西伯利亚蓼rd22基因的克隆与序列分析

以3% NaHCO₃溶液胁迫处理48 h的西伯利亚蓼为试材,利用RACE技术,从其茎部组织克隆了脱水应答蛋白RD22的全长cDNA序列。测序后的结果分析表明,该cDNA序列全长为1 302 bp,5′非翻译区为59 bp,3′非翻译区为25 bp,开放读码框为1 218 bp,编码405个氨基酸。在氨基酸序列的C端含有一个比较保守的BURP结构域,N端含有5个重复序列THV-VGKGGV-V。信号肽检测证明该蛋白为分泌性蛋白,前21个氨基酸区域为信号肽结构。其推演的氨基酸序列与葡萄的同源性最高,达到60%。该基因已在GenBank上注册,基因序列登录号为DQ836050。     

黄瓜花叶病毒山东株(CMV\|SD)RNA2全长cDNA克隆的构建及全序列分析

分别利用5’RACE和3’RACE确定了CMV\|SD RNA2的5’和 3’末端序列,在此基础上,利用RTPCR得到了RNA2的5’端一半的cDNA克隆pC25和3’端一 半的cDNA克隆pC23,并通过拼接构建了RNA2全长cDNA克隆pC2F。通过对pC25和pC23进行序列 测定,得到了RNA2的全序列。序列分析结果表明CMVSD RNA2由3048nt组成,其中存在2个 部分重叠的阅读框ORF1(79～2652nt)和ORF2(2414～2746nt),分别编码858aa的2a蛋白和111 aa的2b蛋白,并在2a蛋白的序列中发现了动植物病毒复制酶所特有的两个保守序列。该株系 RNA2核苷酸序列与分属CMV I亚组的Fny株系和II亚组的Q株系RNA2的核苷酸序列同源性分别 为917%和756%;2a蛋白的氨基酸序列同源性分别为938%和677%,2b蛋白的氨基酸序 列同源性分别为830%和513%。同源性比较的结果表明SD株系属于CMV I亚组。     

西伯利亚蓼钙调蛋白基因的克隆及其在盐胁迫下的表达

已从西伯利亚蓼叶中cDNA文库中获得的钙调蛋白EST序列,采用cDNA末端快速扩增（RACE）技术克隆了具有完整编码区的钙调蛋白基因的cDNA序列（GenBank登录号GQ988382）,命名为PsCaM。该基因全长615bp,编码区为450bp,编码149个氨基酸,5＇非翻译区为63bp,3＇非翻译区为102bp。同源性分析表明,该蛋白与其他植物钙调蛋白高度保守,氨基酸同源性高达98%。用实时荧光定量PCR研究3%NaHCO3胁迫下西伯利亚蓼基因表达的结果显示,自然条件下,该基因在叶中表达量最高,地下茎次之,茎中最低;盐胁迫下CaM在西伯利亚蓼的地下茎、茎和叶中均有表达,表达模式不同。     

鸡含锰超氧化物歧化酶cDNA克隆及序列分析

 为弄清鸡含锰超氧化物歧化酶 (manganese containingsuperoxidedismutase ,MnSOD)的cDNA序列 ,以开展动物锰营养学的深入研究 ,根据已知鸡MnSOD的N端氨基酸序列设计简并引物 ,应用 3′RACE(rapidamplificationofcDNAends)技术 ,扩增克隆了鸡心肌MnSOD 990bp的 3′cDNA片段 .再根据 3′RACE片段测序结果设计引物进行 5′RACE ,结果获取了一个与 3′RACE片段相互重叠的鸡心肌MnSOD 52 1bp的 5′RACE片段 ,并对其进行了克隆测序 .最后根据 3′RACE片段和 5′RACE片段序列信息进行拼接 ,从而获取鸡MnSODcDNA的全序列信息 .研究结果表明 :鸡MnSODcDNA全长为 110 8个核苷酸 ,其中 5′非翻译区 2 5个核苷酸 ,编码区 675个核苷酸 ,3′非翻译区 4 0 8个核苷酸 ,编码一个长 2 2 4个氨基酸残基的蛋白质前体 .其中信号肽长 2 6个氨基酸残基 ,成熟肽长 198个氨基酸残基 ,分子量为 2 2kD .与人、大鼠、线虫、果蝇等真核生物MnSOD氨基酸序列的同源性分别为82 4 %、84 .7%、62 .4 %、59.3% .     

能源植物续随子延伸因子EF1A基因cDNA序列的克隆及分析

利用同源克隆和cDNA末端快速扩增技术(RACE)克隆得到能源植物续随子延伸因子EFIA基因的cDNA序列(命名为ElEF1A,GenBank登录号为KT892703)。序列分析表明,ElEF1A编码序列(CDS)长1344bp,编码447个氨基酸,氨基酸序列具有典型的EF1-alpha、EF1-alpha-Ⅱ和EF1-alpha-Ⅲ结构域。ElEF1A与其他植物EF1A基因的核苷酸序列同源性达到88%以上,推导的氨基酸序列的同源性达95%以上。     

光皮桦中4-香豆酸辅酶A连接酶基因Bl4CL的克隆和表达分析

以从光皮桦茎叶组织提取的mRNA为模板,根据其他已克隆到的阔叶类树种中4-香豆酸辅酶A连接酶(4CL)基因的同源序列设计兼并引物,进行RT-PCR扩增,获得部分基因片段,然后结合5’,3’RACE方法从光皮桦中扩增出1个4CL基因的全长cDNA序列,命名为Bl4CL。该基因cDNA全长为1983bp(GenBank登录号FJ410448),具有完整的开放阅读框架(69～1697bp),编码蛋白为542个氨基酸,包含一个AMP结合功能域和一个含有12个氨基酸的功能基序。与其他植物中的4CL进行同源性比对的结果显示,Bl4CL蛋白与东北白桦的同源性最高,达到了98%。该基因在光皮桦的根和茎中表达量较高,而在花和叶中的表达量低。     

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。     

甘蓝型油菜羧基转移酶α亚基全长cDNA的克隆及在大肠杆菌中表达

通过分析拟南芥与大豆的羧基转移酶α亚基的cDNA序列,运用RT-PCR、RACE和基因组步行(Genome walking)三种技术进行组合克隆,首次从油菜开花后20～29天的幼胚中,克隆到质体定位的羧基转移酶α亚基的全长cDNA.同源性分析表明,其开放阅读框(ORF)编码的氨基酸序列与拟南芥、大豆的羧基转移酶α亚基的同源性分别为85%、59%.将此cDNA去除叶绿体转运肽编码序列的成熟肽编码序列,克隆到表达载体pHBM625上,蛋白质印迹分析,它在大肠杆菌中成功表达.     

绞股蓝法呢基焦磷酸合酶基因的克隆及其序列分析

目的:克隆并分析绞股蓝法呢基焦磷酸合酶(FPS)基因的全长序列。方法:参照罗汉果法呢基焦磷酸合酶基因,设计扩增绞股蓝FPS基因的3′RACE引物,采用3'RACE和5'RACE法克隆绞股蓝FPS基因全长cDNA。结果:获得绞股蓝FPS基因全长cDNA序列共1288个核苷酸,包含一个1026核苷酸的开放读框,编码342个氨基酸残基,推断该蛋白的相对分子质量为3.94×104。NCBI Blast结果显示绞股蓝FPS基因编码蛋白的氨基酸序列与已知的植物FPS氨基酸序列的同源性为91%~74%,核酸序列的同源性为88%~78%。结论:克隆了绞股蓝FPS基因全长cDNA序列,为进一步研究绞股蓝FPS基因的表达及三萜皂苷合成通路关键酶分子的进化奠定了基础。     

STUDY ON THE DIVERSITY OF CYTOCHROME P450 GENES FROM AEDES ALBOPICTUS*

Abstract Several pairs of specific primers according to the obtained cDNA sequence fragment from deltamethrin‐resistant Aedes albopktus were designed to amplify new CYP6 genes from total RNA of Aedes albopictus by rapid amplification of cDNA ends (RACE) technique. The products of RACE were cloned and selected for sequencing. The deduced amino acid sequences were subjected to homologous analysis. The results indicated that the identities of clone GZS331 sequence from 5′‐RACE products and clone GZG033 sequence from 3′‐RACE products to CYP6N3vl ‐ v3 are 83.9% ‐ 84.3% and 98.2% ‐ 99.1% respectively; while the identities of the others from 3′‐RACE products to CYP6N3v1 ‐ v3 are 84.3% ‐ 85.6%. All of these obtained cDNA sequences have a higher homology to CYP3A1 in mouse and CYP9A1 in moth. The dendrogram constructed by PC/GENE software showed similar results to homologous analysis. These obtained sequences were submitted and named by the P450 Nomenclature Committee. The diversity of cytochrome P450 genes in Culicidae species was discussed.     

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.     

Alternative splicing generates multiple transcripts of the inhibitor of apoptosis protein 1 in Aedes and Culex spp. mosquitoes

We determined the sequences of cDNA encoding Inhibitor of Apoptosis Protein 1 (IAP1) homologues from Aedes triseriatus, Aedes albopictus, Aedes aegypti, Culex pipiens and Culex tarsalis. The cDNAs encode translation products that share > or = 84% sequence similarity. The IAP1 mRNA of each mosquito species exists as 3-5 distinct variants due to the presence of heterogeneous sequences at the distal end of their 5'UTRs. Partial genomic sequencing upstream of the 5' end of the Ae. triseriatus IAP1 gene, and analysis of the Ae. aegypti genomic sequence, suggest that these mRNA variants are generated by alternative splicing. Each IAP1 mRNA variant from Ae. triseriatus and Cx. pipiens was detected by RT-PCR in all mosquito life-stages and adult tissues examined, and the relative concentration of each Ae. triseriatus IAP mRNA variant in various tissues was determined.     

cDNA cloning and functional expression in yeast Saccharomyces cerevisiae of beta-naphthoflavone-induced rabbit liver P-450 LM4 and LM6

A cDNA library was constructed from liver mRNA of a beta-naphthoflavone-induced rabbit. Two clones pLM4-1 and pLM6-1 containing 2.2-kbp inserts that hybridized at low stringincy with a mouse P1 P-450 probe were selected. The clone pLM4-1 was fully sequenced and found to contain a full-length cDNA coding for cytochrome P-450 LM4. Partial sequence and restriction mapping made it possible to identify pLM6-1 as coding for the major part of cytochrome P-450 LM6. Cloned LM4-1 cDNA was reformed by deletion of the 5' and 3' non-coding regions before insertion into yeast expression vectors PYe DP1/10. A similar operation was performed on pLM6-1 cDNA after replacement of the missing N-terminus-coding sequences by homologous sequences form the pLM4-1 clone resulting in a chimeric cytochrome P-450 coding sequence. Expression of cloned rabbit cytochrome P-450 into transformed yeast was optimized by studying the effect of the nature of the DNA sequence just preceding the initiation codon on the level of cytochrome P-450 production. Yeast synthesized cytochromes P-450 were characterized by immunoblotting, spectra and catalytic activity determinations. Cloned cytochrome P-450 LM4 was found by all criteria to be identical to the authentic rabbit one. The chimeric cytochrome P-450 that contains the 143 N-terminal amino acids of cytochrome P-450 LM4 and the remaining 375 amino acids of cytochrome P-450 LM6 was found to exhibit most of the authentic cytochrome P-450 LM6 catalytic properties. Enzymatic and evolutionary implications of these results are 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。