This work aims to clarify the structure and expression characteristics of dct gene in Pelodiscus sinensis, which belongs to tyrosinase family. Primers were designed according mRNA sequence of dct gene in Ensembl online database. Opening reading frame(ORF )region was cloned using RT-PCR and the bioinformatics analysis of dct ORF sequence was carried out. At the same time, mRNAs expression levels of lung, kidney, calipash and muscle tissue in P. sinensis were detected via q-PCR method. The results showed that the full length of ORF of dct gene in P.sinensis was 1 578 bp, encoding a putative 525 aa; The sequence- DOI:10.3969/j.issn.1000-1565.2019.06.009中华鳖dct基因克隆及表达分析刘文婷1,许鹏2,张娜1,管越强1,张耀红3(1. 河北大学 生命科学学院,河北 保定 071002;2. 河北省药品检验研究院,河北 石家庄 050011;3. 保定水产技术推广站,河北 保定 071051)摘 要:为了阐明中华鳖酪氨酸酶家族成员dct基因的结构与表达特征,根据Ensembl数据库的中华鳖dct基因序列设计引物,运用RT-PCR技术进行开放阅读框(opening reading frame,ORF)克隆,并进行生物信息学分析,进而采用实时荧光定量PCR(quantitative real-time PCR,q-PCR)技术检测正常体色和黄色中华鳖肺、肾脏、裙边和肌肉组织的mRNA表达水平.结果显示中华鳖dct基因ORF区全长1 578 bp,编码525个氨基酸,2种体色鳖dct基因序列完全一致;氨基酸序列分析得C末端无L-亮氨酸基序;N末端含有1个信号肽结构域;预测蛋白(成熟肽)分子质量为56.449 ku,理论等电点为6.67,不稳定指数为38.71,表明DCT蛋白是酸性稳定蛋白;预测DCT蛋白为亲水性蛋白;含有跨膜结构域、类表皮生长因子结构域、层粘连蛋白型类表皮生长因子结构域和2个铜离子结合结构域.系统发育树分析显示,在dct基因进化过程中,中华鳖与绿海龟和锦龟的亲缘关系较近.q-PCR结果表明:dct基因在同一个体各组织中均有表达,肺组织中相对表达量最高,且极显著高于其他3种组织;黄色中华鳖肺、肾脏和裙边组织dct 表达水平与正常体色鳖的相应组织均存在显著性差异.结果表明,中华鳖体色变异与dct基因突变无关,但dct基因表达改变可能对体色变异起了一定的作用.关键词:中华鳖;dct基因;克隆;生物信息学;q-PCR中图分类号:S917.4 文献标志码:A 文章编号:1000-1565(2019)06-0619-10Cloning and expression analysis of dct gene in Pelodiscus sinensisLIU Wenting1, XU Peng2, ZHANG Na1, GUAN Yueqiang1, ZHANG Yaohong3(1. College of Life Sciences,Hebei University,Baoding 071002, China;2. Hebei Institute for Drug Control and Testing, Shijiazhuang 050011, China;3. Baoding Fishery Technology Extension Station, Baoding 071051, China)Abstract: This work aims to clarify the structure and expression characteristics of dct gene in Pelodiscus sinensis, which belongs to tyrosinase family. Primers were designed according mRNA sequence of dct gene in Ensembl online database. Opening reading frame(ORF )region was cloned using RT-PCR and the bioinformatics analysis of dct ORF sequence was carried out. At the same time, mRNAs expression levels of lung, kidney, calipash and muscle tissue in P. sinensis were detected via q-PCR method. The results showed that the full length of ORF of dct gene in P.sinensis was 1 578 bp, encoding a putative 525 aa; The sequence- 收稿日期:2019-03-19 基金项目:河北省自然科学基金资助项目(C2016201207);河北省现代农业产业技术体系淡水养殖创新团队(HBCT2018180203) 第一作者:刘文婷(1990—),女,安徽合肥人,河北大学在读硕士研究生.E-mail: 280408479@qq.com 通信作者:管越强(1973—),男,河北深泽人,河北大学教授,博士,主要从事水生动物营养与免疫研究.E-mail:guanyueqiang@hbu.edu.cn第6期刘文婷等:中华鳖dct基因克隆及表达分析of dct gene was completely identical in two groups of turtles with different body colours of P. sinensis. The amino acid sequence analysis result showed that L-leocine motif was not observed in the C-terminus. A signal peptide domain was located in the N-terminus. The deduced molecular weight of DCT protein(Mature Peptide)was 56.449 ku, the theoretical isoelectric point was 6.67, and the instability index was 38.71, which indicated it was an acid stable protein. Prediction of hydrophilicity showed that DCT protein was a hydrophilic protein. It contain transmembrane domain, EGF-like domain, laminin-type EGF-like(LE)domain and two copper ion-binding domains. Phylogenetic tree of DCT protein showed that P.sinesis, green sea turtle Chelonia mydas, and Western painted turtle Chrysemys picta clustered into one clade, and the relationship among them was close. The results of q-PCR showed that dct gene was expressed in each detected tissue of the same individual and the expression level of lung tissue was highest and was very significantly higher than the other three detected tissues; In the same tissues of two group with different body colours, there were significant differences in lung, kidney and calipash, respectively.The results showed that the body colour variation of P.sinensis was not related to the mutation of dct gene, and the change of dct gene expression may play a role in the body color variation.
LIU Wenting
,
XU Peng
,
ZHANG Na
,
GUAN Yueqiang
,
ZHANG Yaohong
. Cloning and expression analysis of dct gene in Pelodiscus sinensis[J]. Journal of Hebei University(Natural Science Edition), 2019
, 39(6)
: 619
-628
.
DOI: 10.3969/j.issn.1000-1565.2019.06.009
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