水稻乙醛脱氢酶基因的克隆及其在不育系中的表达

从水稻中分离了乙醛脱氢酶基因（Ｏｓａｌｄｈ）的全长ｃＤＮＡ,序列分析显示Ｏｓａｌｄｈ ｃＤＮＡ含有一个完整的编码５４９个氨基酸的开放阅读框,其编码蛋白ＯＳＡＬＤＨ的Ｎ端为预期的线粒体前导肽,中部具有醛类脱氢酶基因家族的酶催化活性中心,ＯＳＡＬＤＨ与玉米、烟草、人类的线粒体乙醛脱氢酶同源性分别高达８７％、７７％、５９％。Ｎｏｒｈｅｒｎ分析显示,幼根中Ｏｓａｌｄｈ的表达水平高于幼苗,幼穗,不育品种幼重     

甘薯叶片超氧化物歧化酶基因克隆及测序

以甘薯（Ｉｐｏｍｏｅａｂａｔａｔａｓ（Ｌ．）Ｐｏｉｒ）叶为材料提取植物总ＲＮＡ,经反转录后,利用多聚酶链式反应技术,扩增并克隆超氧化物歧化酶基因的ｃＤＮＡ,并进行测序分析。该序列全长４８２ｂｐ,其读码框编码１５２个氨基酸,与国外文献报道的甘薯块根ＳＯＤ基因的ｃＤＮＡ序列相比,具有９９％的同源性。     

番茄花叶病毒外壳蛋白基因及3′端非编码区的克隆、序列分析及其表达

根据已报道的番茄花叶病毒Ｌ株系（ＴｏＭＶＬ）序列人工合成引物,经ＲＴＰＣＲ扩增并克隆了我国番茄花叶病毒分离物（ＴｏＭＶＳ１）的外壳蛋白ＣＰ基因及３′端非编码区。序列测定结果表明,所得ｃＤＮＡ共长６８２个核苷酸,其中ＣＰ基因含４８０个核苷酸,编码１５８个氨基酸,３′端非编码区含２０２个核苷酸,其核苷酸序列与ＴｏＭＶＬ株系具有９９．５％的同源率。将该基因片段克隆到ｐＧＥＭＥＸ１载体中,转入Ｅ．ｃｏｌｉ后诱导表达,经Ｗｅｓｔｅｒｎｂｌｏｔ检测证明,该基因已在大肠杆菌中正确表达。这是我国首次报道ＴｏＭＶＣＰ基因序列。     

导入小麦加倍单倍体植株的玉米DNA在后代中的遗传及序列同源性分析

作者曾报道了一个玉米（Ｚｅａ ｍａｙｓ Ｌ．）基因组特异的重复序列ＤＮＡ（克隆ＭＲ６４）通过小麦（Ｔｒｉｔｉｃｕｍ ｐｅｒｓｉｃｕｍ Ｖａｖ．ｅｘ Ｚｈｕｋ．）与玉米杂交导入一个小麦加倍单倍体（ＤＨ）植株中。利用分析主宰该重复ＤＮＡ序列可以稳定地传递到小麦ＤＨ３代植株。通过Ｉｎｔｅｒｍｅｔ在ＤＮＡ数据库中进行序列相似性搜寻和同源性比较,结果显示,ＭＲ６４的ＤＮＡ序列和玉米的最近报道的两个逆转座子Ｐ     

水稻矮缩病毒最外层外壳蛋白基因（S2）cDNA克隆,序列分析及其在大？…

从水稻矮缩病毒（Ｒｉｃｅｄｗａｒｆｖｉｒｕｓ,ＲＤＶ）中国福建分离物中克隆分离了最外层外壳蛋白基因（Ｓ２）全长ｃＤＮＡ并对其进行序列分析,结果表明ＲＤＶＳ２ｃＤＮＡ全长３５１２ｂｐ,仅含一个３３４８ｂｐ的阅读框架,编码一人含有１１１６个氨基酸的蛋白（Ｐ２）。与基因库中已知基因序列比较,发现它与日本ＲＤＶＨ株系相应片段的核苷酸和氨基酸同源率分别为９４．６％和９５．４％与轮状病毒ＶＰ２氨基酸序列有一定     

拟南芥LFYcDNA的克隆及转化菊花的研究

以野生型拟南芥（Ａｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）为材料,克隆并测序了Ｌｅａｆｙ（ＬＦＹ）基因的全长ｃＤＮＡ;同时构建了ＬＦＹｃＤＮＡ以ＣａＭＶ３５Ｓ为启动子的植物表达载体,并转化菊花（Ｃｈｒｙｓａｎｔｈｅｍｕｍｍｏｒｉｆｏｌｉｕｍ（Ｒａｍａｔ．）Ｔｚｖｅｌ．）。该ｃＤＮＡ全长１２６３ｂｐ,共编码４２０个氨基酸残基,Ｓｏｕｔｈｅｒｎ杂交结果表明,ＬＦＹｃＤＮＡ整合到菊花染色体组中。跟正常植株相比,转基因植株中有３株分别提早６５、６７、７０ｄ开花,２株分别推迟７８、９０ｄ开花。     

稻胚凝集素基因的克隆,序列分析及表达

以水稻基因组ＤＮＡ为模板,以特异引物经聚合酶链式反应方法扩增出稻胚凝集素基因并克隆到Ｅ．ｃｏｌｉ质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ（＋）的ＳｍａⅠ位点。序列分析表明,克隆到的基因片段大小为７８１ｂｐ,没有内含子,编码１条长２２７个氨基酸、分子量约２３ｋＤ的肽链,其中Ｎ－端２８个氨基酸是信号肽。与报道的稻胚凝集素ｃＤＮＡ序列进行顺序同源性比较,发现它们之间有很高的同源性（９９．７４％）,其编码区第１６７     

蝶兰胚珠发育基因的表达及序列分析

对从蝶兰（Ｐｈａｌａｅｎｏｐｓｉｓｓｐ．）成熟胚珠的ｃＤＮＡ文库中获得的ｃＤＮＡ克隆Ｏ１３８的时空表达特性和序列进行了分析。Ｎｏｒｔｈｅｒｎ分子杂交结果显示出该基因的ｍＲＮＡ在成熟胚珠和根中大量积累,但在大孢子母细胞时期的胚珠和子房组织中积累水平很低。ＤＮＡ序列分析表明,该基因含一个编码２８４个氨基酸,分子量为３０ｋＤ蛋白的开放阅读框架。Ｏ１３８是一个与成熟胚珠发育相关的基因。     

人胎肝唾液酸转移酶基因的克隆及测序

根据已克隆的唾液酸转移酶的保守区的序列,以人胎肝ｍＲＮＡ为模板扩增出１５０ｂｐ的片段并测序。其中一个片段（ｓ３８）与已克隆的唾液酸转移酶的活性中心有５７％～９７％的同源性。根据ｓ３８的序列合成寡核苷酸并标记后用作探针筛选人胎肝ｃＤＮＡ文库。从文库中分离了一个编码α２,３唾液酸转移酶的ｃＤＮＡ。该ｃＤＮＡ序列含一个编码３４０个氨基酸的开放读框,推导的氨基酸序列与人颌下腺Ｇａｌβ１,３ＧａｌＮＡｃα２,３唾液酸转移酶相同,与猪颌下腺α２,３唾液酸转移酶有８３２％的同源性。表明从人胎肝ｃＤＮＡ文库中分离的ｃＤＮＡ所编码的蛋白为Ｇａｌβ１,３ＧａｌＮＡｃα２,３唾液酸转移酶     

豌豆钙调蛋白基因的克隆及七种来源钙调蛋白基因的分析

从豌豆（Ｐｉｓｕｍ ｓａｔｉｖｕｍ Ｌ．）中提取总ＲＮＡ, 逆转录出ｃＤＮＡ 第一条链后, 以大麦钙调蛋白结构基因两端的寡聚核苷酸为引物, 用ＰＣＲ方法合成豌豆钙调蛋白基因, 克隆到Ｂｌｕｅ－ｓｃｒｉｐｔ 载体上并测定其全序列。结果与已知的苜蓿（Ｍｅｄｉｃａｇｏ ｓａｔｉｖａ Ｌ．）、水稻（Ｏｒｙｚａ ｓａｔｉｖａＬ．）、大麦（Ｈｏｒｄｅｕｍ ｖｕｌｇａｒｅ Ｌ．）、电鳗（Ｅｌｅｃｔｒｏｐｈｏｒｉｄａｅ）、根瘤（Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｄｕｌａｎｓ）、酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ） 钙调蛋白基因相比,同源性较高（９１．３％ —６０．８％ ）,其不同部分则常常是Ｃ和Ｔ相替换。进一步分析发现,上述七种来源的钙调蛋白基因之间,常有某些核苷酸替换方式占优势,它们对于氨基酸密码子的使用也各有偏爱     

Isolation and Characterization of a cDNA Encoding Maize Cyto solic Malate Dehydrogenase

A cDNA fragment derived from a gene over-expressing in hybrid maize ( Zea mays L. ) was isolated with RT-PCR and used as probe to screen cDNA library of hybrid maize seedlings. A positive cDNA clone ZH02 corresponding to the full-length mRNA sequence was obtained, which was shown to have an open reading frame encoding 332 a.a. DNA and proteinase database search revealed that the deduced amino acid sequence of ZH02 has high similarity with the cMDH of Mesembryaathemum crystallium L. and Arabidopsis thaliana (L.) Heynh. up to 90% and 84%, respectively. This is the first report of the full-length gene coding for the cereal cMDH.     

甘蔗细胞质型苹果酸脱氢酶基因的克隆及其表达分析

对甘蔗(Saccharum officinarum L.)叶片全长cDNA文库进行测序,获得了1个细胞质型苹果酸脱氢酶(cMDH)基因的全长cDNA序列,命名为Sc-cMDH。生物信息学分析表明,该基因全长1314 bp,开放阅读框为999 bp,编码332个氨基酸。Sc-cMDH与其他植物cMDH的氨基酸序列同源性高达86.5%～97.0%。Sc-cMDH包含典型的NAD+结合基元T11GAAGQI17和催化基元I184WGNH188,还有相当保守的6个半胱氨酸残基,因此推断该基因为细胞质型NAD-MDH。定量PCR分析结果表明,该基因在甘蔗叶片和根中的表达量高于茎。     

Prokaryotic Expression and Bioinformatics Analysis of Cytosolic Malate Dehydrogenase from Camellia sinensis(Theaceae)

The complete gene of cytosolic malate dehydrogenase (cMDH) from Camellia sinensis, called Cs cMDH, was obtained by RT PCR and rapid amplification of cDNA ends (GenBank accession number GQ845406). This gene was 1 235 bp in length, encoding a protein of 332 amino acids with the putative molecular weight of 355 kD. The Ecoli Rosetta (DE3) harboring pGEX MDH was induced by 05 mmol·L 1 IPTG at 32℃ for 3 hours, and a 615 kD glutathione Stransferase (GST) fused MDH was obtained in soluble form. The results of NCBI BLAST revealed that Cs cMDH shared 88%-93% of amino acid sequence identity with other cMDH from different higher plants. According to the multiple sequence alignment based on the three dimensional structure of protein, Cs cMDH was predicted to be a dimer with thirteen β sheet and thirteen α helix of each subunit. Cs cMDH contains typical fingerprint sequence (G12AAGQIG18) as all MDHs. The amino acid D43 in Cs cMDH is conserved in all NAD MDHs. Cs cMDH also has some conserved sequence units homologous to other NAD MDHs, such as NAD+ binding sites, catalytic motif and substrate binding sites. Moreover, Cs cMDH contains six Cys which are highly conserved in all plant NAD cMDHs. Therefore, Cs cMDH was inferred to be NAD dependent cMDH. The present study may provide the fundament for the further functional characterization of Cs cMDH.     

Cloning and sequence analysis of cDNAs encoding mammalian cytosolic malate dehydrogenase. Comparison of the amino acid sequences of mammalian and bacterial malate dehydrogenase

A cDNA clone, named ppcMDH-1 and covering a part of the coding region for the porcine cytosolic malate dehydrogenase (cMDH) mRNA, was isolated from a porcine liver cDNA library. Subsequently, mouse cMDH cDNA clones were isolated from mouse liver and heart cDNA libraries, using the ppcMDH-1 cDNA as a probe. The longest clone, named pmcMDH-5, was sequenced and the primary structure of the mouse cMDH deduced from its cDNA sequence showed that the mouse cMDH consists of the 334-amino acid residues. When the amino acid sequence of the mouse cMDH was compared with that of the porcine cMDH, they shared a 93% homology. On the other hand, the amino acid sequences of mouse cMDH and mitochondrial MDH (mMDH) showed about 23% overall homology. Surprisingly, comparison of the amino acid sequences among the mammalian and bacterial MDHs revealed that the homology between the mouse cMDH and thermophilic bacterial MDH, as well as the homology between the mouse mMDH and Escherichia coli MDH, markedly exceeds the intraspecies sequence homology between mMDH and cMDH from mice.     

玉米花粉特异的非编码RNA基因ZM401 cDNA全长的克隆及其发育表达模式

通过差异筛选法并结合冷噬菌斑筛选,从玉米(Zea mays L.)成熟花粉cDNA文库中克隆到一个玉米花粉特异表达的cDNA片段ZM401(663bp).Northern杂交表明ZM401是一个玉米花粉特异表达的基因.本文采用5'RACE,3'RACE及重叠PCR技术获得了ZM401 cDNA的全长(1 149 bp).采用生物学软件对ZM401 cDNA的序列和结构进行分析,结果表明,该基因缺乏明显的开放阅读框架,序列中最长的开放阅读框架仅有89个氨基酸,但具有poly(A)尾部结构,符合非编码RNA基因的特点.推断ZM401基因是一个非编码基因.RT-PCR及Northern blot分析表明ZM401基因从玉米花粉小孢子四分体时期、单核期、双核期、成熟花粉开始表达,而且表达量依次增强,证明ZM401可能与玉米花粉的晚期发育过程相关.同时,Northern杂交显示ZM401基因在玉米花粉发育中有两种转录本存在.     

Clonorchis sinensis: molecular cloning and functional expression of novel cytosolic malate dehydrogenase

The NAD-dependent cytosolic malate dehydrogenase (cMDH, EC 1.1.1.37) plays a pivotal role in the malate-aspartate shuttle pathway that operates in a metabolic coordination between cytosol and mitochondria, and thus is crucial for the survival and pathogenicity of the parasite. In the high throughput sequencing of the cDNA library constructed from the adult stage of Clonorchis sinensis, a cDNA clone containing 1152bp insert was identified to encode a putative peptide of 329 amino acids possessing more than 50% amino acid sequence identities with the cMDHs from other organisms such as fish, plant, and mammal. But low sequence similarities have been found between this cMDH and mitochondrial malate dehydrogenase as well as glyoxysomal malate dehydrogenase from other organisms. Northern blot analysis showed the size of the C. sinensis cMDH mRNA was 1.2 kb. The cMDH was expressed in Escherichia coli M15 as a His-tag fusion protein and purified by BD TALON metal affinity column. The recombinant cMDH showed high MDH activity of 241 U mg(-1), without lactate dehydrogenase and NADP(H) selectivity. It provides a model for the structure, function analysis, and drug screening on cMDH.