猪CCAR1基因细胞定位、表达特性及对细胞增殖的影响

doi:10.11843/j.issn.0366-6964.2020.11.005

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (11): 2651-2664.doi: 10.11843/j.issn.0366-6964.2020.11.005

猪CCAR1基因细胞定位、表达特性及对细胞增殖的影响

张宁芳¹, 吴怡琦¹, 成志敏², 杨晓伟³, 李萌¹, 杨阳¹, 刘宏⁴, 高鹏飞¹, 蔡春波¹, 郭晓红¹, 李步高¹, 曹果清^1*

1. 山西农业大学动物科学学院, 太谷 030801;
2. 山西省高等创新研究院, 太原 030032;
3. 大同海关技术中心, 大同 037000;
4. 大同市种猪场, 大同 037000

收稿日期:2020-05-29 出版日期:2020-11-25 发布日期:2020-11-20
通讯作者: 曹果清,主要从事猪新品种(系)培育与推广和地方猪种特色基因研究与利用,E-mail:anniecao710502@aliyun.com
作者简介:张宁芳(1993-),女,山西晋城人,硕士,主要从事动物遗传育种与繁殖研究,E-mail:1097482023@qq.com;吴怡琦(1996-),女,山西运城人,硕士生,主要从事动物遗传育种与繁殖研究,E-mail:946095068@qq.com
基金资助:
三晋学者支持计划专项经费资助（2016；2017）；国家自然科学基金（31872336）；山西省农业重点研发项目（201803D221022-1）；“山西省1331工程”资助（2017）

Cellular Localization, Expression Patterns of CCAR1 Gene, and Its Effect on Cell Proliferation in Pig

ZHANG Ningfang¹, WU Yiqi¹, CHENG Zhimin², YANG Xiaowei³, LI Meng¹, YANG Yang¹, LIU Hong⁴, GAO Pengfei¹, CAI Chunbo¹, GUO Xiaohong¹, LI Bugao¹, CAO Guoqing^1*

1. College of Animal Science, Shanxi Agricultural University, Taigu 030801, China;
2. Shanxi Academy of Advanced Research and Innovation, Taiyuan 030032, China;
3. Datong Customs Technology Center, Datong 037000, China;
4. Datong Pig Breeding Farm, Datong 037000, China

Received:2020-05-29 Online:2020-11-25 Published:2020-11-20

摘要/Abstract

摘要： 旨在获得猪CCAR1基因的完整CDS序列，研究其亚细胞定位和表达特性，探究其对细胞增殖的影响和作用机制。本研究以1日龄马身猪肾组织cDNA为模板，采用RT-PCR技术分段扩增猪CCAR1基因的CDS区，通过测序和序列拼接获得完整CDS区；采用细胞免疫荧光技术检测CCAR1在PK15细胞中的定位；采用qRT-PCR技术检测猪CCAR1基因的时空表达规律；采用CRISPR/Cas9基因编辑技术敲除PK15细胞的CCAR1基因，通过qRT-PCR、Western blot以及CCK8（cell counting kit 8）技术检测CCAR1基因敲除对细胞增殖能力及细胞增殖和凋亡相关基因表达的影响。结果表明，猪CCAR1基因的完整CDS区长3 459 bp（MH301308.1），在PK15细胞的细胞质和细胞核中均有表达。大白猪和马身猪不同组织CCAR1的表达谱基本相似，在所检测的组织中均有表达，均表现为在肾和小肠中表达量最高，在脾、肝、小脑和肌肉中呈中度表达，在心和皮下脂肪中低表达；CCAR1基因在大白猪和马身猪初生、3月龄和6月龄3个年龄阶段的两种骨骼肌中也均有表达。CRISPR/Cas9基因编辑系统能有效降低CCAR1的表达，在转染48 h后，相比于对照组，试验组都对细胞增殖产生极显著抑制（P<0.01）；CCAR1基因敲除后，细胞增殖标记基因Mki67表达水平显著下降（P<0.05），Wnt通路下游靶基因C-myc表达量显著下降（P<0.05），Wnt通路核心蛋白β-catenin和凋亡标记基因Caspase3的表达量无显著差异。CCAR1基因在猪不同组织和不同发育阶段均有表达，可能通过调控细胞增殖基因Mki67和Wnt通路下游靶基因C-myc的表达而影响细胞增殖，在猪的生长发育过程中起重要作用。

关键词: 猪, CCAR1基因, 细胞定位, 表达特征, 基因敲除, 细胞增殖

Abstract: The aim of this study was to obtain the complete coding sequence (CDS) of CCAR1 gene, and to explore its subcellular localization, expression profile and its effects on cell prolification and action mechanism in pig. In this study, the cDNA from kidney tissue of 1-day-old Mashen pigs were used as the template to obtain the full-length CDS of CCAR1 gene by RT-PCR and sequencing. The cellular immunofluorescence staining was used to explore the subcellular localization of CCAR1 in PK15 cells. The temporal and spatial expression profile of CCAR1 was investigated by qRT-PCR in this study. The CCAR1 gene in PK15 cells was knocked out by using CRISPR/Cas9 gene editing technology, and the effects of CCAR1 gene on cell proliferation and expression of cell proliferation and apoptosis related genes were investigated by qRT-PCR, Western blot and CCK8 (cell counting kit 8) technologies in this experiment. The results showed that the complete CDS region of pig CCAR1 gene was 3 459 bp in length (MH301308.1). CCAR1 protein was localized in both cytoplasm and nucleus of PK15 cells. The expression profiles of CCAR1 mRNA between Large White and Mashen pigs was similar, which was expressed in all detected tissues, with the highest expression in kidney and small intestine, middle expression in spleen, liver, cerebellum and muscle, and the lowest expression in heart and subcutaneous fat. Temporal expression results showed that CCAR1 was expressed in both psoas muscle and longissimus dorsi muscle at 3 developmental stages both in Mashen and Large White pigs. The CRISPR/Cas9 gene editing system effectively reduced the expression of CCAR1. CCK8 results showed that after 48 hours of transfection, compared with the control group, the proliferation of cells in the experimental groups were extremely significantly inhibited (P<0.01). After CCAR1 gene knocked out, the expression level of Mki67, a marker of cell proliferation, was significantly decreased (P<0.05). There was no significant difference in the expression level of Caspase3 and the core protein β-catenin in Wnt pathway between control group and experimental groups, and the expression level of downstream target gene C-myc of Wnt pathway was decreased significantly(P<0.05). CCAR1 gene was expressed almost in all tissues at different developmental stages, and affected cell proliferation by regulating the expression levels of Mki67 and C-myc, and played an important role in growth and development of pig.

Key words: pig, CCAR1 gene, cellular localization, expression patterns, gene knockout, cell proliferation

中图分类号:

S828.2

张宁芳, 吴怡琦, 成志敏, 杨晓伟, 李萌, 杨阳, 刘宏, 高鹏飞, 蔡春波, 郭晓红, 李步高, 曹果清. 猪CCAR1基因细胞定位、表达特性及对细胞增殖的影响[J]. 畜牧兽医学报, 2020, 51(11): 2651-2664.

ZHANG Ningfang, WU Yiqi, CHENG Zhimin, YANG Xiaowei, LI Meng, YANG Yang, LIU Hong, GAO Pengfei, CAI Chunbo, GUO Xiaohong, LI Bugao, CAO Guoqing. Cellular Localization, Expression Patterns of CCAR1 Gene, and Its Effect on Cell Proliferation in Pig[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(11): 2651-2664.

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猪CCAR1基因细胞定位、表达特性及对细胞增殖的影响

Cellular Localization, Expression Patterns of CCAR1 Gene, and Its Effect on Cell Proliferation in Pig

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