差异蛋白质组学在家蚕中的研究进展

差异蛋白质组是蛋白质组学的一个重要分支,通过对蛋白质组表达谱的比较,揭示细胞生理或病理状态的进程与本质,发现具有关键作用的蛋白。近年来,家蚕差异蛋白质组学发展迅速且涉及面广,已然成为家蚕蛋白质组学研究的热点。对差异蛋白质组学的主要研究方法,及在家蚕中的研究进展做一简要评述。     

家蚕保幼激素结合蛋白Bmtol基因的克隆及表达分析

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c DNA为模板利用RT-PCR技术扩增和克隆获得Bmtol基因c DNA全长序列,并提交至Gen Bank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Bm TOL及其它昆虫同源TO的进化树;通过q PCR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c DNA序列(Gen Bank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27.72k Da,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,Bm TOL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。Bm TOL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Bm TOL蛋白与配体结合的核心部位。序列比对结果显示,家蚕Bm TOL序列与其他昆虫TO的氨基酸序列一致性差别较大。家蚕Bm TOL与果蝇Dm TO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Ep TO1、烟草天蛾Ms TO、意大利蜜蜂Am TOL、果蝇Dm TO和黑花蝇Pr TOL聚为一个分支,埃及伊蚊Aa TO、冈比亚按蚊Ag TOL-2和家蚕Bm TOL聚为另一大分支。q PCR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:Bm TOL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,Bm TOL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。     

低能Ar^＋注入家蚕卵的生物学效应研究

家蚕作为鳞翅目昆虫的模式生物,是研究遗传和变异的极好模型.本文以家蚕卵为材料,研究不同能量和剂量的低能Ar+注入家蚕卵的生物学效应,结果表明:在真空10 min时间内,对家蚕卵的孵化无明显影响;在25 keV和30 keV的能量下,2.6～8×2.6×1015 ion/cm2的剂量作用于家蚕卵,对家蚕卵的孵化影响较为显著;在能量为30 keV, 剂量为8×2.6×1015 ion/cm2和9×2.6×1015 ion/cm2的Ar+轰击下,以扫描电镜可观察到蚕卵壳表面有明显的刻蚀痕迹;并且经30 keV, 9×2.6×1015 ion/cm2处理的饲养区至5龄期发现了3例突变性状.     

新霉素抗性基因在家蚕中的插入和表达

构建含新霉素抗性基因（ｎｅｏｍｙｃｉｎｒｅｓｉｓｔａｎｃｅｇｅｎｅ,ｎｅｏＲ）的重组质粒ｐＦＮ,经ＨｉｎｄＩＩ酶切后,用基因枪将ＤＮＡ片段导入家蚕早期受精卵中（Ｇ０代）。孵化的Ｇ１、Ｇ２代蚁蚕均经含新霉素的人工饲料添食２４ｈ后,筛选出新霉素抗性的个体（能正常生长发育的）改为桑叶饲养。于Ｇ２代的５龄第二天从后部丝腺抽提总ＤＮＡ,再以ｎｅｏＲ的ｃＤＮＡ为探针进行Ｓｏｕｔｈｅｒｎ杂交检测。结果表明ｎｅｏＲ基因已转入家蚕ＤＮＡ中,获得了含ｎｅｏＲ的转基因蚕。     

in silico identification of protein-protein interactions in Silkworm,Bombyx mori

The Domesticated silkworm, Bombyx mori, an economically important insect has been used as a lepidopteran molecular model next only to Drosophila. Compared to the genomic information in silkworm, the protein-protein interaction data are limited. Therefore experimentally identified PPI maps from five model organisms such as E.coli, C.elegans, D.melanogaster, H. sapiens, S. cerevisiae were used to infer the PPI network of silkworm using the well-recognized Interlog based method. Among the 14623 silkworm proteins, 7736 protein-protein interaction pairs were predicted which include 2700 unique proteins of the silkworms. Using the iPfam interaction domains and the gene expression data, these predictions were validated. In that 625 PPI pairs of predicted network were associated with the iPfam domain-domain interactions and the random network has average of 9. In the gene expression method, the average PCC value of the predicted network and random network was 0.29 and 0.23100±0.00042 respectively. It reveals that the predicted PPI networks of silkworm are highly significant and reliable. This is the first PPI network for the silkworm which will provide a framework for deciphering the cellular processes governing key metabolic pathways in the silkworm, Bombyx mori and available at SilkPPI (http://210.212.197.30/SilkPPI/).     

家蚕细胞凋亡相关基因BmICAD的克隆、序列和功能分析及其在精巢中特异表达的初步研究

DREP-1基因在果蝇的细胞凋亡过程中对DNA的降解有重要调控作用.本研究将该基因序列在家蚕EST数据库中进行同源性检索,把检索到的EST序列进行电子延伸和克隆重叠群拼接,根据拼接结果设计引物进行PCR扩增并克隆测序验证,首次成功克隆了家蚕第一个ICAD基因BmICAD的cDNA:该cDNA全长844 bp,ORF长522 bp,编码含有174个氨基酸的蛋白;预测分子量为19 6 kDa,等电点为4.23,所编码蛋白与果蝇DREP-1的一致性(identity)为36%;虽然Northern印记杂交未检测到信号,但是RT-PCR结果显示,该基因在精巢中特异表达.     

Isolation and Characterization of Prothoracicotropic Hormone from Silkworm,Bombyx mori

Members of the small family of Arabidopsis PSEUDO-RESPONSE REGULATORS (PRR1/TOC1, PRR3, PRR5, PRR7, and PRR9) play roles close to the circadian clock in Arabidopsis thaliana. We have reported that the rice (Oryza sativa) genome also encodes a set of PRR counterparts (designated OsPRR1, OsPRR37, OsPRR59, OsPRR73, and OsPRR95 respectively). To gain new insight into the molecular functions of OsPRRs, we carried out genetic complementation analyses by introducing two representative rice genes, OsPRR1 and OsPRR37, into the corresponding Arabidopsis loss-of-function mutants (toc1 and prr7 respectively). The results showed that these OsPRR and AtPRR genes are genetically interchangeable at least in part, suggesting the conserved clock-associated function of these OsPRRs.     

家蚕LongSAGE文库鉴定及延长标签的改进GLGI方法

结合标签的基因序列延伸(GLGI)是随着基因表达序列标签(SAGE)应运而生的一种鉴定新标签的技术。为了克服该技术操作繁琐、成本高的弊端,根据已经构建的家蚕限性茧色品种的2个LongSAGE文库,选择30个差异表达的标签(tag)进行GLGI延伸。尝试了几个简化,(1)用totalRNA代替mRNA进行dsDNA的合成;(2)调整磁珠吸附DNA的顺序;(3)在PCR反应中用普通的DNA聚合酶代替高保真酶(Pfu)。结果显示,30个标签有27个获得有效扩增,扩增序列真实包括了SAGE标签的17bp序列,3个标签的结果符合NCBI的家蚕数据库已有注释,13个未注释标签经过延长后得到了注释,11个标签序列为待确定的新基因序列。这种简化能够作为家蚕SAGE文库其他tag和新基因研究的有效简化操作体系。与传统的方法相比,可减少成本30%以上,缩短操作时间20%。     

家蚕耐氟基因RAPD分子标记的筛选及其克隆

为获得家蚕耐氟基因的分子标记,以家蚕耐氟品种T6和高敏感品种733新为材料,并构建了其近等基因,采用300个随机引物进行RAPD扩增,获得了与家蚕耐氟性有关的一个分子标记,此标记由引物S250扩增得到,并在F2代分离个体中得到验证,证明了此分子标记的可靠性,进而将此标记克隆进T载体pmd-18中,完成测序,分析发现此序列是未有报道的新序列。     

Occurrence of Lectin in the Silkgland of the Silkworm, Bombyx mori

Monoclonal antibodies were prepared against the 350 kDa lectin purified from larval hemolymph of the silkworm, Bombyx mori . The antibodies inhibited the hemagglutinating activity (HA activity) and bound specifically to the hemolymph 350 kDa lectin on Western blotting analysis. Immunohistological observations revealed the occurrence of lectin in the cuticular intima of the anterior silk gland, but not the middle or posterior silk glands of fifth instar larvae of Bombyx mori . Extracts from the anterior silk glands showed HA activity and exhibited the same biochemical characteristics as those of the 350 kDa lectin in the hemolymph. These results suggested that lectin-like molecules in epithelial tissues may be important in histolysis during molting and metamorphosis.     

家蚕BmST2基因的克隆与转录活性检测

通过生物信息学分析和生物学试验获得了家蚕糖转运蛋白基因BmST2(GenBank登录号:GQ871755),基因位于家蚕27号染色体,开放阅读框(ORF)长1398 bp,编码465个氨基酸,预测蛋白序列有典型的Sugar_tr结构域和11个疏水的跨膜结构域,与家蚕BmST1蛋白相似性和一致性分别达79%和64%,与登录号为EAT47626、EDS35465、EAA11457和EFA05337的同源蛋白相似性在50%以上。RT-PCR检测基因在5龄第3天家蚕幼虫的9种组织中转录活性,结果显示,BmST2基因除在脂肪体没有表达外,其他组织均有表达。最后成功构建了基因的酵母穿梭表达质粒pG-BKT7-BmST2。     

大豆诱导型启动子驱动类受体蛋白激酶GmSARK转基因植物分析

植物LRR型类受体蛋白激酶在植物生命活动中发挥着重要作用。前期研究发现,大豆（Glycine max）LRR型类受体蛋白激酶基因GmSARK可能参与调控大豆叶片的衰老过程。利用CaMV35S启动子驱动组成型过表达GmSARK基因可导致转基因植株出现致死表型,据此构建了可诱导型启动子GVG驱动GmSARK基因过表达的双元表达载体,转化野生型拟南芥（Arabidopsis thaliana）并获得了多株转基因植株。研究结果表明,外源施加诱导物地塞米松可引起GmSARK基因在转基因植株中过表达,并导致转基因植株出现叶片变黄下卷和生长受抑制等表型;外源细胞分裂素处理可以抑制GmSARK的表达,但是不能逆转GmSARK过表达所引起的上述变化。     

嵌合抗体轻链基因在家蚕细胞中的重组与表达

用家蚕修饰型核多角体病毒为载体,在家蚕细胞获得人鼠嵌合的抗人小细胞肺癌抗体轻链基因的表达。ＰＣＲ和Ｓｏｕｔｈｅｒｎ杂交证明了抗体轻链基因已组建家蚕病毒基因组中。Ｗｅｓｔｅｒｎ ｂｌｏｔ和ＥＬＩＳＡ和分析都检测到在重组病毒感染的家蚕细胞中产生了人鼠嵌合的抗体轻链。     

Ecdysone Metabolism in Diapause Eggs and Non-Diapause Eggs of the Silkworm, Bombyx mori

In order to compare ecdysone metabolism between diapause eggs and non-diapause eggs of the silkworm, Bombyx mori, (3)H-ecdysone and its derivatives ((3)H-3-epiecdysone and (3)H-ecdysone 22-phosphate) were injected into the eggs at various stages during early embryogenesis, and the resultant labelled metabolites were analyzed by high-performance liquid chromatography. From the quantitative and qualitative changes in the labelled metabolites between diapause eggs and non-diapause eggs, it was demonstrated that epimerization of ecdysone occurred during early embryogenesis irrespective of the embryonic stage in both diapause eggs and non-diapause eggs, and that phosphorylation of ecdysone was a major metabolic step in diapause eggs, whereas dephosphorylation of ecdysone 22-phosphate and its subsequent hydroxylation at the C-20 and C-26 positions were characteristic in non-diapause eggs.     

Construction of a BmNPV polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, Bombyx mori

The baculovirus expression vector system is one of the most powerful and versatile eukaryotic expression systems available. However, as the recombinant baculovirus is usually generated by replacing the foreign gene into the polyhedrin locus, the resulting polyhedrin-negative virus is less infectious to the host larvae when administered via oral ingestion. This limits the large-scale production of the recombinant protein, as the host larvae can only be inoculated through dorsal injection, which is a laborious task. In this paper, we describe a new Bombyx mori nucleopolyhedrovirus polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, B. mori. In this system, the foreign gene and the polyhedrin are co-expressed, and polyhedra are produced as in the wild-type virus, and thus the recombinant baculovirus can be used directly via oral infection. It effectively improves the efficiency of the baculovirus expression system and also widens the application of baculovirus in other fields, such as the development of new biological insecticides.     

Expression of Isopentenyl Transferase Gene Controlled by Seed-Specific Lectin Promoter in Transgenic Tobacco Influences Seed Development

The bacterial isopentenyl transferase (ipt) gene involved in cytokinin biosynthesis was fused with a seed-specific lectin promoter from soybean and introduced into tobacco. Under the control of the lectin promoter, the expression of the ipt gene increased cytokinin levels and promoted cell division in the embryo in transgenic tobacco seeds. Compared with controls, the number of plerome cell layers and the cell number of cotyledons and pleromes were significantly increased from 16 DAF (days after flowering); the embryo diameter of transgenic tobacco was enlarged at 16, 19, and 21 DAF (16.1%, 12.7%, and 13.9% increase, respectively). Furthermore, the soluble protein content of the transgenic mature seeds was increased by 9.8–22.2% and the dry weight of transgenic tobacco seeds was increased by 8.8–21.8% compared with that of controls. The transgenic tobacco seedlings also grew quickly and a greater increase in fresh weight compared with controls was observed at 20 and 35 days after germination (average 14% and 8% increase above controls, respectively).