Xu Fei, Peng Guoxiong, Xia Yuxian
Genetic Engineering Research Center of Bioengineering College, Chongqing University, Chongqing Engineering Research Center for Fungal Insecticides, Chongqing 400030, China.
Wei Sheng Wu Xue Bao. 2011 Mar;51(3):360-7.
To clone Ser/Thr protein phosphatase type 5 gene (PP5) from Metarhizium anisopliae, analyze the structure of PP5 gene with its encoding protein and expression profile during two conidiation program (microcycle conidiation and normal conidiation).
The DNA sequence of PP5 was isolated by blasting the expressed sequence tags (EST) 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.
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.
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.
从绿僵菌中克隆5型丝氨酸/苏氨酸蛋白磷酸酶基因（PP5），分析PP5基因及其编码蛋白的结构，以及在两种产孢程序（小循环产孢和正常产孢）中的表达谱。
通过将PP5在消减文库中的表达序列标签（EST）与绿僵菌基因组数据进行比对，分离出PP5的DNA序列。根据该DNA序列设计引物，通过PCR克隆PP5的全长cDNA，并利用在线工具和生物软件分析编码蛋白的特征。通过实时PCR对绿僵菌小循环产孢不同阶段和正常产孢菌丝阶段的PP5表达谱进行定量分析。
基因组DNA长2100 bp，被6个内含子打断。cDNA长1428 bp，编码325个氨基酸残基。对绿僵菌5型丝氨酸/苏氨酸蛋白磷酸酶的分析显示出保守的结构特征。实时定量PCR分析表明，PP5在小循环产孢的不同阶段表达明显不同。在小循环产孢中，16 h后表达急剧上调，24 h时转录水平最高，但在正常产孢中表达较低。
本研究首次报道了昆虫病原真菌PP5的详细序列和结构。小循环产孢和正常产孢表达谱的比较表明，PP5主要参与绿僵菌的小循环产孢，为进一步研究PP5及其分子调控提供了理想的候选对象。
