橡胶树炭疽菌P型ATP酶家族基因的鉴定及其致病性功能研究

Rubber trees (Hevea brasiliensis) are perennial crops that takes important place in our national economie and defense goods, it also brings excellent welfare to local small farmers. Colletotrichum gloeosporioides(Penz) Sac. is one of the major causes of declining yields of rubber tree in most rubber plantations.Despite the fact that C.gloeosporioides is ubiquitous pathogen, little is known about the fungal molecular characters that are important for pathogenicity. P-type ATPases are ubiquitous, and they are important enzymes that participate in cation trans-membrane transport, cell signal transduction, and stability of the cell membrane. Published evidence suggests that a great lot family members of P-type ATPases were looked up, but the molucular identities of these enzymes have not yet been revealed. However, the biological function of the P-type ATPases of Colletotrichum species is still little known.. In previous study, we identified a Ca2+ P-type ATPase gene which was important to pathogenicity of C.gloeosporioides by loss of gene function method. In this progress, we will analyze P-type ATPases in three Colletotrichum species for which complete genome sequence data are available,and identify the pathogenicity related genes of them by the gene-deletion assays and pathogenicity tests. After that, gene complementary assays will be performed to further confirm their functions. We also will investigate the gene expression profiles and protein subcellular localization to known when and where they function. Additionally, we will try to find out conserved sites in their peptide sequences, and create amino acid substitutions to analyze their contributions to functions of gene. After completing the research mentioned above, the members of P-type ATPase family of three Colletotrichum species will be analyzed, and roles of the genes of P-type ATPase in pathogenesis of C.gloeosporioides be elucidated. These would be contributed to develop a new effective control of colletotrichum diseases of crop.

橡胶是关系国民经济及国防建设的重要战略物资，由胶孢炭疽引起的橡胶树炭疽病是影响橡胶产量的重要病害。P型ATP酶家族基因与植物病原真菌的致病性密切相关，但该家族基因在植物病原炭疽菌中的鉴定与功能分析未见报道。本项目拟在胶孢炭疽菌全基因组序列获得及验证其一个钙调P型ATP酶基因参与致病功能，以及胶孢炭疽菌P型ATP酶基因家族分析基础上，采用基因敲除/互补技术逐一观察和分析ATP酶基因敲除突变体与致病性的关系，鉴定出影响致病力和生长发育的P型ATP酶基因；并对其中与致病相关的基因进行功能验证，分析其蛋白亚细胞定位和时空表达模式，解析蛋白序列中保守结构域和保守位点的功能。通过研究，对橡胶树炭疽菌P型ATP酶家族基因进行鉴定，并明确其在致病机制中的作用，这不仅有助于进一步阐明植物病原真菌P型ATP酶家族基因的致病机理，也为作物炭疽病的抗病育种策略的制定提供理论依据。

天然橡胶是关系到国民经济发展和国防安全建设的重要战略性物资，由炭疽菌属真菌引起的橡胶树炭疽病是为害天然橡胶产业发展的重要病害。研究发现，P型ATP酶家族蛋白在生物的离子运输、信号转导和细胞膜形态建成等方面发挥着重要作用，但该家族基因在炭疽菌中的鉴定与功能尚未开展过系统研究。为此，本项目通过多基因系统发育明确了引起我国橡胶树炭疽病菌的优势种为暹罗炭疽菌（胶孢炭疽复合种）和华南炭疽菌（尖孢炭疽复合种），并完成了强致病力的暹罗炭疽菌株HBCG01和华南炭疽菌株GX1655的全基因组测序、组装和功能注释。通过TblastN和保守基序（DKTGTLT）搜索分析发现橡胶树暹罗炭疽菌株HBCG01含有29个P型ATP酶家族基因，华南炭疽菌株GX1655基因组中含有27个P型ATP酶家族基因，两个炭疽菌株基因组中的P型ATP酶家族组成也存在一定的差异，其中暹罗炭疽菌株HBCG01中含有更多的钙离子转运和重金属或阳离子转运蛋白编码基因，而华南炭疽菌株GX1655基因组中含有更多的钠/钾阳离子转运和磷脂转运蛋白编码基因。在此基础上，通过基因敲除技术鉴定并验证了2个与致病性相关的钙离子转运P型ATP酶基因CgATPase和CgATPase01，敲除突变体的生物学特性分析发现CgATPase基因调控HBCg01菌株生长速度、产孢量、菌落形态、孢子大小和维持细胞完整性；CgATPase01基因可能参与生长发育、细胞壁完整性、胁迫应答和侵染致病等一系列过程；利用BUSCA方法对致病相关P型ATP酶蛋白进行亚细胞定位预测，结果发现致病相关P型ATP酶基因CgATPase01表达产物定位于内质网，而CgATPase基因表达产物定位于质膜；致病性相关的P型ATP酶基因的重要功能域分析发现，具有催化酶的磷酸化作用的保守基序MVTGD后第二（R）和第六氨基酸（E）残基可能决定了P型ATP酶蛋白的致病性功能。本项目取得的研究结果进一步阐明植物病原真菌P型ATP酶家族基因的致病机理，也为作物炭疽病的抗病育种和防治新策略的制定提供依据。

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