竹黄菌Ku70基因全长克隆及序列分析

Cloning and Sequence Analysis of the Full Length Ku70 DNA in Shiraia bambusicola P. Hennings

为构建竹黄菌高效敲除载体,以利于竹黄菌基因功能及竹黄菌与寄主竹子之间的相互关系的研究,利用RT-PCR(reverse transcriptase-PCR)及RACE(rapid amplification of c DNA ends)技术克隆获得Ku70基因全长序列,命名为s Ku70。序列分析显示:该序列基因全长为2 306 bp,开放阅读框长度为1 974 bp,编码657个氨基酸,分子质量为73.940 5 k D,理论等电点p I为5.58;序列同源性分析表明:该基因序列和预测的蛋白序列与小麦叶枯病菌SN15(Phaeosphaeria nodorum SN15)的Ku70 m RNA序列(XM_001803174.1)及ATP依赖的DNA解旋酶亚基Ⅱku70(Q0U5F2.1)同源性最高,identity分别75%和78%。二级结构预测表明Ku70蛋白α-螺旋(alpha helix)占36.99%,延伸链(extended strand)占17.05%,无规卷曲(random coil)占36.23%;包含一个Ku-core domain、一个Von Willebrand factor type A(v WA)domain和一个SAP domain,为亲水蛋白;并利用同源模建的方法预测了其三级结构。

In order to construct a high efficient gene disruption strain of Shiraia bambusicola, the complete DNA of Ku70 gene was cloned by RT-PCR(reverse transcriptase-PCR) and RACE(rapid amplification of c DNA ends)and named s Ku70, which facilitating the research of functional verification of genes and interact mechanism between Shiraia bambusicola and its host bamboo. Sequence analysis showed that: the full length genomic sequence of Ku70 is 2 306 bp; the gene contains a open reading frame of 1 974 bp and encodes 657 amino acid residues.The estimated molecular weight and isoelectric point of the putative protein respectively are 73.940 5 k D and 5.58.Sequence homology analysis indicated that the gene sequence and putative protein sequence respectively had the highest 75% and 78% similarity with Ku70 m RNA sequence(XM_001803174.1) and ATP-dependent DNA helicaseⅡsubunit Ku70(Q0U5F2.1) of Phaeosphaeria nodorum SN15. Secondary structure prediction stated that Ku70 protein α-helix(Alpha helix), extended strand and random coil severally accounted for 36.99%, 17.05% and 36.23%.It included a Ku-core domain, a Von Willebrand factor type A(v WA) domain and a SAP domain. It is a hydrophilic protein. And its tertiary structure was predicted by using homology modeling method.