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]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）.[Methods]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.[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.
出处
《微生物学报》
CAS
CSCD
北大核心
2011年第3期360-367,共8页
Acta Microbiologica Sinica
基金
生物源农药创制与技术集成及产业化开发(200903052)
211工程(S-09104)~~
