中华绒螯蟹EsSox21b-like基因干扰载体的构建及 原核表达制备dsRNA

生物技术通报 ›› 2014, Vol. 0 ›› Issue (6): 134-138.

中华绒螯蟹EsSox21b-like基因干扰载体的构建及原核表达制备dsRNA

刘志强, 陈洁, 邱高峰

（上海海洋大学农业部淡水水产种质资源重点实验室，上海 201306）

收稿日期:2013-11-06 出版日期:2014-06-25 发布日期:2014-06-25
作者简介:刘志强，男，硕士，研究方向：中华绒螯蟹生殖与发育；E-mail：liu503212225@sohu.com
基金资助:
国家自然科学基金项目（31272655），国家科技支撑计划项目（2012BAD26B04），上海市科委基础重点科研项目（11JC1404600），上海高校水产学一流学科建设项目（B5005120001）

Construction of Interference Vector of EsSox21b-like Gene from Chinese Mitten Crab（Eriocheir sinensis）and Preparation of dsRNA by Prokaryotic Expression

Liu Zhiqiang, Chen Jie, Qiu Gaofeng

（Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306）

Received:2013-11-06 Published:2014-06-25 Online:2014-06-25

摘要/Abstract

摘要： Sox（SRY-related HMG-box）基因是一类重要的转录因子，在动物的性别分化与决定、器官的发生等方面具有重要的调控作用。旨在利用RNA干扰技术证实该基因的功能，将EsSox21b-like基因617 bp的片段克隆至含有两个T7启动子的L4440载体上，构建了EsSox21b-like-L4440干扰载体，将该重组载体转化至RNaseⅢ缺陷型的HT115菌株中，经IPTG诱导菌株体内转录，获得了EsSox21b-like正义和反义单链RNA，经过变性、退火形成大量双链RNA（EsSox21b-like-dsRNA），能满足以后应用RNA干扰试验对于EsSox21b-like基因功能的研究。

关键词: 中华绒螯蟹, RNA干扰, L4440, HT115, 原核表达, dsRNA

Abstract: Sox（SRY-related HMG-box）gene is a kind of important transcription factor, which functions in sex determination, organogenesis, etc. The gonad-specific expression of this novel gene suggested that it might play an important role in the development of gonad. To determine the exact role of EsSox21b-like gene using RNA interference, a EsSox21b-like-L4440 interference vector was successfully constructed for production of large amount of dsRNA. The target sequence of EsSox21b-like gene was inserted into the L4440 vector containing two T7 promoters. Then the recombinant plasmid was transformed into HT115 bacteria, a RNaseⅢ deficient strain. A large amount of EsSox21b-like sense and antisense RNAs was simultaneously transcripted in vivo when induced by IPTG. After denatured and annealed, EsSox21b-like double strand RNAs（dsRNAs）were produced and the yield of EsSox21b-like-dsRNA was enough for future RNA interference experiment on the role of EsSox21b-like gene.

Key words: Eriocheir sinensis, RNA interference, L4440, HT115, Prokaryotic expression, dsRNA

刘志强, 陈洁, 邱高峰. 中华绒螯蟹EsSox21b-like基因干扰载体的构建及原核表达制备dsRNA[J]. 生物技术通报, 2014, 0(6): 134-138.

Liu Zhiqiang, Chen Jie, Qiu Gaofeng. Construction of Interference Vector of EsSox21b-like Gene from Chinese Mitten Crab（Eriocheir sinensis）and Preparation of dsRNA by Prokaryotic Expression[J]. Biotechnology Bulletin, 2014, 0(6): 134-138.

参考文献

[1] Sinclair A, Berta P, Palmer M, et al. A gene from the human sex-determining region encodes a protein with[J] . Nature, 1990, 346（6281）：240-244.
[2] Gubbay J, Collignon J, Koopman P, et al. A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes[J] . Nature, 1990, 346（6281）：245-250.
[3] 汪桂玲, 朱琴, 李家乐. 斑节对虾 Sox 基因 HMG 盒的 PCR 扩增及 SSCP 分析[J] . 水产学报, 2005, 29（4）：478-481.
[4] 刘艳红, 楼允东, 邱高峰. 锯缘青蟹 Sox 基因的 PCR 扩增（英文）[J] . 上海水产大学学报, 2003, 12（S1）：24-27.
[5] 周荣家, 常重杰. 两种泥鳅中 Sox 基因的检出[J] . 发育与生殖生物学报（英文版）, 1999, 8（2）：11-17.
[6] Wallis M, Waters P, Delbridge M, et al. Sex determination in platypus and echidna：autosomal location of SOX3 confirms the absence of SRY from monotremes[J] . Chromosome Research, 2007, 15（8）：949-959.
[7] Veyrunes F, Waters PD, Miethke P, et al. Bird-like sex chromosomes of platypus imply recent origin of mammal sex chromosomes[J] . Genome Research, 2008, 18（6）：965-973.
[8] Chardard D, Chesnel A, Gozé C, et al. Pw Sox-1：the first member of the Sox gene family in Urodeles[J] . Nucleic Acids Research, 1993, 21（15）：3576.
[9] Bowles J, Schepers G, Koopman P. Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators[J] . Developmental Biology, 2000, 227（2）：239-255.
[10] 常重杰, 杜启艳, 邵红伟. Sox 基因家族研究的新进展[J] . 遗传, 2002, 24（4）：470-476.
[11] 江玲霞, 李纪委, 贺彧, 等. Sox 基因家族功能的研究进展[J] . 生物技术通报, 2008（6）：44-48.
[12] 亓海燕, 邱高峰. 一个在中华绒螯蟹性腺中特异表达的 Sox 基因 HMG 盒区的克隆与鉴定[J] . 自然科学进展, 2009, 19（3）：279-284.
[13] Matzke M, Matzke AJ, Kooter JM. RNA：guiding gene silencing [J] . Science, 2001, 293（5532）：1080-1083.
[14] Plasterk RH. RNA silencing：the genome’s immune system[J] . Science Signaling, 2002, 296（5571）：1263.
[15] Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J] . Nature, 1998, 391（6669）：806-811.
[16] Tenllado F, Martínez-García B, Vargas M, Díaz-Ruíz JR. Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections[J] . BMC Biotechnology, 2003, 3（1）：3.
[17] Ongvarrasopone C, Roshorm Y, Panyim S. A simple and cost effective method to generate dsRNA for RNAi studies in invertebrates[J] . Science Asia, 2007, 33：35-39.
[18] Morley JF, Morimoto RI. Regulation of longevity in Caenorhabditis elegans by heat shock factor and molecular chaperones[J] . Molecular Biology of the Cell, 2004, 15（2）：657-664.
[19] Eschenlauer SC, Page AP. The Caenorhabditis elegans ERp60 homolog protein disulfide isomerase-3 has disulfide isomerase and transglutaminase-like cross-linking activity and is involved in the maintenance of body morphology[J] . Journal of Biological Chemistry, 2003, 278（6）：4227-4237.
[20] Beisson J, Bétermier M, Bré MH, et al. Silencing specific Paramecium tetraurelia genes by feeding double-stranded RNA[J] . Cold Spring Harbor Protocols, 2010, 2010（1）：pdb. prot5363.
[21] Ratzka C, Gross R, Feldhaar H. Systemic gene knockdown in Camponotus floridanus workers by feeding of dsRNA[J] . Insectes Sociaux, 2013, 60：475-484.
[22] Li X, Zhang M, Zhang H. RNA interference of four genes in adult Bactrocera dorsalis by feeding their dsRNAs[J] . PloS One, 2011, 6（3）：e17788.
[23] 王根洪, 祝慧敏, 罗会松, 等.细菌表达dsRNA介导的家蚕FTZ-F1基因的RNA干扰[J] .昆虫学报, 2011, 54（5）：596-601.
[24] 荣光, 王新华, 薄新文, 等.捻转血矛线虫Hc38基因的克隆及其RNAi载体的构建[J] .动物医学进展, 2007, 28（1）：38-41.
[25] 梁艳, Sritunyalucksan KS.一种大规模制备双链 RNA 的简单方法及其在斑节对虾中的应用[J] .水产学报, 2010, 34（7）：1011-1017.
[26] Sarathi M, Simon MC, Ahmed VPI, et al. Silencing VP28 gene of white spot syndrome virus of shrimp by bacterially expressed dsRNA[J] . Marine Biotechnology, 2008, 10（2）：198-206.
[27] Timmons L, Fire A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans[J] . Gene, 2001, 263（1）：103-112.