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.     

Functional Characterization of a Cactus Homolog from the Silkworm Bombyx mori

A cDNA encoding an IκB family protein was identified and the full nucleotide sequence was determined in the silkworm Bombyx mori. The IκB gene, designated BmCactus, was constitutively expressed mainly in the fat body and hemocytes. Transfection experiments on a B. mori cell line, NIAS-Bm-aff3, with expression vectors containing BmCactus, BmRelA, BmRelB, or the active portion of BmRelish1 showed that activation of the CecB1 gene promoter by either BmRelA or BmRelB, but not the active portion of BmRelish1, was strongly inhibited by BmCactus. In addition, activation of CecB1 gene by autoclaved E. coli in the cultured cells was observed regardless of the presence or absence of BmCactus. A gultathione S-transferase pull-down assay and analysis using a yeast two-hybrid system demonstrated that BmCactus interacted with the BmRel Rel homology domain, but not with the BmRelish Rel homology domain. These results suggest that BmCactus is involved in the Toll signal transduction pathway in B. mori.     

Purification,Identification and Functional Analysis of a Novel Immunomodulatory Peptide from Silkworm Pupa Protein

International Journal of Peptide Research and Therapeutics - In this study, we isolated and characterized an immunomodulatory peptide from silkworm (Bombyx mori) pupa protein hydrolysates....     

Control of Protein Synthesis in the Female Pupa of Bombyx mori

Four degraded okaramine B (2) products, 4′,5′-dihydrookaramine B (3), two azetidine ring-opened compounds (4 and 5) and 1′,2′,4′,5′-tetrahydrookaramine B (6), were prepared and their insecticidal activity was examined. Neither compounds 4 nor 5 showed such activity against silkworms, indicating that the azetidine ring moiety played an important role in the insecticidal activity. Moreover, both compounds 3 and 6 exhibited lower activity than 2, which means that the azocine ring moiety was indispensable to form the active conformation.     

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。     

重组人骨形态形成蛋白2在家蚕幼虫中表达及产物纯化

将编码人ＢＭＰ２ｃＤＮＡ基因插入昆虫杆状病毒转移载体ｐＢａｃＰＡＫ１,与修饰的家蚕核形多角体病毒Ｂｍ－ＢａｃＰＡＫＤＮＡ共转染家蚕细胞,通过同源重组得到含有在多角体蛋白基因启动子控制下的ＢＭＰ２ｃＤＮＡ基因的重组病毒Ｂｍ－ＢａｃＰＡＫ－ＢＭＰ２。用重组病毒感染家蚕幼虫,第五天ＢＭＰ２表达率最高,每毫升蚕血淋巴中约１０μｇ表达产物;表达产物在在体内被加工成Ｃ－端１６ｋＤ片段,以二硫键连结成分子量为３０ｋＤ的同源二聚体;经纯化获得９０％以上纯度的成熟ＢＭＰ２,与骨基质胶原结合后植入大鼠皮下,７天后在局部诱导生成软骨组织。     

Diapause Hormone B: Its Selective Extraction and Isolation from the Silkworm,Bombyx mori

A selective extraction procedure of the diapause hormone-B (DH–B) from male silkworm adult heads is described. By this new method a highly active extract (1 DH unit in 30 ~ 60 μg) can be obtained easily without Chromatographic purification. It was further purified by successive gel permeation chromatographies to give finally pure DH–B having an activity of 1 DH unit in 2 μg.     

家蚕滞育激素基因的克隆

利用放射性同位素３２Ｐ－ｄＣＴＰ标记家蚕滞育激素ｃＤＮＡ作为探针,从家蚕基因库大约４×１０~５个噬菌体斑中筛选得到３０个阳性克隆,其中３个克隆具有相同的限制性内切酶图谱,并测知插入ＤＮＡ约１８ｋｂ,用限制性内切酶ＳａｌＩ、ＥｃｏＲＩ切割,进行次克隆。用ＴａｑＤｙｅｐｒｉｍｅｒ序列测定法,得到部分核苷酸序列,经比较,鉴定了这３个克隆确属滞育激素基因。基因组Ｓｏｕｔｈｅｒｎ分析,推定家蚕滞育激素基因属单基因编码。     

家蚕细胞遗传学及其应用

由于家蚕染色体数目较多、着丝粒弥散, 在较长时期内, 家蚕染色体识别、核型分析、染色体结构和功能的研究都受到很大限制。近年来, 应用比较基因组杂交、基因组原位杂交、基于细菌人工染色体克隆的原位杂交技术建立了家蚕的细胞学图, 综合分子连锁图构建高密度的细胞遗传学图已成为可能。分子细胞遗传学的应用正在推动家蚕染色体结构和功能的研究, 揭示出家蚕W染色体密集地分布着嵌套结构的逆转座子, 染色体端粒由重复序列(TTAGG)n以及端粒特异的非长末端逆转座子TRAS1和SART1组成, TRAS1、SART1具有较高的转录活性, 可能与维持染色体的稳定性有关。     

Isolation and Identification of a Pathogen of Silkworm Bombyx mori

A pathogenic bacterial strain, ST-1, was isolated from a naturally infected silkworm. The strain was identified on the basis of its physiological and biochemical properties and the results of sequence analysis of its 16S rRNA gene. The results of the 16S rRNA gene sequence analysis revealed that ST-1 shared the highest sequence identity (more than 99%) with Pseudomonas chlororaphis subsp. aurantiaca. ST-1 bacteria were gram-negative and 0.7-0.9 × 1.3-1.5 μm long, short rods with rounded ends. The strain could utilize sodium citrate, malonate, D-glucose, sucrose, D-fructose, D-mannose, and L-arabinose. Pathogenicity of ST-1 for silkworm could be depicted as a linear regression of the logarithm (y) of ST-1 concentration against probability (x) (y = 0.4040 + 0.0600x). The median lethal concentration (LC(50)) was 2.12 × 10(4) cfu/ml. In conclusion, ST-1 was identified as Ps. chlororaphis subsp. aurantiaca. This is the first report that Ps. aurantiaca is a pathogen for silkworm Bombyx mori.     

家蚕胚胎发育时期的蛋白质变化及构造分析

采用蛋白质双向电泳技术及蛋白质氨基酸序分析技术,从蛋白质水平研究了家蚕胚胎发育时期的基因表达情况。结果表明,从家蚕临界期胚胎直到点青期胚胎的较长一段时间内,蛋白质的双向电泳图变化不大,匹配蛋白质斑点率达６３．０％,卵特异性蛋白质,３０Ｋ蛋白质的含量很大。     

家蚕滞育生物钟蛋白质Ease A4的纯化及其分子结构分析

ＥａｓｅＡ４是家蚕卵的一种滞育生物钟蛋白质．产下后２ｄ的家蚕Ｃ１０８品种滞育性卵,经过丙酮脱脂、８５℃热处理、硫酸铵沉淀和ＳｅｐｈａｄｅｘＧ－２５凝胶过滤层析初步分离,进一步经过Ｓｅｐ－ＰａｋＣ１８脱盐浓缩,ＨＰＬＣ（柱为ＹＭＣ－ＰａｃｋＰｒｏｔｅｉｎ－ＲＰ）分离,通过ＳＤＳ－ＰＡＧＥ和ＭＡＬＤＩＭＳ方法鉴定,纯化得到ＥａｓｅＡ４蛋白质．从１０ｇ蚕卵最终得到了１１．８μｇＥａｓｅＡ４．ＥａｓｅＡ４由从Ｈｉｓ到Ｔｙｒ的１５５个氨基酸残基构成,蛋白质部分的分子量为１６６０１．其２２位氨基酸残基Ａｓｎ处有一个Ａｓｎ－Ｘ－Ｔｈｒ糖基化场所,并有糖基结合在该部位,糖基的分子量约为７６０．ＥａｓｅＡ４的６１位和１５０位的两个Ｃｙｓ氨基酸残基之间存在二硫键．糖基和二硫键的存在不仅有利于酶蛋白的分离,还可能与酶活性有关     

昆虫来源的几丁质酶的分离纯化及酶学性质

几丁质酶在真菌和昆虫的生理和发育过程中起着关键作用,该酶本身及其酶抑制剂是获取生物农药的重要途径。本研究从蚕蛹体内提取几丁质粗酶,经硫酸铵分级沉淀和Sephadex G-150分离得到几丁质酶。用SDS-PAGE测得该酶的分子量为88kDa。水解胶体几丁质的Km值为22.3μmol/L。酶反应的最适温度为45℃,最适pH值为6.0,金属离子和有机试剂对几丁质酶活性都有影响,其中高浓度的Mn2+对酶有较强的激活作用,而Cu2+、SDS则有较强的抑制作用。研究结果为基于几丁质酶的生物农药筛选研究奠定了基础。     

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。     

家蚕生物钟蛋白TIME-EA4的进化踪迹分析

家蚕是卵滞育性鳞翅目模式昆虫,其滞育卵的活化与生物钟蛋白TIME-EA4密切相关。TIME-EA4具备稳定的Cu/Zn SOD活性和瞬时的ATP酶活性。目前,国内外关于家蚕TIME-EA4的研究主要集中在结构和功能上,其分子进化机制研究尚未开展。本文利用生物信息学方法对TIME-EA4进行了进化踪迹分析,结果显示TIME-EA4的重要氨基酸残基(coverage25%)的91.2%都用来维持与Cu/Zn SOD的序列一致性,但TIME-EA4与Cu/Zn SOD的Cu/Zn离子结合位点在氨基酸残基组成、极性、绑定原子方面都存在差异。