_2O_2的分布与积累量均显著多于对照组叶片,说明TaPR1对小麦叶锈菌的致病性具有抑制作用,即TaPR1基因参与了小麦抗叶锈病防御反应。本研究成功构建了能在小麦中瞬时表达的重组载体pLGY-02-TaPR1,并建立了农杆菌介导的在小麦叶片细胞中高效表达外源基因的转化体系,为利用瞬时表达法验证小麦抗性基因功能奠定了基础。This study intends to identify the function of rapid expressed genes in resistance to wheat leaf rust using Agrobacterium transient expression system. One wheat pathogenesis-related protein gene, TaPR1, which was cloned and named in the previous studies, was constructed into wheat overexpression vector pLGY-02, and transformed into Agrobacterium strain GV3101. GV3101 carrying the pLGY-02-TaPR1 recombinant plasmid was injected into susceptible wheat leaves Jinhe 9123, and the empty vector pLGY-02 was used as a control. After 3 days, all the wheat leaves were inoculated with leaf rust pathogens, then the phenotype was observed 14 days after inoculation, and the number of spores on the leaves was counted. In the meanwhile, the wheat leaves were collected at 24 h, 36 h and 48 h after inoculation to observe the changes of H2O2 in leaves by DAB histochemical staining, and histological observation with microscopy was used to analyze and verify the function of TaPR1 enrolled in the interaction between wheat and rust. The results showed that the leaf rust degree was lighter incidence and leaf spores of the recombinant plasmid pLGY-02-TaPR1 was less than those of the control leaves, and the distribution and accumulation of H2O2 in wheat leaves induced by leaf rust infection were higher than those in the control leaves after 48 h inoculation. All these results indicated that TaPR1 did inhibit the pathogenesis in the course of wheat leaf rust and involved in wheat defense in response to leaf rust attack. In this study, the recombinant vector pLGY-02-TaPR1 was constructed successfully, and the Agrobacterium-mediated transformation system was proved to efficiently express foreign genes in wheat leaf cells,which provided a feasible method for functional identification of wheat genes using transient expression.

关 键 词：小麦 叶锈菌 病程相关蛋白基因 农杆菌 TaPR1 H2O2 

分 类 号：S435.12[农业科学—农业昆虫与害虫防治]