实验用西藏小型猪CYP3A基因的克隆及序列分析

目的:克隆西藏小型猪CYP3A基因并与人及其它小型猪品种进行序列比对分析。方法:根据Genebank数据库设计引物,取西藏小型猪新鲜肝脏组织,Trizol法提取肝脏总RNA,应用RT-PCR反转获得肝脏cDNA。分别扩增基因CYP3A46、CYP3A22、CYP3A29及CYP3A39,PCR片段回收后分别连接pMD-18T载体,获得重组质粒pT-CYP3A,阳性重组克隆送测序。采用NCBI Blast及vector NTI软件进行序列比对分析。结果:CYP3A46、CYP3A22、CYP3A29及CYP3A39基因编码序列全长1512 bp,编码503个氨基酸,与人CYP3A4相同。其中CYP3A22和CYP3A39与NCBI记录巴马小型猪序列相同;CYP3A46与巴马小型猪CYP3A46(NM_001134824.1)有6个位点碱基不同。CYP3A29与巴马小型猪CYP3A46(EU918131.1)亦有6个位点碱基不同。对来自2个母本后代猪的基因经过PCR扩增后测序发现CYP3A46、CYP3A29序列完全一致。结论:成功克隆西藏小型猪CYP3A的4个基因,其中CYP3A46与人CYP3A4的序列相似性最高。所得CYP3A46与CYP3A29序列可能为西藏小型猪独有。     

杨树木质素合成酶c3h基因的克隆及其序列分析

根据CYP98A3(GenBank登录号为AY064170)cDNA序列保守区设计引物,从正在分化的2年生‘欧美杨107’次生木质部提取的总RNA经RT-PCR扩增出一基因片段,然后与pMD20-T载体连接。重组质粒经限制性内切酶酶切、特异引物PCR扩增和测序鉴定。结果表明,扩增片段长度为994bp,其中包含一个长为495bp的开放阅读框,编码的氨基酸序列(登录号为CAP47423)与NCBI中AY064170的CYP98A3编码氨基酸序列的相似性为91%,初步断定其为CYP98A3家族中的一员,其cNDA序列GenBank登录号为AM920690。参照国内外已发表的部分植物的c3h基因序列,构建了c3h的遗传进化树,分析了不同植物c3h的遗传进化关系。     

白桦肌动蛋白(Actin)基因全长cDNA克隆与序列分析

以白桦(Betula platyphylla Suk.)次生木质部为材料,用改良CTAB方法提取总RNA。根据植物肌动蛋白(Actin)基因编码区的保守序列设计引物后进行RT-PCR,并采用RACE技术扩增出Actin基因全长序列。该基因cDNA全长1 785 bp,序列分析表明,该基因编码区1 134 bp,编码377个氨基酸,5′非编码区157 bp,3′非编码区495 bp。所得序列与GenBank中注册的其它植物肌动蛋白核苷酸序列的相似性均在80%以上,氨基酸序列的相似性高达96%以上。此基因已在GenBank注册（EU588981）。根据高等植物肌动蛋白相似性构建了进化树,表明白桦肌动蛋白与蓖麻肌动蛋白之间的亲缘关系最为密切,在进化中分化时间最为接近。     

甘蓝型油菜BnCYP79B1基因的克隆与表达

硫苷是十字花科植物的一种次生代谢产物,其合成途径受细胞色素P450的CYP79家族蛋白的调控,该实验采用同源克隆技术在甘蓝型油菜中克隆到了CYP79B1基因,命名为BnCYP79B1(GenBank登录号为JX535391.1)。BnCYP79B1基因cDNA全长1 625bp,编码一个含有541个氨基酸、理论等电点为8.88。序列对比结果显示,BnCYP79B1与花椰菜CYP79B1在DNA序列上的相似性为98.83%,推测蛋白氨基酸序列的相似性为99.26%。通过不同时期不同部位BnCYP79B1基因表达量的分析,发现BnCYP79B1基因在高秆高硫苷品系的根中表达量较高,而对矮秆高硫苷品系则是叶中表达量较高。在BnCYP79B1表达总量上,高秆品系较矮秆品系高,高硫苷品系较低硫苷品系高。     

实时荧光定量检测中国实验用小型猪肝脏CYP3A29 mRNA表达水平

CYP3A29是猪肝脏最重要的药物代谢关键酶。研究中国实验用小型猪肝脏CYP3A29 mRNA的表达特性对于评估其是否适宜于作为人CYP3A4介导的药理学研究动物模型具有一定意义。以b-actin作校正, 利用TaqMan定量技术对巴马香猪、贵州小型香猪肝脏CYP3A29 mRNA表达水平进行检测, 并以荣昌猪作为对照。结果表明, 巴马香猪、贵州小型香猪、荣昌猪肝脏CYP3A29 mRNA表达水平与报道的人肝脏CYP3A4相近; 三品系(种)猪间肝脏CYP3A29 mRNA表达水平较为接近, 但品系(种)内个体间变异较大。提示巴马香猪、贵州小型香猪作为药物评价的实验动物具有一定可行性。     

唐鱼细胞色素P450 1A基因cDNA克隆及分析

鱼类细胞色素P450 1A(CYP1A)基因作为一种有效的生物标志物已被广泛应用于环境污染物的评价,唐鱼在水环境污染监测中有较好的应用优势,为建立唐鱼在水环境的检测的有效生物标志物方法,本研究对其CYP1A基因进行克隆。利用RT-PCR法扩增出一条651bp的唐鱼CYP1A基因cDNA片段,该片段与其他物种的CYP1A基因具有很高的相似性。在此基础上进行5'端扩增和3'RACE获得了唐鱼CYP1A基因的全长cDNA序列,其长度为2177bp,其中编码区长1566bp,编码521个氨基酸残基组成的蛋白质,推测该蛋白质分子量大小为58.5kDa,理论等电点为5.65。所得序列具有CYP1A分子的主要特征和功能域,包括亚铁血红素结合区、酶功能所需的精氨酸密码子和终止密码子。推导出的氨基酸序列与斑马鱼、底鳉、虹鳟、大西洋鳕、人、鼠等脊椎动物同源性分别为87.7%、70.1%、76.2%、62.2%、54.5%、55.7%。     

猪CFL2b 基因cDNA克隆初步分析

利用基因表达谱芯片分析法筛选出与大白猪高肌肉产量-肌纤维形成有关的CFL2b基因.参考人和小鼠CFL2b基因序列,采用SMART-RACE技术结合EST序列拼接技术,从猪骨骼肌肌肉中首次克隆了猪CFL2b的全长cDNA序列（GenBank登录号EU561660,EU561661）,Northern杂交检测CFL2b基因 mRNA.结果表明,猪CFL2b基因含有2个转录本,长转录本3　012 bp,短转录本1　466 bp .CFL2b基因在多种真核生物中都有表达,且编码区序列非常保守,开放式读码框501 bp编码了166个氨基酸的蛋白质.氨基酸序列分析表明,猪CFL2基因与人和小鼠氨基酸同源性分别为100%和99.1%.核苷酸序列相似性分别为88.1%和74.9%.     

NRDRiso酶cDNA的序列测定及生物信息学分析

通过鉴定分析人肝组织中辅酶II依赖性视黄醇脱氢酶不同剪接体全长cDNA核苷酸序列与氨基酸序列的结构特征,为今后进一步研究体内维甲酸的代谢情况奠定基础。根据人、小鼠NRDR编码区的一致性序列,设计一对引物,应用RTPCR方法从人肝组织中得到一条377bp的新的cDNA片段。采用RACE法得到了NRDR新亚型cDNA,并以生物信息学软件分析其生物学特征。 测序得知该cDNA长为1003bp,以NADP-dependent retinol dehydrogenase/reductase short isoform（NRDRiso）登录GenBank。其读码框为525bp,拟编码174个氨基酸的蛋白。     

柿果实多聚半乳糖醛酸酶基因克隆与序列分析

以'富平尖柿'(Diospyros kaki L.cv.Fuping Jianshi)为材料,采用RACE方法,首次获得了柿果实多聚半乳糖醛酶(PG)基因的3个全长cDNA(登录号为EU816197、EU816198、EU816199),分别命名为DKPG1、DKPG2、DKPG3.DKPG1全长1 616 bp,DKPG2全长1 654 bp,DKPG3全长1 545 bp.3个基因均含有一个1 326 bp的开放阅读框,共编码441个氨基酸.通过Blast比对发现该基因核苷酸序列与其他植物已报道的PG基因具有74%～78%的相似性;其氨基酸序列与其他植物的相似性为60%～73%.对GenBank同源性搜索获得的其他植物PG基因氨基酸序列进行系统进化分析,发现其与葡萄、猕猴桃、桃的亲缘关系近,与大豆亲缘关系较远.     

Molecular cloning and sequence analysis of guinea-pig NADPH-cytochrome P-450 oxidoreductase [corrected]

A cDNA clone coding for cytochrome P-450 oxidoreductase was isolated from a guinea-pig liver cDNA library. The cDNA, MSr2, contained a complete coding region of 678 amino acids. The amino acid sequence of the guinea-pig cytochrome P-450 oxidoreductase showed approx. 90% identities with those of rat, human, rabbit, pig enzymes indicating conservation of primary structure of the enzyme during evolutionary divergence of species. The high conservation of acidic residues of the enzyme sustained the importance of them to maintain its function [corrected].     

乌金猪FTO基因的克隆和表达分析

目的:克隆乌金猪脂肪和肥胖相关基因(FTO)编码区序列(CDS),分析其序列组成特征及在乌金猪不同组织中的表达情况。方法:采用反转录PCR方法从乌金猪脂肪组织中克隆FTO基因CDS,利用生物信息学方法分析其序列组成特征;采用实时PCR方法分析乌金猪FTO基因mRNA在不同组织中的表达情况。结果:克隆的FTO基因全长1518 bp(已提交至GenBank数据库,登录号为JQ031263),编码由505个氨基酸残基构成的蛋白,推测的相对分子质量为58.16×103,等电点为5.18;乌金猪与牛、羊、人和大鼠的FTO蛋白的氨基酸序列同源性分别为91%、90%、89%和83%;进化树分析显示,推测的乌金猪FTO蛋白的氨基酸组成与牛、羊的亲缘性较近,其次是人、大鼠;推测的氨基酸组成中无跨膜区,无信号肽,为亲水蛋白;分析发现该蛋白有22个磷酸化修饰位点,包括10个丝氨酸蛋白激酶磷酸化位点、7个苏氨酸蛋白激酶磷酸化位点、5个酪氨酸蛋白激酶磷酸化位点,200、246、302位氨基酸残基有糖基化位点;二级结构预测发现该蛋白共有201个螺旋、33个伸展链和271个卷曲结构;实时PCR检测的组织表达谱表明,FTO基因mRNA在乌金猪肝脏组织中的表达量最高,在脂肪、肾、脾中也有大量表达,在心脏、肌肉中的表达量最少。结论:为深入探讨乌金猪FTO基因的生物学功能奠定了重要基础。     

Cloning of CYP4F7, a kidney-specific P450 in the sea bass Dicentrarchus labrax

A cDNA sequence coding for a cytochrome P450 of the CYP4F subfamily was isolated from total RNA of sea bass kidney by rapid amplification of cDNA ends. The full length sequence coded for a protein of 526 amino acids. The amino acid sequence shared 39% to 56% residue identities with the mammalian CYP4F sequences, and thus was named CYP4F7 (accession number AF123541). RNA blot analysis using CYP4F7 cDNA as a probe indicated that the corresponding mRNA was only detected in kidney. Expression in the kidney was constitutive, and no induction of this mRNA was detected in this or other tissues, with any of the inducers tested, including peroxisome proliferators.     

Cloning and tissue distribution of a novel human cytochrome p450 of the CYP3A subfamily, CYP3A43

On the basis of the detection of an expressed sequence tag ('EST') similar to the human cytochrome P450 3A4 cDNA, we have identified a novel member of the human cytochrome P450 3A subfamily. The coding region is 1512-bp long and shares 84, 83, and 82% sequence identity on the cDNA level with CYP3A4, 3A5, and 3A7, respectively, with a corresponding amino acid identity of 76, 76, and 71%. Quantitative real time based mRNA analysis revealed CYP3A43 expression levels at about 0.1% of CYP3A4 and 2% of CYP3A5 in the liver, with significant expression in 70% of the livers examined. Gene specific PCR of cDNA from extrahepatic tissues showed, with the exception of the testis, only low levels of CYP3A43 expression. The CYP3A43 cDNA was heterologously expressed in yeast, COS-1 cells, mouse hepatic H2.35 cells and in human embryonic kidney (HEK) 293 cells, but in contrast to CYP3A4 which was formed in all cell types, no detectable CYP3A43 protein was produced. This indicates a nonfunctional protein or specific conditions required for proper folding. It is concluded that CYP3A43 mRNA is expressed mainly in liver and testis and that the protein would not contribute significantly to human drug metabolism.     

A novel human cytochrome P450 4F isoform (CYP4F11): cDNA cloning, expression, and genomic structural characterization

By a combination of cDNA library screening and rapid amplification of cDNA ends analysis, a novel human cytochrome P450 4F isoform has been cloned and sequenced. The new 4F isoform is designated CYP4F11 and contains 1765 nucleotides. The coding region encodes 524 amino acid residues, and the heme-binding region is highly conserved. The CYP4F11 amino acid sequence has 80.0, 82.3, and 79.2% identity to CYP4F2, CYP4F3, and CYP4F8 amino acid sequences, respectively. In vitro translation shows the molecular mass of CYP4F11 is approximately 57 kDa, consistent with the calculated molecular mass. CYP4F11 is expressed mainly in human liver, followed by kidney, heart, and skeletal muscle. The genomic structure of CYP4F11 was solved by database searching and computer analysis. The coding region of CYP4F11 has 12 exons. The CYP4F11 gene is located 16 kb upstream of the CYP4F2 gene on chromosome 19. This is consistent with the notion that the human cytochrome P450 4F genes form a cluster on chromosome 19.     

Rat cytosolic aspartate aminotransferase: molecular cloning of cDNA and expression in Escherichia coli

cDNA clones for rat cytosolic aspartate aminotransferase (cAspAT, L-aspartate:2-oxoglutarate aminotransferase) [EC 2.6.1.1] were isolated from a rat cDNA library, and the primary structure of the gene for cAspAT was deduced from its cDNA sequence. Rat cAspAT consists of 412 amino acids and its molecular weight is 46,295. The deduced amino acid sequence of rat cAspAT was compared with the sequences of AspATs from other species. The degree of sequence identities of rat/mouse cAspAT, rat/pig cAspAT, rat/chicken cAspAT, rat/pig mAspAT, and rat/Escherichia coli AspAT were 97.1, 89.6, 81.7, 48.1, and 41.2%, respectively. A coding region of rat cAspAT cDNA was inserted into E. coli expression vector pUC9, and enzymatically active cAspAT was expressed as a beta-galactosidase-cAspAT hybrid protein. This hybrid protein represented about 18% of the soluble proteins in E. coli and its kinetic properties were comparable with those of cAspAT preparations purified from rat liver.     

绵羊fertilin β基因编码区的钓取与结构分析

Fertilin β与精卵的结合和融合有密切关系。为探讨fertilin β蛋白在绵羊受精过程中的作用机理, 采用RACE技术, 首次钓取了该基因的编码区。结果绵羊fertilin β基因的编码区cDNA全长为2,217 bp。同源性分析显示, 绵羊的fertilin β氨基酸序列与牛、猪和人的fertilin β具有79.4%、66.7%和58.1%的同源性。系统发育分析表明, 绵羊fertilin β与牛属于同一分支, 并且也显示了绵羊和牛分类地位最近, 这和传统的分类一致。Fertilin β蛋白结构域分析显示, 绵羊fertilin β去整合素识别序列为TDE, 与牛的序列相同。除了上述三肽序列外, 紧随X-D/E-E的ECD保守序列, 从而形成了X-D/E-ECD五肽保守序列, 在绵羊fertilin β中该五肽序列为TDECE。     

亚心型扁藻叶绿体psbA基因的克隆及序列分析

psbA基因是叶绿体基因组中一个重要的光调控基因,编码光和系统Ⅱ反应中心的D1蛋白。根据叶绿体基因组序列高度保守的特性,利用菜茵衣藻（Chlamydomonasreinhardtii）psbA基因的保守序列（基因登录号：HQ667991．1）设计引物,采用PCR步移的方法从亚心型扁藻（Platymonassubcordiformis）基因组DNA中克隆到psbA基因全长（基因登录号：KF528742）。序列分析表明,亚心型扁藻psbA基因全长1939bp,编码区长度为1062bp,推导编码353个氨基酸,包括4个赖氨酸残基。有效密码子数显示脚删基因具有明显的密码子偏好性,并且偏好使用以A／T结尾的密码子。相对同义密码子使用度表明25个密码子在编码使用时具有偏好性,其中20个密码子以A／T碱基结尾,占到80％。其终止密码子使用了TAG。