家蚕保幼激素结合蛋白<i>Bmtol</i>基因的克隆及表达分析

doi:10.13523/j.cb.20171003

中国生物工程杂志

2017, Vol. 37

Issue (10): 16-25 DOI: 10.13523/j.cb.20171003

研究报告

家蚕保幼激素结合蛋白Bmtol基因的克隆及表达分析

何石宝, 杨成飞, 尚杉, 王凌燕, 唐文超, 朱勇

西南大学生物技术学院重庆 4007715

Cloning and Expression Analysis of Juvenile Hormone Binding Protein Gene Bmtol in Silkworm,Bombyx mori

HE Shi-bao, YANG Cheng-fei, SHANG Sha, WANG Ling-yan, TANG Wen-chao, ZHU Yong

College of Biotechnology, Southwest University, Chongqing 400715, China

全文: PDF(2298 KB) HTML

摘要： 目的：克隆获得家蚕（Bombyx mori）Bmtol基因序列，并对其蛋白结构进行预测，分析其在组织和JHA处理后头部的表达差异，为该基因的功能研究提供参考。方法：以家蚕的全组织cDNA为模板利用RT-PCR技术扩增和克隆获得Bmtol基因cDNA全长序列，并提交至GenBank数据库；利用多种生物信息学软件预测分析其编码蛋白的理化特性和结构特征；采用MREGA5.0软件中的邻接法（neighbor-joining，NJ）构建BmTOL及其它昆虫同源TO的进化树；通过qPCR技术分析Bmtol基因在5龄3天家蚕不同组织的表达情况，及JHA处理5龄蚕后在0 h、24 h、48 h、72 h和96 h家蚕头部的表达情况。结果：克隆获得了家蚕Bmtol基因的cDNA序列（GenBank登录号KY681053），Bmtol基因的开放阅读框（ORF）长度为759 bp，编码252个氨基酸，预测其蛋白分子量为27.72kDa，理论等电点为6.16，有信号肽，无跨膜结构，且第25~251位氨基酸之间存在一个保幼激素结合蛋白家族JHBP保守结构域；N端为疏水区域，可能与保幼激素结合蛋白的核心部位有关。亚细胞定位分析表明，BmTOL属于分泌型蛋白，主要集中在内质网-高尔基体-质膜分泌途径上。BmTOL蛋白具有3个α螺旋，第34位的Cys和第44位Cys形成一个二硫键链接在α1螺旋和N末端，构成BmTOL蛋白与配体结合的核心部位。序列比对结果显示，家蚕BmTOL序列与其他昆虫TO的氨基酸序列一致性差别较大。家蚕BmTOL与果蝇DmTO的相似性为25.10%，与烟草天蛾的相似性为19.69%，与冈比亚按蚊的相似性为25.78%，与埃及伊蚊的相似性为23.53%，与黑花蝇的相似性为28.17%，与意大利蜜蜂的相似性为23.05%，与苹浅褐卷蛾的相似性为21.18%。系统进化树分析表明，所有选用昆虫TO形成两个大的分支：苹浅褐卷蛾EpTO1、烟草天蛾MsTO、意大利蜜蜂AmTOL、果蝇DmTO和黑花蝇PrTOL聚为一个分支，埃及伊蚊AaTO、冈比亚按蚊AgTOL-2和家蚕BmTOL聚为另一大分支。qPCR结果显示，Bmtol基因在家蚕头部、表皮和精巢有较高表达，其他组织表达量很低或没有。在JHA处理的5龄家蚕的头部，Bmtol基因在处理后0 h、24 h、48 h、72 h和96 h的表达量差异不明显。结论：BmTOL蛋白属于JHBP家族，具有JHBP家族的典型结构；组织表达谱和JHA处理结果暗示，BmTOL属保幼激素结合蛋白（JHBP），在家蚕中除保幼激素结合之外还参与其他多种生理功能。

关键词： 家蚕; 基因克隆; JHA; 生物信息学分析; Bmtol

Abstract: Objective:The objective is to clone the cDNA sequence of Bmtol from silkworm Bombyx mori, predict the protein structure, explore its expression profiles in different tissues and at different time of JHA treatment of silkworm, and to provide a fundamental evidence for the future study of the physiological function of this gene.Method:The full-length cDNA sequence of Bmtol was cloned from the entire tissues of silkworm by RT-PCR and submitted to the GenBank database. Physiochemical properties and structure characteristics of the deduced amino acid sequence were analyzed by multiple bioinformatics methods. Phylogenetic tree between BmTOL and its homologous JHBP from other insects was constructed using neighbor-joining of MEGA5.0. The expression levels of Bmtol mRNA of different tissues at day-3 in 5th instar were detected by qPCR.Result:The full-length cDNA sequence of Bmtol was obtained (GenBank is KY681053). The open reading frame (ORF) of Bmtol is 759 bp, encoding a protein of 252 aa with an estimated molecular weight of 27.72 kDa and pI of 6.16. The encoding protein has a signal peptide and no transmembrance structure, possesses a JHBP superfamily domain between 25~251 residues, exists hydrophobic regions in N-terminus and contains two conserved cysteins, suggesting that it is the core part of the family proteins to bind ligand. Subcellular localization results showed that BmTOL was located on the secretory pathway about endoplasmic reticulum, golgi and plasmalemma which belongs to the secretory protein. BmTOL possesses three α-helixs. The thirty-fourth bit Cys and the forty-fourth bit Cys form a disulfide bond in the α1 helix and the N-terminus, which forms the core part of the BmTOL protein binding to the ligand. The results of amino acid sequence alignment showed that the amino acid sequence of BmTOL of silkworm was significantly different from TO of other insect. It was homologous to DmTO with 25.10% amino acid sequence identity, and had 19.69% sequence identity with MsTO, and had 25.78% sequence identity with AgTOL-2, and had 23.53% sequence identity with AaTO, and had 28.17% sequence identity with PrTOL, and had 23.05% sequence identity with AmTOL, and had 21.18% sequence identity with EpTO1. Phylogenetic analysis revealed that BmTOL,EpTO1, MsTO, AmTOL, DmTO and PrTOL were clustered into one group, AaTO, AgTOL-2 and BmTOL were clustered into the other group. Besides, the results of qPCR showed that the Bmtol transcripts were higher expressed in the head, epidermis and testis of the day-3 in 5th instar.The Bmtol gene expression was not significantly different in the head at 0 h, 24 h, 48 h, 72 h and 96 h in 5th silkworm by JHBP treatment.Conclusion:BmTOL belongs to the JHBP family with a JHBP domain. The results of expression profiling and JHA treatment suggested that BmTOL belongs to juvenile hormone binding protein (JHBP) and is involved in not only juvenile hormone binding recognition, but also other physiological functions.

Key words: Bmtol Gene cloning Bioinformatics analysis JHA Bombyx mori

收稿日期: 2017-04-18 出版日期: 2017-10-25

ZTFLH:

Q819

基金资助: 国家农业科技成果转化资金项目（2012GB2F100376）、油蚕ov突变基因的定位克隆研究（Csts2014jcyjA80007）资助项目

通讯作者: 朱勇,zhuy@swu.edu.cn E-mail: zhuy@swu.edu.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	唐文超
	何石宝
	尚杉
	王凌燕
	杨成飞
	朱勇

引用本文:

何石宝, 杨成飞, 尚杉, 王凌燕, 唐文超, 朱勇. 家蚕保幼激素结合蛋白Bmtol基因的克隆及表达分析[J]. 中国生物工程杂志, 2017, 37(10): 16-25.

HE Shi-bao, YANG Cheng-fei, SHANG Sha, WANG Ling-yan, TANG Wen-chao, ZHU Yong. Cloning and Expression Analysis of Juvenile Hormone Binding Protein Gene Bmtol in Silkworm,Bombyx mori. China Biotechnology, 2017, 37(10): 16-25.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20171003 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I10/16

[1]	Whisenton L R, Bowen M F, Granger N A, et al. Brain-mediated 20-hydroxyecdysone regulation of juvenile hormone synthesis by the corpora allata of the tobacco hornworm, Manduca sexta. Gene and Comp Endoc, 1985, 58(2):311-318.
[2]	Orth A P, Lan Q, Goodman W G. Ligand regulation of juvenile hormone binding protein mRNA in mutant Manduca sexta. Mol Cell Endocrinol, 1999, 149(1-2):61-69.
[3]	Orth A P, Tauchman S J, Doll S C, et al. Embryonic expression of juvenile hormone binding protein and its relationship to the toxic effects of juvenile hormone in Manduca sexta. Insect Biochem Molec, 2003, 33(12):1275-1284.
[4]	贺秀婷, 张亚楠, 李飞,等. 灰飞虱保幼激素结合蛋白基因的克隆及表达动态. 南京农业大学学报, 2012, 35(2):59-64. He X T, Zhang Y N, Li F, et al. Cloning, temporal expression of a juvenile hormone binding protein gene in Laodelphax striatellus(Fallén). Journal of Nanjing Agricultural University, 2012, 35(2):59-64.
[5]	Dauwalder B, Tsujimoto S, Moss J, et al. The Drosophila takeout gene is regulated by the somatic sex-determination pathway and affects male courtship behavior. Gene Dev, 2002, 16(22):2879-2892.
[6]	Hagai T, Cohen M, Bloch G. Genes encoding putative takeout/juvenile hormone binding proteins in the honeybee(Apis mellifera) and modulation by age and juvenile hormone of the takeout-like gene GB19811. Insect BiochemMolec, 2007, 37(37):689-701.
[7]	Sarov-Blat L, So W V, Liu L, et al. The Drosophila takeout gene is a novel molecular link between circadian rhythms and feeding behavior. Cell, 2000, 101(6):647-656.
[8]	So W V, Sarov-Blat L, Kotarski C K, et al. takeout, a novel Drosophila gene under circadian clock transcriptional regulation. Mol Cell Biol, 2000, 20(18):6935-6944.
[9]	Gáliková M, Flatt T. Dietary restriction and other lifespan extending pathways converge at the activation of the downstream effector takeout. Aging, 2010, 2(7):387-389.
[10]	Benito J, Hoxha V, Lama C, et al. The circadian output gene takeout is regulated by Pdp1ε. P Natl Acad Sci USA, 2010, 107(6):2544-2549.
[11]	Khericha M, Kolenchery J B, Tauber E. Neural and non-neural contributions to sexual dimorphism of mid-day sleep in Drosophila melanogaster:a pilot study. Physiol Entomol, 2016,41(4):327-334.
[12]	Bohbot J, Vogt R G. Antennal expressed genes of the yellow fever mosquito (Aedes aegypti L.) characterization of odorant-binding protein 10 and takeout. Insect BiochemMolec, 2005, 35(9):961-979.
[13]	Kaneko Y, Hiruma K. Allatotropin inhibits juvenile hormone biosynthesis by the corpora allata of adult Bombyx mori. J Insect Physiol, 2015, 80(1):15-21.
[14]	Vanaphan N, Dauwalder B, Zufall R A. Diversification of takeout, a male-biased gene family in Drosophila. Gene, 2012, 491(2):142-148.
[15]	Wei D, Li H M, Tian C B, et al. Proteome analysis of male accessory gland secretions in oriental fruit flies reveals juvenile hormone-binding protein, suggesting impact on female reproduction. SciRep-UK, 2015, 5:16845.
[16]	于芹, 安利国, 王宪辉. 昆虫Takeout蛋白家族的研究进展. 应用昆虫学报, 2011, 48(2):404-409. Yu Q, An L G, Wang X H. Recent advances in Takeout protein family of insects. Chinese Journal of Applied Entomology, 2011, 48(2):404-409.
[17]	Hamiaux C, Stanley D, Greenwood D R, et al. Crystal structure of Epiphyas postvittana takeout1 with bound ubiquinone supports a role as ligand carriers for takeout proteins in insects. J Biol Chem, 2009, 284(6):3496-3503.
[18]	Fujimoto Z, Suzuki R, Shiotsuki T, et al. Crystal structure of silkworm Bombyx mori JHBP in complex with 2-methyl-2,4-pentanediol:plasticity of JH-binding pocket and ligand-induced conformational change of the second cavity in JHBP. PLoS One, 2013, 8(2):e56261.
[19]	Meunier N, Belgacem Y H, Martin J R.Regulation of feeding behaviour and locomotor activity by takeout in Drosophila.J ExpBiol, 2007, 210(Pt 8):1424-1434.
[20]	Bauer J, Antosh M, Chang C, et al. Comparative tranciptional profiling identifies takeout as a gene that regulates life span. Aging, 2010, 2(5):298-310.
[21]	Du J, Hiruma K, Riddiford L M. A novel gene in the takeout gene family is regulated by hormones and nutrients in Manduca larval epidermis. Insect BiochemMolec, 2003, 33(8):803-814.
[22]	Justice R W, Dimitratos S, Walter M F, et al. Sexual dimorphic expression of putative antennal carrier protein genes in the malaria vector Anopheles gambiae. Insect MolBiol, 2003, 12(6):581-594.
[23]	Chang E S, Coudron T A, Bruce M J, et al. Juvenile hormone-binding protein from the cytosol of Drosophila Kc cells. P Natl Acad Sci USA, 1980, 77(8):4657-4661.
[24]	Shemshedini L, Lanoue M, Wilson T G. Evidence for a juvenile hormone receptor involved in protein synthesis in Drosophila melanogaster. J BiolChem, 1990, 265(4):1913-1918.
[25]	Chang E S, Bruce M J, Prestwich G D. Further characterization of the juvenile hormone-binding protein from the cytosol of a Drosophila cell line:use of a photoaffinity label. Insect Biochem, 1985, 15(2):197-204.
[26]	Ashok M, Turner C, Wilson T G. Insect juvenile hormone resistance gene homology with the bHLH-PAS family of transcriptional regulators.P Natl AcadSci USA, 1998, 95(6):2761-2766.
[27]	Wilson T G, Yerushalmi Y, Donnell D M, et al. Interaction between hormonal signaling pathways in Drosophila melanogaster as revealed by genetic interaction between Methoprene-tolerant and Broad-Complex. Gene, 2006, 172(1):253-264.
[28]	赵亚华. 基础分子生物学教程. 第2版. 北京:科学出版社, 2006. 351-355. Zhao Y H. Fundamental Molecular Biology Course. 2nd ed. Beijing:Science Press, 2006. 351-355.
[29]	彭佳师, 龚继明. 信号肽与蛋白质的分选转运. 植物生理学报, 2011, 47(1):9-17. Peng J S, Gong J M. The mechanisms of protein sorting and translocation regulated by signal peptides. Plant Physiology Journal, 2011, 47(1):9-17.
[30]	Fujikawa K, Seno K, Ozaki M. A novel Takeout-like protein expressed in the taste and olfactory organs of the blowfly, Phormia regina. Febs J, 2006, 273(18):4311-4321.
[31]	Saito K, Su Z H, Emi A, et al. Cloning and expression analysis of takeout/JHBP family genes of silkworm, Bombyx mori. Insect MolBiol, 2006, 15(3):245-251.

[1]	王嘉祯,姚伦广,王峰,阚云超,罗金萍,黄倩倩,段建平. 家蚕中肠特异启动子P56的克隆及活性分析 ^*[J]. 中国生物工程杂志, 2018, 38(2): 13-17.
[2]	柳双双,吴锁伟,饶力群,万向元. 玉米核雄性不育的分子机制研究与应用分析[J]. 中国生物工程杂志, 2018, 38(1): 100-107.
[3]	张艳芳, 孙瑞芬, 郭树春, 侯建华. 向日葵V-ATPase a3亚基基因的克隆及表达分析[J]. 中国生物工程杂志, 2017, 37(5): 19-27.
[4]	项丽, 王申, 田海山, 钟美娟, 周汝滨, 曹定国, 梁朋, 张国平, 何滔, 庞实锋. 小鼠c-Myc基因的克隆表达及其纯化[J]. 中国生物工程杂志, 2017, 37(2): 20-25.
[5]	饶菁菁, 景一娴, 邹明月, 胡小蕾, 廖飞, 杨晓兰. 季也蒙毕赤酵母菌尿酸酶基因的克隆、重组表达及表征[J]. 中国生物工程杂志, 2017, 37(11): 74-82.
[6]	刘婷婷, 梁梓强, 梁士可, 郭技星, 王方海. 利用生物工程技术生产蜘蛛丝的研究进展[J]. 中国生物工程杂志, 2016, 36(5): 132-137.
[7]	张敏, 田银帅, 胡晓乐, 徐莺, 陈放. *麻疯树G蛋白γ亚基JcAGG3基因的克隆及表达分析*[J]. 中国生物工程杂志, 2016, 36(5): 46-52.
[8]	万永青, 杨洋, 张春林, 万东莉, 杨爱琴, 杨杞, 王瑞刚, 李国婧. *中间锦鸡儿DHN1基因克隆及表达分析*[J]. 中国生物工程杂志, 2016, 36(4): 88-96.
[9]	陈晓峰, 胡盼, 李岩松, 郭兴, 邹德颖, 刘楠楠, 卢士英, 周玉, 柳增善, 李兆辉, 任洪林. 小鼠Peroxiredoxin 6基因克隆、原核表达及多克隆抗体的制备[J]. 中国生物工程杂志, 2016, 36(3): 11-16.
[10]	段建平, 李莹, 阚云超, 姚伦广, 夏庆友. *家蚕雄dsx^M3异位表达对下游靶基因表达的影响*[J]. 中国生物工程杂志, 2016, 36(2): 38-42.
[11]	李达, 代鹏, 王伟, 张文涛, 汪钦, 束毅, 祝春来, 纪奇峰, 梁平, 颜真. PLCE1基因及rs2274223和rs3765524单体型的克隆与表达[J]. 中国生物工程杂志, 2016, 36(12): 1-7.
[12]	高飞, 周婧, 刘晓彤, 李成磊, 姚慧鹏, 赵海霞, 吴琦. *苦荞锌指蛋白基因FtLOL1的克隆及其对非生物胁迫的应答*[J]. 中国生物工程杂志, 2015, 35(8): 44-50.
[13]	王世奇, 刘婧莹, 刘晨浪, 李纯, 胡晓凤, 夏立秋, 张友明. sTRAIL蛋白原核表达载体的构建、表达及抗肿瘤活性研究[J]. 中国生物工程杂志, 2015, 35(12): 1-7.
[14]	樊飞飞, 李杰勤, 詹秋文, 王丽华, 刘言龙. 植物肉桂酰辅酶A还原酶基因克隆研究进展[J]. 中国生物工程杂志, 2015, 35(12): 96-102.
[15]	杨杞, 尹佳佳, 王颖, 王瑞刚, 李国婧. *柠条锦鸡儿CkLEA1基因克隆及表达分析*[J]. 中国生物工程杂志, 2013, 33(5): 93-99.

Viewed

Full text

Abstract

Cited

Shared

Discussed