自噬基因PbrATG1在梨轮纹病抗性中的功能及作用机理研究

Pear ring rot, caused by Btryosphaeria dothidea, harms pear stems, leaves and fruits, which is one of the most serious diseases of pear, but the mechanism of pear resist ring rot is still unclear. Previously the applicant found that the expression of PbrATG1 (autophagy-related gene 1) was up-regulated during the infection of B. dothidea in pear, indicating that PbrATG1 was involved in the response to infection progress of B. dothidea, but the function and working mechanism of PbrATG1 in pear resistance to ring rot is still unclear. Firstly, the applicant will clone the gene and promoter sequence of PbrATG1, analyze the subcellular localization，tissue localization and expression mode of PbrATG1. Thereafter, the applicant will overexpress or transiently silence PbrATG1 in pear, then analyze autophagy activity and disease resistance in transgenic pear plants. Finally, yeast two-hybrid and immunoprecipitation were used to identify PbrATG1 interacting proteins, and then the effect of PbrATG1 on the expression and protein activity of interacting proteins was analyzed. The aim of this project was to clarify the function and working mechanism of PbrATG1 in pear resistance to ring rot, and to provide genetic resources for cultivating pear resistance to ring rot varieties.

轮纹病由Btryosphaeria dothidea引起，危害梨枝干、叶片和果实，是梨最严重病害之一，目前梨植株抵御轮纹病的机制尚不清楚。申请人前期研究发现，梨自噬基因PbrATG1在轮纹病侵染期间表达量上调，表明PbrATG1参与响应轮纹病菌侵染过程,但PbrATG1在梨抵御轮纹病菌侵染中的功能及作用机理尚不清楚。首先申请者拟克隆PbrATG1基因及启动子序列，分析PbrATG1的亚细胞定位情况、组织定位情况及表达模式；继而在梨中过表达及瞬时沉默PbrATG1，分析转基因梨植株中的自噬活性及抗病性；最后利用酵母双杂交和免疫沉淀挖掘PbrATG1互作的蛋白，分析PbrATG1对互作蛋白的表达及蛋白活性的影响。本研究旨在明确PbrATG1在梨轮纹病抗性中的功能及作用机理，为培育梨抗轮纹病品种提供基因资源。

轮纹病是梨最严重的病害之一，但梨对轮纹病的抗病机理尚不清楚。自噬在植物抵御病原菌中起到关键作用，其中自噬相关基因ATG1在诱导自噬的产生发挥重要功能，但在梨中对自噬的了解较少。本项目通过全基因组筛选，确定了PbrATG1及其他自噬关键基因家族成员，并对这些基因进行了结构分析、表达分析、启动子序列分析等研究，表明PbrATG1参与梨轮纹病抗性过程中。随后对PbrATG1在梨中进行了瞬时沉默试验，发现沉默PbrATG1后梨植株抗病性减弱，抗病相关基因表达降低，表明PbrATG1在梨轮纹病抗性中起关键作用。随后进行了PbrATG1的互作蛋白筛选，筛选出7个互作蛋白，并挑选出其中对轮纹病侵染响应较明显的PbrDSK2进行后续研究。通过酵母双杂交、双分子荧光互补、Co-IP等试验，明确PbrDSK2基因与PbrATG1基因相互作用，并通过影响自噬活性，提高梨对轮纹病的抗性。综上，本研究探究了PbrATG1基因的生物学功能，并筛选了其互作基因PbrDSK2，通过与ATG1互作，影响自噬活性，提高了梨轮纹病抗性。