玫烟色棒束孢几丁质酶(Ifuchit2)结构基因及上游序列的克隆与分析

Cloning and sequence analysis of structural gene and upstream sequence of chitinases(Ifuchit2) from Isaria fumosorosea

玫烟色棒束孢作为一类可致昆虫死亡的虫生真菌,是生物防治中极具开发应用潜力的种类。运用PCR技术与DNA步移技术,从玫烟色棒束孢中克隆得到几丁质酶的结构基因及其上游序列。结构基因总长1 425 bp,扩出的结构基因DNA与其cDNA比较含有3个内含子,分别位于距离起始密码子的121碱基至176碱基处,277碱基至331碱基处和380碱基至431碱基处,大小分别为54 bp、53 bp和51 bp。在玫烟色棒束孢几丁质酶Ifuchit2结构基因的基础上,克隆得到了其上游序列。该基因上游序列大小为1 794 bp,其中有209 bp与该基因cDNA序列相互重叠。序列分析表明,玫烟色棒束孢几丁质酶Ifuchit2上游序列中,含有真核生物基因典型的TATA-盒,以及多个高度保守的潜在转录因子结合位点,如Oct1、GATA-1、GATA-2和CdxA等。另外,该上游序列还存在一些特异的应答元件,如热激应答元件(HSE)、胁迫应答元件(STRE)等。

Isaria fumosorosea is a kind of entomogenous fungi causing insect death with great potentials of exploitation and application for biological control.The structural gene of chitinases,with 1 425 bp,was cloned from Isaria fumosorosea,and the amplified structural gene have three introns compared with its cDNA.In the current study,the upstream sequence of chitinases（Ifuchit2） from Isaria fumosorosea was cloned by genome walking based on its structural gene.The obtained upstream sequence was 1 794 bp,and 209 bp was overlapped with its cDNA sequences.The sequence analysis indicated that the upstream sequence has a typical eukaryotic gene TATA-box of several hidden highly conserved transcription factor binding sites,such as Oct1,GATA-1,GATA-2,CdxA and contains some specific response elements,such as the heat responsive element（HSE）,stress response elements（STRE）.