湖羊Sp1基因CDS区克隆及其对颗粒细胞增殖和凋亡的影响

doi:10.11843/j.issn.0366-6964.2017.11.011

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (11): 2098-2106.doi: 10.11843/j.issn.0366-6964.2017.11.011

湖羊Sp1基因CDS区克隆及其对颗粒细胞增殖和凋亡的影响

姚一龙^1,3#, 李隐侠^1,2,4#, 安外尔·热合曼³, 张俊^1,2,4, 孟春花^1,2,4, 王慧利^1,2,4, 钱勇^1,2,4, 曹少先^1,2,4*

1. 江苏省农业科学院畜牧研究所, 南京 210014;
2. 江苏省农业科学院动物品种改良和繁育重点实验室, 南京 210014;
3. 南京农业大学动物科技学院, 南京 210095;
4. 江苏省农业种质资源保护与利用平台, 南京 210014

收稿日期:2017-06-15 出版日期:2017-11-23 发布日期:2017-11-23
通讯作者: 曹少先,E-mail:sxcao@jaas.ac.cn
作者简介:姚一龙(1995-),男,山西大同人,硕士生,主要从事动物遗传育种研究,E-mail:1127789951@qq.com;李隐侠(1979-),女,河南固始人,博士,主要从事动物遗传育种研究,E-mail:liyxmh@126.com。
基金资助:
江苏省农业科技自主创新资金（CX （15）1007）；江苏省自然科学基金（BK20140705）

Cloning of Sp1 Gene CDS Region of Hu Sheep and Its Effect on Proliferation and Apoptosis of Granulosa Cells

YAO Yi-long^1,3#, LI Yin-xia^1,2,4#, REHEMAN·Anwaier³, ZHANG Jun^1,2,4, MENG Chun-hua^1,2,4, WANG Hui-li^1,2,4, QIAN Yong^1,2,4, CAO Shao-xian^1,2,4*

1. Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;
2. Key Laboratory of Animal Breeding and Reproduction, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;
3. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China;
4. The Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Nanjing 210014, China

Received:2017-06-15 Online:2017-11-23 Published:2017-11-23
Supported by:

摘要/Abstract

摘要：

旨在探讨湖羊Sp1基因序列特征及其对颗粒细胞增殖及凋亡的影响。采用克隆、测序获得湖羊Sp1基因CDS区并用生物信息学方法分析其序列特征；NJ方法构建基于Sp1氨基酸序列的系统进化树；RT-PCR方法检测Sp1基因湖羊组织表达谱；克隆方法构建湖羊Sp1过表达载体；试剂盒检测Caspase-3活性和CCK-8值；利用流式细胞术检测人卵巢颗粒细胞（KGN）凋亡率。结果表明，湖羊Sp1 CDS区长2 340 bp，编码779个氨基酸残基，与人的核苷酸序列和氨基酸序列的同源性分别为94.28%和95.75%；湖羊与牛的亲缘关系最近；Sp1基因在湖羊卵巢、卵泡等组织中广泛表达。本研究成功构建了湖羊Sp1过表达载体并转染人卵巢颗粒细胞（KGN）；试验组Caspase-3活性显著高于对照组（P<0.05），CCK-8值检测结果相反。表明过表达Sp1基因抑制KGN-细胞增殖（P<0.05）、促进KGN-细胞凋亡（P<0.05）。Sp1基因在哺乳动物中高度保守，湖羊Sp1基因可抑制人卵巢颗粒细胞（KGN）增殖、诱导其凋亡。

Abstract:

The aim of this study was to explore the sequence characteristics of Sp1 gene of Hu sheep and its effect on proliferation and apoptosis in granulosa cells. The CDS region of Sp1 gene was obtained by cloning and sequencing, and was analyzed by bioinformatics methods. Phylogenetic tree was constructed based on Sp1 amino acid sequence through NJ method. The expression profile of Sp1 was detected by RT-PCR. The Sp1 overexpression vector of Hu sheep was constructed by cloning, the activity of Caspase-3 and CCK-8 value was detected by correspoding kits; and the apoptosis rate of human ovarian granulosa cells (KGN) was detect by the flow cytometry. The results showed that the length of Sp1 CDS region was 2 340 bp and encoded 799 amino acid residues, which were 94.28% and 95.75% homology with the nucleotide sequence and amino acid sequence of human, respectively; Hu sheep had closer relationship with cow; Sp1 gene was widely expressed in many tissues of Hu sheep such as ovary, granulosa cells, and so on. Sp1 overexpression vector was constructed and transfected KGN cells successfully; the activity of Caspase-3 in the experimental group was remarkably higher than that in the control group (P<0.05) and CCK-8 value was just the reverse, which suggesting that overexpression of Sp1 gene inhibited the proliferation of KGN cells(P<0.05) and promoted the apoptosis of KGN cells (P<0.05).The Sp1 gene of Hu sheep was highly conserved in mammals, it could inhibit the proliferation of KGN and lead to its apoptosis.

中图分类号:

S826.2

姚一龙, 李隐侠, 安外尔热合曼, 张俊, 孟春花, 王慧利, 钱勇, 曹少先. 湖羊Sp1基因CDS区克隆及其对颗粒细胞增殖和凋亡的影响[J]. 畜牧兽医学报, 2017, 48(11): 2098-2106.

YAO Yi-long, LI Yin-xia, REHEMAN Anwaier, ZHANG Jun, MENG Chun-hua, WANG Hui-li, QIAN Yong, CAO Shao-xian. Cloning of Sp1 Gene CDS Region of Hu Sheep and Its Effect on Proliferation and Apoptosis of Granulosa Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(11): 2098-2106.

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湖羊Sp1基因CDS区克隆及其对颗粒细胞增殖和凋亡的影响

Cloning of Sp1 Gene CDS Region of Hu Sheep and Its Effect on Proliferation and Apoptosis of Granulosa Cells

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