稻瘟菌P-ATPase基因MoCTA3的克隆及表达分析

Cloning and expression analysis of a P-ATPase gene MoCTA3in rice blast pathogen Magnaporthe oryzae

为分析P-ATPase基因与稻瘟菌致病性的相关性,从稻瘟菌中克隆了一个P-ATPase基因MoCTA3,然后对其进行了序列分析和氨基酸序列比对,并利用qRT-PCR检测了该基因在稻瘟菌侵染水稻叶片不同时期以及菌丝阶段的表达情况。结果表明该基因全长3 747 bp,含有6个外显子和5个内含子,编码1 096个氨基酸,含有7个跨膜结构域,第24～98位有一个保守的钙离子转运结构域;其编码氨基酸与朱红丛赤壳、尖孢镰刀菌、蝗绿僵菌、炭疽菌等真菌的P-ATPase氨基酸相似性分别为77%、78%、83%和79%;基因表达结果显示该基因在菌丝阶段的表达量明显高于孢子阶段和侵染阶段,且在孢子侵染水稻过程中随侵染时间延长表达量逐渐升高,72 h时达到高峰,随后降低,说明MoCTA3可能参与了稻瘟菌的侵染过程,在稻瘟菌的致病性方面具有重要作用。

Rice blast, caused by Magnaporthe oryzae, is one of the most severe diseases of cultivated rice throughout the world. And the fungus is widely regarded as a model fungus because of its economic and social significance. To analyze the role of P-ATPase in pathogenesis of Magrtaporthe oryzae, a P-ATPase gene named MoCTA3 was cloned from Magnaporthe oryzae. Sequence analysis and amino acid sequence alignment were done to analyze the gene. Simultaneously quantitative RT- PCR was performed at 0 h, 48 h, 72 h, 96 h post-inoculation and the hyphal phase to examine the MoCTA3 expression. The results showed that full length of the MoCTA3 was 3 747 bp, containing 6 extrons and 5 introns, and encoding 1 096 alnino acids. The gene contained 7 trans-membrane domains, including a conservative calcium ion transporting domain at the site of 24-98. The similarities of the MoCTA3 amino acids sequence between Magnaporthe oryzae and Nectria haematococca, Fusari- um oxysporum, Metarhizium anisopliae, Glomerella graminicola were 77%, 78%, 83% and 79%, respectively. Gene ex- pression analysis suggested that the gene expression level at hwhal phase was siznificantly hizher than those at spore stage andinfection stage. The expression level increased gradually with time during the infection phase, reached peak at 72 h post inoculation, and then decreased. These indicated that MoCTA3 gene might work in Magnaporthe oryzae＇ s infection action and pathogenicity.