家蚕中肠特异启动子BmAPN的克隆及活性分析

doi:10.3864/j.issn.0578-1752.2012.20.018

摘要/Abstract

摘要： 【目的】克隆和鉴定1个家蚕中肠特异启动子，为研究家蚕的免疫应答和应用研究提供新的工具。【方法】从家蚕品种大造中提取基因组总DNA，用PCR方法克隆家蚕中肠特异表达基因BmAPN（GenBank登录号：BAA33715.1）的上游调控序列。利用此序列构建以EGFP为报告基因的转基因表达载体pBac[BmAPN-EGFP-SV40，3×P3-DsRed]，通过显微注射获得转基因家蚕，在个体水平上检测启动子的活性。【结果】所克隆的BmAPN启动子长度为1 598 bp，其驱动的EGFP仅在转基因家蚕中肠特异表达，与内源基因BmAPN的表达特征一致。该启动子在家蚕幼虫时期活性相对较高，且在起蚕期的活性要明显高于眠蚕期，推测该启动子中的特异元件受激素调控。【结论】经克隆获得的BmAPN启动子为有活性的启动子，且为家蚕中肠特异启动子。

关键词: 家蚕, 中肠特异, 启动子, 转基因

Abstract: 【Objective】The objective of this study is to provide a new tool for the immune-response and application research, and to clone and identify a midgut-specific promoter from Bombyx mori.【Method】 The 5′ upstream regulatory sequence of midgut-specific gene BmAPN (GenBank accession: BAA33715.1) was obtained by PCR from B. mori genomic DNA. A transgenic expression vector named pBac[BmAPN-EGFP-SV40, 3×P3-DsRed] was constructed, in which the reporter gene EGFP was driven by the BmAPN promoter. Furthermore, transgenic B. mori lines was obtained by microinjection and the activity of the promoter at the individual level were detected.【Result】The EGFP driven by a 1 598 bp BmAPN promoter was just specifically expressed in B. mori midgut. It was consistent with the expression pattern of endogenous gene BmAPN. The promoter activity was higher in B. mori larvae than other stages. And the activity in newly exuviated larvae was higher than that of molting larvae. It was speculated that specific components of the promoter might be regulated by hormone.【Conclusion】The BmAPN promoter is an active and midgut-specific promoter in B. mori.

Key words: Bombyx mori, midgut-specific, promoter, transgene

陆改, 程廷才, 蒋亮, 金盛凯, 林平, 胡翠美, 夏庆友. 家蚕中肠特异启动子BmAPN的克隆及活性分析[J]. 中国农业科学, 2012, 45(20): 4279-4287.

LU Gai, CHENG Ting-Cai, JIANG Liang, JIN Sheng-Kai, LIN Ping, HU Cui-Mei, XIA Qing-You. Cloning and Activity Analysis of a Midgut-Specific Promoter BmAPN in Silkworm (Bombyx mori)[J]. Scientia Agricultura Sinica, 2012, 45(20): 4279-4287.

0
/ 收藏文章 0 / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.20.018

https://www.chinaagrisci.com/CN/Y2012/V45/I20/4279

参考文献

[1]Hua G, Tsukamoto K, Rasilo M L, Ikezawa H. Molecular cloning of a GPI-anchored aminopeptidase N from Bombyx mori midgut : a putative receptor for Bacillus thuringiensis CryIA toxin. Gene, 1998, 214 (1/2): 177-185.

[2]Yaoi K, Nakanishi K, Kadotani T, Imamura M, Koizumi N, Iwahana H, Sato R. cDNA cloning and expression of Bacillus thuringiensis Cry1Aa toxin binding 120 kDa aminopeptidase N from Bombyx mori. Biochimica et Biophysica Acta, 1999, 1444 (1): 131-137.

[3]Crava C M, Bel Y, Lee S F, Manachini B, Heckel D G, Escriche B. Study of the aminopeptidase N gene family in the lepidopterans Ostrinia nubilalis (Hübner) and Bombyx mori (L.): sequences, mapping and expression. Insect Biochemistry and Molecular Biology, 2010, 40(7): 506-515.

[4]Vadlamudi R K, Weber E, Ji I, Ji T H, Bulla Jr. L A. Cloning and expression of a receptor for an insecticidal toxin of Bacillus thuringiensis. Journal of Biological Chemistry, 1995, 270(10): 5490-5494.

[5]Pacheco S, Gómez I, Arenas I, Saab-Rincon G, Rodríguez-Almazán C, Gill S S, Bravo A, Soberón M. Domain II loop 3 of Bacillus thuringiensis Cry1Ab toxin is involved in a "ping pong" binding mechanism with Manduca sexta aminopeptidase-N and cadherin receptors. The Journal of Biological Chemistry, 2009, 284(47): 32750-32757.

[6]Bravo A, Gómez I, Conde J, Muñoz-Garay C, Sánchez J, Miranda R, Zhuang M, Gill S S, Soberón M. Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. Biochimica et Biophysica Acta, 2004, 1667(1): 38-46.

[7]Bravo A, Gill S S, Soberón M. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon, 2007, 49(4): 423-435.

[8]Bravo A, Likitvivatanavong S, Gill S S, Soberón M. Bacillus thuringiensis: A story of a successful bioinsecticide. Insect Biochemistry and Molecular Biology, 2011, 41(7): 423-431.

[9]Rajagopal R, Sivakumar S, Agrawal N, Malhotra P, Bhatnagar R K. Silencing of midgut aminopeptidase N of Spodoptera litura by double-stranded RNA establishes its role as Bacillus thuringiensis toxin receptor. The Journal of Biological Chemistry, 2002, 277(49): 46849-46851.

[10]Herrero S, Gechev T, Bakker P L, Moar W J, de Maagd R A. Bacillus thuringiensis Cry1Ca-resistant Spodoptera exigua lacks expression of one of four aminopeptidase N genes. BMC Genomics, 2005, 6: 96.

[11]Abraham E G, Donnelly-Doman M, Fujioka H, Ghosh A, Moreira L, Jacobs-Lorena M. Driving midgut-specific expression and secretion of a foreign protein in transgenic mosquitoes with AgAper1 regulatory elements. Insect Molecular Biology, 2005, 14(3): 271-279.

[12]Zhao A C, Zhao T F, Zhang Y S, Xia Q Y, Lu C, Zhou Z Y, Xiang Z H, Nakagaki M. New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm. Transgenic Research, 2010, 19(1): 29-44.

[13]邓党军, 徐汉福, 王峰, 马三垣, 夏庆友. BmLSP基因启动子驱动DsRed在转基因家蚕中的表达分析. 蚕业科学, 2011, 37(2): 200-205.

Deng D J, Xu H F, Wang F, Ma S Y, Xia Q Y. Expression analysis of DsRed gene driven by promoter of BmLSP gene in transgenic silkworm, Bombyx mori. Science of Sericulture, 2011, 37(2): 200-205. (in Chinese)

[14]胡翠美, 王菲, 宋亮, 夏庆友. 家蚕中肠cDNA T7噬菌体展示文库的构建和免疫相关基因的淘选. 蚕业科学, 2011, 37(4): 642-649.

Hu C M, Wang F, Song L, Xia Q Y. Construction of a T7 phage display cDNA library and panning for immune-related genes from midgut of silkworm, Bombyx mori. Science of Sericulture, 2011, 37(4): 642-649. (in Chinese)

[15]马三垣, 徐汉福, 段建平, 赵爱春, 张美蓉, 夏庆友. 家蚕转基因技术中若干因素对转基因效率的影响. 昆虫学报, 2009, 52(6): 595-603.

Ma S Y, Xu H F, Duan J P, Zhao A C, Zhang M R, Xia Q Y. Effect of several factors on the transformation efficiency in the transgenic technology of silkworm, Bombyx mori. Acta Entomologica Sinica, 2009, 52(6): 595-603. (in Chinese)

[16]Wang G H, Xia Q Y, Cheng D J, Duan J, Zhao P, Chen J, Zhu L. Reference genes identified in the silkworm Bombyx mori during metamorphism based on oligonucleotide microarray and confirmed by qRT-PCR. Insect Science, 2008, 15(5): 405-413.

[17]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2－ΔΔCt method. Methods, 2001, 25(4): 402-408.

[18]Fernandes M, Xiao H, Lis J T. Fine structure analyses of the Drosophila and Saccharomyces heat shock factor-heat shock element interactions. Nucleic Acids Research, 1994, 22(2): 167-173.

[19]Bray S J, Kafatos F C. Developmental function of Elf-1: an essential transcription factor during embryogenesis in Drosophila. Genes & Development, 1991, 5(9): 1672-1683.

[20]Akiyama Y, Hosoya T, Poole A M, Hotta Y. The gcm-motif: a novel DNA-binding motif conserved in Drosophila and mammals. Proceedings of the National Academy of Sciences of the United States of America, 1996, 93(25): 14912-14916.

[21]Lecourtois M, Schweisguth F. The neurogenic suppressor of hairless DNA-binding protein mediates the transcriptional activation of the enhancer of split complex genes triggered by notch signaling. Genes & Development, 1995, 9(21): 2598-2608.

[22]Bailey A M, Posakony J W. Suppressor of hairless directly activates transcription of enhancer of split complex genes in response to notch receptor activity. Genes & Development, 1995, 9(21): 2609-2622.

[23]Ekker S C, von Kessler D P, Beachy P A. Differential DNA sequence recognition is a determinant of specificity in homeotic gene action. The EMBO Journal, 1992, 11(11): 4059-4072.

[24]Von Kalm L, Crossgrove K, Von Seggern D, Guild G M, Beckendorf S K. The broad-complex directly controls a tissue-specific response to the steroid hormone ecdysone at the onset of Drosophila metamorphosis. The EMBO Journal, 1994, 13(15): 3505-3516.

[25]Stanojevi? D, Hoey T, Levine M. Sequence-specific DNA-binding activities of the gap proteins encoded by hunchback and Krueppel in Drosophila. Nature, 1989, 341(6240): 331-335.

[26]Treisman J, Desplan C. The products of the Drosophila gap genes hunchback and Krueppel bind to the hunchback promoters. Nature, 1989, 341(6240): 335-337.

[27]Read D, Manley J L. Alternatively spliced transcripts of the Drosophila tramtrack gene encode zinc finger proteins with distinct DNA binding specificities. The EMBO Journal, 1992, 11(3): 1035-1044.

[28]Brinster R L, Allen J M, Behringer R R, Gelinas R E, Palmiter R D. Introns increase transcriptional efficiency in transgenic mice. Proceedings of the National Academy of Sciences of the United States of America, 1988, 85(3): 836-840.

[29]Rafiq M, Suen C K, Choudhury N, Joannou C L, White K N, Evans R W. Expression of recombinant human ceruloplasmin-an absolute requirement for splicing signals in the expression cassette. FEBS Letters, 1997, 407(2): 132-136.

[30]Wang H B, Iwanaga M, Kawasaki H. Activation of BMWCP10 promoter and regulation by BR-C Z2 in wing disc of Bombyx mori. Insect Biochemistry and Molecular Biology, 2009, 39(9): 615-623.

[31]Wang H B, Nita M, Iwanaga M, Kawasaki H. BetaFTZ-F1 and broad-complex positively regulate the transcription of the wing cuticle protein gene, BMWCP5, in wing discs of Bombyx mori. Insect Biochemistry and Molecular Biology, 2009, 39(9): 624-633.

[32]Yaoi K, Nakanishi K, Kadotani T, Imamura M, Koizumi N, Iwahana H, Sato R. Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N. FEBS Letters, 1999, 463(3): 221-224.

[33]Nakanishi K, Yaoi K, Nagino Y, Hara H, Kitami M, Atsumi S, Miura N, Sato R. Aminopeptidase N isoforms from the midgut of Bombyx mori and Plutella xylostella - their classification and the factors that determine their binding specificity to Bacillus thuringiensis Cry1A toxin. FEBS Letters, 2002, 519(1/3): 215-220.

Metrics

Viewed

Full text

181

HTML			PDF

Just accepted	Online first	Issue	Just accepted	Online first	Issue
0	0	0	0	0	181

From	Others	local

Times	138	43
Rate	76%	24%

Abstract

204

Just accepted	Online first	Issue

0	0	204

From	Others	local

Times	184	20
Rate	90%	10%

Cited

Web of Science	Crossref	ScienceDirect	Search for Citations in Google Scholar >>


This page requires you have already subscribed to WoS.

Shared