红鲫Gsk3β基因克隆与分化表达模式

张卉琴; 张永勤; 邓美霞; 桂赛玉; 文苑桧; 刘锦辉; 陈志男; 李晓超; 肖亚梅; 李万程; 刘文彬

doi:doi:10.3969/j.issn.1007-7146.2018.01.007

激光生物学报, 2018, 27 (1): 45, 网络出版: 2018-04-23

红鲫Gsk3β基因克隆与分化表达模式

A Study on Cloning and Differential Expression of Gsk3β in Red crucian carp

论文大纲

张卉琴张永勤邓美霞桂赛玉文苑桧刘锦辉陈志男李晓超肖亚梅李万程刘文彬

作者单位

省部共建淡水鱼类发育生物学重点实验室, 湖南师范大学生命科学学院, 湖南长沙 410081

基因克隆 Gsk3β Gsk3β gene cloning PCR PCR western blot western blot

摘要

目的: 本文以糖原合成酶激酶3β(glycogen synthase kinase 3 beta, Gsk3β)为研究对象, 探讨Gsk3β基因在红鲫胚胎发育及成体组织中的表达模式, 以及在镉金属胁迫下的分子应答初探。方法: 采用RACE克隆技术获得红鲫Gsk3β cDNA全长, 半定量RT-PCR技术和Western Blot技术分析Gsk3β在mRNA水平和蛋白水平的表达情况, 最后通过特定浓度的镉处理分析Gsk3β的表达情况。结果: 通过克隆发现Gsk3β基因全长为2 195 bp, 5′端非编码区(UTR)共692 bp, 3′非编码区有237 bp。编码含421个氨基酸的蛋白质, 蛋白分子量约为46 846.40, 等电点为9.05, 且与斑马鱼和青鳉的亲缘关系最近。Gsk3β基因在胚胎发育和成体组织中呈现不同程度的表达, 在不同组织中, Gsk3β在大脑组织表达最丰富, 其次在眼睛, 肌肉和精巢中表达相对较高, 在卵巢、肝脏、肾脏和皮肤组织表达量相对较低。在不同发育时期, Gsk3β在两细胞期没有表达, 之后在后续的不同时期呈现差异表达, 出膜期表达最强, 其次为肌效期、体节期、神经胚、囊胚期表达次之, 在黑色素期和体色素期蛋白表达较弱。在镉处理后的红鲫肝脏中检测Gsk3β基因的表达, 发现Gsk3β表达下调。结论: Gsk3β基因在红鲫胚胎发育中起着重要作用, 且参与了镉金属胁迫应答, 但其具体的分子机制仍有待进一步研究。

Abstract

Objective: To investigate the expression pattern of Gsk3β gene in different developmental stages and adult tissues of red crucian carp, as well as its molecular response under cadmium stress. Methods: The full length of Gsk3β cDNA was obtained by RACE cloning technique, which Gsk3β mRNA and protein was analyzed by semi-quantitative RT-PCR and Western Blot.The expression of Gsk3β was analyzed by specific concentration of cadmium. Results: The total length of Gsk3β gene was 2 195 bp,the 5′untranslated region (UTR) was 692 bp, and the 3′ untranslated region was 237 bp. Encoding 421 amino acids containing protein and protein molecular weight was 46 846.40, its isoelectric point was 9.05,and has the closest relationship with zebrafish and medaka.Gsk3β gene was expressed in different embryonic developmental stages and adult tissues.The expression of Gsk3β was most abundant in the brain tissue, and the expression of Gsk3β in the muscle and testis was higher than in the liver and kidney, ovaries and skin tissues. At different developmental stages, Gsk3β was not expressed in the two-cell stage, and then expressed at different stages of follow-up. The expression of Gsk3β was the strongest in the last stage, followed by the duration of muscle, In the eye pigment period and somatic stage protein expression is weak. The expression of Gsk3β related genes were detected in the liver of red crucian carp after cadmium treatment, and the result showed that expression level of Gsk3β was in down-regulation. Conclusion: Gsk3β gene plays an important role in different embryonic developmental stages of red crucian carp and participates in cadmium stress response, but its specific molecular mechanism remains to be further studied.

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张卉琴, 张永勤, 邓美霞, 桂赛玉, 文苑桧, 刘锦辉, 陈志男, 李晓超, 肖亚梅, 李万程, 刘文彬. 红鲫Gsk3β基因克隆与分化表达模式[J]. 激光生物学报, 2018, 27(1): 45. ZHANG Huiqing, ZHANG Yongqin, DENG Meixia, GUI Saiyu, WEN Yuanhui, LIU Jinhui, CHEN Zhinan, LI Xiaochao, XIAO Yamei, LI Wancheng, LIU Wenbin. A Study on Cloning and Differential Expression of Gsk3β in Red crucian carp[J]. Acta Laser Biology Sinica, 2018, 27(1): 45.

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