蓝点马鲛两种Wap65基因克隆及蛋白功能研究

发布时间：2018-05-01 10:01

本文选题：蓝点马鲛 + Wap65　；参考：《中国计量大学》2016年硕士论文

【摘要】：Wap65蛋白(Warm temperature acclimation 65k Da protein)是一种血浆蛋白,最开始是在金鱼、鲤鱼体内发现的。实验结果表明,金鱼和鲤鱼在高温诱导下,有一种分子量为65 k Da的蛋白大量存在于各组织中,被命名为Wap65,此蛋白与哺乳动物的血红素结合蛋白高度同源。蓝点马鲛(Scomberomorus niphonius)属于辐鳍亚纲,鲈形目,鲭亚目、鲭科、马鲛属,广泛分布于北西太平洋部、我国产于东海、黄海和渤海,最为著名的是在南海的海南文昌铺前的附近海域。因为近些年来,人们对蓝点马鲛的肆意捕捞和环境的不断恶劣,致使蓝点马鲛的产量快速减少,为了减少对蓝点马鲛的伤害,当前已经在中国宁波象山建立了蓝点马鲛的保护基地。蓝点马鲛属近海温水性洄游鱼类,所以高温是蓝点马鲛的生存及生产的重要因素之一。1.蓝点马鲛头肾cDNA文库的构建提取蓝点马鲛头肾的总RNA,逆转录合成cDNA,运用了SMART技术构建蓝点马鲛头肾的cDNA文库。使用NCBI的Vec Screen程序识别所测序列,去除载体,利用DNAStar软件的Seq Man程序对多序列进行了比对,将获得的EST序列在Gen Bank上进行BLAST比对分析,得到一些具有特殊功能的基因。2.两种不同的Wap65基因cDNA克隆以及mRNA的表达特征分析通过NCBI找出同源性较大的几种鱼类的Wap65-1和Wap65-2的cDNA序列,在保守区设计简并性引物,克隆两种Wap65的部分片段。然后依据已得到Wap65-1和Wap65-2基因的部分片段分别设计3’和5’RACE特异性引物,利用Gene Racer TM Kit扩增得到cDNA。Wap65-1和Wap65-2基因的cDNA长度分别为1659 bp和1725 bp,各自编码426和436个氨基酸。其中,Wap65-1基因5’和3’非翻译区的碱基长度分别为71 bp和307 bp,开放阅读框(ORF)为1281 bp;Wap65-2基因5’和3’的非翻译区的碱基长度分别为83 bp和331 bp,开放阅读框(ORF)为1311 bp。通过生物信息学软件分析了Wap65基因编码氨基酸序列的相似性,发现两种不同的Wap65基因与其它鱼类的的氨基酸序列具有相似。将蓝点马鲛的幼体在20℃,25℃,30℃,35℃的温度下进行1 h的热诱导,然后提取总RNA,并逆转录为cDNA,用于荧光定量表达分析。q RT-PCR结果表明:蓝点马鲛Wap65-1基因和Wap65-2基因在20℃,25℃,30℃,35℃分别都有表达,而Wap65-2随着温度的增高,其表达量明显增高,这一结果与所查文献所得出的结果类似,在蓝点马鲛体内,主要参与温度调节的基因是Wap65-2。3.蓝点马鲛两种Wap65基因的原核表达及蛋白功能研究设计特异性引物,扩增两个基因ORF,双酶切后进行重组质粒的构建,并导入表达宿主菌E.coli BL21(DE3)IPTG诱导,将诱导的菌液进行45℃的热刺激,当处理15,30,45和60min后,Wap65-1细菌存活率分别是40%,22%,15%,7%,Wap65-2细菌的存活率为75%,60%,45%和30%,而空白组(含pCold TF)细菌存活率在上述各时间点分别是30%,17%,10%和5%。这说明热刺激条件下,Wap65-2与空白对照组比较具有显著性差异,说明Wap65-2对细菌起到了一定程度的保护作用。总之,本研究中蓝点马鲛的头肾文库的构建,帮助我们发掘了一些具有特殊功能的基因序列,具有一定的参考价值。克隆了蓝点马鲛两种Wap65基因的cDNA全长,并对其进行耐热性分析,以及原核表达,丰富了水生生物Wap65的研究,为以后更深入的研究提供了基础,也希望可以为蓝点马鲛耐热品种的培育提供帮助。
[Abstract]:The Wap65 protein (Warm temperature acclimation 65K Da protein) is a plasma protein, which was first found in goldfish and carp. Experimental results showed that a protein with a molecular weight of 65 K Da was found in a large number of tissues and was named as Wap65 with the heme of mammals. Scomberomorus Niphonius is a subclass of radiant fins, bass, mackerel, mackerel, and mackerel, widely distributed in the North West Pacific, I am home to the East China Sea, the Yellow Sea, and Bohai, most famous in the vicinity of the Hainan Wenchang paving in the South China Sea. Because in recent years, people are wanton to the blue mackerel. In order to reduce the damage to the blue point mackerel, the production of the blue point mackerel has been rapidly reduced. In order to reduce the damage to the blue point mackerel, the blue dot mackerel has been established in Xiangshan, Ningbo, China. The blue point mackerel is a warm water migratory fish in the near sea, so the high temperature is one of the important factors for the survival and production of the blue point mackerel,.1. blue point horse. The cDNA Library of the mackerel kidney is constructed to extract the total RNA of the head kidney of the blue point mackerel, reverse transcriptional synthesis of cDNA, use the SMART technology to construct the cDNA Library of the head kidney of the blue point mackerel, use the Vec Screen program of NCBI to identify the sequence, remove the carrier, and compare the multisequence with the Seq Man program of the DNAStar software, and the EST sequence is obtained. In the BLAST comparison analysis, we got some special functions of gene.2. two different Wap65 gene cDNA clones and mRNA expression characteristics analysis through NCBI to find the cDNA sequence of Wap65-1 and Wap65-2 of several species of homologous fish. In the conservative region, we designed the degeneracy primers and cloned part of the part of the Wap65 of the two species. 3 'and 5' RACE specific primers were designed for the partial fragments of the Wap65-1 and Wap65-2 genes. The cDNA lengths of cDNA.Wap65-1 and Wap65-2 genes were amplified by Gene Racer TM Kit, respectively, 1659 BP and 1725 BP, respectively, each encoding 426 and 436 amino acids, of which the length of the base of the 5 'and 3' non translation regions was 71 and 307, respectively. BP, the open reading frame (ORF) is 1281 BP, the base length of the non translated region of the Wap65-2 gene 5 'and 3' is 83 BP and 331 BP respectively. The open reading frame (ORF) is 1311 bp. to analyze the similarity of the sequence of the amino acid sequence of the Wap65 gene through the bioinformatics software, and the amino acid sequence of two different Wap65 genes and other fish is found. Similar. Heat induced by 1 h at 20, 25, 30 and 35 degrees centigrade of the blue point mackerel, then the total RNA was extracted and cDNA was extracted. The.Q RT-PCR results showed that the Wap65-1 gene and Wap65-2 gene of the blue point mackerel were expressed at 20, 25, 30, 35, respectively, and Wap65-2 with the increase of temperature. The results were similar to those obtained in the literature. In the blue point mackerel, the genes involved in the temperature regulation were the prokaryotic expression of two Wap65 genes in the Wap65-2.3. blue point mackerel and the specific primers of protein function, the amplification of two genes ORF, and the construction of the recombinant plasmid after double enzyme digestion. Induction of E.coli BL21 (DE3) IPTG induced by host bacteria, the induced bacterial fluid was heated at 45 degrees C. After 15,30,45 and 60min, the survival rate of Wap65-1 bacteria was 40%, 22%, 15%, 7%, and the survival rate of the bacteria was 75%, 60%, 45% and 30%, while the survival rate of the blank group (including pCold TF) was 30%, 17%, 10% and 5%. at the above time points, respectively. This shows that Wap65-2 has a significant difference compared with the blank control group, indicating that Wap65-2 has a certain protective effect on the bacteria. In a word, the construction of the head kidney Library of the blue point mackerel in this study helps us to discover some special functions of the base sequence, which has a certain reference value. The full length of the two Wap65 genes of mackerel and the analysis of its heat resistance and prokaryotic expression have enriched the study of aquatic organisms Wap65, which provides a basis for further research, and hopes to provide help for the breeding of the heat-resistant varieties of the blue point mackerel.

【学位授予单位】：中国计量大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78

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