通过优化的5'RACE技术鉴定牛脂肪组织KLF4基因转录起始位点  

Identifying KLF4 Gene Transcription Initiation Site by Optimized 5’RACE Technique in Bos taurus Adipose Tissue

作　　者：郝瑞杰[1] 张燕玲 徐淑雨 李敏 张铭铭 李星雨 陈亚珍 郭允 昝林森[2] HAO Rui-Jie;ZHANG Yan-Ling;XU Shu-Yu;LI Min;ZHANG Ming-Ming;LI Xing-Yu;CHEN Ya-Zhen;GUO Yun;ZAN Lin-Sen(College of Life Science,Xinyang Normal University,Xinyang 464000,China;College of Animal Science and Technology,Northwest A&F University,Yangling 712100,China)

机构地区：[1]信阳师范学院生命科学学院,信阳464000 [2]西北农林科技大学动物科技学院,杨凌712100

出　　处：《农业生物技术学报》2023年第4期856-866,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31802043);河南省自然科学基金(162300410227);河南省高校重点研究项目(17A230007);信阳师范学院青年科研基金项目(2018-QN-044);国家级大学生科创项目(S201810477010);信阳师范学院生科院特色骨干学科培育项目。

摘　　要：5’cDNA末端快速扩增(5’rapid amplification of cDNA ends,5’RACE)技术是基因转录表达调控研究的关键手段之一,其操作复杂,对样品要求较高。牛(Bos taurus)脂肪组织杂质含量高,单位体积RNA含量低,经常造成5’cDNA末端扩增的失败,影响脂肪沉积分子机制的研究。Kruppel样因子4(Kruppellike factor 4,KLF4)是调控牛脂肪沉积的关键基因,为了准确鉴定牛KLF4基因在脂肪组织中表达的转录起始位点,揭示脂肪沉积关键基因的表达调控机理,以郏县红牛皮下脂肪组织为研究对象,对经典5’RACE技术进行改进。首先采用特异性反转录引物,仅反转录总RNA中的特定mRNA,以提高目标基因的反转效率,耐受更低浓度的RNA;然后,调整反转录体系,简化加尾前cDNA纯化步骤,减少目标cDNA的损失,保证后续RACE扩增中的模板浓度;最后,采用适当靠近mRNA 5’端的基因特异性引物(产生较短的RACE片段),降低RACE扩增中聚合酶延伸能力,模板二级结构等因素的干扰。通过上述改进措施,成功获得了KLF4基因的RACE片段。对该片段进行测序和比对分析后,发现郏县红牛KLF4基因的转录起始位点与NCBI数据库中以胞嘧啶C作为起始核苷酸的KLF4基因(NM_001105385)不同,其向5’端延伸了2个碱基(GA),以鸟嘌呤作为起始核苷酸,该结果更符合转录起始位点通常为一个嘌呤(A或G)的特征。分析不同长度RACE片段对PCR扩增的影响,结果发现较短的RACE片段(<500 bp)可以顺利扩增成功;而对于大于1000 bp的RACE片段,试剂盒法和优化法均没有获得有效片段。本研究通过优化5’RACE技术,成功鉴定了郏县红牛皮下脂肪KLF4基因的转录起始位点,该新位点证实了我国地方牛种的遗传特异性,为进一步研究该基因在脂肪沉积中的作用提供了参考。5’rapid amplification of cDNA ends(5’RACE)is usually used to identify transcription initiation sites(TIS)of target gene for gene expression regulation research.However,it usually suffers unexpected failure when processing RNA from adipose tissue because high content of impurities and low content of RNA disturbs RNA extraction in fat,leading to evident declines in yield,quality and purity of extracted RNA compared to other tissues.Kruppel-like factor 4(KLF4)is a key regulatory gene of bovine adipose deposition.And it is of great significance to determine KLF4 gene TIS in bovine adipose tissue for study of molecular mechanism of fat accumulation.“Classical 5’RACE”technique was considered to have great improvement potential for complex samples by means of comparison and analysis of various 5’RACE techniques.And it was adopted to identify KLF4 gene TIS with RNA extracted from subcutaneous fat tissue of Jiaxian Red Cattle(Bovine)as samples,of which some processes were modified according to sample characteristics.Firstly,specific reverse transcription primers were employed to produce cDNA of special gene,which could significantly increase the cDNA concentration of target gene compared to reverse transcription of total RNA;Then,cDNA purification before tailing was simplified to reduce c DNA losses,which was particularly necessary when adipose tissue with low amount of initial mRNA was confronted;Finally,appropriate GSP primers close to 5’end of mRNA ensured successful amplification of RACE fragment,reducing as many uncertainties as possible in PCR.In addition,in view of the controversy surrounding the best size of RACE fragment,the effects of amplified fragments on PCR in 5’RACE were also analyzed with different lengths.In this study,the RACE fragment of KLF4 from bovine adipose tissue was successfully obtained by the above improvement measures.By sequencing 5’-terminal nucleotide sequence of the amplified KLF4 fragment was confirmed to extend additional 8 bp of CGTATCGA to 5’end of KLF4 mRNA of NCBI

关 键 词：5’cDNA末端快速扩增(5’RACE)技术 特异性反转录 RACE片段大小 牛脂肪组织 Kruppel样因子4(KLF4)基因 

分 类 号：S823.81[农业科学—畜牧学]