鲤鱼肝胰脏cDNA 文库的表达序列标签分析及Lb-Fabp mRNA的特征

本文构建了鲤鱼肝胰脏cDNA 文库,共获得了1016条有效的表达序列标签。拼接组装成115 个contigs和282 个singletons。其中215个拼接序列在GenBank公共数据库中寻找到相对应的基因。对它们进行功能性分类和比较分析为鲤鱼肝胰脏的研究提供了基因表达信息的基础。文库中1016条表达序列标签有11条代表了鲤鱼肝基本型脂肪酸结合蛋白(Lb-FABP)。通过序列比较我们获得了两个具有相同开放阅读框长度的Lb-Fabp cDNAs。开放阅读框全长381bp,编码126个氨基酸。半定量RT-PCR结合Southern blot技术研究了Lb-Fabp mRNA 在成鱼不同组织以及早期发育不同时期的表达图式。结果表明,Lb-Fabp mRNA 在肝胰脏、中肠和后肠中表达量较高。同时在精巢和皮肤中有低水平的表达。脑、肌肉、卵巢、肾脏、脾脏、鳃和心脏等组织中其表达量更低。而在脂肪和前肠中则没有检测到Lb-FabpmRNA表达。Lb-Fabp mRNA 最早在胚体形成期检测到有低水平表达,随后的发育阶段中表达量逐渐升高。鲤鱼Lb-Fabp基因的表达图式提示在肝脏和肠等器官开始发育后,它可能在脂肪代谢中具有重要作用。     

家蚕母性基因的表达序列标签分析

为了研究家蚕Bombyx mori卵中储存的母性遗传信息,以未受精的肾脏型和正常型蚕卵为材料构建了cDNA文库,并对其随机测序,共获得391条表达序列标签(EST)序列,经拼接后得到374条非重复序列。生物信息学分析发现,172个非重复序列与国际数据库GenBank和silkbase中的已知基因具有较高的相似性。除3个已知母性基因外,其余主要与DNA复制、能量代谢、信号转导、转录、蛋白质结构以及胚胎发育中个体形成等相关。该结果可为进一步研究家蚕胚胎早期发育的调控提供基础数据。     

铝胁迫下柱花草SSH文库构建及表达序列标签分析

柱花草栽培种热研2号（Stylosanthes guianensis‘Reyan2’）对铝毒有较强的耐受性。为了鉴定其在铝胁迫下的诱导基因,利用抑制消减杂交（SSH）技术构建在300μmol·L-1铝胁迫下正向cDNA文库。挑选插入片段大于300bp的600个克隆进行测序,共获得504条表达序列标签（EST）。序列重复性分析表明,其中12.1%的EST只有1次重复,61.4%的EST有2-16次重复,重复出现次数较高的EST是细胞色素P450（53次,占10.5%）、病原诱导型胰蛋白酶抑制剂（44次,占8.7%）和衰老相关蛋白（37次,占7.3%）。BLASTX分析显示,504条EST中有97种非冗余基因,其中包括46条功能已知基因和51条功能未知序列。46条功能已知EST中有30个为已报道铝胁迫相关基因,16个是新发现的铝胁迫相关基因。SSHcDNA文库提供的信息为阐明柱花草耐铝毒的分子机制提供了重要线索。     

铝胁迫下柱花草SSH文库构建及表达序列标签分析

柱花草栽培种热研2号(Stylosanthes guianensis ‘Reyan 2’)对铝毒有较强的耐受性。为了鉴定其在铝胁迫下的诱导 基因, 利用抑制消减杂交(SSH)技术构建在300 μmol·L^–1铝胁迫下正向cDNA文库。挑选插入片段大于300 bp的600个克隆进行测序, 共获得504条表达序列标签(EST)。序列重复性分析表明, 其中12.1%的EST只有1次重复, 61.4%的EST有2–16次重复, 重复出现次数较高的EST是细胞色素P450(53次, 占10.5%)、病原诱导型胰蛋白酶抑制剂(44次, 占8.7%)和衰老相关蛋白(37次, 占7.3%)。BLASTX分析显示, 504条EST中有97种非冗余基因, 其中包括46条功能已知基因和51条功能未知序列。46条功能已知EST中有30个为已报道铝胁迫相关基因, 16个是新发现的铝胁迫相关基因。SSH cDNA文库提供的信息为阐明柱花草耐铝毒的分子机制提供了重要线索。     

高山植物黄管秦艽cDNA文库构建与表达序列标签（EST）分析

黄管秦艽（Gentiana officinalis）是一种重要的藏药高山植物,本研究构建了该物种开花期的eDNA文库。经检测达到中等cDNA文库水平,文库滴度为1．2×10^7pfu／ml,重组率95．9％,插入片段平均长度大于500bp。对343个随机挑选的重组克隆进行部分测序,获得的ESTs经编辑后共有181条有效序列。经生物信息学方法分析181条表达序列标签（EST）代表144个单克隆序列,其中55个与已鉴定的基因同源,35个序列与未鉴定的EST匹配,54个未找到同源序列;后两者共有89个EST序列未发现功能相似的蛋白。对已鉴定的EST进行功能分析发现,相关基因主要编码以下蛋白：与蛋白表达相关的占35％;光合作用相关的占笠％;新陈代谢相关的占18％;抗性相关的占11％;质膜运输和细胞分裂相关的分别占5％;染色体变化和细胞信号转导的分别占2％。根据有效EST序列设计引物,通过RT-PCR进一验证了所得EST的准确性。这些研究结果为将来研究黄管秦艽的功能基因以及该物种与相关物种的群体遗传学、进化生物学等方面提供了基础。     

基于单碱基延伸标签反应的常见食源性致病菌基因芯片检测方法的建立

针对8种常见的食源性致病菌(金黄色葡萄球菌、副溶血弧菌、单核细胞增生李斯特菌、沙门氏菌、阪崎肠杆菌、志贺氏菌、肠出血性大肠杆菌O157:H7和空肠弯曲杆菌),建立了基于单碱基延伸标签反应原理的基因芯片检测方法。筛选和整合8种食源性致病菌基因组中的特异性序列和相应PCR引物,致病菌靶DNA片段被扩增和纯化作为单碱基延伸标签反应的模板,反应产物在DNA芯片上与探针进行杂交反应,然后通过扫描基片的荧光强度进行判断。实验结果表明,可采用基于单碱基延伸标签反应的基因芯片方法同时特异性检测8种食源性致病菌,基因组DNA多重检测灵敏度可达到0.1pg,以鼠伤寒沙门氏菌为单一检测对象的细菌纯培养物灵敏度可达到5×102CFU/mL。本方法可以快速灵敏地检测食源性致病菌,为食源性疾病的诊断和防治提供了一个有效的方法。     

一种新的人端粒相关锌指蛋白cDNA的克隆及鉴定

为了从早期胚胎寻找与发育分化有关的新基因,本文构建了3周龄人胚cDNA文库,并应用EST技术对该文库中随机挑选的47个低丰度克隆进行测序,结果发现了一个与人亚端粒DNA和锌指基因同源的cDNA克隆(L30),该基因长约3.8kb,5＇端序列有明显的阅读框架(ORF),3＇端序列有加尾信号(AAUAGA)和有39个A组成的Poly(A)尾巴;通过Northern杂交确认在早期人胚胎中有表达,应用地高辛染色体原位杂交技术将其定位于人第12号染色体长臂端部.     

应用改进的多聚组氨酸标签T7表达载体构建肺癌cDNA文库

应用噬菌体C端展示系统构建的cDNA文库缺乏开放阅读框筛选机制,文库中多数噬菌体克隆展示框外非天然短肽,给后期蛋白质的筛选带来了不便. 为实现噬菌体的ORF筛选功能,利用PCR技术对已有载体T7Select10-3b进行改造,在MCS处外源cDNA插入位点的3′端引入6聚组氨酸筛选标签,经包装后挑取成功表达的单克隆构建肺癌cDNA文库. 经镍柱亲和层析后,收集文库中表达组氨酸的克隆,利用化学发光免疫试验进行筛选效果鉴定. 结果显示,改造的新型载体可成功表达组氨酸标签,以此构建的肺癌cDNA文库经筛选后,含ORF插入的克隆由筛前的6 %提高至70 %,本研究为提高cDNA文库的质量提供了一种简便可行的方法.     

抗FLAG标签单克隆抗体的制备、鉴定及初步应用

用碳化二亚胺法合成FLAG完全抗原,利用杂交瘤技术制备出5株分泌抗FLAG标签单克隆抗体的杂交瘤细胞株。经鉴定,腹水效价均高于1：106 ,且5株单抗与其它融合蛋白标签无交叉反应性,并在免疫亲和层析实验中取得了满意的效果,为含标签的融合蛋白的纯化和研究应用提供了重要的工具。     

中国地方品种香猪的肌肉特异组织表达序列标签(ESTs)的

通过构建香猪肌肉组织cDNA文库,并在文库中随机挑选克隆进行测序的方法,获得了131个香猪肌肉EST序列.在这131个EST序列所代表的109个单一克隆中,有99个为人类及其他物种的同源序列,3个为已知的猪的ESTs,7个为未知ESTs.对这10个已知、未知ESTs进行开放阅读框预测并进行B1ast分析,没有找到高度同源的氨基酸序列.对上述EST所对应的基因功能分析结果表明,除去27.27%的EST未能分类外,克隆到的EST大多来自与基因/蛋白的表达调控相关的基因(占45.46%).来自具有其他功能的基因的EST依次是细胞代谢占10.10%、细胞结构/迁移占10.10%、细胞/机体防御占5.05%和细胞信号/传导占2.02%.没有发现和细胞分裂相关的已知功能基因.本研究结果为中国地方品种香猪提供了第一个骨骼肌的基因表达谱,为今后寻找猪肌肉生长和肉用品质的候选基因奠定了基础.     

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.     

桑蚕品种纯度序贯抽样检验方案设计

桑蚕一代杂交种杂交率检验存在抽样方案和检验方法两方面的缺陷,无法准确、及时地检验一批蚕种的品种真实性和纯度．本文提出了使用DAN检验方法进行品种纯度检验,研究设计了适合的序贯抽样检验方案．该方案保证了目前行业标准检验方法的可靠性和精确度．使用该方案,在蚕种纯度很高的情况下,只需检验很小的样本就能通过,非常适合个体检验准确而成本相对较高的DNA检测方法．     

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。     

Dietary Sterol Preference in the Silkworm,Bombyx mori

Since insects are unable to biosynthesize sterols de novo, sterols must be obtained from dietary sources. Although it has been reported that β-sitosterol is crucial for larval growth in the silkworm, Bombyx mori, little has been investigated concerning the dietary selection of sterols by Bombyx larvae. Here, we demonstrate that Bombyx larvae have the following sterol preference: β-sitosterol >> ergosterol > cholesterol = stigmasterol. Interestingly, Bombyx larvae preferred ergosterol, an inhibitory sterol on larval growth, indicating that sterol selection following first contact of the diet with the mouthpart might be different from the sterol recognition mechanism present in sterol metabolism.     

Internal ultrastructure of spores of microsporidans isolated from the Silkworm,Bombyx mori

Nosema bombycis, two Nosema spp., and a Pleistophora sp. were propagated in the silkworm and the fine structures of their spores were studied. The morphology of the polaroplast, the appearance of the nucleus, and the number of coils in the polar filament differed among the spores of the four species. The spores of the three Nosema species, however, had several identical components; e.g., the polaroplast was made up of two parts, they had two nuclei, and the ribosome arrangement was similar. On the other hand, the spore of Pleistophora sp. had a polaroplast composed of three parts, a single nucleus, and ribosomes arranged around the polar filament. Thus the fine structures of the spore differentiate microsporidan species.     

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.     

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。     

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/).     

Molecular characterization of a sodium channel gene from the Silkworm Bombyx mori

The voltage-gated sodium channel mediates the rapid rising phase of action potentials in almost all excitable cells and is a molecular target of a variety of neurotoxins including pyrethroid insecticides. Most studies have focused on the expression of sodium channel genes in the adult stage, information on other developmental stages, however, is limited. In this study, we characterized the para sodium channel orthologous gene (BmNa(v)) of the silkworm Bombyx mori, a model insect of Lepidopteran species. The BmNa(v) gene covers a 31 kb genome region and contains 36 exons. The longest ORF contained 6258 bp and encoded 2085 amino acid residues, which shares 74%, and 77% overall amino acid sequence identities with the sodium channel proteins from Drosophila melanogaster and Blattella germanica, respectively. Using high-throughput Solexa sequence technology we conducted sequence analysis of BmNa(v) cDNAs from embryos, larvae, pupae and adults of the silkworm, identified alternative splicing sites and determined the frequencies of these splicing events in four developmental stages. Three optional exons, two sets of mutually exclusive exons, and one internal spliced exon were identified. One optional exon is unique to BmNa(v), while the others are conserved in other insect sodium channel genes. Interestingly, the expression of the mutually exclusive exons is developmentally regulated.     

Isolation of Three Main Sericin Components from the Cocoon of the Silkworm,Bombyx mori

To characterize the sericin components of the cocoon of silkworm Bombyx mori, fresh cocoon shells were dissolved in saturated aqueous lithium thiocyanate containing 2-mercaptoethanol, and fractionated by ethanol precipitation. Cocoon sericin was found to mainly consist of three polypeptides having molecular masses of the 400, 250, and 150 kDa estimated by SDS-PAGE, which corresponds to the sericin present in the middle, anterior, and posterior part of the middle silk gland. The amino acid compositions of the 400 and 150 kDa components were similar to each other, but that of the 250 kDa component was different. This suggests differences in the coding gene and properties of the 250 kDa sericin from the other two.