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【目的】克隆绿僵菌第五类Ser/Thr蛋白磷酸酶(PP5)基因,了解该基因及其编码产物的结构特征和两种产孢模式(微循环产孢和正常产孢)中的表达特征。【方法】通过绿僵菌中PP5基因EST序列与全基因组数据库比对,获得PP5基因DNA序列;通过同源蛋白比对预测PP5基因的DNA结构并设计引物,PCR扩增获取PP5全长cDNA序列;通过在线分析工具及生物软件进行蛋白结构分析。采用实时荧光定量PCR检测PP5基因在两种产孢模式中的表达特征。【结果】PP5基因长2100 bp,含7个外显子和6个内含子;cDNA开放阅读框长为1428 bp(GenBank登录号HQ317137),编码475个氨基酸;一级、二级及三级结构分析均显示较保守的蛋白磷酸酶结构特征。实时荧光定量PCR分析表明,PP5基因在绿僵菌微循环产孢的不同阶段表达水平具有显著性差异,特别是在孢子接种16、24、32 h高表达,而在正常产孢模式下表达量非常少。【结论】克隆了绿僵菌的PP5基因,详细了解了该基因及其编码产物的结构特征,发现了该基因在微循环产孢孢子形成后期高表达的重要特征,为进一步研究该基因在微循环产孢中的功能奠定了基础。
【Objective】 The objective of this study is to clone the Ser / Thr protein phosphatase (PP5) gene of Metarhizium anisopliae to understand the structural characteristics of the gene and its encoded product and the expression in two sporulation patterns (microcirculation sporulation and normal sporulation) feature. 【Method】 The DNA sequence of PP5 gene was obtained by comparing the EST sequence of PP5 gene in Metarhizium anisopliae with the genome-wide database. The PP5 gene DNA sequence was predicted by homologous protein alignment and the primers were designed. The full-length cDNA sequence of PP5 Protein structure analysis with online analysis tools and bio-software. The expression characteristics of PP5 gene in two sporulation patterns were detected by real-time fluorescence quantitative PCR. 【Result】 The PP5 gene was 2,100 bp in length and contained 7 exons and 6 introns. The open reading frame of PP5 gene was 1428 bp (GenBank accession number HQ317137), encoding 475 amino acids. The first, second and third level Structural analysis showed more conserved protein phosphatase structural features. Real-time quantitative PCR analysis showed that the expression level of PP5 gene was significantly different at different stages of sporulation of Metarhizium anisopliae, especially at 16, 24 and 32 h after inoculation, very few. 【Conclusion】 The PP5 gene of Metarhizium anisopliae was cloned and its structure was characterized in detail. The gene was highly expressed in the late stage of microcirculation conidia formation. To further study the effect of this gene on micro Circulation sporulation function laid the foundation.