红花花青素合成酶基因的克隆及表达分析

根据红花转录物测序结果中得到的中间序列,采用R11-PCR和RACE方法从红花花瓣中克隆到1个4嬲基因的全长cDNA,该基因全长序列1226bp,具有完整的开放阅读框（ORF）,共1050bp,编码349个氨基酸。生物信息学软件分析显示,该基因编码的蛋白理论分子量约为82．27kDa,等电点为5．09,序列里含有典型的加尾信号序列AATAA和Poly（A）。保守结构域预测表明,该基因编码的蛋白具有典型的ANS蛋白功能结构域,其保守结构域中含有铁离子及2．0-酮戊二酸结合位点。结合其他物种的臌因构建系统树表日月,红花ANS蛋基因与其他物种氨基酸具有一定的同源性,其中与芍药的亲缘关系最近。应用实时荧光定量PCR分析表明,ANS基因在红花的初花期和盛花期的表达量最高。     

文蛤HDAC1基因克隆、时空表达及生长相关SNP位点筛查

为探索HDAC1基因在文蛤生长发育中的作用, 研究通过已构建的文蛤转录组文库, 利用SMARTRACE技术扩增得到文蛤HDAC1 (Mm-HDAC1)基因的cDNA全长序列, 分析了其生物信息学、组织及发育阶段表达特征, 并用直接测序法在外显子中筛查生长相关的SNP位点。结果显示, Mm-HDAC1基因的cDNA全长3065 bp, 开放阅读框1599 bp, 编码532个氨基酸; 氨基酸序列比对发现, 文蛤与其他物种同源性为74.3%78.7%。荧光定量PCR (qRT-PCR)结果表明, Mm-HDAC1基因在文蛤6个组织中均有表达, 其中外套膜中的表达量相对最高, 并与其他组织间有极显著差异(P0.01); 不同发育时期的表达差异结果表明, Mm-HDAC1基因在壳顶幼虫期表达量最高, 显著高于其他发育时期(P0.05)。SNP位点筛查结果表明, 在Mm-HDAC1基因的外显子区域发现了19个SNP位点, 其中有3个SNP位点(627AT、924TC和1266TC)与文蛤生长相关。     

甘蔗细胞色素C基因的电子克隆与分析

以甘蔗类似细胞色素C的EST序列CF576943.1为探针,通过电子克隆技术获得了甘蔗细胞色素C基因（Cytochrome C,Cyt C）的一条cDNA全长序列,命名为ScCyt C.用生物信息学方法对该基因氨基酸序列与组成、亚细胞定位、跨膜区与信号肽、疏水性/亲水性、蛋白质二、三级结构以及功能等进行分析与预测.结果表明：Cyt C基因全长1 073 bp,编码112个氨基酸,该基因位于细胞质,为非分泌型蛋白,无规卷曲为主要二级结构原件,含有1个保守功能域,主要功能为翻译并且在不同植物中具有高度保守性.电子表达分析结果显示,该基因在甘蔗各个组织均有表达,其中在茎和根中的表达量比其他组织类型中表达量高,此外,该基因的表达可能受到低温的调控.为甘蔗细胞色素C基因的结构及其功能的研究奠定了一定的基础.     

家蚕CyPA基因的克隆、表达谱及进化分析

CyP蛋白家族在蛋白质折叠过程中起着重要作用。本研究克隆了家蚕Bombyx mori CyPA基因(BmCyPA),该基因由2个外显子和1个内含子组成,推导开放阅读框编码165个氨基酸,分子量为19.4 kD,等电点为8.79。序列分析表明BmCyPA在不同物种间具有高度的保守性,含有肽酰脯氨酸顺反异构酶活性位点及与CsA侧链结合的氨基酸,提示BmCyPA可能具有肽酰脯氨酸顺反异构酶活性和与CsA结合的特性。组织表达谱及EST数据分析显示,BmCyPA在丝腺中高丰度表达。通过对不同物种来源的CyP基因的进化分析,进一步预测了BmCyP基因的功能,BmCyP可能与丝蛋白的正确折叠相关。     

牛性别决定新基因Fgf9的克隆及生物信息学分析

Fgf9基因是脊椎动物性别决定中重要的信号因子,它在睾丸发育过程中参与Sertoli细胞的增殖和睾丸索的形成。基于表达序列标签（expressed sequence tags, ESTs）克隆原理,采用序列拼接和 RTPCR方法获得了荷斯坦奶牛Fgf9基因的cDNA序列,并对其组织表达特征进行分析。利用生物信息学方法对Fgf9基因序列和蛋白结构进行分析。结果显示：Fgf9基因定位于牛12号染色体上,cDNA全长为697bp,开放阅读框为627bp,编码208个氨基酸,分子量23.38245kDa,等电点7.0600。RTPCR证实该开放阅读框正确,在牛的各组织中均有表达,且与牛其他cDNA无同源性,获得GenBank登陆号为：EU693028。功能结构分析显示Fgf9蛋白具有典型的FGF家族保守结构域,包括受体相互作用位点和肝素结合位点。信号肽预测显示牛Fgf9蛋白可能不存在信号肽序列。
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甘薯苯丙氨酸解氨酶基因IbPAL的克隆与表达分析

本研究基于甘薯(Ipomoea batatas(L.) Lam.)与绿原酸合成代谢相关的转录组数据克隆了苯丙氨酸解氨酶基因Ib PAL,通过蛋白序列比对、系统进化分析、二级和三维蛋白结构预测、跨膜结构域以及启动子分析,对该基因的氨基酸序列和启动子序列的结构特征进行了解析;通过qRT-PCR技术分析该基因在不同甘薯品种的根、茎、茎尖和叶中的表达情况,并对其进行亚细胞定位验证。结果显示,Ib PAL的CDS序列全长2130 bp,编码709个氨基酸。其氨基酸序列与已知物种的氨基酸序列同源性在80%以上。Ib PAL蛋白以α螺旋和随机卷曲为主,具有1个可能的跨膜螺旋区。该基因启动子中包含多个顺式作用元件。表达模式分析结果表明,Ib PAL主要在茎和茎尖中表达,其在甘薯品种‘EC16’4个组织器官中的表达量均显著高于其他品种。亚细胞定位结果显示该基因在细胞膜和细胞核上均可以表达。     

中华蜜蜂Malvolio基因 Acmvl 的克隆及组织表达分析

【目的】本研究克隆了中华蜜蜂Apis cerana cerana Malvolio (Mvl)基因的cDNA序列,分析了其编码蛋白的结构特点,并探讨其mRNA在内勤蜂、采蜜蜂和采粉蜂各部位组织中的表达差异,以期为该基因的生物学功能研究提供参考。【方法】利用RT-PCR技术从中华蜜蜂内勤蜂头部组织中扩增和克隆获得Acmvl的全长序列,并采用多种生物信息学软件分析Acmvl蛋白的结构特征;采用Real-time PCR对中华蜜蜂Acmvl在内勤蜂、采蜜蜂和采粉蜂各组织中的表达特征进行分析。【结果】Acmvl基因cDNA全长为2 130 bp（GenBank登录号：KP662686）,编码587个氨基酸,预测该蛋白分子量为65.86 kD,等电点为6.03,无信号肽,存在11个跨膜结构域、9个糖基化位点和14个潜在磷酸化位点;系统发育树分析结果显示,中华蜜蜂Acmvl与其他膜翅目昆虫Malvolio聚为一支,与小鼠Mus musculus和人Homo sapiens Nramp家族的Nramp2聚为另一大分支,且与小鼠、水稻 Oryza sativa 、黑腹果蝇 Drosophila melanogaster 和酵母Saccharomyces cerevisiae的Nramp家族同源体在跨膜区、跨膜区带电残基及转运蛋白特征结构域上有很高的保守性,尤其是与Nramp2。Acmvl 基因在中华蜜蜂各部位组织中均有表达,但高表达于内勤蜂的胸部及采蜜蜂和采粉蜂的腹部和足部,提示该基因表达的差异影响采集行为。【结论】Acmvl 属于Nramp基因家族,可能为Nramp2的同源基因,该基因影响采集行为可能与转运Cu²⁺, Mn²⁺和Fe²⁺（尤其是Fe²⁺）有关。     

苏太猪ELOVL7基因的生物信息学分析

不同于人、鼠等物种ELOVL7基因的高相似度,不同品种的猪ELOVL7基因相似度较低。为了探究该基因的特性,本研究运用生物信息学的方法对苏太猪ELOVL7基因及其氨基酸序列的同源性、理化性质、保守结构域、亚细胞定位、信号肽、跨膜结构域、亲水性/疏水性、二级结构、功能预测以及磷酸化位点等进行预测分析。结果表明：在苏太猪中,ELOVL7全长2 324 bp,编码区为846 bp,共编码281个氨基酸。其结构稳定,分子量为33 387.4 Da,带正电荷,偏碱性。该基因所编码的蛋白质最可能位于细胞膜上,主要的功能是运输和结合,为跨膜、非分泌型疏水蛋白质,含有1个GNS1/SUR4家族的保守结构域,并有15个丝氨酸激酶、15个苏氨酸激酶和20个酪氨酸激酶潜在磷酸化位点。α螺旋是ELOVL7二级结构和三级结构中最主要的结构元件。另外ELOVL7与大部分物种的氨基酸序列相似性达90%以上,且亲缘关系较近。分析ELOVL7基因及其氨基酸序列的特征,能够为进一步挖掘该基因内的突变对长链脂肪酸表型的影响以及合成、代谢机理提供分子依据。     

羊流产嗜衣原体ompA基因克隆及生物信息学分析

分析羊流产嗜衣原体ompA基因结构并预测其编码蛋白的结构和功能。采用DNA Star、DNA MAN、vector NTI suite11.5序列分析软件和在线网站ExPASy分析该基因的结构和预测其编码蛋白的理化性质、亚细胞定位、一级结构修饰位点、二级结构特征及三维空间构象、潜在抗原表位等。结果显示,该基因全长1 170 bp,可编码389个氨基酸,编码蛋白理化性质较稳定,无各种亚细胞定位序列,含有多个能被其他酶修饰的位点,该蛋白以无规则卷曲为主,大部分氨基酸残基包埋在分子内部,含5个跨膜区,3个亲水性较强的抗原表位。ompA基因生物信息学分析结果为ompA蛋白功能的深入研究和新型多价疫苗的开发提供了基础数据。     

A phylogenetic analysis based on the gene encoding phosphoglycerate kinase

Summary We have determined the nucleotide sequence of both genomic and complementary DNA (cDNA) for the gene encoding the glycolytic enzyme phosphoglycerate kinase from the ciliated protozoan Tetrahymena thermophila. The amino acid sequence for the enzyme has also been derived from the cDNA sequence. The gene contains an open reading frame of 1260 nucleotides encoding 420 amino acids. Coding sequence in genomic DNA is interrupted by two introns at positions corresponding to introns 3 and 4 in mammalian phosphoglycerate kinase genes. The derived amino acid sequence was used to prepare a phylogeny by aligning the Tetrahymena sequence with 25 other phosphoglycerate kinase amino acid sequences. The Tetrahymena sequence is a typical eukaryotic sequence. There is recognizable and clear homology across species that cover nearly the complete range of life forms. The phylogenetic reconstruction of these sequences generally supports the conclusions that have been reached using rRNA sequences.Offprint requests to: R.E. Pearlman     

Molecular characterization of a cDNA encoding vitellogenin in the banana shrimp, Penaeus (Litopenaeus) merguiensis and sites of vitellogenin mRNA expression

In order to determine the primary structure of banana shrimp, Penaeus merguiensis, vitellogenin (Vg), we previously purified vitellin (Vt) from the ovaries of vitellogenic females, and chemically analyzed the N-terminal amino acid sequence of its 78 kDa subunit. In this study, a cDNA from this species encoding Vg was cloned based on the N-terminal amino acid sequence of the major 78 kDa subunit of Vt and conserved sequences of Vg/Vt from other crustacean species. The complete nucleotide sequence of Vg cDNA was achieved by RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) approaches. The full-length Vg cDNA consisted of 7,961 nucleotides. The open reading frame of this cDNA encoding a precursor peptide was comprised of 2,586 amino acid residues, with a putative processing site, R-X-K/R-R, recognized by subtilisin-like endoproteases. The deduced amino acid sequence was obtained from the Vg cDNA and its amino acid composition showed a high similarity to that of purified Vt. The deduced primary structure, of P. merguiensis Vg was 91.4% identical to the Vg of Penaeus semisulcatus and was also related to the Vg sequences of six other crustacean species with identities that ranged from 86.9% to 36.6%. In addition, the amino acid sequences corresponding to the signal peptide, N-terminal region and C-terminal region of P. merguiensis Vg were almost identical to the same sequences of the seven other reported crustacean species. Results from RT-PCR analysis showed that Vg mRNA expression was present in both the ovary and hepatopancreas of vitellogenic females but was not detected in other tissues including muscle, heart, and intestine of females or in the hepatopancreas of mature males. These results indicate that the Vg gene may be expressed only by mature P. merguiensis females and that both the ovary and hepatopancreas are possible sites for Vg synthesis in this species of shrimp.     

一色齿毛菌锰过氧化物酶2基因克隆及生物信息学分析

摘要 锰过氧化物酶（manganese peroxidase,MnP）是由一系列同功酶组成的木质素降解酶。我们前期工作克隆了一色齿毛菌（Cerrena unicolor) MnP1基因序列。在此基础上,本研究采用简并PCR、染色体步移和RACE等技术对C. unicolor mnp2基因(Cu-mnp2)序列进行克隆。同时,采用生物信息学软件对Cu-mnp2的基因结构、Cu-MnP2的蛋白质结构及多物种MnPs蛋白质序列的系统进化关系进行分析。克隆得到3 053 bp的Cu-mnp2 DNA序列(GenBank:JX270806.1)和1 429 bp的Cu-mnp2 cDNA序列(GenBank: JQ782580.1)。序列分析结果显示,Cu-mnp2 DNA序列包含14个外显子和13个内含子,启动子区域包含TATA-BOX、SP1和AP1等作用元件;Cu-mnp2 cDNA序列包含71 bp的5′UTR、230 bp的3′ UTR以及1 128 bp的开放阅读框(ORF)。Cu-mnp2 ORF序列的BLAST比对结果表明,Cu-mnp2与Trametes versicolor FP-101664 SS1 mnp序列覆盖度为53%,序列相似性为65%;与Heterobasidion irregulare mnp、C. unicolor mnp1等cDNA序列都有较高的序列相似性。Cu-mnp2的ORF编码(GenBank:AFK91530.1)由340个氨基酸残基组成的多肽链(Cu-MnP2)。Cu-MnP2蛋白质序列的BLAST比对和蛋白质三维结构均显示,Cu-MnP2包含Mn ²⁺ 、Ga ²⁺ 、血红素及芳香底物结合位点。对包含Cu-MnP1、Cu-MnP2蛋白质序列在内的多物种MnPs蛋白质序列的系统发育分析表明,多物种的MnPs分为两大类群,分别为包含4个二硫键的短MnPs和包含5个二硫键的长MnPs。其中,Cu-MnP1与Cu-MnP2均属于短MnPs,Cu-MnP2与Trametes versicolor MrP 的蛋白质序列亲缘关系最近。通过Cu-mnp2基因的克隆和序列分析,对继续研究C. uniclor的MnP同工酶基因结构和功能奠定基础。     

cDNA sequence analysis of ribosomal protein S13 gene in Plutella xylostella （Lepidoptera： Plutellidae）

Ribosomal protein S 13 gene has been cloned and analyzed in many organisms,but there are few documents relating to insects. In this communication, the full-length cDNA sequence of ribosomal protein S 13 gene in the diamondback moth, Plutella xylostella(Lepidoptera: Plutellidae), was determined by using PCR amplification technique. The features of the ribosomal protein S 13 gene sequence were analyzed and the deduced amino acids sequence was compared with those from other insects. The results of multi-alignment of the amino acid sequences between the diamondback moth and other insect species revealed that this gene sequence is highly conserved in insects. Based on maximum likelihood method, a phylogenetic tree was constructed from 10 different species using PHYLIP software. It showed that nematode is one separate lineage and the five insect speciesbe long to another lineage, whereas those species higher than insects form the third one. The pattern of this phylogenetic tree evidently represented the evolution of different species.     

草鱼自噬相关基因Beclin1的克隆及其在MC-LR胁迫下的表达特征

为评估微囊藻毒素-LR (MC-LR)对鱼类自噬的影响, 根据转录组测序结果得到的EST序列, 采用RACE技术获得了草鱼(Ctenopharygodon idella)自噬基因Beclin1 (CiBeclin1)的cDNA全长序列。该基因序列全长1590 bp, 包括1344 bp的开放阅读框, 编码447个氨基酸, 分子量为51.2 kD, 理论等电点为4.88。CiBeclin1包含1个BH3和1个ECD结构域。CiBeclin1氨基酸序列与其他物种的相似性为88%—98%, 构建的进化树显示CiBeclin1与稀有鲫(Gobiocypris rarus)的Beclin1亲缘关系最近。实时荧光定量PCR (qRT-PCR)检测结果表明, CiBeclin1在肝、肾、脾等10种不同组织中均广泛表达, 但在肝脏组织中的相对表达量最丰富, 显著高于相对表达量最低的头肾组织(P<0.05)。在不同剂量(25和100 μg MC-LR/kg BW) MC-LR胁迫24h、48h、72h和96h后, 草鱼肝脏中CiBeclin1表达量均显著低于对照组的表达水平, 研究结果表明, MC-LR能抑制草鱼CiBeclin1的表达, 但MC-LR是否在该剂量下诱导了草鱼的自噬还有待于进一步研究。     

Isolation and Sequence Characterization of Mammary Derived Growth Inhibitor Gene of Riverine Buffalo (Bubalus Bubalis)

In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5′ UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3′ UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI//fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.     

Isolation and sequence characterization of mammary derived growth inhibitor gene of riverine buffalo (Bubalus bubalis)

In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5' UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3' UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI/fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.     

Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase.

The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues.     

Vitellogenin of Blattella germanica (L.) (Dictyoptera, blattellidae): nucleotide sequence of the cDNA and analysis of the protein primary structure

The cloning and sequencing of a cDNA of the vitellogenin gene from the cockroach Blattella germanica is reported. It is 5,749 nucleotides long and encodes an amino acid sequence of 1,862 residues (including a putative signal peptide of 17 residues). The vitellogenin sequence includes a long serine-rich stretch between amino acids 322 and 349, and two other stretches between amino acids 1691 and 1740. The vitellogenin of B. germanica shows a notable similarity (between 32 and 42%) to those described in other insects, and its alignment shows a high number of motifs conserved in all species, especially in the subdomains I-V. Non-parsimony methods (Neighbor Joining) of phylogenetic analysis of the insect vitellogenin sequences gave a tree showing a topology that is, in general, congruent with the currently accepted insect phylogenetic schemes. Arch.