MEI1基因可变剪切事件对蒙古马精子生成的调控作用

doi:10.11843/j.issn.0366-6964.2022.04.010

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (4): 1096-1108.doi: 10.11843/j.issn.0366-6964.2022.04.010

MEI1基因可变剪切事件对蒙古马精子生成的调控作用

崔迎迎¹, 芒来^1*, 李蓓^1*, 特日格乐¹, 赵一萍¹, 任秀娟¹, 宋连杰², 苏少锋^1,3

1. 内蒙古农业大学动物科学学院, 内蒙古自治区马属动物遗传育种与繁殖学重点实验室, 呼和浩特 010018;
2. 承德市农林科学院, 承德 067000;
3. 内蒙古自治区农牧业科学院, 呼和浩特 010031

收稿日期:2021-08-10 出版日期:2022-04-23 发布日期:2022-04-25
通讯作者: 李蓓,主要从事马属动物遗传育种与繁殖研究,E-mail:mulanlb@163.com;芒来,主要从事马属动物遗传育种与繁殖研究,E-mail:dmanglai@163.com
作者简介:崔迎迎(1994-),女,山东临沂人,硕士生,主要从事马属动物遗传育种与繁殖研究,E-mail:398612133@qq.com
基金资助:
内蒙古农业大学动物科学学院青年基金(QN202012；QN202105)；内蒙古自治区自然科学基金重大项目(2019ZD03)；国家自然科学基金(3191101008；31960657；31860636)；国家重点研发计划项目(2017YFE0108700)；自治区应用技术研究与开发资金项目(2019GG242);内蒙古农业大学科技成果转化专项资金动植物新品种选育(培育)项目(YZGC2017001)

Regulatory Role of MEI1 with Alternative Splicing Event on Spermatogenesis in Mongolian Horse

CUI Yingying¹, DUGARJAVIIN Manglai^1*, LI Bei^1*, Terigele¹, ZHAO Yiping¹, REN Xiujuan¹, SONG Lianjie², SU Shaofeng^1,3

1. Inner Mongolia Key Laboratory of Equine Genetics, Breeding and Reproduction, College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China;
2. Chengde Academy of Agriculture and Forestry Sciences, Chengde 067000, China;
3. Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences, Hohhot 010031, China

Received:2021-08-10 Online:2022-04-23 Published:2022-04-25

摘要/Abstract

摘要： 旨在探索发生可变剪切事件的MEI1基因对蒙古马精子生成的调控作用，实现利用分子调控技术提高马的精子数量和精液品质，有效提高繁殖效率，降低生产成本，加快遗传进展，促进种群繁衍。本研究以两岁蒙古马的睾丸支持细胞(SC)作为研究对象，构建未发生外显子跳跃(exon skip,ES)事件的MEI1-1-pIRES2-EGFP重组质粒和发生ES事件的MEI1-2-pIRES2-Dsred重组质粒，并分别转染睾丸支持细胞；每组3个重复，分别在转染0、24、48、72 h时用CCK-8检测转染细胞的增殖及活性；G418抗生素筛选转染细胞；收集转染细胞并提取总RNA，经反转录后qRT-PCR检测减数分裂相关基因在两种细胞中的差异表达情况等。成功构建了MEI1-1-pIRES2-EGFP载体和MEI1-2-pIRES2-Dsred载体，经琼脂糖凝胶电泳和测序试验检测到载体构建成功并绘制重组质粒质谱图；CCK-8检测发现转染两种质粒的细胞在转染48 h时细胞活性最高、增殖情况最好且MEI1-2-pIRES2-Dsred转染的细胞活性最高；经G418筛选转染细胞发现转染效率增加；检测结果发现，减数分裂通路部分基因(BUB1、CUL1、CCNB1、PPP2R5C、CCNB2等)及支持细胞信号通路中调节精子发生减数分裂的部分因子(ARID4A、FSH、GDNF、Stra8、NRG1等)在两类细胞中的表达量差异显著，且均在转染MEI1-2-pIRES2-Dsred质粒的SC内表达量高。结果表明，发生ES事件的MEI1基因更有利于调控减数分裂及精子发生，为探索可变剪切事件影响蒙古马精子生成调控机制奠定了理论基础。

关键词: 蒙古马, MEI1基因, 可变剪切, 精子生成

Abstract: The purpose of this study was to explore the regulatory effect of MEI1 gene with alternative splicing event on spermatogenesis in Mongolian horses, improve sperm quantity and semen quality of horses by using molecular regulation techniques, effectively improve reproductive efficiency, reduce production costs, accelerate genetic progress, and promote population reproduction. The testicular sertoli cells(SC) of two-year-old Mongolian horses were used as the subjects, the MEI1-1-pIRES2-EGFP recombinant plasmids without exon skip (ES) events and MEI1-2-pIRES2-Dsred recombinant plasmids with ES events were constructed and transfected into testicular sertoli cells, respectively. There were 3 repeats in each group. The proliferation and activity of transfected cells were detected by CCK-8 at 0, 24, 48, 72 h of transfection, the transfected cells were screened by G418. The transfected cells were collected and total RNA was extracted, and the differential expression of meiosis related genes in the two cells was detected by qRT-PCR after reverse transcription. The MEI1-1-pIRES2-EGFP and MEI1-2-pIRES2-Dsred vectors were successfully constructed. Agarose gel electrophoresis and sequencing experiments showed that the vectors construction was successful, and the recombinant plasmid mass spectrum was drawn. CCK-8 assay showed that the cells transfected with two plasmids had the highest cell activity and proliferation level at 48 h after transfection, and cells transfected with MEI1-2-pIRES2-Dsred had the highest cell activity. The transfection efficiency of transfected cells increased by G418 screening; The test results showed that some genes in meiosis pathway (BUB1, CUL1, CCNB1, PPP2R5C, CCNB2, etc.) and some factors regulating sperm meiosis in sertoli cell signaling pathway (ARID4A, FSH, GDNF, Stra8, NRG1, etc.) had significant differences in expression in the two types of cells, and all had higher expressions in SC transfected MEI1-2-pIRES2-Dsred plasmids. The MEI1 gene with ES events occurence is more conducive to regulating the meiosis and spermatogenesis, which has laid a theoretical foundation for exploring the mechanism of alternative splicing events affecting spermatogenesis in Mongolian horses.

Key words: Mongolian horse, MEI1 gene, alternative splicing, spermatogenesis

中图分类号:

S821.3

崔迎迎, 芒来, 李蓓, 特日格乐, 赵一萍, 任秀娟, 宋连杰, 苏少锋. MEI1基因可变剪切事件对蒙古马精子生成的调控作用[J]. 畜牧兽医学报, 2022, 53(4): 1096-1108.

CUI Yingying, DUGARJAVIIN Manglai, LI Bei, Terigele, ZHAO Yiping, REN Xiujuan, SONG Lianjie, SU Shaofeng. Regulatory Role of MEI1 with Alternative Splicing Event on Spermatogenesis in Mongolian Horse[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1096-1108.

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MEI1基因可变剪切事件对蒙古马精子生成的调控作用

Regulatory Role of MEI1 with Alternative Splicing Event on Spermatogenesis in Mongolian Horse

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