粉棒束孢几丁质酶基因cDNA全序列克隆及结构特征分析

Cloning and characterization of full-length cDNA of chitinase gene from Isaria farinosa

通过设计基因保守区的特异性简并引物,运用SMARTRACERT-PCR技术,首次从粉棒束孢中克隆出完整的几丁质酶基因。该基因cDNA全长1549bp,5'端非翻译区89bp,3'端非翻译区有188bp,开放阅读框(ORF)1272bp,编码423个氨基酸。信号肽长度为22个氨基酸。信号肽很可能需要两次剪切。成熟的蛋白理论分子量为43.9kDa,理论等电点为5.67。氨基酸序列具有几丁质酶18族的两个高度保守的活性区域,一个是酶作用活性位点,另一个是几丁质结合区域。该蛋白可归于几丁质酶18族V类。成熟蛋白的氨基酸序列与裂虫壳AAV98691、白色扁丝霉CAA45468、菌生轮枝孢AAP45631、莱氏野村菌AAP04616和球孢白僵菌AAN41261的同源性分别为91%,89%,80%,76%和75%。

The full-length cDNA coding the chitinases produced by the biocontrol agent Isaria farinosa using SMART RACE RT-PCR was reported in this paper. Analysis of the cloned complete cDNA, with a whole sequence of 1549bp, showed that it encompassed an open reading frame （ORF） of 1272bp encoding 423 amino acids with a stretch of 22 amino acid residues displaying characteristics of signal peptide. The result showed that the mature chitinase （without signal sequence） had a molecular mass of 43.9 kDa with a calculated pI of 5.67. This sequence contained two highly conserved regions of the active domain of the family 18 glycosyl hydrolases including a presumed enzymatic active site and a potential chitin-binding domain. The cloned Isaria chinitase belongs to the class V in 18 family of glycosyl hydrolase. Alignments with the deduced amino acid of mature proteins in 5 species of fungi showed 91%, 89%, 80%, 76% and 75%, respectively, identical with those of Torrubiella confragosa （AAV98691）, Aphanocladium album （CAA45468）, Verticillium fungicola （AAP45631）, Nomuraea rileyi （ AAP04616） and Beauveria bassiana （AAN41261）, respectively.