PG基因在莲腐败病菌致病过程中的作用和机制研究

Lotus (Nelumbo nucifera Gaertn.) is cultivated as an important crop in Jiangxi province. Rhizome rot of lotus, caused by Fusarium oxysporum f.sp. nelumbicola (Nis.&Wat.) Booth (FON), is a soil-borne disease, which has become the most destructive disease of lotus in recent years, and cause enormous economic loss. However, to date, little is known about the pathogenic factors and molecular pathogenic mechanisms of FON. Polygalacturonase (PG) is one of the main enzymes that degrade the pectin of plant cell wall, playing an important role in the pathogenicity of plant pathogenic fungi. F. oxysporum have at least four PG genes encoding different PG types (Pg1, pg5, pgx1 and pgx4). However, not all the PG deletion mutants show reduced virulence, the understanding of the role of PG genes of F. oxysporum is not still clear. Our previous studies demonstrated that FON secreted a set of cell wall-degrading enzymes in lotus after inoculated with FON, and the PG activities were the strongest. The PG activities of FON with strong virulence were obviously higher than that with relatively weaker virulence. PG activities are verified to be related to the virulence of FON. Six PG genes (pg1, pg5, pgx4, pgx1, pgA and pgB) were identified from FON. The relative transcript level of each PG in lotus after inoculated with FON was analyzed by using quantitative RT-PCR (qRT-PCR). The results revealed that the transcript levels of pg1, pgB and pg5 were higher than the other PG genes, suggesting these three PG genes may play an important role in the pathogenicity of FON. In this study, we try to investigate genes FONpg1, FONpgB and FONpg5 in the pathogenic role by using Pichia pastoris expression system and gene knock-out techniques. To date, little is known about the function of the cell wall degrading enzymes or other pathogenic factors from FON, the result will provide usefull evidences for understanding the pathogenic mechanism and the materials for the resistive breeding.

尖孢镰刀菌莲专化型引起的莲腐败病是一种毁灭性病害，迄今对该病的研究几乎为空白，对其致病因子及致病机理尚不明确。我们前期研究发现该病原菌致病力与多聚半乳糖醛酸酶（PG）活性密切相关，并克隆获得6个PG基因，这是首次从莲腐败病菌上获得PG基因，而且有2个是未知的新PG基因。其中pg1、pg5和新基因pgB可被诱导高效表达，表明这3个基因可能与病原菌致病性密切相关。但它们是如何参与病原菌致病过程的？究竟哪一个基因发挥主要的降解细胞壁功能？目前尚不清楚。 本项目拟在现有基础上，对这3个基因进行深入研究。通过真核表达技术，比较酶动力学特性和致病性差异，并通过电镜观察对细胞壁超微结构的降解差异；通过基因敲除获得单/双突变体，比较野生型和突变体在酶活性、致病性、生物学性状的差异；最终从分子、蛋白质和细胞水平，解析PG基因在病原菌致病过程中的作用和致病机制，为开发莲腐败病防治策略提供理论依据。

尖孢镰刀菌莲专化型引起的莲腐败病是一种毁灭性病害，迄今对该病的研究几乎为空白，对其致病因子及致病机理尚不明确。本项目对pg1，pg5和pgB 3个基因进行深入研究。通过真核表达技术，比较酶动力学特性和致病性差异，并通过电镜观察对细胞壁超微结构的降解差异；通过基因敲除获得突变体，比较野生型和突变体在酶活性、致病性、生物学性状的差异；最终从分子、蛋白质和细胞水平，解析PG基因在病原菌致病过程中的作用和致病机制，为开发莲腐败病防治策略提供理论依据。项目资助发表论文4篇，待发表2篇。培养硕士生3名，其中2名已经取得硕士学位，1名在读。项目投入经费22万元，支出15.1939万元，各项支出基本与预算相符。剩余经费6.8061万元，剩余经费计划用于本项目