山羊卵巢褪黑素信号特异性载体pcDNA3.1-Pro/GDF9-ASMT与pcDNA3.1-Pro/Cyp17-MTNR1A/B的构建

doi:10.16431/j.cnki.1671-7236.2020.12.005

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (12): 3833-3843.doi: 10.16431/j.cnki.1671-7236.2020.12.005

山羊卵巢褪黑素信号特异性载体pcDNA3.1-Pro/GDF9-ASMT与pcDNA3.1-Pro/Cyp17-MTNR1A/B的构建

王晓东¹, 杨婵¹, 刘清华¹, 李振宇¹, 吴昊², 刘国世², 李翔¹, 何长久¹

1. 华中农业大学动物科技学院动物医学院, 农业动物遗传育种与繁殖教育部重点实验室, 国家级国际联合研究中心, 武汉 430070;
2. 中国农业大学动物科学技术学院, 北京 100193

收稿日期:2020-06-15 出版日期:2020-12-20 发布日期:2020-12-18
通讯作者: 何长久 E-mail:chungjoe@mail.hzau.edu.cn
作者简介:王晓东(1996-),男,河南洛阳人,硕士生,研究方向:动物繁殖生理调控,E-mail:1759818000@qq.com
基金资助:
农业部转基因重大专项（2018ZX08008-01B）；中央高校科研基本业务费（2662016QD038）

The Construction of the Goat Ovary Melatonin Signal Specific Vectors pcDNA3.1-Pro/GDF9-ASMT and pcDNA3.1-Pro/Cyp17-MTNR1A and MTNR1B

WANG Xiaodong¹, YANG Chan¹, LIU Qinghua¹, LI Zhenyu¹, WU Hao², LIU Guoshi², LI Xiang¹, HE Changjiu¹

1. National Center for International Research;Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education;College of Animal Sciences & Technology/College of Veterinary Medicine, Huazhong Agriculture University, Wuhan 430070, China;
2. College of Animal Science and Technology, China Agricultural University, Beijing 100083, China

Received:2020-06-15 Online:2020-12-20 Published:2020-12-18

摘要/Abstract

摘要： 本研究旨在构建褪黑素（melatonin，MLT）合成酶乙酰血清素甲基转移酶（acetylserotonin methytransferase，ASMT）的卵母细胞特异表达载体GDF9-ASMT和MLT受体（MTNR1A与MTNR1B）的颗粒（黄体）细胞特异表达载体Cyp17-MTNR1A、MTNR1B，以期为进一步生产高繁殖力转基因山羊提供载体材料。首先，分别扩增出山羊生长分化因子9（GDF9）与细胞色素P450家族17亚家族A成员1（Cyp17a1）启动子序列备用；然后，扩增ASMT、MTNR1A及MTNR1B基因CDS全长序列，并通过酶切将ASMT连接至GDF9启动子下游，将MTNR1A和MTNR1B连接至Cyp17a1启动子下游，制备出Pro/GDF9-ASMT、Pro/Cyp17-MTNR1A与Pro/Cyp17-MTNR1B表达元件；最后通过酶切连接将上述表达元件整合至pcDNA3.1（+）载体中，从而构建出pcDNA3.1-Pro/GDF9-ASMT、pcDNA3.1-Pro/Cyp17-MTNR1A和pcDNA3.1-Pro/Cyp17-MTNR1B真核表达载体。经测序验证，本研究扩增出的GDF9和Cyp17a1启动子长度分别为2 496和2 497 bp，与GenBank数据库中山羊GDF9和Cyp17a1基因起始密码子上游2 500 bp区域的同源性均为98%；扩增出的ASMT、MTNR1A与MTNR1B的CDS序列长度分别为1 037、1 100和1 130 bp，与GenBank所递交的标准序列同源性分别为98%、98%和99%，氨基酸序列同源性分别为97%、98%和99%。上述载体的成功构建可为进一步生产褪黑素高繁殖力转基因山羊提供参考。

关键词: 褪黑素; 黄体; 卵母细胞; ASMT; MTNR1A; MTNR1B

Abstract: This study intended to construct an oocyte-specific expression vector (GDF9-ASMT) for melatonin (MLT) synthetase acetylserotonin methytransferase (ASMT),and granule (luteal) cell-specific expression vector (Cyp17-MTNR1A,MTNR1B) for MLT receptors MTNR1A and MTNR1B,the construction of the above vectors provided carrier materials for further production of transgenic sheep with high fertility.First,the goat GDF9 and Cyp17a1 promoter sequences were amplified separately.Then,the full-length CDS sequences of ASMT,MTNR1A and MTNR1B genes were amplified,and ASMT gene was connected to the downstream of GDF9 promoter by enzyme-cutting,with MTNR1A and MTNR1B respectively connected to the downstream of Cyp17a1 promoter to prepare Pro/GDF9-ASMT,Pro/Cyp17-MTNR1A and Pro/Cyp17-MTNR1B expression elements.Finally,the above-mentioned expression elements were integrated into the pcDNA3.1(+) vector by enzyme digestion,thereby constructing pcDNA3.1-Pro/GDF9-ASMT,pcDNA3.1-Pro/Cyp17-MTNR1A and pcDNA3.1-Pro/Cyp17-MTNR1B eukaryotic expression vectors.After sequencing verification,the GDF9 and Cyp17a1 promoters amplified in this study were 2 496 and 2 497 bp in length respectively,and they were 98% homologous to the 2 500 bp region upstream of the start codons of the goat GDF9 and Cyp17a1 genes in GenBank database.The amplified ASMT,MTNR1A and MTNR1B CDS sequence lengths were 1 037,1 100 and 1 130 bp,and the homology with the standard sequences submitted by GenBank were 98%,98% and 99%.The amino acid sequence homologies was 97%,98%,99% respectively.The construction of the above vectors provided references for further production of melatonin transgenic sheep with high fertility.

Key words: melatonin; corpus luteum; oocyte; ASMT; MTNR1A; MTNR1B

中图分类号:

王晓东, 杨婵, 刘清华, 李振宇, 吴昊, 刘国世, 李翔, 何长久. 山羊卵巢褪黑素信号特异性载体pcDNA3.1-Pro/GDF9-ASMT与pcDNA3.1-Pro/Cyp17-MTNR1A/B的构建[J]. 中国畜牧兽医, 2020, 47(12): 3833-3843.

WANG Xiaodong, YANG Chan, LIU Qinghua, LI Zhenyu, WU Hao, LIU Guoshi, LI Xiang, HE Changjiu. The Construction of the Goat Ovary Melatonin Signal Specific Vectors pcDNA3.1-Pro/GDF9-ASMT and pcDNA3.1-Pro/Cyp17-MTNR1A and MTNR1B[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(12): 3833-3843.

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山羊卵巢褪黑素信号特异性载体pcDNA3.1-Pro/GDF9-ASMT与pcDNA3.1-Pro/Cyp17-MTNR1A/B的构建

The Construction of the Goat Ovary Melatonin Signal Specific Vectors pcDNA3.1-Pro/GDF9-ASMT and pcDNA3.1-Pro/Cyp17-MTNR1A and MTNR1B

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