柳蚕卵黄原蛋白基因的克隆与表达分析
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摘要
柳蚕是一种尚未开发的野生绢丝昆虫,国内外对柳蚕的研究并不多,且大部分都是关于形态学和生物习性方面的报道和记载。柳蚕的卵黄原蛋白是由大小两个亚基组成,分子量分别是175 KDa和45 KDa。卵黄原蛋白作为昆虫卵黄发生的主要蛋白之一,除了具有营养功能外,在生物体内还具有许多其他重要的生物学功能。近些年来,关于昆虫卵黄原蛋白基因的研究是热点,不断有蚕卵黄原蛋白的一级结构被解析,但柳蚕卵黄原蛋白全基因序列至今未解析。该研究结果可为进一步研究卵黄原蛋白在柳蚕中的功能及其应用提供参考。
本文根据已解析的柳蚕卵黄原蛋白cDNA序列和家蚕、天蚕、柞蚕卵黄原蛋白5′端调控区的序列,设计特异性引物,其中有10对引物从柳蚕基因组DNA中扩增出了其卵黄原蛋白基因的10个DNA片段,经克隆、测序和分析后获得了柳蚕卵黄原蛋白的基因序列(GenBank登录号:GU361974),基因全长7329 bp,包含6个外显子,5个内含子以及一段206 bp的5′端调控区序列,其中外显子的大小分别为2246 bp、205 bp、982 bp、879 bp、184 bp和863 bp;内含子的大小分别为107 bp、84 bp、626 bp、706 bp和230 bp。与其它已解析的昆虫卵黄原蛋白基因比较发现,柳蚕和柞蚕的卵黄原蛋白基因结构更为相似,它们均在起始密码子ATG后31 bp处有一个大的内含子缺失。在5′端调控区发现了BmDsx结合位点以及CdxA和GATA-X调控元件。
同时,提取柳蚕雌蚕不同时期和不同组织的总RNA,以M-MLV反转录第一链cDNA,以柳蚕18S rRNA基因做内参,摸索条件后进行了半定量RT-PCR。实验结果显示,柳蚕卵黄原蛋白基因在5龄第1、4、7、11天时未表达,预蛹期达到一个相当高的水平,随后又开始下降,直至滞育期其量又稍有上升;且在中肠、头、马氏管中不表达,在血液、脂肪体和卵巢中有所表达,但表达量无明显差异。这说明柳蚕卵黄原蛋白的表达存在明显的时期和组织的特异性。
PCR法扩增了柳蚕卵黄原蛋白的小亚基,并连接至pET-28a(+)表达载体,转入大肠杆菌BL21(DE3)菌株中,用IPTG进行了不同浓度和不同时间的诱导表达,经SDS-PAGE电泳和Western blot检测,表明被诱导的目的蛋白均得到了稳定的表达,这为以后对卵黄原蛋白的真核表达及其功能的进一步研究奠定基础。
Actias selene Hubner is an undeveloped wild silk insect. Few reports about it were found and most of them are on the morphology and biological behavior. Vitellogenin from Actias selene Hubner is composed of two subunits, the big one is 175 KDa and the small one is 45 KDa.
Vitellogenin has many other important biological functions in organism as well as nutritional functions. In resent years, researches on insect vitellogenin are very popular, and there are many primary structures of vitellogenin were sequenced continuously, while the vitellogenin gene of Actias selene Hubner has not been analysed yet. Results of this experiment will provide some informations for further study on function and application of vitellogenin in Actias selene Hubner.
In this study, ten pairs of specific primers were designed to amplify the DNA sequence of vitellogenin according to the vitellogenin cDNA sequence of Actias selene Hubner and 5′regulation region of the Bombyx mori, Antheraea pernyi and Antheraea yamamai. A sequence of 7329 bp long (GenBank accession number: GU361974) including 6 extrons and 5 introns with an open reading frame encoding a 1774 amino acids peptide was obtained by PCR. A BmDsx binding site (ACATTGT) and some conserved signatures such as CdxA and GATA-X were found in 206 bp long 5′- flanking region of vitellogenin gene in Actias selene Hubner. The size of exons were 2246 bp、205 bp、982 bp、879 bp、184 bp and 863 bp respectively, and introns were 107 bp、84 bp、626 bp、706 bp and 230 bp respectively. Compared with other insects, we discovered that the structure of vitellogenin gene of Actias selene Hubner is more similar to that of Antheraea pernyi. They both have a large intron loss in 31 bp after ATG.
Meanwhile, the total RNA of female Actias selene Hubner was extracted to analyse the expression of vitellogenin in different developmental stages and tissues, then reversely transcribed and synthesized the first strand cDNA by M-MLV. Take the 18sRNA of Actias selene Hubner as a reference gene, we carried the semi-quantitative PCR. The result showed that vitellogenin of Actias selene Hubner did not express in the whole larva stages, and its expression reached the maximum in prepupa stage. During the whole pupation, it decreased slightly in the first day and declined a lower level in the forth day. During diapause stage, it kept almost the level of the first day of pupation. In addition, vitellogenin of Actias selene Hubner did not express in midintestine, head and malpighian, while expressed in blood, fat body and ovary with no obvious quantitative difference. It confirmed that the vitellogenin gene of Actias selene Hubner expressed specifically in different stages and tissues.
The small subunit was amplified by PCR, ligated to pET- 28a(+) expression vector and transformed into Escherichia coli BL21(DE3), induced by IPTG under different concentrations and different time length. Through SDS-PAGE electrophoresis and Western blot analysis, the induced fusion protein was successfully expressed. This will provide the foundation for further study on eukaryotic expression and function of vitellogenin gene.
本文根据已解析的柳蚕卵黄原蛋白cDNA序列和家蚕、天蚕、柞蚕卵黄原蛋白5′端调控区的序列,设计特异性引物,其中有10对引物从柳蚕基因组DNA中扩增出了其卵黄原蛋白基因的10个DNA片段,经克隆、测序和分析后获得了柳蚕卵黄原蛋白的基因序列(GenBank登录号:GU361974),基因全长7329 bp,包含6个外显子,5个内含子以及一段206 bp的5′端调控区序列,其中外显子的大小分别为2246 bp、205 bp、982 bp、879 bp、184 bp和863 bp;内含子的大小分别为107 bp、84 bp、626 bp、706 bp和230 bp。与其它已解析的昆虫卵黄原蛋白基因比较发现,柳蚕和柞蚕的卵黄原蛋白基因结构更为相似,它们均在起始密码子ATG后31 bp处有一个大的内含子缺失。在5′端调控区发现了BmDsx结合位点以及CdxA和GATA-X调控元件。
同时,提取柳蚕雌蚕不同时期和不同组织的总RNA,以M-MLV反转录第一链cDNA,以柳蚕18S rRNA基因做内参,摸索条件后进行了半定量RT-PCR。实验结果显示,柳蚕卵黄原蛋白基因在5龄第1、4、7、11天时未表达,预蛹期达到一个相当高的水平,随后又开始下降,直至滞育期其量又稍有上升;且在中肠、头、马氏管中不表达,在血液、脂肪体和卵巢中有所表达,但表达量无明显差异。这说明柳蚕卵黄原蛋白的表达存在明显的时期和组织的特异性。
PCR法扩增了柳蚕卵黄原蛋白的小亚基,并连接至pET-28a(+)表达载体,转入大肠杆菌BL21(DE3)菌株中,用IPTG进行了不同浓度和不同时间的诱导表达,经SDS-PAGE电泳和Western blot检测,表明被诱导的目的蛋白均得到了稳定的表达,这为以后对卵黄原蛋白的真核表达及其功能的进一步研究奠定基础。
Actias selene Hubner is an undeveloped wild silk insect. Few reports about it were found and most of them are on the morphology and biological behavior. Vitellogenin from Actias selene Hubner is composed of two subunits, the big one is 175 KDa and the small one is 45 KDa.
Vitellogenin has many other important biological functions in organism as well as nutritional functions. In resent years, researches on insect vitellogenin are very popular, and there are many primary structures of vitellogenin were sequenced continuously, while the vitellogenin gene of Actias selene Hubner has not been analysed yet. Results of this experiment will provide some informations for further study on function and application of vitellogenin in Actias selene Hubner.
In this study, ten pairs of specific primers were designed to amplify the DNA sequence of vitellogenin according to the vitellogenin cDNA sequence of Actias selene Hubner and 5′regulation region of the Bombyx mori, Antheraea pernyi and Antheraea yamamai. A sequence of 7329 bp long (GenBank accession number: GU361974) including 6 extrons and 5 introns with an open reading frame encoding a 1774 amino acids peptide was obtained by PCR. A BmDsx binding site (ACATTGT) and some conserved signatures such as CdxA and GATA-X were found in 206 bp long 5′- flanking region of vitellogenin gene in Actias selene Hubner. The size of exons were 2246 bp、205 bp、982 bp、879 bp、184 bp and 863 bp respectively, and introns were 107 bp、84 bp、626 bp、706 bp and 230 bp respectively. Compared with other insects, we discovered that the structure of vitellogenin gene of Actias selene Hubner is more similar to that of Antheraea pernyi. They both have a large intron loss in 31 bp after ATG.
Meanwhile, the total RNA of female Actias selene Hubner was extracted to analyse the expression of vitellogenin in different developmental stages and tissues, then reversely transcribed and synthesized the first strand cDNA by M-MLV. Take the 18sRNA of Actias selene Hubner as a reference gene, we carried the semi-quantitative PCR. The result showed that vitellogenin of Actias selene Hubner did not express in the whole larva stages, and its expression reached the maximum in prepupa stage. During the whole pupation, it decreased slightly in the first day and declined a lower level in the forth day. During diapause stage, it kept almost the level of the first day of pupation. In addition, vitellogenin of Actias selene Hubner did not express in midintestine, head and malpighian, while expressed in blood, fat body and ovary with no obvious quantitative difference. It confirmed that the vitellogenin gene of Actias selene Hubner expressed specifically in different stages and tissues.
The small subunit was amplified by PCR, ligated to pET- 28a(+) expression vector and transformed into Escherichia coli BL21(DE3), induced by IPTG under different concentrations and different time length. Through SDS-PAGE electrophoresis and Western blot analysis, the induced fusion protein was successfully expressed. This will provide the foundation for further study on eukaryotic expression and function of vitellogenin gene.
引文
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