褶皱臂尾轮虫（Brachionus plicatilis）β-actin基因克隆分析
发布时间：2019-06-05 20:12
【摘要】：轮虫(Rotifer)属于动物界中一类无脊椎假体腔多细胞动物,个体微小却正适合幼鱼口径,营养丰富且易于人工培养。作为生物饵料,轮虫在控制水质、提高海洋生物幼体成活率等方面也具有明显的优势,因而是鱼类幼苗极佳的开口饵料。轮虫种类较多,其中褶皱臂尾轮虫是唯一可以进行人工海水养殖的轮虫种,大多生活于半咸水区域。长期以来对轮虫的分子生物学方面的研究较少,本文选择系统进化中极其保守的β-actin基因为目的基因,通过比对其它物种的β-actin基因,设计简并引物进行PCR扩增,从而获得轮虫β-actin基因cDNA全长,后经染色体步移得其启动子序列,并对其进行了生物信息学分析以及初步的载体构建,为进一步利用该基因的启动子开展转基因相关研究提供了参考。本研究现已获得褶皱臂尾轮虫β-actin基因总长2987bp,编码区无内含子。其中cDNA序列全长1377bp,ORF(开放读码框)1131bp,编码氨基酸376个；5’UTR为76bp,3'UTR为170bp；启动子区序列1706bp。将褶皱臂尾轮虫β-actin基因的ORF序列与日本三角涡虫、金线虫、单环刺嚑及贝类等物种进行Blastp比对分析,结果显示无论进化关系远近,各物种β-actin基因序列以及蛋白序列相似度都较高,达75%以上。表明β-actin基因在漫长的生命进化过程中,长期保持着高度的遗传稳定性。此外,由于褶皱臂尾轮虫基因组尚未报道研究,在基因克隆方面的研究极少。因而,对进化较为低等的动物进行基因克隆方面的研究,管家基因β-actin是极佳的首选基因。目前,关于转基因动物生物反应器的研究还存在理论基础薄弱和技术不完善等问题,使得目的基因转入后的有效整合、稳定遗传及高效表达等问题难以攻克。此外,目的产物的分离、纯化及生物安全性等方面也存在着诸多问题。本文旨在使培养成本低、生命周期短,但繁殖效率高,且可作为幼鱼首选饵料的轮虫——这一小型无脊椎动物成为表达诸多外源目的产物的生物反应器,从而获得大量人们预期的活性产物,使转基因后的轮虫成为更加改良优质的开口饵料。本文首先由简并引物经PCR扩增得褶皱臂尾轮虫β-actin cDNA全长,再经染色体步移技术进一步获得其启动子核心序列,经生物信息学预测分析后,可作为目的载体,以轮虫β-actin核心启动区构建pEGFP。后期预将此启动子序列融合外源目的基因如抗菌肽、生长激素等,进行体外载体重组构建,经电穿孔、显微注射等转基因手段进行轮虫转基因技术的探索,从而获得预期的转基因轮虫体。将此轮虫体进行人工扩大培养,大量繁殖时表达出大量目的药用产物,并喂食给幼鱼以改善其生长发育状况,成为更加优化的开口饵料。
[Abstract]:Rotifer (Rotifer) belongs to a class of invertebrate cavity multicellular animals in animal kingdom. The individual is small but suitable for juvenile fish caliber, rich in nutrition and easy to be cultured artificially. As a biological feed, rotifer also has obvious advantages in controlling water quality and improving the survival rate of marine living larvae, so it is an excellent open feed for fish seedlings. There are many species of rotifer, among which Brachiontail rotifer is the only rotifer species which can be cultured in artificial seawater, most of which live in brackish water area. There has been little research on the molecular biology of rotifer for a long time. In this paper, the extremely conservative 尾-actin gene in systematic evolution was selected as the target gene, and the degenerate primers were designed for PCR amplification by comparing the 尾-actin gene of other species. The full length of 尾-actin gene cDNA of rotifer was obtained, and its promoter sequence was obtained by chromosome step, and its bioinformatics analysis and preliminary vector construction were carried out. It provides a reference for the further study of transgenic research by using the promoter of the gene. In this study, the total length of 尾-actin gene was 2987bp, and there was no intron in the coding region. The full length of cDNA sequence was 1377bp. the open reading frame (ORF) 1131bp encoded 376 amino acids, 5'UTR was 76bpand 3 鈮，
本文编号：2493796
[Abstract]:Rotifer (Rotifer) belongs to a class of invertebrate cavity multicellular animals in animal kingdom. The individual is small but suitable for juvenile fish caliber, rich in nutrition and easy to be cultured artificially. As a biological feed, rotifer also has obvious advantages in controlling water quality and improving the survival rate of marine living larvae, so it is an excellent open feed for fish seedlings. There are many species of rotifer, among which Brachiontail rotifer is the only rotifer species which can be cultured in artificial seawater, most of which live in brackish water area. There has been little research on the molecular biology of rotifer for a long time. In this paper, the extremely conservative 尾-actin gene in systematic evolution was selected as the target gene, and the degenerate primers were designed for PCR amplification by comparing the 尾-actin gene of other species. The full length of 尾-actin gene cDNA of rotifer was obtained, and its promoter sequence was obtained by chromosome step, and its bioinformatics analysis and preliminary vector construction were carried out. It provides a reference for the further study of transgenic research by using the promoter of the gene. In this study, the total length of 尾-actin gene was 2987bp, and there was no intron in the coding region. The full length of cDNA sequence was 1377bp. the open reading frame (ORF) 1131bp encoded 376 amino acids, 5'UTR was 76bpand 3 鈮，
本文编号：2493796
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