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

Abstract

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

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.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2014.23.018

https://www.chinaagrisci.com/EN/Y2014/V47/I23/4736

References

[1] 朱勃, 沈中元, 曹喜涛. 微孢子虫起源和进化研究进展. 江西农业学报, 2007, 19(1): 154-158.

Zhu B, Shen Z Y, Cao X T. Recent progress in origin and evolution of microsporidia. Acta Agriculturae Jiangxi, 2007, 19(1): 154-158. (in Chinese)

[2] 沈中元, 黄可威, 徐莉. 从蓝萤叶甲分离出的大型微粒子孢子对家蚕的胚种传染性研究. 蚕业科学, 1994, 20(4): 231-234.

Shen Z Y, Huang K W, Xu L. Studies on the transovarian transmission in the silkworm of large microsporidia isolated from the Phyllobrotica armata Baly. Science of Sericulture, 1994, 20(4): 231-234. (in Chinese)

[3] Zhu F, Shen Z Y, Guo X J, Xu X F, Tao H P, Tang X D, Xu L. A new isolate of Nosema sp. (Microsporidia, Nosematidae) from Phyllobrotica armata Baly (Coleoptera, Chrysomelidae) from China. Journal of Invertebrate Pathology, 2011, 106(2): 339-342.

[4] Tsai S J, Kou G H, Lo C F, Wang C H. Complete sequence and structure of ribosomal RNA gene of Heterosporis anguillarum. Diseases of Aquatic Organisms, 2002, 49(3): 199-206.

[5] Nath B S, Gupta S K, Bajpai A K. Molecular characterization and phylogenetic relationships among microsporidian isolates infecting silkworm, Bombyx mori using small subunit rRNA (SSU-rRNA) gene sequence analysis. Acta Parasitologica, 2012, 57(4): 342-353.

[6] Dong S N, Shen Z Y, Xu L, Zhu F. Sequence and phylogenetic analysis of SSU rRNA gene of five microsporidia. Current Microbiology, 2010, 60(1): 30-37.

[7] Liu H D, Pan G Q, Luo B, Li T, Yang Q, Vossbrinck C R, Debrunner-Vossbrinck B A, Zhou Z Y. Intraspecific polymorphism of rDNA among five Nosema bombycis isolates from different geographic regions in China. Journal of Invertebrate Pathology, 2013, 113(1): 63-69.

[8] Pombert J F, Xu J, Smith D R, Heiman D, Young S, Cuomo C A, Weiss L M, Keeling P J. Complete genome sequences from three genetically distinct strains reveal high intraspecies genetic diversity in the microsporidian Encephalitozoon cuniculi. Eukaryotic Cell, 2013, 12(4): 503-511.

[9] Arisue N, Sanchez L B, Weiss L M, Muller M, Hashimoto T. Mitochondrial-type hsp70 genes of the amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and two microsporidians. Parasitology International, 2002, 51(1): 9-16.

[10] Inagaki Y, Susko E, Fast N M, Roger A J. Covarion shifts cause a long-branch attraction artifact that unites microsporidia and archaebacteria in EF-1α phylogenies. Molecular Biology and Evolution, 2004, 21(7): 1340-1349.

[11] Murakami S. Hepatitis B virus X protein: structure, function and biology. Intervirology, 1999, 42(2/3): 81-99.

[12] Reeb V, Lutzoni F, Roux C. Contribution of RPB2 to multilocus phylogenetic studies of the euascomycetes (Pezizomycotina, fungi) with special emphasis on the lichen-forming Acarosporaceae and evolution of polyspory. Molecular Phylogenetics and Evolution, 2004, 32(3): 1036-1060.

[13] Froslev T G, Matheny P B, Hibbett D S. Lower level relationships in the mushroom genus Cortinarius (Basidiomycota, Agaricales): a comparison of RPB1, RPB2, and ITS phylogenies. Molecular Phylogenetics and Evolution, 2005, 37(2): 602-618.

[14] Carris L M, Castlebury L A, Huang G M, Alderman S C, Luo J F, Bao X D. Tilletia vankyi, a new species of reticulate-spored bunt fungus with non-conjugating basidiospores infecting species of Festuca and Lolium. Mycological Research, 2007, 111(Pt 12): 1386-1398.

[15] 高飞, 高利, 刘太国, 高继国, 陈万权. 小麦矮腥黑粉菌及其近缘种的RPB2基因片段序列分析. 植物保护, 2010, 36(1): 42-46.

Gao F, Gao L, Liu T G, Gao J G, Chen W Q. Sequence analysis of RPB2 gene of Tilletia controversa and its related species. Plant Protection, 2010, 36(1): 42-46. (in Chinese)

[16] Cheney S A, Lafranchi-Tristem N J, Bourges D, Canning E U. Relationships of microsporidian genera, with emphasis on the polysporous genera, revealed by sequences of the largest subunit of RNA polymerase II (RPB1). Journal of Eukaryotic Microbiology, 2001, 48(1): 111-117.

[17] Ku C T, Wang C Y, Tsai Y C, Tzeng C C, Wang C H. Phylogenetic analysis of two putative Nosema isolates from Cruciferous Lepidopteran pests in Taiwan. Journal of Invertebrate Pathology, 2007, 95(1): 71-76.

[18] Ironside J E. Diversity and recombination of dispersed ribosomal DNA and protein coding genes in microsporidia. PLoS One, 2013, 8(2): e55878.

[19] Ironside J E. Multiple losses of sex within a single genus of microsporidia. BMC Evolutionary Biology, 2007, 7: 48.

[20] Hirt R P, Logsdon J J, Healy B, Dorey M W, Doolittle W F, Embley T M. Microsporidia are related to fungi: evidence from the largest subunit of RNA polymerase II and other proteins. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(2): 580-585.

[21] Chen Y P, Pettis J S, Zhao Y, Liu X Y, Tallon L J, Sadzewicz L D, Li R H, Zheng H Q, Huang S K, Zhang X, Hamilton M C, Pernal S F, Melathopoulos A P, Yan X H, Evans J D. Genome sequencing and comparative genomics of honey bee microsporidia, Nosema apis reveal novel insights into host-parasite interactions. BMC Genomics, 2013, 14: 451.

[22] Corradi N, Pombert J F, Farinelli L, Didier E S, Keeling P J. The complete sequence of the smallest known nuclear genome from the microsporidian Encephalitozoon intestinalis. Nature Communications, 2010, 1: 77.

[23] Kornberg R D. The molecular basis of eukaryotic transcription. Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(32): 12955-12961.

[24] Westover K D, Bushnell D A, Kornberg R D. Structural basis of transcription: nucleotide selection by rotation in the RNA polymerase II active center. Cell, 2004, 119(4): 481-489.

[25] Davis J A, Takagi Y, Kornberg R D, Asturias F A. Structure of the yeast RNA polymerase II holoenzyme: Mediator conformation and polymerase interaction. Molecular Cell, 2002, 10(2): 409-415.

[26] Cramer P, Bushnell D A, Kornberg R D. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution. Science, 2001, 292(5523): 1863-1876.

[27] Severinov K, Mustaev A, Kukarin A, Muzzin O, Bass I, Darst S A, Goldfarb A. Structural modules of the large subunits of RNA polymerase. Introducing archaebacterial and chloroplast split sites in the β and β' subunits of Escherichia coli RNA polymerase. The Journal of Biological Chemistry, 1996, 271(44): 27969-27974.

Related Articles 14

[1]	KE DanXia, LI XiangYong, WANG Lei, CHENG Lin, LIU YongHui, LI XiaoYan, WANG HuiFang. Isolation of GmHAT5 from Glycine max and Analysis of Saline Tolerance for Transgenic Lotus japonicus [J]. Scientia Agricultura Sinica, 2017, 50(9): 1559-1570.
[2]	ZHENG XueWei, SHAH Syed Tariq, FAN ShuLi, WEI HengLing, PANG ChaoYou, LI HongBin, YU ShuXun. Molecular Cloning and Functional Analysis of GhNAC7 in Upland Cotton (Gossypium hirsutum L.) [J]. Scientia Agricultura Sinica, 2017, 50(3): 426-436.
[3]	YAN QingDi, ZHAO YaLin, ZHANG Hao, GAO Jing, GAO MengZhu, WANG Qian, WANG Rui, WANG FengRu, DONG JinGao. Analysis on Gene Feature and Function of the Subfamily Members Containing START Domain Only in Arabidopsis thaliana [J]. Scientia Agricultura Sinica, 2017, 50(17): 3274-3285.
[4]	ZHANG Yang, WU Jia-yan, WU Ya-ni, TANG Ming-wei, XIE Li-nan. Progresses and Perspective of the Function of MYB Transcription Factor MIXTA and Its Orthologous Gene [J]. Scientia Agricultura Sinica, 2016, 49(7): 1230-1241.
[5]	NIE Rui-qiang, YANG Yu-jing, XIE Jian-shan, FAN Rui-wen, XU Dong-mei, YU Xiu-ju, DUAN Zhi-cheng, DONG Chang-sheng. Influences of Pax6 PAI Subdomain on MITF, TYR, TYRP1 and TYRP2 in Melanocytes [J]. Scientia Agricultura Sinica, 2016, 49(17): 3433-3442.
[6]	LIU Bei-bei, ZHANG Xiao, XIE Ya-jing, JIAO Ling-xia, LIU Yuan, ZHANG Cun-zheng, ZHAO Yan-yan, WU Ai-hua, LIU Xian-jin. Analysis, Expression and Identification of the Common Structural Domain of Bacillus thuringiensis (Bt) Cry1 Toxins [J]. Scientia Agricultura Sinica, 2016, 49(16): 3130-3139.
[7]	XIAO Rui-xia, WANG Xin-guo, XIA Guo-jun, LI Yong-chun, NIU Hong-bin, WANG Xiang, YIN Jun, REN Jiang-ping. Cloning and Expression Analysis of A Stress-Related TaC2DP1 Gene from Wheat [J]. Scientia Agricultura Sinica, 2015, 48(8): 1463-1472.
[8]	LI Ming-Na-1, 2 , LONG Rui-Cai-1, YANG Qing-Chuan-1, 2 , SHEN Yi-Xin-2, KANG Jun-Mei-1, ZHANG Tie-Jun-1. Cloning and Function Analysis of a Salt-Stress-Induced HD-Zip Trascription Factor MsHB2 from Alfalfa [J]. Scientia Agricultura Sinica, 2014, 47(4): 622-632.
[9]	GUO Rui, LIU Wei-Xing, PAN Zhong-Hua, XUE Ren-Yu, CAO Guang-Li, ZHU Yue-Xiong, GONG Cheng-Liang. Construction of Recombinant BmNPV Infecting Ecotropis oblique [J]. Scientia Agricultura Sinica, 2012, 45(16): 3288-3296.
[10]	TANG Ru-chun,YANG Yu-heng,FAN San-hong,GUO Ai-guang . Cloning of Triticum turgidum L. ramosa2 and DNA Binding Activity Assay of the Recombinant Protein [J]. Scientia Agricultura Sinica, 2011, 44(3): 439-446 .
[11]	GONG Lei,ZHANG Wen-na,XU Hai-yan,YANG Yu-yan,HU Jian-fang,LI Tian-zhong . Molecular Cloning of the NAC Domain Protein of Pear and Research on Its mRNA Transport in Phloem [J]. Scientia Agricultura Sinica, 2010, 43(11): 2307-2314 .
[12]	MENG Fan-rong,LI Yong-chun,LING Na,WANG Xiao,SI Zhi-fei,ZHANG Yan-xia,YIN Jun . Cloning of Full-Length cDNAs Encoding Two Methyl-Binding Domain Proteins and Their Expression Patterns in Wheat Seeds [J]. Scientia Agricultura Sinica, 2009, 42(12): 4132-4138 .
[13]	Yun-Xiang ZHAO Zhao-shi XU Ming CHEN Lian-Cheng LI Yao-Feng CHEN You-Zhi MA. Analysis of specific binding and subcellular localization of wheat ERF transcription factor W17 [J]. Scientia Agricultura Sinica, 2008, 41(6): 1575-1582 .
[14]	,,,,. Toxicity Analysis of Truncated Insecticidal Crystal Protein Cry1Ba3 from Bacillus thuringiensis [J]. Scientia Agricultura Sinica, 2005, 38(08): 1585-1590 .

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

Sequencing and Phylogenetic Analysis of the RPB1 Gene of Nosema sp. PA

RichHTML

PDF