犏牛L-PGDS基因克隆及其在各组织和睾丸不同发育阶段的表达研究

doi:10.16431/j.cnki.1671-7236.2020.12.021

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (12): 3985-3992.doi: 10.16431/j.cnki.1671-7236.2020.12.021

犏牛L-PGDS基因克隆及其在各组织和睾丸不同发育阶段的表达研究

穆松银^1,2, 熊显荣^1,2, 马鸿程^1,2, 海卓^1,2, 李键^1,2,3

1. 西南民族大学生命科学与技术学院, 成都 610041;
2. 青藏高原动物遗传资源保护与利用重点实验室, 成都 610041;
3. 西南民族大学国家民委动物科学重点实验室, 成都 610041

收稿日期:2020-04-20 出版日期:2020-12-20 发布日期:2020-12-18
通讯作者: 李键 E-mail:jianli_1967@163.com
作者简介:穆松银(1992-),男,河南范县人,硕士生,研究方向:动物细胞和胚胎工程,E-mail:musongyin@163.com
基金资助:
国家"十三五"重点研发专项（2018YFD0502304）；西南民族大学中央高校基本科研业务费专项资金项目（2020PTJS15005）

Cloning of L-PGDS Gene and Its Expression Pattern in Different Tissues and Testis at Different Development Stages in Cattle-yak

MU Songyin^1,2, XIONG Xianrong^1,2, MA Hongcheng^1,2, HAI Zhuo^1,2, LI Jian^1,2,3

1. College of Life Science and Technology, Southwest Minzu University, Chengdu 610041, China;
2. Key Laboratary of Qinghai-Tibetan Plateau Animal Centis Resource Reservation and Utolization of Ministry of Education, Chengdu 610041, China;
3. Key Laboratary of Animal Science, State Ethnic Affairs Commission, Southwest Minzu University, Chengdu 610041, China

Received:2020-04-20 Online:2020-12-20 Published:2020-12-18

摘要/Abstract

摘要： 本研究旨在克隆犏牛前列腺素D合成酶（prostagland D synthase，L-PGDS）基因序列，并检测其在不同组织及不同发育时期睾丸中的表达水平，从而为深入研究L-PGDS基因在犏牛睾丸发育过程中的作用机制提供依据。采集成年健康犏牛心脏、肝脏、脾脏、肺脏、肾脏、大肠、小肠、胃、大脑及不同发育时期犏牛睾丸组织：胎牛期（5~6月）、幼年期（1~2岁）、青春期（2.5~4岁）、老年期（7~9岁），Trizol法提取各组织总RNA，利用RT-PCR技术扩增、克隆L-PGDS基因序列并利用相关生物学软件进行分析，利用实时荧光定量PCR检测犏牛各组织及4个不同发育阶段睾丸中L-PGDS基因mRNA的表达水平。结果显示，L-PGDS基因序列全长624 bp，包括完整的开放阅读框576 bp，编码191个氨基酸。同源性比对发现，犏牛与黄牛和绵羊的同源性最高，分别为99.28%和94.0%。系统进化树分析结果显示，犏牛与黄牛亲缘关系最近，其次是绵羊、马。L-PGDS蛋白为不稳定疏水蛋白，分子质量为21.229 ku，分子式为C₉₅₃H₁₄₇₁N₂₅₁O₂₈₁S₉，等电点为6.43，不稳定指数为47.25，不含跨膜区及信号肽，蛋白质二级结构预测显示α-螺旋、无规则卷曲、延伸链分别占25.65%、53.40%和20.94%。实时荧光定量PCR检测结果显示，L-PGDS基因在犏牛睾丸组织中特异性高表达，极显著高于其他组织（P<0.01）；随着年龄增长L-PGDS基因mRNA在犏牛睾丸中呈先上升后下降趋势，其中在青春期相对表达量最高，极显著高于其他时期（P<0.01）。本研究结果为深入探究L-PGDS基因在犏牛睾丸发育过程中的作用机制提供了基础资料。

关键词: L-PGDS基因; 犏牛; 睾丸; 组织表达谱

Abstract: The purpose of this study was to clone the prostaglandin D synthase (L-PGDS) gene and identify its expression pattern in various tissues and testis at different development stages in cattle-yak,in order to provide experimental basis for studying the mechanism of L-PGDS gene in testis development of cattle-yak.The total RNA of heart,liver,spleen,lung,kidney,large intestine,small intestine,gastric,brain and testis at different development stages (fetal,calve tuberty and old) of healthy cattle-yak were extracted by Trizol method.The L-PGDS gene sequence was amplified and cloned by RT-PCR,and analyzed by related biological software.The expression of L-PGDS gene mRNA in different tissues and testicular tissues at different development period of in cattle-yak were detected by Real-time quantitative PCR.The results showed that the length of L-PGDS gene was 624 bp,including the ORF of 576 bp,which encoded 191 amino acids.Homology alignment results showed that L-PGDS gene in cattle-yak had high homology with Bos taurus and Ovis aries,which were 99.28% and 94.00%,respectively.Phylogenetic tree results showed that the relation between cattle-yak and Bos taurus was the closest,followed by Ovis aries and Equus caballus.The L-PGDS protein was an unstable hydrophobic protein,the molecular weight was 21.229 ku,the molecular formula was C₉₅₃H₁₄₇₁N₂₅₁O₂₈₁S₉,the isoelectric point was 6.43,and the instability index was 47.25.The L-PGDS protein had no transmembrane region and no signal peptide.The secondary structure of L-PGDS protein contained alpha helix,random coil and extended chain,the rats of them were 25.65%,53.40% and 20.94%,respectively.L-PGDS gene was highly expressed in testis of cattle-yak,which was extremely significantly higher than that in other tissues (P<0.01).The expression of L-PGDS gene mRNA in testis of cattle-yak increased firstly and then decreased along with age,and with the highest expression in puberty stage,which was extremely significantly higher than that at other periods (P<0.01).This study provided basic data for further study on the mechanism of L-PGDS gene in the development of testis in cattle-yak.

Key words: L-PGDS gene; cattle-yak; testis; tissue expression pattern

中图分类号:

穆松银, 熊显荣, 马鸿程, 海卓, 李键. 犏牛L-PGDS基因克隆及其在各组织和睾丸不同发育阶段的表达研究[J]. 中国畜牧兽医, 2020, 47(12): 3985-3992.

MU Songyin, XIONG Xianrong, MA Hongcheng, HAI Zhuo, LI Jian. Cloning of L-PGDS Gene and Its Expression Pattern in Different Tissues and Testis at Different Development Stages in Cattle-yak[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(12): 3985-3992.

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犏牛L-PGDS基因克隆及其在各组织和睾丸不同发育阶段的表达研究

Cloning of L-PGDS Gene and Its Expression Pattern in Different Tissues and Testis at Different Development Stages in Cattle-yak

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