绿僵菌微循环产孢基因Pyk的克隆及RNA干扰分析

Cloning and analysis by RNAi of a pyruvate kinase gene expressed during microcycle conidiation in Metarhizium ansopliae

摘要:

微循环产孢是真菌遭遇逆境时发生的一种产孢机制, 具有繁殖快速及抗逆性强等诸多优点。研究绿僵菌的微循环产孢机制并加以利用, 对增强该菌在生物防治中的应用效果具有重要的意义。采用绿僵菌基因组数据库与NCBI数据库中同源蛋白比对获得Pyk基因DNA序列; 分析Pyk基因结构并设计引物, 通过RT-PCR扩增克隆Pyk全长cDNA序列。序列分析显示该基因cDNA全长1 934 bp, 开放阅读框长为1 752 bp (GenBank登录号: HQ153828), 编码产物为583个氨基酸的丙酮酸激酶, 该酶与子囊菌门中其他真菌具有较高的相似性(57%?77%)。构建Pyk基因的RNAi载体, 基因枪转化野生型绿僵菌获得3个突变菌株, RT-PCR证实3个干扰突变菌株中Pyk基因的干扰效率分别为: 51%、56%、33%。对突变菌株的微循环产孢模式做了进一步分析, 结果显示: 与野生菌株相比, 突变菌株产生更多的孢子形态类型, 菌落周边的白色菌丝也相对较少。

Abstract:

Microcycle conidiation has been proposed as an advantageous?conidiation pattern for fungus with rapid propagation and strong resistance to stress factors. Deeply understand the microcycle conidiation mechanism will greatly enhance the potential of entomopathogenic fungi in biocontrol. Here, we isolated a microcycle conidiation associated gene, Pyk, in Metarhizium anisopliae and obtained its full length of DNA and cDNA sequence. Pyk gene in M. anisopliae encoded a homolog of pyruvate kinase with 583 amino acid residues, which showed a highly resemblance to that of other species in ascomycota (57%?77% identity). To clarify its role in microcycle conidiation, we constructed a Pyk-RNAi vector to knockdown Pyk transcript. RT-PCR demonstrated a reduced expression in mRNA level in three mutants. Furthermore, we analyzed suppression effect to Pyk in mutations. The mutants showed more varied shapes of conidia and less white hypha in colonial morphology compared to wild type.