.蝗绿僵菌氰化物水合酶基因MaChy的克隆及表达分析

Cloning and characterization of a cyanide hydratase gene from Metarhizium acridum

采用筛选cDNA文库的方法,首次克隆了蝗绿僵菌氰化物水合酶基因MaChy的全长cDNA序列和DNA序列,序列分析表明,蝗绿僵菌氰化物水合酶基因MaChy不含内含子,开放阅读框(ORF)为1,074bp,编码357个氨基酸,推测蛋白的分子量为39.78kDa,等电点(pI)为5.53;利用在线软件分析表明,该蛋白既不是分泌型蛋白也不是膜蛋白;同源性比对结果表明,该蛋白与其他真菌中的氰化物水合酶蛋白的同源性较高,具有高度保守的催化三联体特征。用qRT-PCR方法分析了该基因在蝗绿僵菌侵染昆虫过程中的表达情况,结果表明,MaChy在蝗绿僵菌侵染穿透昆虫体壁阶段的表达量最高,约为体内阶段表达量的2.5倍,推测该基因可能在蝗绿僵菌穿透昆虫体壁的过程中起重要作用。

By screening a cDNA library, the full length cDNA sequence and DNA sequence of a cyanide hydratase gene was isolated from Metarhizium acridum. Sequence analysis showed that the MaChy gene had no intron and the open reading frame was 1,074bp which encodes a protein with 357 amino acid residues with a calculated Mr of 39.78kDa and pl of 5.53. The characters of this putative protein were analyzed by several online bioinformatics tools, including signal peptide sequence, transmembrane segment, and secondary structure. The results indicated that this protein was neither a secreted protein nor a membrane protein. The putative amino acid sequence of MaChy shared high identities with the Chy proteins from other fungi. The highly conserved catalytic triad constituted by Glu-Lys-Cys could be found in the protein. A quantativeRT-PCR analysis showed that the gene expression level was highly up-regulated at the appressorium formation stage, and it was about 2.5 times over the colonization of host hemolymph stage.