缺铁玉米根cDNA文库的构建及蛋白和cDNA差异比较

采用异硫氰酸胍酚氯仿法分离铁胁迫玉米根总ＲＮＡ,然后采用寡聚ｄＴ 纤维素层析柱法纯化Ｐｏｌｙ（Ａ） ＋ ＲＮＡ．用含有ＸｈｏＩ位点的ｌｉｎｋｅｒｐｒｉｍｅｒ 锚定合成ｃＤＮＡ 第一条链后,采用替代法合成第二条链,接着连接上ＥｃｏＲＩＡｄａｐｔｅｒ 以便定向重组到载体上．ｃＤＮＡ 经过ＸｈｏＩ消化和分级分离后,带有ＸｈｏＩ和ＥｃｏＲＩ粘性末端的ｃＤＮＡ 定向重组到λＺＡＰ表达载体的ＸｈｏＩ和ＥｃｏＲＩ位点上．经体外包装,重组的噬菌体转导宿主菌ＸＬ１Ｂｌｕｅ ＭＲＦ,最终获得滴度为４－５ ×１０５ ｐｆｕ／ｕｇ 的λＺＡＰ 表达ｃＤＮＡ 文库．通过差异筛选法和质膜双向电泳验证了缺铁和加铁条件下ｃＤＮＡ 和质膜蛋白的差异     

鲫血清转铁蛋白cDNA克隆及其序列分析

从GenBank数据库查询发表的鱼类转铁蛋白cDNA或基因序列，根据铁离子结合转运功能位点，设计并合成了两对引物P1、P4以及P2、P3，克隆出鲫血清转铁蛋白cDNA中的核心片段，长度为866bp。再根据克隆出的核心片段分别设计上游及下游两对引物P5、P6以及P7、P8，随后用RACE方法分别克隆出鲫血清转铁蛋白cDNA的5’端(787bp)和3’端(1081bp)以及全长cDNA，最后用计算机程序排列出鲫血清转铁蛋白全长cDNA，长度为2444bp。比较了12种鱼类血清转铁蛋白cDNA序列的同源性。     

cDNA cloning and expression of earthworm fibrinolytic enzyme component A

Earthworm fibrinolytic enzyme component A(EFEa),a protein with dual fibrinolytic activity ,is one of the major therapeutically important earthworm fibrinoltic enzyme components .The cDNA fragment encoded the mature protein was cloned from earthworm (Eisenia fetida )by the RT-PCR technique,The deduced amino acid sequence of the EFE component A show high homology with some members of serine proteases trypsin family,and the amino acid residues constituting the active sites are conserved in the EFEa as compared with the other proteins of the trypsin family ,The cDNA fragment was subcloned into the expression vector pQE31 and pMAL-c2X of E.coli.The resulting expression plasmids,pQE-efea and pMAL-efea ,were used to transform the E.coli strain M15.Recombinant protein bands corresponding with calcuated molecular witht were induced .The induced His6-EFEa fusion protein with pQE-efea was accumulated into inclusion body ,while the induced MBP-EFEa fusion protein with pMAL-efea was soluble and showed fibrinoloytic activities.     

Construction and characterization of a normalized whole-life-cycle cDNA library of rice

A cDNA library with genomic complete coverage is a powerful tool for functional genomic studies.For studying the functions of rice genes on a large scale,a normalized whole-life-cycle cDNA library is constructed based on the strategy of saturation hybridization with genomic DNA using rice cultivar Minghui 63,an elite restorer line for a number of rice hybrids that are widely cultivated in China,This library consists of cDNA from 15 directionally cloned cDNA libraries constructed with different tissues from 9 developmental stages.For normalization,the denatured plasmids purified from the 15 directionally cloned libraries are mixed and hybridized with saturated genomic DNA labeled with magnetic beads in two complementary systems. Well-matched plasmids are captured from the hybridized genomic DNA and electroporated into competent DH10B E. coli for construction of the normalized whole-life-cycle cDNA library.This library consists of 62000 clones with an average insert length about 1.4kb.Inverse Northern blotting shows that this cDNA library included many rarely expressed genes and tissue-specific genes.Sequencing of 10750 cDNA clones of this library reveals 6399 unique EST s(expressed sequence tags),indicating that the non-redundancy of the library is about 59.5%.This library has been used to make cDNA microarrays for functional genomic studies.     

生物素标记的PSTV互补DNA探针分子杂交的研究

用~(32)P标记的互补DNA探针进行分子杂交是基因重组和分子克隆中常用技术,然而由于~(32)P的半衰期短,从而为供应和保存带来不便,致使这一技术的应用受到一定限制。近年来国内外均十分重视研究应用非同位素标记DNA探针的技术。我们用Biotin—11—dUTP(Bethesda Research Laboratories)以缺口平移(Nick translation)标记了pST-B14和pST-B5 DNA中的PSTV cDNA插入顺序,制备出生物素标记的DNA     

人肿瘤坏死因子cDNA衍生物在大肠杆菌中的表达及活性测定

应用基因工程方法构建了含人肿瘤坏死因子ＣＤＮＡ衍生物的重组体．转化大肠杆菌后酶切鉴定．转化菌经摇瓶培养及温度调控，获得高效表达．ＳＤＳ－ＰＡＧＥ结果表明，表达的ＴＮＦ分子量约为１７ｋＤ，蛋白量约为菌体可溶性蛋白的２０％．用Ｌ９２９细胞毒性测定法测得菌液ＴＮＦ的表达为２×１０８ｕ／ｍＬ．抗ＴＮＦ的单克隆抗体可以中和大肠杆菌表达的ＴＮＦ对Ｌ９２９细胞的细胞毒性．     

用cDNA芯片技术筛选中华绒鳌蟹卵巢发育相关基因

为了克隆中华绒螯蟹卵巢发育相关基因,我们构建了中华绒螯蟹卵巢差减cDNA文库.本研究用cDNA芯片技术对中华绒螯蟹卵巢差减cDNA文库进行了初步筛选,并从cDNA芯片筛选的正向差减文库中随机选取50个克隆进行序列测定.结果共获得2倍以上差异表达克隆167个,其中Ⅲ期高表达克隆104个,Ⅱ期高表达克隆63个.正向差减文库中共获得7个独立的EST(expressed sequence tag, EST).同源性分析结果表明,这些EST皆为新的EST, dbEST登录号分别为:CA591892、CA591893、CA591894、CA591895、CA591896、CA591897和CA591898.本研究结果为进一步克隆中华绒螯蟹卵巢发育相关基因的全长cDNA序列并研究其功能,阐明中华绒螯蟹卵巢发育的分子机理奠定了重要的前期工作基础.     

玉米PDE191基因全长cDNA的克隆及其生物信息学分析

为研究玉米白化相关基因PDE191序列信息和基因功能,以玉米叶片总RNA逆转录后的c DNA为模板,采用RT-PCR和RACE技术克隆出玉米PDE191基因全长c DNA。DNA测序及生物信息学分析表明,玉米PDE191基因c DNA全长1229 bp,编码333个氨基酸的蛋白质,亚细胞定位于线粒体和叶绿体。软件预测PDE191蛋白分子量为37.79 k D,理论等电点为9.12,该蛋白二级结构主要构成元件为?螺旋和无规则卷曲,包含7个MTERF结构域。三级结构预测显示该蛋白为球状结构,预测结果与二级结构预测的结果相符。对该蛋白进行多重序列比对和聚类分析显示,PDE191属于植物MTERF家族,在不同植物中存在其同源蛋白质,其中与拟南芥PDE191氨基酸序列的相似性高达99%。本研究为进一步研究玉米PDE191基因的功能奠定了基础。     

毕赤酵母△~9-脂肪酸脱氢酶基因的克隆和表达

根据真菌Δ9-脂肪酸脱氢酶保守的氨基酸序列设计简并引物进行PCR,获得一个741 bp的毕赤酵母cDNA片段,再根据获得的部分序列设计基因特异性引物,通过cDNA末端扩增技术(RACE)获得该cDNA的3′和5′序列,从而得到全长为1 689 bp的cDNA序列.序列分析结果表明,该序列具有一个长度为1 194 b p、编码397个氨基酸的开放阅读框(PPD9).与报道的Δ9-脂肪酸脱氢酶一样,推测的氨基酸序列具有膜整合脂肪酸脱氢酶特异性的3个组氨酸保守区和疏水结构,在其氨基酸序列的C-末端具有类似于细胞色素b5的血红素结合区.该序列为一个新的编码Δ9-脂肪酸脱氢酶的基因,为了验证其功能,把开放阅读框序列PPD9亚克隆到表达载体pYES 2.0,构建重组表达载体pYPPD9,并转化到酿酒酵母的Δ9-脂肪酸脱氢酶缺陷型菌株DTY-11A中进行表达。通过平板互补实验结果表明,该序列在酿酒酵母中获得表达。所编码的蛋白具有Δ9-脂肪酸脱氢酶活性,能弥补DTY-11A中由于Δ9-脂肪酸脱氢酶的缺失而引起的致死性突变。