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1.
从广西产眼镜王蛇(Ophiophagus hannah)毒腺中抽提总RNA,经mRNA纯化后构建眼镜王蛇毒腺cDNA文库。从所构建的cDNA文库中,随机筛选200个克隆测序,得到两个在进化上高度保守的基因:泛素融合蛋白基因(GenBank登录号为AF297036)和核糖体蛋白L30基因(GenBank登录号是AF297033)。前者cDNA的开放阅读框为387bp,后者为348bp。前者编码128个氨基酸残基组成的泛素融合蛋白前体;后者编码115个氨基酸残基组成的核糖体蛋白L30前体。由cDNA序列推导出的氨基酸序列分析表明,泛素融合蛋白前体包括N-末端的泛素结构域(76个氨基酸残基)和C-末端的核糖体蛋白L40结构域(52个氨基酸残基)。该蛋白为一高碱性蛋白,C末端含有一个“锌指”模式结构。与16个物种比较的结果表明,眼镜王蛇与脊椎动物的泛素融合蛋白氨基酸序列相似度较高,具有高度的保守性。  相似文献   

2.
家蝇攻击素(Attacin)基因的克隆与表达   总被引:9,自引:0,他引:9  
耿华  安春菊  郝友进  李德森  杜荣骞 《遗传学报》2004,31(12):1344-1350
通过同源克隆策略并结合cDNA末端快速扩增(RACE)技术,克隆到了编码家蝇攻击素(Attacin)基因的全长cDNA序列(GenBank登陆号:AY460106),并对相应的氨基酸序列进行了序列比对,对系统关系等进行了生物信息学分析。家蝇Attacin的cDNA全长778bp,其中包括627bp的开放式读码框(ORF),44bp的5’末端非编码区(5’UTR)和107bp的3’末端非编码区(3’UTR),相应的蛋白产物含208个氨基酸,与其他双翅目昆虫的Attacin具有很高的相似性,约50%-70%。以邻接法(Neighbor-Joining,NJ)所构建的系统关系表明,家蝇的Attacin与其他双翅目昆虫的Attacin起源于共同的祖先。应用半定量RT-PCR的方法,研究家蝇幼虫在受到外源刺激后Attacin基因的表达,结果表明,在家蝇幼虫体内Attacin基因呈诱导型表达,表达水平随诱导时间和诱导源的不同而变化。  相似文献   

3.
猪骨骼肌快肌肌钙蛋白C2基因的cDNA克隆与表达分析   总被引:3,自引:0,他引:3  
从人骨骼肌快肌肌钙蛋白C2(TNNC2)基因出发,在dbEST数据库中进行同源性搜索,找到一个有较高同源性且在猪背最长肌中表达EST(BM083186)。通过电子克隆和进一步RT-PCR实验验证,获得猪TNNC2基因全长cDNA序列,其全长843bp,开放阅读框为201~683bp,编码有160个氨基酸。同源性分析结果表明,与人、鼠的骨骼肌快肌肌钙蛋白C2基因cDNA编码区(CDS)同源性分别为93.6%、90.5%,蛋白序列同源性均为97.5%。多种组织的半定量RT-PCR研究表明,该基因在骨骼肌中表达,并且在杜洛克猪背最长肌中的表达比兰塘猪高。  相似文献   

4.
根据真菌△^6 -脂肪酸脱氢酶基因保守的组氨酸Ⅱ区和Ⅲ区附近保守序列设计兼并引物进行RT-PCR,得到雅致枝霉(Thamnidium elegans)As3.2806△^6 -脂肪酸脱氢酶基因459bp部分cDNA序列,然后通过快速扩增cDNA末端技术(RACE)向两端延伸得到1504bp的△^6 -脂肪酸脱氢酶基因全长cDNA序列。序列分析表明有一个1377bp、编码459个氨基酸的开放阅读框TED6。推测的氨基酸序列与已知其他真菌的△^6 -脂肪酸脱氧酶基因的氨基酸序列比对,具有3个组氨酸保守区、2个疏水区及N末端细胞色素b5融合区。将此编码区序列亚克隆到酿酒酵母缺陷型菌株INVSel的表达载体pYES2.0中,构建表达载体pYTED6,并在酿酒酵母INVSel中异源表达。通过气相色谱(GC)和气相色谱,质谱(GC-MS)分析表明,该序列在酿酒酵母中获得表达,产生γ-亚麻酸(GLA)的含量占酵母总脂肪酸的7.5%。证明此序列编码的蛋白能将外加的亚油酸转化为γ-亚麻酸,是一个新的有功能的△^6 -脂肪酸脱氢酶基因(GenBank.AY941161)。  相似文献   

5.
植物Rac是植物中特有的小分子G蛋白,我们从苎麻转录组中获得一个小分子G蛋白基因eDNA,.5部分序列,设计引物后采用RT-PCR结合RACE技术克隆了该基因的cDNA。序列分析表明,所克隆的RaclcDNA全长为l043bp,包括594bp开放阅读框、214bp的3’端非编码区和235bp的5’端非编码区,能编码一个197氨基酸的推导蛋白。该蛋白包含G蛋白典型的效应因子结合位点、GTP/GDP结合位点和碱性氨基酸区,c末端具有保守的异戊烯基化位,CSIL。采用半定量RT-PCR分析了该基因在5个苎麻品种及不同组织器官中的表达情况,结果表明Racl基因在苎麻根、茎、叶中均有表达,其中在叶中的表达量最高。纤维木质素含量不同的品种中,Racl基因的表达量存在明显差异。木质素含量高的品种具有较高的Racl基因表达,表明该基因可能在苎麻木质素合成过程中发挥作用。  相似文献   

6.
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。  相似文献   

7.
猪L-FABP基因的克隆、表达特征及遗传多态性研究   总被引:10,自引:0,他引:10  
FABPs属于脂结合蛋白超家族成员,是一类分子量较小而对脂肪酸有高亲和力的蛋白质,广泛存在于脊椎动物和非脊椎动物的细胞质中.FABPs担当细胞内脂肪酸的运输任务,它们与脂肪酸结合将其运输到脂肪酸氧化的位置、脂肪酸脂化成甘油三醋或磷脂的位置,或者进入细胞核内发挥其可能的调控功能.因此FABPs对脂类代谢具有重要的调控作用.本研究把L-FABP基因作为影响猪肌内脂肪含量的候选基因.为此,利用cDNA末端快速扩增(RACE)和PCR技术,克隆到猪肝脏型脂肪酸结合蛋白基因(L-FABP)的全长cDNA序列(GenBank登录号AY960623)和部分基因组序列(GenBank登录号DQ182323).猪L-FABP基因的cDNA序列全长518 bp,该序列包括起始密码子TGA和38 bp的5'末端非编码区(5'URT),终止密码子TAG和99 bp的3'末端非编码区(3'URT),在3'URT结构区域中包含polyA加尾信号序列AATAAA.猪L-FABP基因与其他FABPs基因一样,也由4个外显子(67 bp、173 bp、93 bp和51 bp)和3个内含子组成,内含子1和3的大小是1 679bp和565 bp,没有获得内含子2的序列,外显子和内含子剪接处符合GT/AG规律.应用Clustal W/X程序对猪L-FABP与其他物种的L-FABP进行多重序列比对,发现猪L-FABP与人、大鼠、鸡的L-FABP的相似性分别为89.8%、81.9%和72.4%.亲水性分析表明,猪L-FABP也是一个潜在的跨膜蛋白,在氨基酸残基57-65之间有一个明显的跨膜α螺旋.应用半定量RT-PCR分析发现,猪L-FABP在猪体组织中广泛存在,但在肝脏和小肠组织中表达量最为丰富.分析还发现,所克隆得到的编码区核苷酸序列与已知猪L-FABP基因的编码区核苷酸序列存在一定的变异,分别是外显子2中T→C(116位)、C→T(231位)、C→A(236位)和A→C(258位),演绎成氨基酸在Leu74Met存在差异.为进一步证实这些突变位点在猪群中真实存在,利用PCR-SSCP检测方法对4个猪种(藏猪、大河猪、雅南猪和约克夏)的157头个体的外显子2全序列进行SNP位点多态性片段的基因型分型,结果发现一个C→T的单核苷酸多态,等位基因频率在中国地方猪种(藏猪、大河猪、雅南猪)与国外约克夏猪种间存在极显著的差异(P<0.01).连锁分析发现,基因型CC的肌内脂肪含量(4.86±0.22%)显著的高于基因型CT(4.16±0.23%)和TT(4.05±0.27%)的肌内脂肪含量(P<0.05).因此,推测L-FABP基因可能是影响猪肌内脂肪含量的主效基因或与主效基因紧密连锁的标记基因,并且能够在分子标记辅助选择中用于对猪肌内脂肪含量的遗传改良.  相似文献   

8.
采用同源克隆、染色体步移和RT-PCR技术,首次克隆到苦荞查尔酮合酶基因(CHS)的全长DNA序列和cDNA开放阅读框(ORF)序列. 序列分析表明,苦荞CHS DNA序列(GU172165)全长1 632 bp,含1个445 bp的内含子;cDNA编码区(HM852753)全长1 188 bp,编码395个氨基酸,命名为FtCHS. 生物信息学分析表明,FtCHS和推导的氨基酸序列与其它植物CHS基因同源率在95%以上,含有CHS多基因家族的标签序列(GFGPG)、活性位点、底物结合口袋位点和环化反应口袋位点. 半定量RT-PCR分析苦荞花期FtCHS空间表达模型表明,其表达量未成熟种子>叶>茎>花>根>成熟种子,与苦荞芦丁含量的分布基本一致,具有组织特异性.  相似文献   

9.
α1-微球蛋白和Bikunin是由同一基因翻译表达出的两种功能不相关联的血浆蛋白。本文通过快速扩增cDNA末端的方法,首次从草鱼肝脏组织克隆了α1-微球蛋白和Bikunin前体蛋白(α1-microglobulin/Bulinin precursor, AMBP)基因全长cDNA。其cDNA全长1230bp,包含5′非翻译区23 bp,3′非翻译区160 bp和开放读码框1047 bp。开放读码框编码348个氨基酸,包含182个氨基酸的α1-微球蛋白和145个氨基酸的Bikunin。草鱼AMBP与其他物种的氨基酸序列分析结果表明,它们具有较高的同源性(44.7%-84.4%),其中草鱼与斑马鱼同源性最高(84.4%)。结果表明AMBP序列结构和α1-微球蛋白与Bikunin共翻译表达特点在动物机体中具有着重要的生理意义。  相似文献   

10.
少根根霉△^6-脂肪酸脱氢酶基因的克隆和表达   总被引:1,自引:0,他引:1  
根据真菌△^6-脂肪酸脱氢酶保守的氨基酸序列设计简并引物进行RT-PCR,获得一个593 bp的cDNA片段,再根据获得的部分序列设计基因特异性引物,通过cDNA末端扩增技术(RACE)获得该cDNA的3’和5’序列,从而得到全长为1482bp的cDNA序列。序列分析结果表明,该序列具有一个长度为1377bp、编码458个氨基酸的开放阅读框,所编码蛋白质的大小为52kD。与报道的△^6-脂肪酸脱氢酶一样,推测的氨基酸序列具有膜整合脂肪酸脱氢酶特异性的3个组氨酸保守区和疏水结构,在其氨基酸序列的N-末端具有类似于细胞色素b5的血红素结合区。该序列为一个新的编码△^6-脂肪酸脱氢酶的基因,为了验证其功能,把开放阅读框序列RAD6亚克隆到表达载体pYES2.0,构建重组表达载体pYRAD6,并转化到酿酒酵母的缺陷型菌株INVScl进行表达。通过气相色谱(GC)和气相色谱/质谱(GC-MS)分析表明,该序列在酿酒酵母中获得表达。所编码的酶具有△^6-脂肪酸脱氢酶活性,能将外源性的底物亚油酸转化为γ-亚麻酸,γ-亚麻酸的含量占酵母总脂肪酸的3.85%。  相似文献   

11.
12.
Fatty acid-binding proteins (FABPs) are members of a superfamily of lipid-binding proteins, occurring intracellularly in invertebrates and vertebrates. This study was designed to clone and characterize the genes of heart fatty acid-binding protein and intestine fatty acid-binding protein in the chicken. PCR primers were designed according to the chicken EST sequences to amplify cDNA of H-FABP and I-FABP genes from chicken heart and intestinal tissues. Analysis of sequence showed that the cDNA of the chicken H-FABP gene is 75 to 77% homologues to human, mouse, and pig H-FABP genes, and the chicken I-FABP gene is 71 to 72% homologues to human, mouse, and pig I-FABP genes. In addition, Northern blot analysis indicated that of the two genes, similar to the copartner of the mammal, H-FABP gene was expressed in a wide variety of tissues, and I-FABP gene was expressed only in intestinal tissues. The expression levels of the chicken H-FABP mRNA in heart and I-FABP mRNA in intestine had significant differences between the broilers from fat line and Bai'er layers at six weeks of age. The results of this study provided basic molecular information for studying the role of two FABPs in the regulation of fatty acid metabolism in avian species.  相似文献   

13.
Wang Q  Li H  Liu S  Wang G  Wang Y 《Animal biotechnology》2005,16(2):191-201
Fatty acid-binding proteins (FABPs) are members of a superfamily of lipid-binding proteins, occurring intracellularly in invertebrates and vertebrates. This study was designed to clone and characterize the genes of heart fatty acid-binding protein and intestine fatty acid-binding protein in the chicken. PCR primers were designed according to the chicken EST sequences to amplify cDNA of H-FABP and I-FABP genes from chicken heart and intestinal tissues. Analysis of sequence showed that the cDNA of the chicken H-FABP gene is 75 to 77% homologues to human, mouse, and pig H-FABP genes, and the chicken I-FABP gene is 71 to 72% homologues to human, mouse, and pig I-FABP genes. In addition, Northern blot analysis indicated that of the two genes, similar to the copartner of the mammal, H-FABP gene was expressed in a wide variety of tissues, and I-FABP gene was expressed only in intestinal tissues. The expression levels of the chicken H-FABP mRNA in heart and I-FABP mRNA in intestine had significant differences between the broilers from fat line and Bai'er layers at six weeks of age. The results of this study provided basic molecular information for studying the role of two FABPs in the regulation of fatty acid metabolism in avian species.  相似文献   

14.
We have cloned a cDNA from zebrafish (Danio rerio) that contains an open-reading frame of 132 amino acids coding for a fatty acid binding protein (FABP) of approximately 15 kDa. Multiple sequence alignment revealed extensive amino acid identity between this zebrafish FABP and intestinal-like FABPs (I-FABP) from other species. The zebrafish I-FABP cDNA hybridized to single restriction fragments of total zebrafish genomic DNA digested with the restriction endonucleases PstI Bg/II or EcoRI suggesting that a single copy of the I-FABP gene is present in the zebrafish genome. An oligonucleotide probe complementary to the zebrafish I-FABP mRNA hybridized to an mRNA of approximately 800 bases in Northern blot analysis. In situ hybridization revealed that the I-FABP mRNA was expressed exclusively in the intestine of the adult zebrafish.  相似文献   

15.
16.
SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp (Cyprinus carpio L.) was isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp SLP-76 was 2007 bp, consisting of a 5'-terminal untranslated region (UTR) of 285 bp, a 3'-terminal UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34-73% to that of other species homologues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts.  相似文献   

17.
Heterologous cDNA clones were used as hybridization probes to define the temporal expression of intestinal functions during fetal and postnatal development in the pig. Northern hybridization analysis revealed the presence of the mRNAs for the cellular retinol binding protein CRBP II, for the digestive enzyme aminopeptidase N, and for the microvillar proteins villin and ezrin in the small intestine of both weaned and 40-day fetal pigs. The presence of these mRNAs suggests that at the end of the first third of gestation the pig fetal intestine is already exhibiting some characteristics of a differentiated epithelium. The mRNAs for the two fatty acid-binding proteins I-FABP and L-FAPB, both involved in the metabolism of long chain fatty acids, were detected only in the intestinal mRNA extracted from weaned animals, while that for the cellular retinol-binding protein CRBP I was expressed only in the fetal tissue. The temporal limits of expression of intestinal genes in the pig epithelium seem therefore more easily defined than in other experimental animals with shorter times of fetal development. To isolate pig genes expressed at different developmental stages during intestinal epithelial cell differentiation, a cDNA library was constructed from poly(A) + RNA extracted from mature pig intestine. This library was employed in the isolation of clones encoding CRBP II and L-FABP. The nucleotide sequence of the two pig cDNA clones was determined, and the sequences of the deduced proteins compared with their homologues from other species. The results of this analysis showed that the two pig clones share a high level of homology with human and rat homologues both at the DNA and at the protein level.  相似文献   

18.
猪CuZnSOD基因的克隆、表达及功能分析   总被引:2,自引:0,他引:2  
Du JF  Zeng YQ  Chen W  Cui JX  Chen QM  Yang L  Hu YX 《遗传》2010,32(10):1037-1042
为了进一步了解和认识CuZnSOD基因的结构和功能,揭示CuZnSOD对猪抗氧化机能的影响,寻找与肉质性状相关联的分子标记,文章采用RACE(Rapid amplification of cDNA end)方法,对莱芜猪CuZnSOD基因cDNA进行克隆测序,分析其结构和功能,并用Real-timePCR检测CuZnSOD基因的表达.结果表明,CuZnSOD基因cDNA序列全长658 bp(GenBank登录号:GU944822),包含76 bp的5'UTR和120 bp的3'UTR序列.全部CDS序列462 bp,编码153个氨基酸,分子量为15.9 kDa,等电点为6.03.CuZnSOD基因编码的氨基酸序列中,第3氨基酸残基处存在1个O-糖基化位点,第86氨基酸残基处存在1个N-糖基化位点.二级结构中α螺旋仅占1.31%.在进化过程中高度保守,与人、牛、小鼠和褐鼠的编码区同源性分别为87.74%、87.66%、83.44%和83.23%;氨基酸序列同源性分别为90.26%、94.12%、92.21%和91.50%.CuZnSOD存在典型的金属结合配体结构域(GFHVHQFGDNT).基于蛋白序列所构建的分子进化树表明猪与牛的亲缘关系最近.在mRNA水平上,CuZnSOD是一个广谱表达基因,在大脑、心脏、脾脏、肝脏、肾脏、肺、大肠、小肠、脊髓,肌肉、背膘和胃中都能检测到,其在肾脏,小肠和肺中表达量较高,在心脏和肌肉组织中表达量较低.  相似文献   

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Zhang X  Dai L  Wu Z  Jian J  Lu Y 《Marine Genomics》2011,4(3):143-150
Heat shock protein 10 (HSP10) gene of humphead snapper (Lutjanus sanguineus), designated as ByHSP10, was cloned by rapid amplification of cDNA ends (RACE) techniques with the primers designed from the known EST sequence identified from the subtracted cDNA library of the head kidney of humphead snapper. Sequence analysis showed the full length cDNA of ByHSP10 was 529 bp, containing a 5′ terminal untranslated region (UTR) of 51 bp, a 3′ terminal UTR of 181 bp, and an open reading frame (ORF) of 297 bp encoding a polypeptide of 99 amino acids. Based on the deduced amino acid sequence, the theoretical molecular mass of ByHSP10 was calculated to be 10.92 kDa with an isoelectric point of 9.46. Moreover, chaperonins hsp10/cpn10 signature was found in the amino acids sequence of ByHSP10 by PredictProtein. BLAST analysis revealed that the amino acids of ByHSP10 had the highest homology of 88% compared with other HSP10s. Fluorescent real-time quantitative RT-PCR was used to examine the expression of ByHSP10 gene in eight kinds of tissues of humphead snapper after the challenge with Vibrio harveyi. There was a clear time-dependent expression pattern of ByHSP10 in head kidney, spleen and thymus after bacteria challenge. The expression of mRNA reached the maximum level at the time point of 9 h, 6 h and 24 h, respectively and then returned to control level in 36 h. The up-regulated mRNA expression of ByHSP10 in humphead snapper after bacteria challenge indicated that the HSP10 gene was inducible and might be involved in immune response. A phylogenetic tree was constructed based on the ORF nucleotide sequences of HSP10 for 30 species. The relatonships among them were generally in agreement with the traditional taxonomy which suggested that HSP10 genes could aid in the system classification research.  相似文献   

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