泰国斗鱼酪氨酸羟化酶基因cDNA的克隆及表达分析  

The cDNA Cloning and Expression Analysis of Tyrosine Hydroxylase Gene from Betta Splendens

作　　者：马细兰 林志远 蓝筱敏 肖博怡 崔语 MA XiLan;LIN Zhiyuan;LAN Xiaomin;XIAO Boyi;CUI Yu(School of Life Science,Huizhou University,Huizhou 516007,Guangdong,China)

机构地区：[1]惠州学院生命科学学院,广东惠州516007

出　　处：《惠州学院学报》2021年第6期44-51,共8页Journal of Huizhou University

基　　金：惠州学院重点学科培育项目(HZU201807)。

摘　　要：泰国斗鱼(Betta splendens)雄性天生好斗,适合作为鱼类好斗性研究的模型生物.首先通过RT-PCR从泰国斗鱼的全脑中克隆并鉴定出酪氨酸羟化酶基因(tyrosine hydroxylase gene,th)的部分cDNA片段,并进行序列分析;然后通过镜像斗争法和配对斗争法观察泰国斗鱼的打斗行为,最后用实时定量PCR检测比较其在斗争前、后及优、劣势个体间th的mRNA表达差异.克隆获得了261 bp泰国斗鱼th部分核苷酸序列,编码86个氨基酸;氨基酸同源性显示,泰国斗鱼th与同为鲈形目、攀鲈亚目龟壳攀鲈(Anabas testudineus)的同源性最高为98.82%,遗传距离最小,为0.14168;而与爬行纲、有鳞目的鬓狮蜥(Pogona vitticeps)遗传距离最大,为0.37119;与泰国斗鱼th的亲缘关系由近至远分别为鱼纲、哺乳纲、鸟纲和爬行纲.行为学观察显示,镜子放入烧杯后,斗鱼立即表现出张鳃、咬镜、撞镜等好斗性行为;配对斗争更为剧烈,两鱼相互撕咬、撞击、追逐,直止分出胜负.实时定量PCR检测结果显示,th在优势个体中的表达水平显著高于劣势个体;经配对斗争或镜像斗争后的个体其th的mRNA表达水平均高于斗争前个体,由此推测泰国斗鱼th可能与其好斗性正相关.Aggression is an adaptive behavior characteristic of animals,which plays a very important role in animal mate selection,food competition,territory occupation,social class establishment and so on.Betta splendens males are naturally aggressive.They are suitable to be used as a model organism to study the aggressiveness of fish.To clone and identify the cDNA fragment of tyrosine hydroxylase gene(th)from the brain of Betta splendens.The homologous analysis of th related sequence needed to be carried out with the tentative exploration of the difference of mRNA expression between individuals before and after the struggle,dominant individuals and inferior individuals,which could lay a foundation for further study of the molecular regulatory mechanism of th in Betta splendens.Firstly,using Trizol to extract and purify the total RNA which served as the template for later reverse transcription from the whole brain of Betta splendens.Secondly,designing several pairs of primers based on th sequence of several fish having close relationship with Betta splendens.Thirdly,amplifying target cDNA fragment by PCR.Fourthly,comparing homology in target nucleotide and amino acid sequence by BLAST,MEGA7 and DNA Star.Lastly,observing the fighting behavior of Betta splendens by mirror struggle method and pairing struggle method and exploring preliminarily the difference of m RNA expression between different individuals by RT-qPCR.Partial nucleotide sequence with 261 bp of th in Betta splendens,coding for 86 amino acids,was cloned correctly.The molecular size of the peptide was 9447.35 Dalton and the isoelectric point was 3.81.Amino acid homology showed that the homology of th between Betta splendens and Anabas testudineus was the highest,98.82%.They were both Perciformes and subperca,and the genetic distance of th between them was the smallest,0.14168;The genetic distance from Reptilia,Pogona vitticeps was the largest,0.37119;The phylogenetic relationships of th gene in Betta splendens from near to far are fish,Mammalia,birds and Reptili

关 键 词：泰国斗鱼 好斗性 酪氨酸羟化酶基因 基因表达 

分 类 号：S917.4[农业科学—水产科学]