太平洋鳕抗冻基因的研究

发布时间：2018-04-11 23:31

  本文选题：太平洋鳕 + 转录组　； 参考：《大连海洋大学》2017年硕士论文

【摘要】：太平洋鳕(Gadus macrocephalus)是我国北方重要的海水鱼类,具有较高的经济价值,近年来由于生存环境的恶化以及需求量的增高导致太平洋鳕野生数量锐减,本研究室已经开始致力于太平洋鳕人工繁育研究,并取得阶段性成果。许多海洋鱼类在冬季能够在极地以及北方沿海水域生存,因为他们能够分泌抗冻蛋白,在一定程度上能够有效的减少低温对鱼类的伤害,本研究采用转录组技术挖掘太平洋鳕体内与抗冻相关基因。实验利用Illumina2000测序技术对太平洋肝组织进行转录组测序分析。数据总量为61,778,789,组装后获得77,561个unigenes,unigenes长度分布范围从201 bp到16,370 bp,平均长度为1,118 bp。对获得的转录组数据进行分析,筛选出于抗冻糖蛋白基因(AFGP)和IV型抗冻蛋白基因(AFP-IV)。将太平洋鳕AFGP基因与大西洋鳕AFGP基因比较发现,测序结果相似,都未出现编码区序列,可能是由于重复序列在测序过程中被自动剔除,导致测序结果中不包含编码区序列。通过PCR技术克隆得到Ⅳ型抗冻蛋白基因,并对其进行生物信息学分析。结果表明:AFP-ⅣcDNA全长689 bp(GenBank登录号:KX430014.1),具有完整的开放阅读框(Open Reading Frame ORF),长度为375 bp,编码125个氨基酸。预测分子量(MW)为13.98 kDa,理论等电点(PI)为4.68。序列比对发现太平洋鳕AFP-IV基因的编码序列与其他已知物种AFP-IV基因编码的氨基酸序列同源性较高,相似性范围从40%到70%,其中与大西洋鳕(Gadus morhua)的同源性高达99%。通过生物信息学软件对AFPⅣ基因结构进行分析发现,AFP-IV在N端存在一个信号肽序列,在成熟肽的的亲水性分析中发现在30(Met)-85(Glu)区域的碱基内存在一个跨膜区域,在二级结构预测中发现AFP-IV的结构与载脂蛋白家族结构高度相似。预测AFP-IV可能具有载脂蛋白的功能。以β-actin为内参基因,采用Real-time PCR技术分别对太平洋鳕不同发育阶段的仔鱼及成鱼不同组织进行表达模式分析,结果发现:10 dph仔鱼的表达量最高,而在成鱼组织中肝脏的表达量最高。将太平洋鳕AFP-IV基因连接到原核表达载体pET-32a中,构建原核表达质粒pET-32a-AFP-IV,在表达条件优化后重组蛋白得到大量表达,利用ProteinISO Ni-NTA Resion对重组蛋白进行纯化,利用纯化的重组蛋白免疫新西兰大白兔制备多克隆抗体,ELISA检测显示多克隆抗体效价达到1:1600000,应用Western-blot技术对融合蛋白的免疫原性进行检测,结果表明重组蛋白具有较高亲和力。利用纯化后的抗冻蛋白对大肠杆菌进行耐低温实验,结果表明重组蛋白具有一定的生物活性,抗冻效果不明显。综上所述,本研究利用转录组技术筛选出抗冻糖蛋白及IV型抗冻蛋白基因,并对其进行分析,研究结果为太平洋鳕抗冻基因的研究及应用提供参考。
[Abstract]:The Pacific cod Gadus macrocephalus is an important marine fish in the north of China, which has high economic value. In recent years, due to the deterioration of the living environment and the increase of demand, the wild population of Pacific cod has decreased sharply.The lab has begun to study the artificial breeding of Pacific cod, and has achieved periodic results.Many marine fish survive in polar and northern coastal waters in the winter, because they can secrete antifreeze proteins and, to a certain extent, reduce damage to fish at low temperatures.In this study, transcriptome technique was used to excavate antifreeze related genes in Pacific cod.The transcriptome sequencing of Pacific liver tissue was carried out by Illumina2000 sequencing technique.The total amount of data was 61778789. After assembly, 77561 unigenes were obtained, ranging from 201 BP to 16370 BP, with an average length of 1118 BP.The obtained transcriptome data were analyzed to screen the antifreeze glycoprotein gene (AFGPP) and the type IV antifreeze protein gene (AFP-IVP).Comparing the AFGP gene of Pacific cod with the AFGP gene of Pacific cod, it is found that the sequence results are similar and no coding region sequence appears. It may be that the repeat sequence is eliminated automatically in the process of sequencing, so the sequence result does not contain the coding region sequence.Type IV antifreeze protein gene was cloned by PCR and analyzed by bioinformatics.The results showed that the full length of cDNA: AFP- 鈪，

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