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为了研究ve A基因在竹黄菌中的调节机制,利用RT-PCR(reverse transcriptase-PCR)、RACE(rapid amplification of c DNA ends)及染色体步移技术克隆获得了ve A基因的全长序列。序列分析显示:该基因序列全长为1 780 bp,其开放阅读框长度为1 104 bp,编码367个氨基酸;其编码的蛋白质分子量为40.887 5 k D,理论等电点p I为8.95,含42个负电荷氨基酸和46个正电荷氨基酸,是一种弱酸性蛋白;序列同源性分析结果显示:该基因编码的蛋白序列与偃麦草核腔菌(Pyrenophora tritici-repentis)的ve A蛋白序列(XM_001933944.1)及异旋孢腔菌(Cochliobolus heterostrophus)的velvet-like protein(JF826791.1)的同源性最高,其identity分别为78%和79%;对该基因编码的蛋白质进行二级结构预测,结果显示:该蛋白α-螺旋(alpha helix)占11.72%,延伸链(extended strand)占24.52%,β-转角(beta turn)占9.54%,无规卷曲(random coil)占54.22%;对其保守结构域分析显示,该蛋白含有一个Velvet superfamily结构域;利用同源模建方法预测其三级结构,结果显示该蛋白的三级结构模型与隔孢腔菌目中其他物种的ve A异源二聚体的相似性为46%。
In order to study the regulatory mechanism of ve A gene in the bamboo, the full-length sequence of ve A gene was cloned by RT-PCR, rapid amplification of c DNA ends and chromosome walking. Sequence analysis showed that the full length of this gene was 1 780 bp, with an open reading frame of 1 104 bp encoding a protein of 367 amino acids. The encoded protein had a molecular weight of 40.887 5 kD and a theoretical isoelectric point p I of 8.95. 42 negatively charged amino acids and 46 positively charged amino acids, which is a weakly acidic protein. Sequence analysis revealed that the protein sequence encoded by this gene interacts with the ve A protein sequence of Pyrenophora tritici-repentis (XM_001933944.1) and velvet-like protein (JF826791.1) of Cochliobolus heterostrophus had the highest identities of 78% and 79%, respectively. The proteins encoded by this gene were identified as secondary The results showed that alpha helix accounted for 11.72%, extended strand accounted for 24.52%, β-turn accounted for 9.54%, random coil accounted for 54.22% ; Its conserved domain analysis showed that the protein contains a Velvet superfamily domain; the use of homology modeling method to predict the tertiary structure, the results showed that the tertiary structure of the protein model and the other species of Moniliales other species ve The similarity of A heterodimers was 46%.