抗黄矮病小麦易位系cDNA文库的构建

对小麦-中间偃麦草易位系HW642一周龄幼苗接种饲黄矮病毒蚜虫,接种48 h后取幼叶提取mRNA构建了一个cDNA文库,文库的滴度为1.5×105pru/mL.随机挑取103个重组克隆,用限制内切酶EcoR I、Xho I消化,统计分析结果表明插入片段在150～4000 bp之间,平均插入片段大小为2000 bp左右,重组率为95.15 ％.文库可用于筛选抗病候选基因及EST研究等.     

黄孢平革霉（Phanerochaete chrysosporium）木质素过氧化物酶基因的克隆与分析

用大肠杆菌克隆载体ｐＵＣ１０构建白腐丝状担子真菌黄孢平革霉（Ｐｈａｎｅｒｏｃｈａｅｔｅｃｈｒｙｓｏｓｐｏｒｉｕｍ）ＭＥ４４６基因组文库，用分离自ＢＫＭ－Ｆ１７６７菌株的木质素过氧化物酶ｃＤＮＡ片段ＣＬＧ４和ＣＬＧ５为探针，从构建的基因组文库中分离到一个含木质素过氧化物酶基因的重组子ｐＧＬＧ－Ｍ１。对该重组子插入片段所作的限制酶谱分析结果表明，它与来自ＢＫＭ－Ｆ１７６７的ＧＬＧ２片段的限制酶谱十分相似。     

日本七鳃鳗口腔腺分泌蛋白BGSP-2的序列进化分析、基因克隆及诱导表达

从实验构建的七鳃鳗口腔腺cDNA文库中获取BGSP-2(buccal gland secretion protein)基因片段,并对其进行生物信息学分析.RT-PCR扩增,已获得日本七鳃鳗口腔腺BGSP-2蛋白的全序列cDNA,将目的基因与pET23b载体连接,进一步转化至Rosetta表达菌株中,筛选阳性克隆,对包涵体进行变性、复性与纯化.结果显示已成功获得成分均一的活性蛋白.     

Genome research profile of two <Emphasis Type="Italic">Cordyceps sinensis</Emphasis> cDNA libraries

Cordyceps sinensis (CS) is well known as an ancient Chinese herb. It is used to expand bronchial smooth muscles, inhibit tumor growth and decrease blood pressure. Cordyceps sinensis is composed of two parts. One is the dead larvae body; the other is the stroma like withered grass. Up to now, few genome database articles about Cordyceps sinensis have been reported. In this study, two cDNA libraries were constructed using the worm part and grass part respectively for the first time. 12192 and 15456 clones fro...     

人18周胎儿脑cDNA文库的构建及鉴定

许多脑基因的特异性表达对人脑的发育，分化有着重要的作用，为了研究这些基因的结构和功能，构建了一个人１８周胎儿脑ＣＤＮＡ文库，抽提总ｍＲＮＡ后，经过一系列的酶促反应合成ｃＤＮＡ，分有分离柱除去小片段后克隆到λｇｔ１０载体中，转染宿主菌Ｃ６００ｊｆｌ后文库包装效率为４．６×１０＾６ｐｆｕ／μｇ，ｃＤＮＡ平均郫在于１．２ｋｂｐ已知序列设计引物，从ｃＤＮＡ文库中扩增出神经生长因子（ＮＧＦ）的生长编码基因     

蝎α-毒素的分子进化:加速突变与功能趋异

蝎α-毒素为一类空间结构高度相似而药理学功能趋异的蛋白质家族.cDNA序列分析表明蝎α-毒素5′非翻译区和信号肽编码区序列的保守性显著高于成熟毒素编码区,3′非翻译区在药理学组内高度保守.而且,成熟毒素编码区密码子第1,2和3位核苷酸突变率相似.非翻译区内每个位点的核苷酸替代数(K)小于成熟毒素编码区内每个同义位点的核苷酸替代数(Ks),而且成熟毒素编码区内每个非同义位点的核苷酸替代数(Ka)接近或大于Ks值.上述数据表明,蝎α-毒素成熟肽编码区内的突变为加速进化方式.这一结论得到进化树结果的支持.此外,研究还表明稳定3D结构的15个保守性残基靠近分子内部,这可能作为蝎α-毒素结构性限制因素在进化过程中维持CSαβ结构的恒定;4个热点突变区分布于分子表面的潜在功能区,表明正性Darwin选择驱动了蝎α-毒素的加速进化.研究结果合理地解释了蝎α-毒素保守性3D结构与趋异的药理学功能之间的关系.     

Molecular cloning of a full-length cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1-25 amino acid sequence is a predicted signal peptide and the other 26-216 amino acid sequence is a mature peptide. The 26-45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca2+- dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.     

Cloning and sequence analysis of a gene encoding polygalacturonase-inhibiting protein from cotton

Polygalacturonase-inhibiting proteins (PGIP) play important roles in plant defense of pathogen, especially fungi. A pair of degenerated primers is designed based on the conserved sequence of 20 other known pgip genes and used to amplify Gossypium barbadense cultivation 7124 cDNA library by touch-down PCR. A 561 bp internal fragment of the pgip gene is obtained and used to design the primers for rapid amplification of cDNA ends. A composite pgip gene sequence is constructed from the products of 5′ and 3′ RACE, which are 666 bp and 906 bp respectively. Analysis of nucleic acid sequence shows 69.2% and 68.7% similarity to Citrus and Poncirus pgip genes, respectively. Its open reading frame of the gene encodes a polypeptide of 330 amino acids, in which 10 leucine-rich repeats arrange tandemly. A new set of primers is designed to the 5′ and 3′ ends of the gene, which allows amplification of the full-length gene from the cotton cDNA library. Genomic DNA analysis reveals that this gene has no intron.     

石斑鱼卵巢cDNA文库构建及脂肪酸结合蛋白克隆

应用SMARPTM技术构建了斜带石斑鱼Epinephelus coioides卵巢cDNA文库。所构建的cDNA文库滴度为4.5×106 pfu/mL,重组率为99％,扩增后文库的滴度为1.2×1010pfu/mL。把phagemid转化为质粒后,随机挑选60个阳性克隆进行酶切鉴定和测序。酶切结果表明:插入片段长度均在500~3 000 bp之间。测序结果经过生物信息学分析,发现其中一个克隆为脂肪酸结合蛋白(FABP)基因全长,并与人肝型(L)脂肪酸结合蛋白同源性最高,为70％。