稻曲病菌PMK1类同源基因克隆及在稻瘟病菌遗传互补中的功能验证

Cloning of a Homologous Gene of Magnaporthe grisea PMK1 Type MAPK from Ustilaginoidea virens and Functional Identification by Complement in Magnaporthe grisea Corresponding Mutant

【目的】克隆稻曲病菌PMK1类MAPK(Mitogen-activated protein kinase)同源基因。【方法】根据丝状真菌MAPK蛋白保守性设计简并引物扩增稻曲病菌MAPK基因部分片段,进而利用TAIL-PCR进行染色体步移和RT-PCR获得UVMK1基因全长和cDNA全长。构建互补载体,交叉互补稻瘟病菌?PMK1突变体菌株nn78进行功能验证,包括附着胞分化和致病性测定。【结果】UVMK1基因全长1435bp,包含3个内含子,编码355氨基酸的蛋白。UVMK1推导蛋白与丝状真菌Magnaporthe grisea PMK1,Fusarium oxysporum FMK1,Fusarium solani FSMAPK,Colletotrichum lagenarium CMK1,Botrytis cinerea BMK1,Claviceps purpurea CMPK1等编码蛋白高度同源。转化稻瘟病菌菌株nn78,获得5个转化子。其中选取的转化子恢复了稻瘟病菌正常的附着胞分化和对大麦叶片的致病能力。【结论】本研究成功分离了首个稻曲病菌MAPK基因,而且UVMK1基因是稻瘟病菌PMK1的同源基因。

[Objective] Cloning of a Homologous Gene of PMK1 Type Mitogen-Activated Protein Kinase （MAPK） from the rice false smut fungus Ustilaginoidea virens. [Methods] According to the conserved amino acid sequence of several filamentous fungus MAPKs, which were homologous to Magnaporthe grisea PMK1, degenerate PCR primers were designed to amplify the MAPK internal DNA fragment from Ustilaginoidea grisea. The complete UVMK1 DNA and cDNA sequences were obtained using Thermal Asymmetric Interlaced–PCR （TAIL-PCR） and RT-PCR methods. Functional Identification was done by using the M. grisea ΔPMK1 mutant stain nn78, including appressoria differentiation assay and barley infection test. [Results] The total length of UVMK1 was 1435 bp. It contained 3 introns and encoded 355 amino acids. The induced amino acid sequence showed identical to Magnaporthe grisea PMK1, Fusarium oxysporum FMK1, Fusarium solani FsMAPK, Colletotrichum lagenarium CMK1, Botrytis cinerea BMK1, Claviceps purpurea CMPK1. After transformation of the ΔPMK1 mutant of M. grisea using a complement vector with the complete cDNA of UVMK1 （under the M. grisea MPG1 promoter）, five transformants were obtained. Furthermore, the selected two transformants fully restored their ability to form appressoria and infect a barley leaf. [Conclusion] In this study, we characterized the frst MAPK protein from U. virens, and that UVMK1 is a homologue of M. grisea PMK1.