绿僵菌第五类Ser/Thr蛋白磷酸酶基因的克隆及表达特征分析

Cloning and expression analysis of Ser/Thr protein phosphatase type 5 during microcycle conidiation in Metarhizium ansopliae

摘要:

摘要: 【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

关键词:关键词: 绿僵菌; 微循环产孢; Ser/Thr蛋白磷酸酶; 实时荧光定量PCR

Abstract:

Abstract: [Objective] To clone Ser/Thr protein phosphatase type 5 gene (PP5) from Metarhizium anisopliae, analyze the structure features of PP5 gene with its encoding protein and expression profile during two conidiation program (microcycle conidiation and normal conidiation). [Methods] The DNA sequence of PP5 was isolated by Blasting the EST sequence of PP5 in subtracted library with genomic data of M. anisopliae. Primers were designed based on the DNA sequence to clone the full length cDNA of PP5 by PCR, and the characteristics of the encoded protein was analyzed by online tools and biological softwares. The PP5 expression profile was quantified by real time PCR at different stages of microcycle conidiation and hyphal stage of normal conidiation in M. anisopliae. [Results] The genomic DNA, which was interrupted by six introns, was 2100 bp long. The cDNA, encoding 325 amino acid residues, is 1428 bp. Analysis to Ser/Thr protein phosphatase type 5 in M. anisopliae show a conserved structure features. Quantitative real time PCR analysis showed that PP5 expression varied obviously in different stages of microcycle conidiation. Expression was sharply up-regulated after 16 h, with the highest transcript levels at 24 h in microcycle conidiation, but lowly expressed in normal conidiation. [Conclusion] This work presents the first report about the detailed sequence and structure of PP5 from entomopathogenic fungi. Comparison of expression profile of microcycle conidiation and normal conidiation reveals that PP5 is principally involved in microcycle conidiation in M. anisopliae, and it provides ideal candidate for further studies to PP5 and its molecular regulation.

Key words:Keywords: Metarhizium anisopliae; Microcycle conidiation; Ser/Thr protein phosphatase type 5; Quantitative real time PCR