粉拟青霉类枯草杆菌蛋白酶基因的cDNA克隆及其序列和蛋白质分析

Cloning of sublitisin-like protease gene cDNA from Paecilomyces farinosus and characterization of its sequence and deduced protein

粉拟青霉是一种常见的昆虫病原真菌,被广泛用于生物防治。根据GenBank中已登录的淡紫拟青霉,球孢白僵菌和金龟子绿僵菌的类枯草杆菌蛋白酶基因序列的同源性比较,以它们高度保守的核苷酸序列设计一对引物,采用RT-PCR和3’/5’-RACE相结合的方法,首次从粉拟青霉中克隆出完整的类枯草杆菌蛋白酶cDNA基因。其全序列为2060bp,该序列5’-端和3’-端的非编码序列长度分别为209bp和252bp,开放阅读框为1599bp,编码532个氨基酸,信号肽长度为16个氨基酸。成熟蛋白的氨基酸序列和金龟子绿僵菌小孢变种(CAB63913),玉米赤霉菌(EAA68914),金龟子绿僵菌蚱蜢变种(CAC95047)及柄孢壳(AAC03564)的同源性分别为69%、71%、68%和68%。成熟蛋白具有典型的丝氨酸蛋白酶活性位点,同时有5个半胱氨酸,3个潜在的N-糖基化位点和2个可能的O-糖基化位点。该基因在的密码子第三位碱基的使用上,显示出明显的对C的偏爱。

Paecilomyces farinosus is one of the most common species of entomopathogenic fungi, and has been used as biocontrol agents against pests. In order to clone a complete cDNA of sublitisin-like protease from P. farinosus, a pair of primers was designed according to the high conserved nucleotide sequences in GenBank, and RT-PCR, 3＇/5＇-RACE PCR were used. Analysis of the cloned complete cDNA, with a whole sequence of 2060bp, showed that it encompassed a open reading frame （ORF） with 1599bp encoding 532 amino acid （aa） residues, including 18-aa signal peptide, 135-aa propeptide and 341-aa mature polypeptide. Alitmments with the deduced amino acid of mature proteins in 4 species of fungi showed 69%, 71%, 68%, and 68%, respectively, identical with those of Metarhizium anisopliae var. anisopliae （CAB63913）, Gibberella zeae （EAA68914） , M. anisopliae var. acridum（CAC95047 ）, and Podospora anserina （AAC03564）, respectively. Meanwhile, the active site residues for serine proteases could be readily identified in the mature protean. The sublitisin-like protease of P. farinosus had 5 cycteines, 5 putative N-glycosylation sites, and 2 putative O- glycosylation sites. T was preferred atthe third positions in this gene.