暗纹东方鲀CD8α全长cDNA的克隆及序列分析

根据红鳍东方鲀(Takifugu rubripes)的CD8α序列设计引物,用RACE技术克隆并测定了暗纹东方鲀(Takifugu fasciatus)CD8α全长cDNA序列,经序列比对两者同源性为97%.序列分析显示657 nt的开放读码框编码218个氨基酸残基的跨膜糖蛋白的前体蛋白,蛋白结构预测包括信号肽区、免疫球蛋白样结构域区、茎区、跨膜区和胞浆区.对其中胞外的免疫球蛋白样结构域区和茎区进行了原核表达,表达产物为22 ku左右的重组蛋白.     

烟草叶片全长cDNA文库构建及EST序列分析

【目的】构建烟草叶片全长cDNA文库并测序,发掘与烟叶发育、代谢和抗逆相关的基因,为进一步研究功能基因奠定基础。【方法】以烟草苗期叶片为试验材料,利用改进的Cap-trapper法构建烟草叶片全长cDNA文库。利用测序技术获得大量的EST序列,采用生物信息分析手段,对所测EST进行拼接和功能注释。【结果】构建了烟草叶片的全长cDNA文库,该文库滴度为1.2×106 pfu•mL-1,平均插入片段在1.4 kb左右。利用该文库测序了5 280个克隆,获得5 233条高质量表达序列标签（EST）,序列拼接出3 922个单一基因;同源比对分析表明,89.7%的基因与已知功能基因具有较高的同源性,这些基因涉及细胞生长、信号转导、翻译合成、抗逆反应和能量代谢等功能;并详细鉴定了部分与烟碱合成和抗病相关的基因。【结论】通过Cap-trapper法成功构建了高质量的烟草幼苗叶片全长cDNA文库;大规模EST测序分析挖掘到大量与烟草幼苗叶片发育、抗逆、抗病、烟碱代谢相关的侯选基因。     

黄瓜幼果cDNA文库构建与EST测序分析

将黄瓜授粉前后多个发育阶段的幼果组织等量混合后提取总RNA和mRNA,以λTriplEx2为栽体、XL1-Blue为宿主茵,构建了1个黄瓜幼果cDNA文库;其滴度为1.165×106pfu/mL,重组率在94.4％左右.测序获得116条EST,92.2％的长度在400 bp以上,19％为重叠序列.在GenBank中进行BLAST分析后确认与已知功能基因相似的EST序列有71条,有相似序列而功能未知的基因和没有相似序列的EST序列各占19.83％和18.97％.从对文库的检验结果看,构建的cDNA文库重组率较高,库容达到预期要求.     

反向遗传技术在传染性法氏囊病病毒研究中的应用

传染性法氏囊病是鸡的最严重疾病之一,其病原是鸡传染性法氏囊病病毒(IBDV),IBDV基因组为两片段双链RNA,变异株和超强毒株的出现,给传统的疫苗免疫带来了新的挑战,因此,加强对IBDV的基础研究是控制该病的关键.RNA的结构不稳定成为阻碍RNA病毒研究的主要障碍,近年来兴起的反向遗传技术将RNA病毒的基因组转化为cDNA,从而使RNA病毒的基因操作成为可能,也使RNA病毒的研究获得了快速的发展.文章就传染性法氏囊病病毒反向遗传操作研究的意义、方法、概况及相关分子生物学进行了全面综述.     

葡萄果实cDNA文库的构建及鉴定

构建葡萄果实cDNA文库是研究果实发育相关基因表达调控的基础工作.本实验从巨峰葡萄果实中提取了高质量的RNA,利用MMLV反转录酶把总RNA中的mRNA反转录成cDNA第一链,用特异引物进行LD-PCR扩增合成双链cDNA.将分级纯化的带有粘性末端双链cDNA与λTrip Ⅰ Ex2载体连接,重组载体体外包埋成为cDNA原始文库.初步鉴定原始文库的滴度为1.32×106 pfu/mL,文库扩增后滴度达1.45×1010 pfu/mL.从扩增文库中随机挑取250个克隆进行PCR鉴定,结果表明重组率为98.7%,插入片段大小在0.5～2.0 kb之间,因此,本研究成功构建了葡萄果实cDNA文库.     

柑橘裂皮类病毒广东分离物YC全长cDNA序列克隆及分析

柑橘裂皮类病毒(Citrus exocortis viroid, CEVd)是危害柑橘的重要病原之一,认清该类病毒的分子特性是进一步研究其致病机制的前提.本文利用RT-PCR技术对广东分离物YC的全长cDNA序列进行了扩增、克隆,获得了全长372bp的片段,对该片段进行测序及分析显示:CEVd广东分离物YC的分子结构域包括5个功能区即左手末端区(TL)、致病区(P)、中央保守区(C)、可变区(V)和右手末端区(TR).通过与GenBank已登陆序列的比对分析发现:与来自西班牙的分离物CEVd-f-1(登陆号:EF494677.1)的同源性高达98%;与湖北分离物CEVd-HB(登陆号:AY456136)的同源性仅为92.7%;与强度株系CEVd-A(登陆号:M30868)的同源性为96.8%,与弱毒株系CEVd-de26(登陆号:K00965)的92.4%.说明该广东分离物YC分子特性更接近强毒株系.这是国内首次对柑橘裂皮类病毒cDNA的分子特性进行报道.     

Molecular Cloning and Characterization of a Novel Gene Involved in Fatty Acid Synthesis in Brassica napus L.

Based on the sequence of a novel expressed sequence tag (EST), the full-length cDNA of 1 017 nucleotides was cloned from Brassica napus cv. Xiangyou 15 through rapid amplification of cDNA ends (RACE). The gene was designated as Bnhol34 (HQ585980), encoding a protein of 338 amino acids. BLAST analysis showed no high degree of sequence identity to any known gene. The calculated molecular weight of the Bnhol34 protein was 36.23 kDa, and the theoretical isoelectric point was 8.74. The Bnhol34 was also cloned from a high oleic acid mutant 854-1 through homologous cloning. There was no difference between the two Bnhol34 genes. Bnhol34 was localized in a tissue-specific manner in B. napus, and its expression level was about eight-fold greater in Xiangyou 15 seeds than in 854-1. The promoter region sequences of Bnhol34 were then isolated from Xiangyou 15 and 854-1, and a 93-bp deletion was found to occur in the Bnhol34 promoter region of 854-1. Three abscisic acid-responsive cis-elements (ABRE) were identified in the promoter region of Xiangyou 15. Real-time PCR analyses revealed that exogenous abscisic acid increased Bnhol34 expression by about four-fold in Xiangyou 15 seeds, yet did not change Bnhol34 expression in 854-1. It appeared that Bnhol34 might be abscisic acid insensitive in 854-1.     

应用PCR技术检测兽医生物制品中污染病毒的研究

本文阐述了PCR(基因扩增 )技术的原理、基本操作方法 ,及其在兽医生物制品疫苗污染病毒检测中的应用。着重介绍了应用PCR技术检测新城疫病毒和瘟病毒的方法 ,具有快速、敏感、特异性高等优点 ,对兽医生物制品的研究、生产和临床应用具有一定的指导意义     

Expression of RANTES mRNA in skin lesions of feline eosinophilic plaque

Abstract One of the mechanisms of eosinophil infiltration is its induction by chemoattractants such as regulated upon activation, normal T-expressed and secreted (RANTES) which is a cysteine–cysteine chemokine that mediates chemotaxis and activation of eosinophils in humans and mice. Skin lesions of feline eosinophilic plaque are characterized by a predominant infiltration of eosinophils. The mechanism(s) of eosinophilic infiltration in the skin and/or mucosa of cats is unknown. It is possible that RANTES is involved. To investigate the presence of RANTES in the skin of cats with eosinophilic plaques and nonaffected skin, we cloned and sequenced the full-length feline RANTES cDNA gene, in order to determine whether it is present in the skin of cats with eosinophilic plaques and/or if it is present in normal adjacent skin. We were able to document the the expression of RANTES mRNAs in skin with feline eosinophilic plaque as well as in normal cat skin. The full-length cDNA sequence of the RANTES gene (742 bp) contained a single open reading frame of 276 bp encoding a protein of 92 amino acids. The amino acid sequence of feline RANTES shared 67 and 74% sequence identity with that of bovine and mouse RANTES genes, respectively. RT–PCR analysis on RANTES mRNA in the skin of cats with eosinophilic plaque revealed that its expression was higher in the eosinophilic plaque skin lesions than in the normal skin. The result suggested that RANTES might play a role to induce eosinophil infiltration in feline eosinophilic plaque lesions.     

应用cDNA微阵列芯片技术筛选猪蛔虫性别差异表达基因的研究

采用cDNA微阵列芯片技术，从所构建的猪蛔虫雌、雄成虫cDNA消减文库分别挑取1044和1119个克隆，PCR扩增其插入片段，经纯化后点样于预先处理好的基片上（双点杂交），制备成cDNA微阵列芯片。将分别标记荧光素Cy3-dUTP和Cy5-dUTP的雌虫和雄虫cDNA探针，与制备好的cDNA芯片杂交（平行进行反标杂交试验）。根据每个点杂交后的Ratio值，筛选出双点杂交和正反标中都同时具有表达差异的基因克隆共1559个。将表达差异最明显的前831个克隆进行测序，获得720个有效序列，经生物信息学分析发现，雄虫特异表达的主要精于蛋白和雌虫特异表达的卵巢信息蛋白的基因序列多数与新杆属线虫存在同源性，有31个可能是新的ESTs。性别差异表达基因及其相关生物信息的获得为下一步研究基因功能奠定了基础。