白纹伊蚊细胞色素P450 CYP6家族基因多样性的研究(英文)

根据已获得的白纹伊蚊CYP6家族某成员cDNA序列片段AEDR ,设计基因特异性引物 ,以白纹伊蚊总RNA为模板 ,进行cDNA末端快速扩增 ,扩增产物经T -A克隆、测序。结果显示 :通过 5’ RACE获得 1个非全长cDNA序列 (GZS331 ) ,其与CYP6N1、CYP6N2的同源性分别为 59 8%和 59 1 % ,与CYP6N3v1 -v3同源性最高 ,达 83 9% - 84 3% ;通过 3’ RACE获得 6个非全长cDNA序列 ,其中来自抗性株的GZG0 33序列与CYP6N3v1 -v3的同源性达 98 2 % - 99 1 % ,而其余 3’ RACE克隆与CYP6N3v1 -v3的同源性则达 84 3% - 85 6%。上述所有非全长cDNA序列均与哺乳动物CYP3A1以及夜蛾CYP9A1有较高的同源性 ,分别为 2 3% - 36 1 %和 2 7 6% - 34 1 %。用PC/GENE软件所绘制的系统树显示出与同源性分析相一致的结果。所得非全长cDNA序列上报国际P450命名委员会进行统一的命名 ,并对蚊虫中细胞色素P450基因多样性及其形成原因进行了分析     

MOLECULAR CLONING AND SEQUENCE ANALYSIS OF THREE NEW FULL‐LENGTH cDNAs OF CYTOCHROME P450 (CYP6N3v1‐v3)*FROM AEDES ALBOPICTUS**

Abstract The three new full‐length cDNA sequences including the complete 5′‐and 3′‐ untranslated regions (UTR) coding for cytochrome P450s from Aedes albopictus have been obtained. The P450 proteins deduced from the nucleotide sequences shared 58.6% ‐ 62.4% amino acid identity with CYP6N1 and CYP6N2 from Anopheles gambiae, and 99% with each other. The three new complete sequences have been submitted and named as CYP6N3v1, CYP6N3v2 and CYP6N3v3 by the P450 Nomenclature Committee. The original cDNAs were obtained by rapid amplification of cDNA ends (RACE) approach with several pairs of gene specific primers based on the cDNA fragment previously obtained from deltamethrin‐resistant strain of Ae. albopictus. Further analysis showed that the three new sequences are present in both resistant strain and susceptible strain and might be effectively translated. In addition, the 5′‐ and 3′‐UTRs were compared between the CYP6N3vl‐v3 and other known insect P450s. The multiplicity of trans‐lational control of insect P450 genes was discussed.     

小型猪CYP3A88基因克隆与其他动物的序列比较

目的克隆我国资源小型猪品系巴马香猪肝脏中的CYP3A88基因,并进行生物信息学分析。方法应用RACE（Rapid Amplification of cDNA Ends）技术对其全长进行扩增,测序,利用Internet和GenBank数据库对其序列进行生物信息学分析。结果首次克隆并鉴定了我国资源小型猪品系巴马香猪肝脏中CYP3A88（GenBank登录号：EF625347）的编码区,获得大小为1965bp的全长cDNA,编码区长为1512bp,编码503个氨基酸;比较核苷酸序列,与小型猪CYP3A39相似性高达94%,而与人等其它动物的CYP3A相似性则在86%以下;推导和分析氨基酸序列表明,与小型猪CYP3A其它成员（CYP3A39、CYP3A29、CYP3A22）进行对比,其相似性分别为92%,89%,80%,而将小型猪与人的CYP3A分别比对,小型猪CYP3A88与人CYP3A4相似性最高,为77%;对其二级结构预测,它可能含12个α螺旋,4个β折叠;经NCBI上的CDD程序分析可知,其39～491氨基酸区域为P4503A亚家族保守结构区域;经聚类分析,小型猪和狗的CYP3A与人有较近的进化关系;通过同源建模法对其在线建模,人CYP3A4晶体结构作为其模建模型,得到了其经典的三维结构。结论在猪CYP3A家族四个基因中,CYP3A88在序列和高级结构上均与人CYP3A4的最为相似。     

Cloning, sequencing, heterologous expression, and characterization of murine cytochrome P450 3a25*(Cyp3a25), a testosterone 6beta-hydroxylase

A full-length cDNA clone encoding a novel form of the cytochrome P450 3A subfamily (Cyp3a-25) has been isolated from a mouse liver cDNA library. The sequence contained 2010 base pairs and encoded a protein with 503 amino acids. The amino acid sequence shared greater identities with rat CYP3A18 (90%) and golden hamster CYP3A10 (81%) sequences than with known mouse sequences (Cyp3a-11, Cyp3a-13, Cyp3a-16, and Cyp3a-41 [68--70%]). CYP3A25 was expressed in the Escherichia coli PCWori(+) expression vector following slight modifications of the N- and C-terminals of the cDNA. The purified CYP3A25 was recognized on an immunoblot by CYP3A1 antibody and has a molecular weight of 50 kD. CYP3A25 was catalytically active in the 6 beta-hydroxylation of testosterone and the N-demethylation of benzphetamine and erythromycin. It was demonstrated by RT-PCR that the CYP3A25 mRNA is present in both fetal and adult tissues, including liver, lung, intestines, kidney, and brain. Northern blotting demonstrated that expression is greatest in the liver and small intestine.     

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%。     

吸血诱导的埃及伊蚊海口株后期胰蛋白酶基因测序及与美国株同源性比较

应用逆转录-聚合酶链式反应（RT-PCR）技术从吸血后24 h埃及伊蚊海口株总RNA中扩增出了后期胰蛋白酶编码区cDNA序列。采用自动DNA分析仪进行序列分析,并与已知埃及伊蚊美国株后期胰蛋白酶基因及推导的氨基酸序列进行了同源性比较。结果表明：埃及伊蚊海口株后期胰蛋白酶基因序列与美国株同源性达98%,有11个碱基发生变异;氨基酸同源性达99%,仅有3个氨基酸发生变异,但与催化位点密切相关的氨基酸及N末端氨基酸序列完全一致。以上结果显示,埃及伊蚊胰蛋白酶不同地理株间存在微小的差异。     

Cloning,expression in yeast,and functional characterization of CYP71D14, a root-specific cytochrome P450 from <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Alkaloids, which are naturally occurring amines, are biosynthesized and accumulated in plant tissues such as roots, leaves, and stems. Many alkaloids have pharmacological effects on humans and animals. Cytochrome P450 (P450 or CYP) monooxygenases are known to play key roles in the biosynthesis of alkaloids in higher plants. A cDNA clone encoding a P450 protein consisting of 502 amino acids was isolated from Petunia hybrida. The deduced amino acid sequence of the cDNA clone showed a high level of similarity with the other P450 species in the CYP71D family; hence, this novel P450 was named CYP71D14. Among plant P450 species, CYP71D14 had 45.7% similarity in its amino acid sequence to CYP71D12, which is involved in the biosynthesis of the indole alkaloids vinblastine and vincristine. Expression of CYP71D14 mRNA in Petunia plants was examined by Northern blot analysis by using a full-length cDNA of CYP71D14 as a probe. CYP71D14 mRNA was expressed most abundantly in the roots. The nucleotide sequence of CYP71D14 has been submitted to the DDBJ, EMBL, and GenBank nucleotide databases under the accession number AB028462. An erratum to this article can be found at     

Cloning, Sequencing, and Phylogenetic Analysis of Complementary DNA of Novel Cytochrome P-450 CYP1A in Japanese Eel (Anguilla japonica)

Complementary DNA of cytochrome P-450 CYP1A, in addition to CYP1A1, has been isolated from Japanese eel (Anguilla japonica) liver treated with 3-methylcholanthrene. The cDNA contained a 5′ untranslated region of 66 bp, an open reading frame of 1554 bp coding for 517 amino acids and a stop codon, and a 3′ untranslated region of 1166 bp. The predicted molecular weight of the Japanese eel CYP1A was approximately 58.5 kDa. The nucleotide sequence exhibited identities with the reported CYP1A1 sequences of 77% for Japanese eel, 75% for rainbow trout, 72% for scup, plaice, and butterfly fish, and 71% for toadfish. The deduced amino acid sequence exhibited identities with the reported CYP1A1 sequences of 78% for Japanese eel, 77% for rainbow trout, 75% for scup, 74% for toadfish, 73% for plaice, and 72% for butterfly fish. The novel eel CYP1A obtained had less similarity to the other teleost CYP1A1 proteins (72%–78%) than that of the eel CYP1A1 (74%–80%). When compared with mammalian CYP proteins, the novel eel CYP1A was more similar to the CYP1A1 proteins (54%–56%) than to the CYP1A2 proteins (50%–53%). The phylogenetic tree of the teleost CYP1A genes constructed using the maximum likelihood method suggested that the novel eel CYP1A is ubiquitous among the Anguilliformes. Received August 25, 2000; accepted November 30, 2000     

绿盲蝽P450基因的克隆及增效醚对P450酶活性的抑制作用

为探讨P450介导的绿盲蝽Apolygus lucorum(Meyer-Dür)抗药性机制,合理使用杀虫药剂,本研究通过活体和离体抑制实验发现,增效醚(PBO)对绿盲蝽P450酶活性有显著的抑制作用:在处理时长为24h时,P450酶活性由未处理时的12.02pmol/min/mgPro.下降至1.63pmol/min/mgPro.,PBO对P450酶的抑制中浓度为0.256mmol/L。生物测定结果表明,PBO对三氟氯氰菊酯具有显著增效作用,增效7.2倍,而对吡虫啉、灭多威、马拉硫磷无显著增效作用。利用RT-PCR及RACE技术对绿盲蝽P450基因进行克隆,获得了2条CYP4家族基因,全长均为1631bp,含有完整的开放阅读框,编码501个氨基酸;序列比对表明这是一对等位基因,含有CYP4家族所有保守特征序列;同源性比较及系统发育分析显示这2个基因编码的氨基酸序列与褐飞虱Nilaparvata lugens CYP4CE1亲缘关系最近,同源性分别为41.5%和41.1%。     

Cloning and Sequencing of Cytochrome P450 1A Complementary DNA in Eel (Anguilla japonica)

Cytochrome P450 1A (CYP1A) complementary DNA was isolated from eel (Anguilla japonica) liver treated with 3-methylcholanthrene. The cDNA contained a 5′ untranslated region of 163 bp, an open reading flame of 1560 bp coding for 519 amino acids and a stop codon, and a 3′ untranslated region of 1730 bp. The predicted molecular weight was approximately 58.4 kDa. The deduced amino acid sequence exhibited identities with reported CYP1A sequences of 80% for rainbow trout, 79% for scup, 76% for plaice and butterfly fish, and 74% for toadfish. When compared with mammalian CYP proteins, the eel CYP1A was more similar to CYP1A1 (54%–56%) than to CYP1A2 (49%–52%). Northern and Southern blot analyses showed two distinct bands, suggesting the existence of another 3-methylcholanthrene-inducible CYP1A gene in eel. Received December 19, 1998; accepted February 18, 1999     

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.