大鼠睾丸特异表达基因Ube1的分离鉴定及生物学特征

本研究采用抑制性消减杂交(suppression subtracfive hybridization, SSH)和cDNA快速扩增(rapid amplification of cDNA ends, RACE)技术从大鼠A型精原细胞和粗线期精母细胞中成功克隆出大鼠泛素激活酶(ubiquitin-activating enzyme)基因Ube1 (GenBank登录号EF690356).该基因序列全长3433 bp,其中开放阅读框有3171 bp,编码一个含1057个氨基酸的蛋白质.Blast比对显示,Ube1与小鼠泛素激活酶基因Ubely1的同源性为93%,与人泛素激活酶基因UBE1的同源性为82%.Ube1基因编码的蛋白质含泛素激活酶信号位点和泛素激活酶活化位点,这些位点也存在于人类和小鼠的泛素激活酶1中.RT-PCR分析显示,Ube1在睾丸中大量表达,而在心、肝、脾、肺、肾、肌肉、脑、卵巢中没有表达.荧光定量PCR分析不同生精细胞中Ube1的表达,显示Ube1在A型精原细胞中大量表达,在粗线期精母细胞、圆形精子细胞和支持细胞中微弱表达.以上结果提示,Ube1是大鼠睾丸特异表达基因,可能通过参与泛素/蛋白酶体途径来影响精子发生.     

Cloning and characterization of nanos gene in silkworm Bombyx mori

Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5＇-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mor/and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5.38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee （Apis mellifera）. This gene has been registered in the GenBank under the accession number EF647589. One encoding sequence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells.     

家蚕BmBrat基因的克隆与鉴定

NHL家族蛋白具有调控细胞增殖与分化的功能,在哺育动物中被广泛研究。本文克隆得到家蚕NHL蛋白家族成员BmBrat基因,通过RACE技术获得该基因cDNA全长序列为3 614 bp,其ORF为2 580 bp,编码859个氨基酸,预测其蛋白分子量为94.3 kDa,等电点为6.65。利用RT-PCR技术检测其在五龄3 d家蚕各组织表达情况,结果表明其在幼虫各组织均有表达,包括丝腺、中肠、脂肪体、马氏管等,且卵巢和头部表达量最高;胚胎时期表达谱分析显示其在胚胎发育第4天和第5天有高量表达。经原核表达、蛋白纯化及免疫小鼠后获得家蚕BmBrat多克隆抗体,且Western blotting及免疫荧光检测显示该抗体可以特异检测家蚕BmBrat蛋白;免疫荧光结果表明BmBrat蛋白定位于家蚕血细胞胞质中,为进一步研究BmBrat基因的生物学功能奠定了基础。     

家蚕吡哆醛激酶基因干扰降低转氨酶基因的转录表达

【目的】维生素B₆在氨基酸代谢中是多种酶的辅酶,维持氨基酸代谢的正常运行。磷酸吡哆醛（pyridoxal-5′-phosphate, PLP）是维生素B₆的主要辅酶形式,吡哆醛激酶（pyridoxal kinase, PLK）是PLP的重要生成酶,本研究试图明确PLK基因与PLP依赖酶之间转录水平的调节关系。【方法】本研究采用RNA干扰（RNA interference, RNAi）方法对家蚕 Bombyx mori 的PLK基因进行干扰,通过体外合成PLK基因的3个干扰片段（siRNA1, siRNA2和siRNA3）,将siRNA从体腔注入5龄第3天的家蚕幼虫体内诱导RNAi。利用荧光定量PCR测定不同干扰片段、不同时间点及不同组织中PLK基因表达量的变化;并测定家蚕体内磷酸丝氨酸转氨酶（phosphoserine aminotransferase, SerB）和天门冬氨酸氨基转移酶（asparate aminotransferase, AST）基因的表达量。【结果】注射干扰片段后48 h干扰效果达到最佳。3个干扰片段干扰效果从高到低依次为siRNA1, siRNA2和siRNA3。RNAi效果最好的是中肠组织,其PLK基因的相对表达量下降了55%。RNA干扰PLK基因后,后部丝腺中SerB和AST基因相对表达量分别下降了90%和29%。【结论】本研究通过RNAi实现了家蚕PLK基因干扰,并进一步证明了家蚕PLK基因和SerB基因及AST基因存在联动调节关系。     

家蚕Bm Tpi基因Z染色体定位

采用实时定量PCR技术,以家蚕Bombyx mori第11号染色体上的DH-PBAN基因为参照基因,检测家蚕不同个体间Bm Tpi基因与常染色体上DH-PBAN基因的拷贝数之比,雄体Bm Tpi∶DH-PBAN=1.0,雌体Bm Tpi∶DH-PBAN=0.5;并用已经定位于Z染色体上的Bm Kettin基因为参照,检测Bm Tpi基因的拷贝数与Bm Kettin基因的拷贝数之比,雄体Bm Tpi∶Bm Kettin=1.0,雌体Bm Tpi∶Bm Kettin=1.0,证明Bm Tpi基因在家蚕基因组中的拷贝数与Bm Kettin基因相同,说明Bm Tpi基因位于Z染色体上。     

家蚕细胞周期蛋白基因BmCcnl1的克隆及表达分析

家蚕Bombyx mori由受精卵到完成胚胎发育孵化的过程中, 细胞进行大量的分裂和分化, 然而滞育性卵的胚胎细胞分化至G2期便停滞在此阶段。为了探索这一发育阶段细胞内的分子调控, 本研究以人Homo sapiens的细胞周期蛋白基因cyclin L1为模板, 成功克隆了家蚕同源基因BmCcnl1（GenBank登录号: FJ889988）。BmCcnl1基因开放阅读框（open reading frame, ORF）全长1 254 bp, 编码417个氨基酸。利用Protean软件分析得出BmCcnl1蛋白预测分子量为49 kDa, 等电点为9.84。利用DNA重组技术构建了BmCcnl1基因的重组表达载体pET-21d-BmCcnl1, 对其进行原核表达, 其表达的蛋白以包涵体形式存在。利用RT-PCR技术分析了BmCcnl1基因在胚胎发育过程中的转录水平, BmCcnl1基因在非滞育性卵的胚胎发育阶段基本保持相对稳定的转录表达, 而滞育性卵从蛾体产下经过72 h后已经检测不到BmCcnl1基因的转录。结果提示, BmCcnl1基因与胚胎期滞育及非滞育性卵的发育调控相关。对该基因的克隆和表达分析为今后研究家蚕胚胎发育及细胞周期调控奠定了基础。     

Expression analysis of decapentaplegic gene in the silkworm,Bombyx mori

The decapentaplegic (dpp) gene in Drosophila is involved in multiple developmental processes, and is a highly conserved among various eukaryotic species, including Bombyx mori. Although the gene has well been characterized in Drosophila species the B. mori dpp has not yet been functionally analyzed. In this study, we analyzed the expression pattern of B. mori dpp in 12 different developmental days/stages (7 days for fifth instar larvae, 2 days for spinning stage, 2 days for pupal stages, and 1 day for adults) in both male and female silkworms using quantitative real‐time RT‐PCR (qRT‐PCR). mRNA expression of B. mori dpp was much higher in the female larvae up to the mid‐stage of the fifth instar compared with the corresponding male larvae. Similarly, dpp expression also was much higher in females during the eclosion period than that in the corresponding male pupae. During the embryonic stage, the expression level of the dpp gene was much higher compared to that of adult stage in both male and female silkworms. These results suggest that the B. mori dpp gene plays multiple roles in the developmental of B. mori.     

Expression analysis of chlorophyllid a binding protein a secretory,red fluorescence protein in the midgut of silkworm,Bombyx mori

Chlorophyllid a binding protein （ chbp） was recently characterized by its ability to bind the prosthetic group of chlorophylls and little information is known regarding its expression. In the present study, we found that chpb was expressed highly and exclusively in the midgut of silkworm, Bombyx mori. The expression level of chbp was very high in the newly molted fifth instar larvae followed by gradual decline in the same instar. Our results demonstrated that CHBP was a secretory protein and located mainly in the apical of midgut epithelial cells. Real-time polymerase chain reaction analysis results showed that chpb highly expressed in the anterior midgut, threefold and sixfold higher compared with that of the middle midgut and posterior midgut, respectively, and chpb expression declined in darkness. In addition, the expression of chbp was affected by high-dose virus or bacterium infection.     

家蚕保幼激素结合蛋白Bmtol基因的克隆及功能分析

家蚕头部是一个神经中枢和感受的器官,其头部含有触角和感觉毛,感受外界的信号,并将外界信号传送到大脑进行反应。保幼激素主要是由咽侧体合成和分泌的,而保幼激素结合蛋白是保幼激素转运和发挥功能的载体,在昆虫体内具有极其重要的功能。文中通过Silk DB和NCBI数据库筛选并鉴定到一个新的具有保幼激素结合蛋白家族保守结构的蛋白Bm TOL,其编码基因编号为BGIBMGA003404(Gen Bank登录号:KY681053)。利用原核表达系统成功表达了该蛋白,通过Ni-NTA亲和层析的方法获得了Bm TOL的重组蛋白并制备了多克隆抗体。组织表达分析发现无论是转录水平还是蛋白水平Bm TOL在头部都是高量表达,且Bmtol基因在起蚕时表达量较高,在5龄和蛹期表达量较低,而在化蛾后表达量又开始上调。免疫组化结果显示Bm TOL蛋白定位在头部的皮层、触角和脑中,推测其可能与头部信息传递有关,为家蚕的生长发育和行为调控提供重要的信息来源。     

家蚕3-羟酰辅酶A脱氢酶基因全长cDNA克隆及其在质型多角体病毒感染家蚕的差异表达

家蚕质型多角体病毒(Bombyx mori cytoplasmic polyhedrosis virus,BmCPV)是家蚕的重要病毒病原之一,往往给养蚕业生产造成极大危害。我们以前的研究运用基因芯片技术在感染质型多角体病毒的家蚕中肠中鉴定出一个差异表达的3-羟酰辅酶A脱氢酶蛋白基因(Bombyx mori3-hydroxyacyl-CoA dehyrogenase protein gene-Bm3HAD)。本研究利用cDNA末端快速扩增技术(RACE)克隆了该基因,其全长cDNA序列为1168bp,包含一个83bp5’端非翻译区序列(5’-UTR)、一个930bp的开放阅读框(ORF)和一个155bp的3’端非翻译区序列(3’-UTR);基因结构分析发现该基因由5个外显子和4个内含子组成。RT-PCR结果显示该基因在家蚕中肠、脂肪体、血液、丝腺及生殖体中均有表达。荧光定量PCR结果表明该基因在BmCPV感染初期为上调表达,随着病毒感染的进展,该基因的表达水平逐渐降低,并转变为下调表达。研究结果为进一步研究BmCPV对家蚕致病的分子机制提供了有益的信息。     

家蚕吡哆醛激酶cDNA的克隆、表达和基因结构分析

吡哆醛激酶（pyridoxal kinase,PLK, EC2.7.1.35）是维生素B₆关键代谢酶,其cDNA的克隆在昆虫类还未见报道。利用生物信息学原理和使用PCR方法,克隆出编码家蚕Bombyx mori吡哆醛激酶的cDNA (GenBank登录号DQ452397),体外原核表达成功,并对表达粗提产物进行了酶活检测。克隆到的cDNA含有一894 bp的完整可读框,编码一条分子量为33.1 kD,含298个氨基酸残基的蛋白质。序列比对显示此蛋白质与人类吡哆醛激酶具有52.84%的同一性,包含吡哆醛激酶家族共有的特征保守序列,但比哺乳动物和植物克隆到的吡哆醛激酶均少10多个氨基酸残基,几个有关键功能且在哺乳动物和植物中均保守的氨基酸残基在此蛋白中被替换。依据家蚕基因组数据库信息和PLK的cDNA,家蚕PLK基因包含5个外显子和4个内含子,跨越10 kb DNA序列,所有外显子/内含子交接点都遵从gt/ag剪接规则,基因的5′端启动子调控区发现有TATA-box和CAAT-box保守基序。     

家蚕先天免疫基因 Bmimd 的克隆、表达及序列分析

昆虫的先天免疫应答由一组基因通过级联网络调控实现。果蝇 Drosophila 免疫缺陷(immune deficiency, imd)基因在体液免疫信号传递途径中起着重要的作用。我们利用生物信息学方法进行电子克隆,成功地找到了 imd 基因在家蚕 Bombyx mori 中的同源体,命名为 Bmimd。该基因全长1 092 bp,由 4 个外显子和 3 个内含子组成,开放阅读框(open reading frame, ORF)长 750 bp,编码 250 个氨基酸,预测蛋白质分子量为 28.6 kD。Bmimd 序列中含有一个致死结构域,经聚类分析表明该结构域与哺乳动物的受体相互作用蛋白(receptor interacting protein, RIP)相似。将该基因亚克隆到 PET-50b 载体进行原核表达,表达出了带有 2 个 6×His tag 和 1 个 Nus·Tag 标签的重组蛋白。Western blotting 结果表明 Bmimd 蛋白在 5 龄 4 天家蚕的头、脂肪体 、生殖腺、表皮和中肠中都有表达,但丝腺中没有检测到表达。     

Isolation and reconstitution of the RNA replicase of the cytoplasmic polyhedrosis virus of silkworm,Bombyx mori

Summary After irradiation of the virus particles of CPV, the RNA replicase associated with the virion was isolated in the form of a genome-replicase complex with DEAE-Sephadex A-25 chromatography. This complex was then treated with Triton X-100 and purified by phosphocellulose column chromatography. The RNA replicase reconstituted with the doublestranded RNA of CPV showed both the enzyme activity of RNA polymerase and methyltransferase. The single-stranded RNA could not serve as the template for the RNA replicase. The role of the RNA replicase of CPV is discussed.     

Antiviral activity of selected medicinal plants and marine seaweeds on the grasserie infected larvae of silkworm,Bombyx mori

Nuclear polyhedrosis virus (NPV) is the most harmful virus responsible for the manifestation of grasserie disease in the larvae of silkworm, Bombyx mori. It causes a huge economic loss in the sericulture industry. An attempt was made in the present investigation for the screening of antiviral activity using medicinal plants such as Lantana camara, Phyllanthus amarus and marine seaweeds such as Sargassum wightii, Turbinaria ornata against BmNPV. Crude extracts were prepared using different solvents, such as hexane, ethyl acetate, methanol and water. The silkworm feeding bioassay study was carried out with the crude extracts to investigate the presence of anti-BmNPV activity after inoculating fifth instar larvae of silkworm with occlusion bodies (OBs) of BmNPV. Each extract was tested for their anti-BmNPV activity using various concentrations of crude extracts ranging from 200 μg to 1000 μg. Among the crude extracts tested, methanol and aqueous extracts of P. amarus showed significant anti-BmNPV activity.     

家蚕丝素蛋白轻链基因（fib-L）启动子序列的克隆及其活性分析

家蚕Bombyx mori丝素蛋白轻链（fibroin light-chain, fib-L）基因fib-L具有在后部丝腺组织专一性、高效性表达的特点。为了利用其启动子构建能够表达外源基因的丝腺生物工厂,本实验对fib-L启动子活性进行了研究。通过PCR法克隆了fib-L启动子元件,序列分析显示fib-L启动子由位于-33 ～ -25处的TATA盒元件和位于－128～－121处的特征性序列GTCAATTT共同组成。用fib-L启动子控制报告基因DsRed进行家蚕BmN细胞和蚕体内的瞬时表达研究,结果表明fib-L启动子可以驱动DsRed报告基因在BmN细胞和家蚕后部丝腺组织中瞬时表达。     

家蚕丝素P25蛋白基因启动子顺式作用元件的功能分析

为了阐明蚕丝蛋白基因表达调控的分子机制, 利用Bac-to-Bac杆状病毒表达系统及实时荧光定量PCR等技术, 对家蚕Bombyx mori丝素P25基因启动子上游1 233 bp的活性及其调控元件的功能进行了分析。结果表明： 在P25启动子上游-423~-1 233区和-127~-238区存在正调控元件, 在-238~-423区段存在负调控元件; PSGF和BMFA两结合元件在P25基因表达中起负调控作用。PSGF结合元件对A3启动子在后部丝腺的活性具有一定的增强作用, 进一步验证了PSGF调控元件的功能。通过对P25基因启动子的活性分析, 尤其是对PSGF和BMFA调控元件的功能分析, 有利于进一步了解P25基因表达调控的精细机制。