十字花科植物SAMDC基因同源序列的克隆与进化分析

腺苷甲硫氨酸脱羧酶(SAMDC)是参与植物多胺合成的一个关键酶。根据GenBank中已报道的SAMDC基因编码序列保守区域设计特异引物, 运用PCR技术分别从十字花科6属14个物种中新克隆分离了SAMDC基因的同源序列。比较分析结果表明: 这些同源序列的相似性达87%以上, 所推导的氨基酸序列相似性达90%以上, 且两者种间差异分别为0.2%~10.1%和0.3%~6.6%, 属间差异除“圆白”萝卜外分别是4.9%~13.6%和3.1%~10.3%; SAMDC基因的核苷酸及其可能编码的氨基酸序列差异属间较种间大, 可用于属间的分类等级研究; 且氨基酸序列间的差异比核苷酸序列间的差异小的多, 因而根据核苷酸序列构建了NJ与ME分子系统树。进化树直观地表明在亲缘进化关系上芸薹属与萝卜属较近, 其他依次为山芥属、蔊菜属、拟南芥属, 与荠菜属最远。研究结果有助于从分子水平阐明十字花科植物间的亲缘进化关系, 可为其种质资源利用提供理论依据。     

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。     

谷子肌动蛋白基因的克隆及序列分析

以谷子（Setaria italica）为材料,提取总RNA。根据植物肌动蛋白基因编码区的两端的保守序列设计了简并引物,用5'RACE方法扩增出了谷子肌动蛋白基因编码区序列。以豌豆肌动蛋白cDNA作探针进行的Southern杂交分析表明扩增出了目的基因。将所获得的片段克隆到T载体后进行测序,序列分析结果表明：谷子肌动蛋白基因的编码区长1131个核苷酸,编码了377个氨基酸;所得序列(命名为MIAc)与GenBank中注册的肌动蛋白基因序列的相似性均在60%以上,与其它肌动蛋白氨基酸序列的相似性达89%以上。根据高等植物肌动蛋白序列相似性重建了进化树,表明谷子肌动蛋白与水稻肌动蛋白异型体RAc2和RAc3之间的亲缘关系 最为密切,在进化过程中分化时间最为接近。     

谷子肌动蛋白基因的克隆及序列分析

以谷子 (Setariaitalica)为材料 ,提取总RNA。根据植物肌动蛋白基因编码区的两端的保守序列设计了简并引物 ,用 5’RACE方法扩增出了谷子肌动蛋白基因编码区序列。以豌豆肌动蛋白cDNA作探针进行的Southern杂交分析表明扩增出了目的基因。将所获得的片段克隆到T载体后进行测序 ,序列分析结果表明 :谷子肌动蛋白基因的编码区长 1 1 3 1个核苷酸 ,编码了 3 77个氨基酸 ;所得序列 (命名为MIAc)与GenBank中注册的肌动蛋白基因序列的相似性均在 6 0 %以上 ,与其它肌动蛋白氨基酸序列的相似性达 89%以上。根据高等植物肌动蛋白序列相似性重建了进化树 ,表明谷子肌动蛋白与水稻肌动蛋白异型体RAc2和RAc3之间的亲缘关系最为密切 ,在进化过程中分化时间最为接近     

红纹黄单胞菌a-氨基酸酯水解酶的克隆、序列分析及热稳定性提高

【目的】克隆红纹黄单胞菌α-氨基酸酯水解酶基因全序列,对序列进行生物信息学分析,并提高酶的热稳定性。【方法】利用多聚酶链式反应(PCR)克隆α-氨基酸酯水解酶基因全序列;应用生物信息学软件对获得的基因序列及编码的蛋白序列进行分析;通过同源建模,预测红纹黄单胞菌α-氨基酸酯水解酶的三维结构;通过定点突变替换氨基酸序列中高度柔性的位点,提高该酶的热稳定性。【结果】从红纹黄单胞菌(Xanthomonas rubrillineans)中扩增得到α-氨基酸酯水解酶基因aeh(GenBank登录号JF744990),核苷酸序列长度1 917 bp,编码638个氨基酸。序列比对和同源性分析显示,该酶与白纹黄单胞菌Xanthomonas albilineans str.GPE PC73的肽酶及地毯草黄单胞菌Xanthomonas axono-podis pv. citri str. 306的戊二酰-7-氨基头孢烷酸酰化酶氨基酸序列相似性最高,分别为91%和83%,系统进化分析表明,该酶与白纹黄单胞菌Xanthomonas albilineans str. GPEPC73的肽酶亲缘性最高。基于预测的三维模型,对高度柔性的位点进行饱和突变,从282株突变体中筛选得到3株T50较野生型高5°C以上的突变体。【结论】对红纹黄单胞菌AEH的氨基酸序列分析有助于探索同源蛋白的进化过程。对高度柔性位点进行饱和突变的策略可以用于提高热稳定性。     

孟氏隐唇瓢虫细胞色素P450基因的克隆与序列分析

采用同源克隆和RACE技术,从孟氏隐唇瓢虫Cryptolaemus montrouzieri Mulsant中克隆到细胞色素P450CYP9Z401基因的c DNA全序列(Gen Bank number:KP164813)。c DNA全长1722 bp,包含3'非编码区域(UTR)为86 bp和5'UTR为49 bp,开放阅读框(ORF)长1587 bp,编码528个氨基酸。预测的分子量为61.27 k D,理论等电点为6.58,无信号肽结构,在2-20氨基酸处存在跨膜螺旋。该基因拥有细胞色素P450特征序列螺旋C、螺旋K和Meander区域,另外还有血红素结合区(1个氨基酸差异)和螺旋I区(2个氨基酸差异)。与其他昆虫的CYP9家族基因比较,与赤拟谷盗Tribolium castaneum同源性最高,为47%,系统发育分析也表明两者的亲缘关系最近。CYP9Z401基因的克隆和比较分析为进一步深入研究孟氏隐唇瓢虫的细胞色素P450基因功能及其进化具有重要意义。     

柳树β微管蛋白基因家族的克隆和序列分析

该研究克隆鉴定了旱柳和龙爪柳β微管蛋白基因,并对其进行了序列相似性、系统发育、染色体定位以及表达模式的分析。结果显示,2种柳树β微管蛋白基因家族各有20个成员,家族内部成员间核酸和氨基酸序列相似性分别在74.0%和86.6%以上,种间同源蛋白氨基酸序列相似性在85.8%以上,柳树与其它植物β微管蛋白间的氨基酸序列相似性在81.5%以上。系统发育分析显示,柳树β微管蛋白家族被分为4个亚组,结合杨树β微管蛋白基因染色体定位,推测柳树β微管蛋白基因家族经历了杨柳科全基因组重复事件和串联重复事件,而柳树TUB11和TUB12可能来源于区段重复或者转座。基因表达模式分析发现,该家族成员的表达具有一定的组织特异性,并且部分重复基因对在所检测组织中表达差异较大。柳树β微管蛋白基因家族成员序列的高度相似性、成员数量的进化扩张、以及表达模式的多样性可能赋予了细胞分裂与生长更高的灵活性,这对多年生木本植物的生长发育习性意义重大。     

中华鳖4个Sox基因保守区的序列分析

采用PCR技术,扩增和克隆了中华鳖Sox基因（TSSox）。经DNA序列分析显示,Sox基因在系统进化上十分保守,其中TSSox4与鸟类LF4基因编码的氨基酸序列完全相同、与人类SOX4和Sox4编码的序列仅一个氨基酸的差异;TSSox5与鸟类的LF5基因的编码也仅一个氨基酸发生了改变;TSSox2与海龟的TSox2相似性最高。4条TSSox序列中,TSSox与人SRY基因序列相似性最高,达75％;序列上的相似性可能暗示了它们在功能上的保守性。     

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。     

九台晚李PGIP基因的克隆及生物信息学分析

以九台晚李叶片基因组为模板,PGIP基因保守序列设计引物,PCR扩增到1条全长1192bp的目的片段(GenBank登录号:GU068978)。该基因包含有1个完整的开放阅读框,由2个外显子和1个内含子构成,外显子总长990bp,编码330个氨基酸,其编码的氨基酸序列中含有一段典型的亮氨酸重复序列。序列分析表明:该基因与中国李、杏、桃、马哈利樱桃、梅等李属植物的PGIP基因序列一致度达95%~99%。系统进化分析显示出属内亲缘关系较近、属间亲缘关系较远的特点。该序列为植物分子抗病育种提供了1条新的基因资源。     

Complexity and sequence identification of 24 rat V beta genes

Twenty-four TCR V beta genes were cloned by anchored PCR from the Lewis rat strain and identified by nucleotide and amino acid sequence comparisons to known mouse V beta genes. Rat V beta genes exist in 17 single-member and 3 multimember subfamilies and exhibit 86 to 94 and 72 to 92% nucleotide and amino acid sequence similarities, respectively, to their mouse counterparts. A single rat gene, designated V beta 20, having no previously known mouse counterpart was identified; a closely related gene was subsequently isolated from the C57BL/6 mouse strain. Characterization of the rV beta genes provides the basis for addressing rat T cell tolerance mechanisms and V beta gene usage in immunologically mediated rat disease models.     

Complete mitochondrial genome of the Bewick's swan Cygnus columbianus bewickii (Aves, Anseriformes, Anatidae)

The mitochondrial genome of Bewick's swan Cygnus columbianus bewickii was completely sequenced and then the resultant data were compared with those of the whistling swan Cygnus columbianus columbianus. The complete mitochondrial genome sequence of C. c. bewickii was 16,727?bp in length and its gene arrangement pattern, gene content, and genome organization were identical to those of Cygnus species. The similarities of nucleotide and amino acid sequences between the two swans were 99.1% and 99.6%, respectively. Out of the 13 protein-coding genes and 2 rRNA genes, COIII showed the lowest nucleotide sequence similarity with 98.0%. On the other hand, in amino acid sequence similarities, both COII and ATP6 showed the lowest with 98.7% in common. The control region has the 97.8% nucleotide sequence similarity.     

Cloning, sequencing, and characterization of CYP1A1 cDNA from leaping mullet (Liza Saliens) liver and implications for the potential functions of its conserved amino acids

A 2,037 bp CYP1A1 cDNA (GenBank AF072899) was cloned through screening of a lambdaZipLox cDNA library constructed from the liver of a leaping mullet (Liza saliens) fish captured from Izmir Bay on the Aegean coast of Turkey using rainbow trout CYP1A1 cDNA as a probe. This clone has a 130 bp 5'-flanking region, a 1,563 bp open reading frame (ORF) encoding a 521-amino acid protein (58,972 Da), and a 344 bp 3'-untranslated region without a poly (A) tail. Alignment of the deduced amino acids of CYP1A1 cDNAs showed 58% and 69-96% identities with human and 12 other fish species, respectively. Southern blot analysis suggested that this CYP1A1 cDNA was from a single-copy gene. Based on the comparison with CYP1A1 genes reported for fish and mammals, the leaping mullet CYP1A1 gene is probably split into 7 exons. The intron insertion sites were predicted. Alignment of the CYP1A1 cDNA encoded amino acids from 13 fish and 7 mammalian species disclosed differences in highly conserved amino acids between aquatic and land vertebrates. The possible associated secondary structure; conserved motifs and substrate-binding sites were discussed. The phylogenetic relationships of CYP1A1s among 13 fish species were analyzed by a distance method.     

Sequence variability of Borna disease virus open reading frame II found in human peripheral blood mononuclear cells.

A cDNA fragment of the Borna disease virus (BDV) open reading frame II (ORF-II), which encodes a 24-kDa phosphoprotein (p24 [P protein]), was amplified from total RNA of peripheral blood mononuclear cells (PBMC) from three psychiatric inpatients. The amplified cDNA fragments were cloned, sequenced, and analyzed. A total of 15 clones, 5 from each patient, were studied. Intrapatient divergencies of the BDV ORF-II nucleotide sequence were 4.2 to 7.3%, 4.8 to 7.3%, and 2.8 to 7.1% for the three patients, leading to differences of 7.7 to 14.5%, 10.3 to 17.1%, and 6.0 to 16.2%, respectively, in the deduced amino acid sequence for BDV p24. Interpatient divergencies among the 15 clones were 5.9 to 12.7% at the nucleotide level and 12.8 to 28.2% at the amino acid level. Thus, in p24, BDV in human PBMC of the patients undergoes mutation at high rates in vivo. Additionally, we found that the nucleotide sequence of the 15 human BDV ORF-II cDNA clones differed from those of the horse strains V and He/80-1 by 4.2 to 9.3%. However, comparison of the consensus amino acid sequence deduced from the 15 human clones with those of the horse strains revealed no human-specific amino acid residue, suggesting that the BDV infecting humans may be related to that infecting horses.     

Development of PCR primers specific for the amplification and direct sequencing of gyrB genes from microbacteria, order Actinomycetales

PCR primer sets were developed for the specific amplification and sequence analyses encoding the gyrase subunit B (gyrB) of members of the family Microbacteriaceae, class Actinobacteria. The family contains species highly related by 16S rRNA gene sequence analyses. In order to test if the gene sequence analysis of gyrB is appropriate to discriminate between closely related species, we evaluate the 16S rRNA gene phylogeny of its members. As the published universal primer set for gyrB failed to amplify the responding gene of the majority of the 80 type strains of the family, three new primer sets were identified that generated fragments with a composite sequence length of about 900 nt. However, the amplification of all three fragments was successful only in 25% of the 80 type strains. In this study, the substitution frequencies in genes encoding gyrase and 16S rDNA were compared for 10 strains of nine genera. The frequency of gyrB nucleotide substitution is significantly higher than that of the 16S rDNA, and no linear correlation exists between the similarities of both molecules among members of the Microbacteriaceae. The phylogenetic analyses using the gyrB sequences provide higher resolution than using 16S rDNA sequences and seem able to discriminate between closely related species.     

苹果蠹蛾细胞色素P450基因CYP332A19和CYP337B19的克隆及表达分析

【目的】本研究旨在通过克隆苹果蠹蛾Cydia pomonella细胞色素P450基因CYP332A19和CYP337B19,并对其进行序列和表达分析,以更好地了解这两个P450基因在植物次生物质解毒方面的作用,为进一步的功能研究提供依据。【方法】采用本地BLAST搜索苹果蠹蛾转录组数据库获得细胞色素P450基因cDNA序列,采用RT-PCR技术克隆目的基因的编码区。利用生物信息学软件分析目的基因的序列特征及与其他近缘物种的P450基因的系统进化关系。采用RT-qPCR技术测定目的基因在苹果蠹蛾不同发育阶段(卵、1-5龄幼虫、蛹和成虫)、4龄幼虫不同组织(头部、表皮、脂肪体、中肠和马氏管)以及4龄幼虫分别取食添加0.1%香豆素和0.5%槲皮素的人工饲料2 d后的表达水平。【结果】克隆获得苹果蠹蛾细胞色素P450基因CYP332A19(GenBank登录号: MF574708)和CYP337B19(GenBank登录号: MF574697)的全长cDNA序列,开放阅读框(ORF)分别长1 518和1 491 bp,分别编码505和496个氨基酸,其蛋白质分子量分别为58.586和57.734 kD,理论等电点分别为8.99和7.61。结构域分析显示,CYP332A19和CYP337B19中均包含包括血色素结合区在内的5个保守的细胞色素P450结构域。系统发育树显示,苹果蠹蛾CYP332A19与苹淡褐卷蛾Epighyas postvittana CYP332A9等CYP332A基因聚在一枝,而CYP337B19与稻纵卷叶螟Cnaphalocrocis medinalis CYP337B12和六星灯蛾Zygaena filipendulae CYP337B11等CYP337B基因聚在另一枝。RT-qPCR分析结果表明,CYP332A19和CYP337B19在苹果蠹蛾幼虫期的表达水平高于卵期的,分别在4龄幼虫脂肪体和中肠中的表达量最高。取食分别含0.1%香豆素和0.5%槲皮素的人工饲料2 d后,4龄幼虫体内的CYP332A19和CYP337B19相对表达量显著高于对照组(取食含2%DMSO的人工饲料)。【结论】CYP332A19和CYP332B19分别在苹果蠹蛾幼虫脂肪体和中肠中高表达,且在取食含香豆素和槲皮素的人工饲料的苹果蠹蛾幼虫体内表达量升高,说明这两个基因可能参与苹果蠹蛾对外源物质的解毒代谢过程。本研究的结果有助于我们了解苹果蠹蛾对寄主次生物质解毒代谢机理,为苹果蠹蛾防治提供新思路。     

Characterisation of aminoglycoside acetyltransferase-encoding genes of neomycin-producing Micromonospora chalcea and Streptomyces fradiae

Two genes (aac) encoding aminoglycoside-N-acetyltransferase from Streptomyces fradiae and Micromonospora chalcea were cloned: the former identified by hybridization with a homologous gene from Streptomyces rimosus forma paromomycinus, the second by direct expression in Streptomyces lividans using pIJ702 as a vector. These two genes showed pronounced nucleotide and amino acid sequence similarities between themselves and also between previously described streptomycetes aac genes. Comparison of the flanking sequence of actinomycetes aac genes indicates considerable divergence, contrary to the notion that clustered biosynthetic genes for structurally related antibiotics were disseminated in their entirety between microbial species.     

Characterisation of small double stranded RNA molecule in Cryptosporidium hominis, Cryptosporidium felis and Cryptosporidium meleagridis

Coding regions of double stranded RNA molecules from 3 human faecal samples containing Cryptosporidium hominis, C. felis and C. meleagridis were characterised by sequencing and compared with that previously obtained for C. parvum. Sequences outside the coding regions were also obtained. Overall similarities of between 86% and 92% and between 86% and 93% were observed in the nucleotide and amino acid sequences respectively between these species. These larger sequences will allow further molecular tools for detection, identification and characterisation of Cryptosporidium spp.