Isolation,Identification and Tissue Expression Profile Analysis of One Novel Differentially Expressed Sequence Tag in the Longissimus dorsi Muscle from Meishan,Meishan × Large White Hybrid and Large White Pigs

In order to detect the molecular mechanism of heterosis in pigs, the mRNA differential display technique was performed to investigate the differences of gene expression in the Longissimus dorsi tissue from Meishan, Meishan × Large White hybrid and Large White pigs with nine 3'-end anchored primers in combination with ten 5'-end arbitrary primers and nearly 3 000 reproducible bands were examined. One novel expressed sequence tag (EST4,GenBank accession number: AY553914) that was differentially expressed in Meishan,Meishan × Large White hybrid and Large White pigs was isolated from the Longissimus dorsi muscle tissue and identified through semi-quantitative RT-PCR. BLAST analysis revealed that the 350 bp long EST (EST4) was not homologous to any of the known porcine genes.Tissue expression profile analyses showed that the EST4 was expressed in most of tissues.     

Isolation, Identification of Differentially Expressed Sequence Tags in the Backfat Tissue from Meishan, large White and Meishan×Large White Cross Pigs

In order to detect the molecular mechanism of heterosis in pigs, the mRNA differential display technique was performed to investigate the differences of gene expression in the backfat tissue from Meishan,Large White and Meishan×Large White cross pigs.Nine 3'-end anchored primers in combination with ten 5'-end arbitrary primers were used to perform the differential display PCR and nearly 3 000 reproducible bands were examined .Fifteen expressed sepuence tags that were differentially expressed were isolated and then isdentifide through semi-quantitative RT-PCR.BLAST analysis revealed that the fifteen expressed sequence tags (ESTs) were ntt homologous to any of the known porcine genes or ESTs. These novel ESTs were then submitted to GenBank.     

猪L-Myc基因的分子克隆及在细胞重编程中的应用

【目的】克隆猪L-Myc基因,并在蛋白水平表达的情况下探索其在细胞重编程中的作用,为深入研究猪L-Myc基因替代c-Myc诱导多能干细胞（induced pluripotent stem cell,iPSC）奠定基础。【方法】先通过NCBI序列比对,采用RT-PCR克隆猪L-Myc基因cDNA,生物信息学分析猪L-Myc基因与人和小鼠的同源性,构建融合表达载体pEGFP/L-Myc-C1,通过载体转染和免疫印记检测猪L-Myc基因cDNA的蛋白水平表达;再将L-Myc基因装入逆转录病毒载体中,分别使用不同的转录因子诱导猪胎儿成纤维细胞（porcine embryo fibroblast,PEF）,通过形态变化和碱性磷酸酶（AP）染色验证猪L-Myc基因在细胞重编程中的作用。【结果】①获得了1 113 bp的猪L-Myc基因cDNA,编码364个氨基酸,理论分子质量为40 kD;②生物信息学分析显示猪L-Myc基因与人和小鼠高度同源;③免疫印记检测结果说明猪L-Myc基因cDNA能够在蛋白水平表达;④细胞诱导试验和AP染色结果显示转录因子Oct4、Sox2、Klf4和L-Myc（OSKL）组合诱导的细胞阳性克隆率明显高于Oct4、Sox2和Klf4（OSK）组合的阳性克隆率。【结论】获得了猪L-Myc基因,并且该基因在蛋白水平表达且在细胞重编程过程中起到了重要的作用。     

Differential Gene Expression Between Cross-Fertilized and Self-Fertilized Kernels During the Early Stages of Seed Development in Wheat

In order to understand molecular basis of cross-fertilized kernel advantage and heterosis, improved differential display of mRNA was used in this study to analyze alterations in gene expression between cross-fertilized and self-fertilized kernels at 2, 4, 6, 8, 10 and 12 days after pollination (DAP) by using 3 wheat hybrids with different level of heterosis. Four patterns of differential expression were observed: (i) bands observed in cross-fertilized kernels but not in self-fertilized kernels (BCnS); (ii) bands occurring in only self-fertilized kernels but not in cross-fertilized kernels (BSnC); (iii) cDNA over-expressed in cross-fertilized kernels compared to self-fertilized kernels (OEC);(iv) cDNA under-expressed in cross-fertilized kernels compared to self-fertilized kernels (UEC). Further analysis showed that BCnS is positively correlated with heterosis, but BSnC is negatively correlated with heterosis. Four differentially expressed cDNA fragments were verified by reverse-northern blot and sequence homology search in GenBank showed that one of them was new sequences; the other exhibited higher similarity to NBS-LRR type resistance protein, 1;6-bisphosphatase and photosystem Ⅱ chlorophyll a-binding protein psbB, respectively, which indicated diverse pathways may be involved in heterosis formation.     

猪HK2基因的克隆及序列分析

为克隆猪己糖激酶2基因,分析其生物功能,采用已报道的人及小鼠HK2的cDNA序列为依据,利用电脑克隆策略获得的ESTs设计引物,扩增新的猪HK2基因cDNA序列。将PCR产物克隆测序,分析获得的核苷酸序列及其编码蛋白的特性,分离的开放阅读框全长2754bp,编码917个氨基酸,与人和小鼠的核苷酸序列同源率分别为90%和87%,与人和小鼠的氨基酸序列同源率分别为96%和94%,利用生物信息学软件分析出此蛋白的理化特性并预测了其蛋白结构,为进一步开展猪HK2基因的结构功能、表达调控的相关研究奠定了基础。     

北京鸭IGF-1cDNA部分序列的克隆及其在胸肌组织表达的发育性变化

为探讨IGF-1基因对北京鸭胸肌发育的影响,试验克隆了北京鸭IGF-1 cDNA的部分序列,分析了其核苷酸及编码的氨基酸序列与其他物种的同源性,并检测了IGF-1 mRNA在不同日龄北京鸭胸肌中表达的发育性变化。结果表明,克隆的鸭IGF-1 cDNA部分序列长507 bp,编码168个氨基酸,核苷酸序列与鸡、家鸭、鹌鹑、火鸡、鸵鸟、人和猪的同源性分别为98％,99％,97％,98％,96％,84％和82％,编码的氨基酸序列与家鸭、鸡、火鸡、鹌鹑、人、挪威鼠和猪的同源性分别为99％,98％,97％,97％,84％,79％和84％。表明北京鸭与家鸭的亲缘关系最近,与鸡、鹌鹁、火鸡、鸵鸟也有较近的亲缘关系;北京鸭胸肌中IGF-1 mRNA表达丰度在7,14,21 日龄间和28, 35,42日龄间的差异均不显著(P>0．05),但28,35,42 日龄显著高于7,14,21日龄(P<0．05),说明在北京鸭胸肌发育较快的时期,IGF-1 mRNA表达量也较高,IGF-1 mRNA的表达量与胸肌的发育呈正相关。     

猪TAF7基因的克隆、染色体定位和组织表达谱分析

 【目的】通过对猪TAF7基因初步的研究,为猪分子遗传育种提供基础分子生物学信息,为猪的遗传育种提供分子标记。【方法】以五指山猪为研究对象,克隆TAF7基因,分析该基因结构特点,然后利用IMpRH（the INRA-University of Minnesota porcine radiation hybrid,法国农业科学院-明尼苏达大学的辐射杂种克隆板）分析该基因在猪染色体上定位信息,利用半定量RT-PCR方法,分析该基因在成年五指山猪16个不同组织（心脏、背肌、淋巴、脾脏、肝脏、肾脏、肺脏、子宫、睾丸、胃、小肠、大肠、卵巢、胸腺、脑、脂肪）的表达谱信息。【结果】克隆得到长1 701 bp的五指山猪TAF7基因序列,其中包括1 050 bp完整CDS（Coding Sequence,编码序列）区域,分析表明其编码含349个氨基酸的蛋白质。利用IMpRH分析结果表明,猪TAF7基因与分子标记SW1879和IL4（interleukin-4,白细胞介素-4）紧密连锁,LOD（Limit of Detection,检测极限）值分别为6.69和6.15。组织表达谱分析结果显示该基因在大多数组织中均有表达,其中在睾丸表达量较高,而在心脏、背肌中表达很低。【结论】猪TAF7基因5’UTR中有一个短的内含子,而在该基因CDS区域没有内含子。猪TAF7蛋白序列与人TAF7蛋白序列的相似性较高,二者在生物系统发育树中的距离最接近。猪TAF7基因在大多数组织中均表达,在猪睾丸中表达量较高,证实它调节目的基因转录具有广泛性。     

猪CISD1基因电子克隆与验证

对通过抑制差减杂交技术所筛选的猪前脂肪细胞诱导分化后差异表达的CISD1基因的EST序列为探针进行电子克隆,用克隆序列设计引物,通过RT-PCR、克隆测序、同源比对证实所获序列为猪CISD1基因的mRNA序列,并将序列提交到Genebank,登录号为GQ913654。所获猪CISD1基因的mRNA序列全长为631 bp,CDS包含321 bp,由其编码的CISD1蛋白包含106个氨基酸残基,理论等电点为9.20,属脂溶性不稳定蛋白。猪CISD1蛋白的1~23位氨基酸可能是信号肽,53~91位氨基酸形成CDGSH锌手指域,为该蛋白的重要功能域,猪CISD1蛋白的三级结构与人CISD1蛋白相似度为89.333%。     

猪干扰素IFNE1基因克隆及重组表达载体的构建

依据电子延伸序列设计一对克隆引物,用RT-PCR法从猪胃组织扩增出猪干扰素epsilon1(IFNE1)基因的完整编码区并进行序列分析;再根据克隆的序列设计一对表达引物,用PCR法从重组克隆载体中扩增出EcoRI/XhoI酶切位点的猪IFNE1片段,插入原核表达栽体,转化至宿主菌,诱导表达,SDS-PAGE鉴定融合蛋白.结果表明,克隆的猪IFNE1基因包含完整的开放阅读框架,长为586bp.ORF为582bp,编码193个氨基酸,与人、小鼠的同源性分别为83.6%和69.2%,推测的氨基酸序列与人、小鼠的同源性分别为76.2%和55.2%,表达的融合蛋白分子量约为47kD.     

小麦逆境胁迫相关基因TaC2DP1的克隆及表达分析

【目的】克隆与逆境胁迫相关的基因,并对其序列特征、进化关系和表达特性进行分析,探讨该基因在小麦抗逆调控过程中的生物学功能,为进一步解析植物的抗逆机制提供候选基因和理论依据。【方法】以cDNA芯片数据获得的水分胁迫诱导上调表达基因EST序列为探针,对小麦EST数据库进行搜索,筛选与探针同源性在97％以上的EST序列,通过电子克隆结合RT-PCR获得该基因cDNA全长,采用生物信息学软件分析比较克隆基因的保守结构域及序列特征;采用MEGA6.0软件构建该基因的系统进化树;将测序正确的该基因片段通过EcoRⅠ和HindⅢ限制性内切酶酶切连接至原核表达载体pMAL-c2X,重组质粒转化大肠杆菌BL21,经终浓度为0.3 mmol·L^-1 IPTG诱导1-5 h后,用SDS-PAGE分析融合蛋白的表达;采用实时荧光定量PCR（qRT-PCR）分析该基因在小麦不同组织间的表达差异及其在低温、干旱、高温和ABA处理下的表达模式。【结果】成功获得小麦cDNA全长序列,命名为TaC2DP1。该基因序列全长为1 356 bp,包含一个1 209 bp的开放阅读框（ORF）,5′端非编码区50 bp,3′端非编码区97 bp,编码402个氨基酸,推导编码蛋白质的预测分子量为43.41 kD,等电点为4.30,属于酸性蛋白,BLAST分析表明,该蛋白含有一个与钙离子结合的结构域,称为C2结构域（C2-domain）。多序列比对及进化树分析表明,TaC2DP1与乌拉尔图小麦TuC2亲缘关系最近,二者具有高度的同源性,其编码的氨基酸一致性达到91%;蛋白质结构预测分析显示TaC2DP1无跨膜螺旋和双硫键,亚细胞定位于细胞质中;成功构建了该基因的原核表达载体pMAL-c2X-TaC2DP1,在IPTG诱导下得到90 kD左右的蛋白,与理论值一致。通过实时荧光定量PCR进行TaC2DP1表达分析,显示该基因在小麦的根、茎、叶、幼穗、未成熟籽粒、胚及胚乳中均有表达,其中在幼穗中表达量最高,在花后5 d籽粒中表达量最低。TaC2DP1可被植物激素ABA诱导而上调表达;干旱胁迫过程中,TaC2DP1受胁迫诱导呈稳定上调表达趋势;高温和低温胁迫过程中,TaC2DP1均在胁迫后的0.5 h迅速诱导上调表达,分别为对照的21和17倍。推测该基因可能参与小麦ABA 信号通路中对逆境胁迫的抗性反应。【结论】获得小麦TaC2DP1的全长序列,其编码蛋白含有与钙离子结合的C2结构域;在低温、干旱、高温和ABA逆境胁迫下,TaC2DP1属于依赖于ABA胁迫响应基因调控网络,可能在干旱、低温和热胁迫中发挥重要作用。     

Molecular Characterization and Expression Pattern of Rheb Gene in Inner Mongolia Cashmere Goat （Capra hircus）

As one member of the Ras super family, Rheb is an upstream regulator of mTOR signaling pathway, which regulates the process of cell-growth, proliferation and differentiation. In order to study the relationship between Rheb and mTOR in Inner Mongolian Cashmere goat (Capra hircus) cells, Ras homolog enriched in brain (Rheb) gene cDNA was amplified by RT-PCR. It is 555 bp in length and includes the complete ORF encoding 184 amino acids (GenBank accession no. HM569224). The full cDNA nucleotide sequence has a 99% identity with that of sheep, 98% with cattle and 93% with human while their amino acids sequence shares identity with 98, 97 and 97% of them, correspondingly. The bio informatics analysis showed that Rheb has a Ras family domain, two casein kinase II phosphorylation sites, two ATP/GTP-binding sites motif A (P-loop), a prenyl group binding site (CAAX box). Tissue-specific expression analysis performed by semi-quantitative RT-PCR. The Rheb gene was expressed in all the tested tissues and the highest level of mRNA accumulation was detected in brain, suggesting that Rheb played an important role in goat cells.     

三元杂交猪IL-18全基因的序列测定及分析

白细胞介素-18（IL-18）又称γ干扰素诱导因子（IGIF）,研究表明IL-18是一种重要的新型免疫佐剂分子。为探讨猪IL-18的免疫佐剂作用,该研究根据已发表的猪IL-18基因序列,设计并合成1对特异性引物,从猪脾脏中直接扩增得到猪IL-18全基因,并进行克隆和序列测定、分析。     

山羊SDHD基因cDNA编码序列的克隆与分析

利用NCBI中GenBank里查询到已登录的人、猪、牛和绵羊的SDHD mRNA序列,通过多重同源比较,从而获得高度保守区域序列,设计了同源引物,并首次对山羊SDHD编码序列进行了分子克隆。经过PCR扩增和测序,获得山羊SDHD基因共4段cDNA序列,分别为:451 bp4、20 bp5、29 bp和698 bp。所获得的四段序列测序结果经La-sergene7.0软件SeqMan拼接后,获得一条1238 bp长的cDNA序列。在GenBank数据库中进行BLAST/nr比对,发现其与绵羊、牛、猪和人相应序列相似性分别达98%、97%、85%和81%。用NCBI的ORF Finder软件对已经克隆的山羊SDHD基因cDNA进行开放阅读框分析,发现该序列包含一个480 bp的开放阅读框,编码159个氨基酸残基,计算机分析表明(Compute pI/Mw tool),该蛋白的分子量约为17 224.11 Da,等电点为8.92。通过DNAMAN软件分析发现,山羊SDHD蛋白保守性很高,其与绵羊、牛、猪和人SDHD蛋白在氨基酸序列上的相似性分别达到97%、96%、87%和85%。此山羊SDHD基因cDNA序列和SDHD蛋白质序列已于2010年1月31日登录在NCBI的GenBank上,登录号为GU338978和ADB92501。本项研究为进一步研究山羊的SDHD基因作为山羊肉品质性状候选基因,提供了相应的序列信息。     

鲤鱼白细胞介素10(IL-10)基因cDNA克隆及序列分析(英文)

[目的]获取鲤鱼全长IL-10(interleukin 10)cDNA,并对其序列进行分析。[方法]利用DD-RTPCR(differential display RT-PCR)方法获得差异表达IL-10cDNA片段,以地高辛标记做为探针,对有丝分裂原刺激的鲤鱼外周血白细胞cDNA文库进行核酸杂交筛选,克隆鲤鱼IL-10全长cDNA,并对该序列进行序列分析和同源性比较。[结果]鲤鱼全长IL-10cDNA共1117bp,包含55bp的5’端非编码区,一个540bp的编码179个氨基酸的开放阅读框及522bp的3’端非编码区。其中,3’非编码区包含3个mRNA不稳定基序"ATTTA";该蛋白序列具有IL-10家族的典型序列特征;序列同源性比较表明,所获得的序列与GenBank上登录的鲤鱼IL-10基因同源性为89.1%。[结论]该试验为进一步研究IL-10在体内的表达方式、功能特点、调控机理及其在炎症反应和免疫应答中的作用机制奠定了基础。     

Identification,Characterization, and Expression of P450 Gene Encoding CYP6BQ13v2 from the Red Flour Beetle,Tribolium castaneum （Herbst） （Coleoptera： Tenebrionidae）

An allele of CYP6BQI3, named CYP6BQ 13v2 （GenBank accession no. FJ209361）, was isolated from the red flour beetle, Tribolium castaneum （Herbst） （Coleoptera： Tenebrionidae） by RT-PCR. The cDNA sequence of CYP6BQ13v2, 1 563 bp in length, contains an open reading frame of 1 554 nucleotides encoding a putative protein of 518 amino acid residues with a predicted molecular weight of 59.92 kDa and a theoretical pl of 7.60. The putative protein contains the classic hemebinding sequence motif F××G×××C×G （residues 456-465） conserved among all P450 enzymes as well as other characteristic motifs of all cytochrome P450s. It shares 98% identity with the previously published sequence of CYP6BQ13 （GenBank accession no. XP967146） from the T. castaneum genome project. Phylogenetic analysis of amino acid sequences from members of various P450 families indicated that there was closer phylogenetic relationship of CYP6BQ 13v2 with CYP302A1 and CYP307A1 mediating synthesis of the insect molting hormone, distant relationship with CYP6B1 metabolizing plant allelochemicals, CYP6D 1 linking to pyrethroid resistance and other members of CYP6 family. Expression test of the gene in the adults and immature stages of T. castaneum by quantitative real-time PCR revealed that CYP6BQ13v2 is expressed in all life stages investigated. The mRNA expression level in 1st instar larvae was 14.9- and 3.86-fold higher than those in pupae and adults, respectively. The CYP6BQ13v2 expression levels appeared in the order of 1st instar larvae, followed by 4th instar larvae, 7th instar larvae, adult, and pupae from high to low. The more bioinformation of CYP6BQ 13v2 was also analyzed.     

猪生长激素cDNA克隆及在大肠杆菌中的表达

用逆转录－聚合酰链式反应（RT－PCR）方法，从猪脑垂体总RNA中扩增出编码猪生长激素（GH）成熟及基因序列，定向克隆至质粒pUC18，序列分析表明，克隆的猪GH cDNA长573bp，不含信号肽序列，并在该序列之前加入一起始密码子ATG。将猪GHcDNA定向克隆至原核表达载体pBV220，构建成重组GH基因表达载体pBVpGH7。SDS－PAGE和薄层扫描分析表明：经42℃诱导，pBVpGH7在大肠杆菌中可表达一分子量约2200的特异蛋白，表达量约占细胞总蛋白的20．5％。     

鲤鱼白细胞介素10(IL-10)基因cDNA克隆及序列分析

[目的]获取鲤鱼全长IL-10(interleukin 10)cDNA,并对其序列进行分析。[方法]利用DD-RTPCR(differential display RT-PCR)方法获得差异表达IL-10 cDNA片段,以地高辛标记做为探针,对有丝分裂原刺激的鲤鱼外周血白细胞cDNA文库进行核酸杂交筛选,克隆鲤鱼IL-10全长cDNA,并对该序列进行序列分析和同源性比较。[结果]鲤鱼全长IL-10 cDNA共1 117 bp,包含55 bp的5'端非编码区,一个540 bp的编码179个氨基酸的开放阅读框及522 bp的3'端非编码区,其中,3'非编码区包含3个mRNA不稳定基序"ATTTA";该蛋白序列具有IL-10家族的典型序列特征;序列同源性比较表明,所获得的序列与GenBank上登录的鲤鱼IL-10基因同源性为89.1%。[结论]该试验为进一步研究IL-10在体内的表达方式、功能特点、调控机理及其在炎症反应和免疫应答中的作用机制奠定了基础。     

日本脑炎病毒WHe株NS1基因的克隆、测序及表达

以日本脑炎病毒(Japanese encephalitis virus,JEV)WHe株的基因组RNA为模板,采用RT-PCR技术,克隆了JEV WHe株的NS1基因,并对其进行了测序和序列分析。构建了pET28b-NS1表达载体,转化表达宿主大肠杆菌BL21(DE3),并对其进行了诱导表达,对表达产物进行检测。结果表明,NS1基因全长1 145 bp,其核酸序列与JEV P3株同源性为99．4％,与SA14和SA14-14-2等27个JEV毒株的核苷酸序列同源性为98％,表明NS1基因的保守性很高;pET28b-NS1表达产物的相对分子质量约为43 ku,大小与预期结果相符。NS1基因克隆表达成功。     

甘蔗S-腺苷蛋氨酸脱羧酶基因Sc-SAMDC的克隆和表达分析

【目的】克隆甘蔗(Saccharum officinarum)S-腺苷蛋氨酸脱羧酶(S-adenosylmethionine decarboxylase,SAMDC)基因,并进行序列特征、原核表达和不同逆境胁迫下表达特性分析。【方法】通过对甘蔗茎全长cDNA文库测序和分析,获得SAMDC基因cDNA全长,命名为Sc-SAMDC,并进行序列分析。随后将编码S-腺苷蛋氨酸脱羧酶的cDNA片段克隆到原核表达载体pET29a(+)中,构建融合表达质粒,转化至Esherichia coli BL21(DE3)中进行表达。最后利用定量PCR技术分析甘蔗幼苗中该基因在不同外源胁迫下的表达特性。【结果】序列分析显示,甘蔗Sc-SAMDC基因(GenBank Accession number:GQ246459)cDNA全长1968bp,存在3个读码框(袖珍读码框tORF、上游读码框uORF和主读码框mORF),mORF长1200bp,编码399个氨基酸的SAMDC酶原,预测分子量为43.6kD,该酶原含有两个高度保守的功能结构域(酶原剪切位点和PEST结构域)。原核表达产物经SDS-PAGE表明,Sc-SAMDC以融合蛋白形式表达,相对分子量约为50kD。定量PCR分析表明,Sc-SAMDC基因在聚乙二醇(PEG)、NaCl、水杨酸(SA)和H2O2外源胁迫下表达特性不同,受PEG和NaCl的诱导,受SA和H2O2的抑制。【结论】本研究成功地克隆了Sc-SAMDC基因并在原核生物中进行了表达研究,分析了该基因的表达特性,为其进一步的生物学功能研究及其应用奠定了基础。