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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 an efficient knockout vector for Pseudomonas solanacearum in order to facilitate the gene function of Pseudomonas solanacearum and the relationship between Pseudomonas solanacearum and host bamboo, the reverse transcriptase-PCR and rapid amplification of cDNA ends) was cloned to obtain the full-length Ku70 gene sequence, named s Ku70. Sequence analysis showed that the full length of this gene was 2 306 bp, the open reading frame was 1 974 bp, encoding 657 amino acids with a molecular mass of 73.940 5 kD and a theoretical isoelectric point p I of 5.58. Sequence homology analysis The results showed that the sequence and the predicted protein sequence were homologous to the Ku70 m RNA sequence (XM_001803174.1) and the ATP-dependent DNA helicase subunit Ⅱku70 (Q0U5F2.1) of the wheat leaf spot disease Phagocytide SN15 (Q15U5F2.1) Highest, identity respectively 75% and 78%. Secondary structure prediction showed that the Ku70 protein accounted for 36.99% of alpha helix, extended strand accounted for 17.05%, random coil accounted for 36.23%; contained a Ku-core domain, a Von Willebrand factor type A (v WA) domain and a SAP domain as hydrophilic proteins. The tertiary structure was predicted by homology modeling.