蓝叶虫微孢子虫RPB1基因片段序列的测定及其系统发育分析

doi:10.3864/j.issn.0578-1752.2014.23.018

摘要/Abstract

摘要： 【目的】克隆蓝叶虫微孢子虫RPB1（RNA聚合酶II大亚基）基因片段，对其基因及编码的蛋白序列特征进行生物信息学分析，进一步明确蓝叶虫微孢子虫的分类学地位，为深入探究RPB1基因编码蛋白的具体生物学功能奠定基础。【方法】根据家蚕微孢子虫RPB1基因序列，采用Primer Premier 5.0软件设计6对同源引物，利用PCR技术克隆蓝叶虫微孢子虫RPB1基因片段；通过 GSDS、SMART、DNAstar、MEGA4.1等生物信息学分析软件对蓝叶虫微孢子虫RPB1基因片段进行系统发育分析。【结果】克隆得到了蓝叶虫微孢子虫RPB1基因片段序列，在GenBank登录号为KJ728831。该基因片段的长度为2 933 bp，包含一个长为2 922 bp的开放读码框，预测编码的蛋白质由974个氨基酸组成，蛋白分子量为109.38 kD，等电点为7.087。蓝叶虫微孢子虫RPB1基因片段结构为单外显子结构，编码的蛋白含有4个结构域：RPOLA_N、RNA_pol_Rpb1_4、RNA_pol_Rpb1_5和RNA_pol_Rpb1_6。其中，RPOLA_N结构域在原核和真核生物中都是非常重要的结构域。该蛋白质的二级结构主要由4种形式组成：α螺旋、无规则卷曲、延伸链和β转角。α-螺旋和无规则卷曲所占比例相当高，延伸链主要位于α-螺旋和无规则卷曲之间。N-末端以α-螺旋的形式存在。多序列比对与系统进化分析发现，蓝叶虫微孢子虫和Nosema bombycis、N. trichoplusiae、Nosema sp. CPP、N. fumiferanae、N. disstriae、N. tyriae、N. granulosis 7种Nosema属微孢子虫聚类在同一进化支。蓝叶虫微孢子虫RPB1基因编码的蛋白与同一进化支上的其他7种微孢子虫RPB1基因编码的蛋白相似度在81.9%—99.6%，分化度在0.004—0.049。蓝叶虫微孢子虫与N. bombycis的RPB1基因序列相似性高达99.6%，并与N. bombycis具有非常近的亲缘关系。【结论】成功克隆了蓝叶虫微孢子虫RPB1基因片段，对其系统进化分析进一步证实了蓝叶虫微孢子虫确实属于Nosema属。

关键词: 蓝叶虫微孢子虫, RPB1基因, 结构域, 系统发育分析

Abstract: 【Objective】 The objective of this study is to clarify the taxonomic status of Nosema sp. PA and provide a new foundation for the further study of its biological function by cloning the RPB1 (largest subunit of RNA polymeraseII) gene of Nosema sp. PA and analyzing the gene sequence by bioinformatics methods. 【Method】 Six pairs of homologous primers were designed by Primer Premier 5.0 software for the RPB1 gene of Nosema sp. PA based on the RPB1 gene of Nosema bombycis. Partial sequence of the RPB1 gene of Nosema sp. PA was cloned by PCR amplification. Then, bioinformatics analysis on the RPB1 gene of Nosema sp. PA and its encoding protein were conducted by bio-softs as GSDS, SMART, DNAstar and MEGA4.1. 【Result】 Partial sequence of the RPB1 gene of Nosema sp. PA was cloned by PCR amplification (GenBank accession number KJ728831). The partial sequence of the RPB1 gene of Nosema sp. PA had 2 933 nucleotides which contained an ORF with 2 922 bp encoding a polypeptide of 974 amino acids with a molecular weight of 109.38 kD and an isoelectric point of 7.087. The structure of the partial sequence of the RPB1 gene was a single exon. The encoded protein contained four domains: RPOLA_N, RNA_pol_Rpb1_4, RNA_pol_Rpb1_5 and RNA_pol_Rpb1_6. RPOLA_N domain is a very important domain both in prokaryotes and eukaryotes among the four domains. The encoded protein contained four main secondary structures: α-helix, random coil, β-turn and extended strand. The proportion of α-helix and random coil was quite high. Extended strand was mainly located between the α-helix and random coil. Sequence comparison and phylogenetic analysis showed that the encoded protein of Nosema sp. PA was 81.9%-99.6% identity and 0.004-0.049 divergence with those of N. bombycis, N. trichoplusiae, Nosema sp. CPP, N. fumiferanae, N. disstriae, N. tyriae and N. granulosis. Nosema sp. PA and other seven kinds of “true’’ Nosema species above-mentioned clustered in the same clade. The encoded protein was 99.6% identical to N. bombycis and had a close relationship with N. bombycis. 【Conclusion】The partial sequence of the RPB1 gene of Nosema sp. PA is successfully cloned. The results confirmed that Nosema sp. PA is a member of Nosema species in the aspect of molecular biology.

Key words: Nosema sp. PA, RPB1 gene, domain, phylogenetic analysis

燕薇，沈中元，唐旭东，徐莉，李前龙，肖圣燕，乐亚杰，付绪亮. 蓝叶虫微孢子虫RPB1基因片段序列的测定及其系统发育分析[J]. 中国农业科学, 2014, 47(23): 4736-4744.

YAN Wei1, SHEN Zhong-yuan, TANG Xu-dong, XU Li, LI Qian-long, XIAO Sheng-yan, YUE Ya-jie, FU Xu-liang. Sequencing and Phylogenetic Analysis of the RPB1 Gene of Nosema sp. PA[J]. Scientia Agricultura Sinica, 2014, 47(23): 4736-4744.

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