柑橘采后绿霉菌的致病机理解析

It can be dangerous for the infection of green mold during storage and keep-freshing in citrus, and it exited many problems such as persistent pathogenicity and fungicide- resistence. So it greatly restricted the development of storage and keep-freshing in citrus. T-DNA insertional mutants pool from P. digitatum was constructed through Agrobacterium tumefaciens mediated transformation (ATMT) based on preliminary work in our lab. One strain with loss of pathogenic ability in T-DNA insertional mutant P66-1976 from green mold in citrus was screened by in vivo experiment, and the mutant P66-1976 was a perfect experimental material to study the pathogenic mechanism of citrus green mold. Difference of green mold and its mutant P66-1976 was studied in the release of volatile substances (Ethylene, limonene etc), citric acid production, and antiviral protein expression. Target gene of mutant P66-1976 was cloned by TAIL-PCR, and we carried out target gene function analysis by yeast double hybrid, RNAi and Over-expression. And the role of the gene was revealed in the pathogenic mechanism of citrus green mold. Along with interaction of the upstream and downstream of the gene, the more pathogenic gene were found, and the mechanism of gene regulation networks were revealed between gene and gene, gene and environment. At last, it would lay the foundation for analyzing the the pathogenic mechanism of green mold in citrus.

柑橘绿霉病在贮藏保鲜过程中危害严重，且该菌具有持续的致病性和抗药性等问题，这就极大地制约了柑橘贮藏保鲜的发展。本课题组在前期利用根癌农杆菌介导的转化（ATMT）构建了柑橘绿霉菌的T-DNA插入突变体库，经in vivo果实接种实验筛选出一株丧失致病能力的绿霉菌突变体P66-1976，该突变体为研究绿霉菌致病机理提供了很好的切入点。本项目拟通过致病力分析比较柑橘绿霉菌株及突变菌株在乙烯、柠檬烯等挥发性物质的释放量、柠檬酸等酸性物质的产生以及抗病蛋白的表达差异；应用TAIL-PCR等技术分离绿霉菌的致病基因，以酵母单双杂交分离其上下游的调控基因， RNAi及超量表达研究该基因功能，进而揭示这个基因在柑橘果实致病中的作用，顺着上下游基因的相互作用，找到更多致病相关的基因并揭示基因与环境、基因与基因网络调控的机理，为最终解析柑橘绿霉菌的致病机制奠定基础。

柑橘绿霉病在贮藏保鲜过程中危害严重，且该菌具有持续的致病性和抗药性等问题，这就极大地制约了柑橘贮藏保鲜的发展。本课题组在前期利用根癌农杆菌介导的转化（ATMT）构建了柑橘绿霉菌的T-DNA插入突变体库，经in vivo果实接种实验筛选出一株丧失致病能力的绿霉菌突变体P66-1976，该突变体为研究绿霉菌致病机理提供了很好的切入点。本项目从生理水平分析其与野生菌株的表型差异，结果表明：（1）扫描电镜下，观察到野生株明显的帚状结构，突变株只有菌丝体，没有帚状结构。透射电镜下，野生株分生孢子细胞结构完整，细胞壁、隔膜、细胞膜及内部的细胞器明显，突变株细胞壁外围明显加黑加粗；（2）接种野生菌温州蜜柑伤口处第三天pH从5.5下降到3.9，纽荷尔脐橙伤口pH从5.3下降到3.3，但是接种突变菌的和接种无菌水的结果保持一致，都没有明显的变化；（3）分析野生菌与突变菌的生长速率与孢子产量，培养第8d时，野生菌菌苔的生长直径是突变菌的5倍，第13d时野生菌菌苔直径是突变菌的近4倍，野生菌的孢子数量是突变菌的近7倍，发现突变菌的生长速率和孢子产量都明显低于野生菌。在分子水平上确定导致表型变化的突变基因，针对突变基因进行定点敲除，分析基因功能：应用TAIL-PCR等技术分离绿霉菌的致病基因，基因敲除验证Pd269不参与柑橘绿霉病菌的致病调控，Pd275基因会导致柑橘绿霉病菌丧失致病毒力，Pd275基因在柑橘绿霉病菌的致病机制中起着关键作用。此外，通过比较分析及转化菌株的表型观测，筛选获得6株表型特殊的突变子：N1310，菌丝生长速率加快；N2130，孢子为白色且产孢量下降；N2165，产孢量下降；N2327，接种果实不致病；N2354，臭味；N2535，菌丝稀薄、生长速率和产孢量均显著降低。为最终解析柑橘绿霉菌的致病机制奠定基础。