斑鱾dmrt1的克隆及其表达分析  

Cloning and expression of dmrt1 gene in Girella punctata

作　　者：温思民 翟毅 黄洋[1,2] 朱春华 李广丽[1,2,3] WEN Simin;ZHAI Yi;HUANG Yang;ZHU Chunhua;LI Guangli(Fisheries College, Guangdong Ocean University,Zhanjiang Guangdong 524088, China;Guangdong Research Center on Reproductive Control and Breeding Technology of Indigenous Valuable Fish Species, Zhanjiang Guangdong 524088, China;Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Zhanjiang Guangdong 524088, China;Guangdong Provincial Engineering Laboratory for Mariculture Organism Breeding, Zhanjiang Guangdong 524088, China)

机构地区：[1]广东海洋大学水产学院,广东湛江524088 [2]广东省名特优鱼类生殖调控与繁育工程技术研究中心,广东湛江524088 [3]广东省水产经济动物病原生物学及流行病学重点实验室,广东湛江524088 [4]广东省海水养殖生物育种工程实验室,广东湛江524088

出　　处：《海洋渔业》2022年第2期187-200,共14页Marine Fisheries

基　　金：湛江市科技计划项目(2017A03019)。

摘　　要：克隆了斑鱾(Girella punctata)dmrt1(doublesex and mab-3 related transcription factor 1)基因,用DNAMAN 9软件比较了Dmrt1蛋白的氨基酸同源性,用MEGAX软件构建了Dmrt1系统进化树,并应用实时荧光定量PCR方法检测dmrt1 mRNA在斑鱾雌雄性腺和不同胚胎发育阶段的表达。结果显示,斑鱾dmrt1的开放阅读框(ORF)全长894 bp,编码297个氨基酸,氨基酸序列含有典型的DM结构域。斑鱾Dmrt1氨基酸同源性比对分析发现,它与黄金鲈(Perca flavescens)的同源性最高,为85.71%;与人类(Homo sapiens)同源性最低,为32.89%。斑鱾Dmrt1与其他鲈形目鱼类在系统进化树上聚为一支,与传统分类地位一致。斑鱾dmrt1在精巢中有较高表达(P<0.05),在卵巢中微弱表达。此外在胚胎发育过程中,dmrt1的表达水平呈先升后降的趋势,在桑葚期表达水平最高(P<0.05)。综上所述,dmrt1可能参与了斑鱾性腺分化和早期胚胎发育。Doublesex and mab-3 related transcription factors(dmrt)is a family of genes,which are highly homologous with Drosophila melanogaster doublesex(dsx)and Caenorhabditis elegans male abnormal-3(mab-3).Dmrt proteins have a highly conserved doublesex and male aberrant-3 relative domain(DM domain),which can combine with the specific DNA sequences and determine the regulation of the expression of downstream genes.The doublesex and mab-3 related transcription factor 1(dmrt1)is one of dmrt family and has the classical DM domain.This gene is widely present in mammals,birds,reptiles and fish,and determines gender differentiation.In this study,3 male and 3 female Girella punctata were killed humanely using tricainemethanesulfonate(MS-222,Sigma,St.Louis,MO,USA)for samples of the gonad tissues;in addition,embryos at different developmental stages were observed,and collected for samples.All experimental procedures were carried out by the Guidelines for the Care and Use of Laboratory Animals in China,and the protocol was approved by the Experimental Animal Ethics Committee of Guangdong Ocean University,China.Embryo samples and part of fresh gonad tissues were frozen immediately in liquid nitrogen to extract total RNA,and the rest of the ganod tissues were fixed in Bouin’s solution for subsequent dehydration with alcohol,transparent with xylene,embedded in paraffin,sectioned,and stained with hematoxylin-eosin(HE)for histological observation.Total RNA was extracted using Trizol reagent kit(Invitrogen,Carlsbad,CA,USA),and synthesized the fist-strand cDNA following the PrimeScript^(TM) RT reagent kit with gDNA eraser manufacturer’s protocol(TaKaRa,RR047A,Japan).The relative expression of dmrt1 gene in different tissues was calculated usingβ-actin as an internal reference.The specific primers of dmrt1 gene were designed by Primer Premier 5.0.The dmrt1 gene of G.punctata was cloned and bioinformatics were analyzed in its base sequence.The full-length open reading frame(ORF)of dmrt1 gene of G.punctata was 894 base pairs,encoding 29

关 键 词：斑鱾 DMRT1 基因克隆 性腺 胚胎发育 

分 类 号：S917.4[农业科学—水产科学]