通过优化的5'RACE技术鉴定牛脂肪组织<em>KLF4</em>基因转录起始位点

doi:10.3969/j.issn.1674-7968.2023.04.018

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Abstract 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 cDNA 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 (NM_001105385). Among the 8 extended nucleotides, two adjoining ones of GA at 3' end exactly matched with counterpart sequence in 5' end region of KLF4 gene of bovine genome, which indicated a new TIS of KLF4 gene to be found in Chinese native breeds. Under the same experimental conditions, short RACE fragment (<500 bp) was well achieved by PCR, while long RACE fragment (>1000 bp) suffered failed amplification in both kit method and optimized method. In summary, the optimized RACE method was proved to apply to adipose tissue, by which, a new TIS of KLF4 was successfully identified at 2 bp upstream of the 5’end of published KLF4 sequence of NCBI in Jiaxian Red Cattle, implying the genetic specificity of Chinese cattle breeds, and provides a reference for further studying KLF4 gene function in fat deposition.

Key words： 5' RACE technique Specific reverse transcription Size of RACE fragment Bovine adipose tissue Kruppel-like factor 4 (KLF4) gene

Received: 31 March 2022

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Corresponding Authors: * zanlinsen@163.com

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	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

Cite this article:

HAO Rui-Jie,ZHANG Yan-Ling,XU Shu-Yu, et al. Identifying KLF4 Gene Transcription Initiation Site by Optimized 5' RACE Technique in Bos taurus Adipose Tissue[J]. 农业生物技术学报, 2023, 31(4): 856-866.

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http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2023.04.018 OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2023/V31/I4/856

[1] 邓雪柯, 殷建华, 曹毅. 2007. 3 种 5' RACE 技术的比较与优化[J]. 成都医学院学报, 2(01): 20-25.
(Deng X K, Yin J H, Cao Y. 2007. Comparison and improving of three 5' RACE methods[J]. Journal of Chengdu Medical Col-lege, 2(01): 20-25. )
[2] 董哲, 任桂敏, 李燊, 等. 2016. 利用 3' RACE 和 5' RACE 技术克隆日本刺沙蚕中枢神经系统 TPH 基因全长[J]. 辽宁医学院学报, 37(006): 1-4.
(Dong Z, Ren G M, Li S, et al. 2016. 3' RACE and 5' RACE used to clone trypto-phan hydroxylase gene in central nervous system of Ne-reis (N. japonica)[J]. Journal of Liaoning Medical Uni-versity, 37(006): 1-4. )
[3] 贺斌, 黄兴奇, 余腾琼, 等. 2012. cDNA 末端快速扩增技术及其方法的改进[J]. 浙江农业科学, 09: 1352-1357.
(He B, Huang X Q, Yu T Q, et al. 2012. Rapid amplifica-tion of cDNA ends and improvement of its method[J]. Zhejiang Agricultural Sciences, 09: 1352-1357. )
[4] 冯丽, 任茂智, 罗洪发, 等. 2006. 分离植物目的基因全长 cDNA 和启动子的新方法 -快速定位转录起始位点 (RITIS)[J]. 分子植物育种, 4(001): 23-28.
(Feng L, Ren M Z, Luo H F, et al. 2006. A novel method for isolating full-length cDNA and promoters of target genes in plants: Rapid localization of transcription start sites (RITIS)[J]. Molecular Plant Breeding, 4(001): 23-28. )
[5] 李杰, 张义全, 高鹤, 等. 2014. 利用优化的 5'-RACE 实验定位副溶血弧菌基因的转录起始位点[J]. 生物技术通讯, 025(003): 328-332.
(Li J, Zhang Y Q, Gao H, et al. 2014. The transcription start sites of Vibrio parahaemo-lyticus genes were mapped using the optimized 5' RACE assay[J]. Biotechnology Newsletter, 025(003): 328-332. ) 李飞, 韩召军. 2005. 棉蚜乙酰胆碱受体亚基的选择性剪接和多重转录起始位点分析(英文)[J]. 动物学报,(05): 867-878., 51(5): 867-878. )
[6] 李志英, 牟红珍, 高丁梅, 等. 2014. 本氏烟Ⅰ型启动子的克隆及其转录起始位点分析[J]. 中国生物工程杂志, 34(001): 28-35.
(Li Z Y, Mou H Z, Gao D M, et al. 2014. Ben's type smoke Ⅰ promoter cloning and transcription start site analysis[J]. Chinese Journal of Biological Engi-neering, 34(001): 28-35. )
[7] 栾爱萍, 何业华, 林文秋, 等. 2016. 菠萝 AcSERK1 启动子的转录起始位点及胚性细胞特异性鉴定[J]. 园艺学报, 43(11): 2251-2256.
(Luan A P, He Y H, Lin W Q, et al. 2016. Transcriptional start site and embryogenic cell-specific identification of Acserk1 promoter in pineapple[J]. Journal of Horticulture, 43(11): 2251-2256. )
[8] 罗聪, 何新华, 陈虎, 等. 2011. 一种高效获取基因 5'末端的 RACE 方法[J]. 植物生理学报, 47(04): 409-414.
(Luo C, He X H, Chen H, et al. 2011. An efficient RACE method for obtaining the 5' end of gene[J]. Plant Physi-ology Communications, 47(04): 409-414. )
[9] 田勇, 张聪聪, 张红艳, 等. 2012. 应用 RACE 技术扩增鸭 A-FABP 基因及序列分析[J]. 中国畜牧杂志, 48(21):17-21.
(Tian Y, Zhang C C, Zhong H Y, et al. 2012. Amplifi-cation and sequence analysis of duck A-FABP gene by RACE technique[J]. Chinese Journal of Animal Science, 48(21): 17-21. )
[10] 王津津, 匡昉哲, 庄华, 等. 2016. Prss37 基因转录起始位点的确定及其转录调控的初步探讨[J]. 实验动物与比较医学, 036(002): 93-100.
(Wang J J, Kuang F Z, Zhuang H, et al. 2016. Identification of transcription start site of Prss37 gene and preliminary study on its transcriptional regulation[J]. Experimental Animals and Comparative Medicine, 036(002): 93-100. )
[11] 王景艳, 张高华, 苏乔, 等. 2007. RLM-RACE 法扩增獐茅液泡膜 Na+/H+ 逆向转运蛋白基因 5'-cDNA 末端序列[J]. 西北植物学报, 27(5): 903-907.
(Wang J Y, Zhang G H, Su Q, et al. 2007. RLM-RACE was used to amplify the 5'-cDNA terminal sequence of Na+/H+ antiporter gene in the vacuolar membrane of Swertia chinensis[J]. Acta Bo-tanica Boreali-Occidentalia Sinica, 27(5): 903-907. )
[12] 王新楠, 宋智刚, 吴媛, 等. 2003. 人肝刺激因子基因 5′-侧翼序列克隆和结构分析[J]. 遗传学报, 12: 1171-1176.
(Wang X N, Song Z G, Wu Y, et al. 2003. Cloning and structural analysis of 5'-flanking sequence of human he-patic stimulator substance (hHSS) gene[J]. Acta Geneti-ca Sinica, 12: 1171-1176. )
[13] 张建军, 黄爱龙, 史小玲, 等. 2005. SARS-CoV5'-UTR 相应 cDNA 作启动子时转录起始位点研究[J]. 重庆医科大学学报, 030(006): 849-851.
(Zhang J J, Huang A L, Shi X L, et al. 2005. Study on the transcription starting site of SARS-CoV5'-UTR corresponding cDNA as promoter[J]. Journal of Chongqing Medical University, 030(006): 849-851. )
[14] 左秀美, 秦伟, 周爱红, 等. 2011. RLM-RACE 法确定胰岛素降解酶基因的转录起始位点[J]. 脑与神经疾病杂志, (06): 401-403.
(Zuo X M, Qin W, Zhou A H, et al. 2011. RLM-RACE method was used to determine the tran-scription start site of insulin degrading enzyme gene[J]. Journal of Brain and Nerve Diseases, (06): 401-403. )
[15] Ansaroila M, Fraser V N, Megraw M, et al. 2020. Accurate transcription start sites enable mining for the cis-regula-tory determinants of tissue specific gene expression[J]. BioRxiv, 9(10): 32-49.
[16] Arezi B, Hogrefe H H. 2007. Escherichia coli DNA poly-merase Ⅲ epsilon subunit increases Moloney murine leu-kemia virus reverse transcriptase fidelity and accuracy of RT-PCR procedures[J]. Analytical Biochemistry, 360(1): 84-91.
[17] Bower N I, Johnston I A. 2010. Targeted rapid amplification of cDNA ends (T-RACE) -an improved RACE reaction through degradation of non-target sequences[J]. Nucleic Acids Research, 38(21): 1-7.
[18] Cai Y M, Kalyani K, Henry T, et al. 2020. Rational design of minimal synthetic promoters for plants[J]. Nucleic Ac-ids Research, 1-12.
[19] Chen D, John P T. 2001. Reverse transcriptase adds nontem-plated nucleotides to cDNAs during 5'-RACE and prim-er extension[J]. Biotechniques, 30(3): 574-580.
[20] Clauwaert J, Menschaert G, Waegeman W. 2020. An in-depth evaluation of annotated transcription start sites in E. Co-li using deep learning[J]. Cold Spring Harbor Laborato-ry, 1-15.
[21] Flouriot G, Brand H, Gannon F. 1999. Identification of differ-entially expressed 5'-end mRNA variants by an im-proved RACE technique (PEETA)[J]. Nucleic Acids Re-search, 27(15): E8.
[22] Fromont-Racine M, Bertrand E, Pictet R, et al. 1993. A highly sensitive method for mapping the 5' termini of mRNAs[J]. Nucleic Acids Research, 21(7): 1683-1684.
[23] Frohman M A. 1993. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: Thermal RACE[J]. Methods in Enzymology, 9(218): 340-356.
[24] Green M R, Joseph S. 2019. Rapid amplification of sequences from the 5' ends of mRNAs: 5'-RACE[J]. Cold Spring Harbor Protocols, 5: 416-426.
[25] Helm M. 2006. Post-transcriptional nucleotide modification and alternative folding of RNA[J]. Nucleic Acids Re-search, 34(2): 721-733.
[26] Huang J C, Chen F. 2006. Simultaneous amplification of 5' and 3' cDNA ends based on template-switching effect and inverse PCR[J]. Biotechniques, 40(2): 187-189.
[27] Imashimizu M, Tokunaga Y, Afek A, et al. 2020. Control of transcription initiation by biased thermal fluctuations on repetitive genomic sequences[J]. Biomolecules, 10(9): 1-19.
[28] Josef K, Stefan B. 2002. RT-PCR analysis of 5′ to 3′-end-ligat-ed mRNAs identifies the extremities of cox2 transcripts in pea mitochondria[J]. Nucleic Acids Research, 30(2): 439-446.
[29] Li A, Zhao Z, Zhang Y, et al. 2015. Tissue expression analy-sis, cloning, and characterization of the 5′-regulatory re-gion of the bovine fatty acid binding protein 4 gene[J]. Journal of Animal Science, 93: 5144-5152.
[30] Liu F, Zheng K, Chen H C, et al. 2018. Capping-RACE: A simple, accurate, and sensitive 5' RACE method for use in prokaryotes[J]. Nucleic Acids Research, 46(21): 129 (1-8).
[31] Liu X, Gorovsky M A. 1994. Mapping the 5' and 3' ends of Tetrahymena thermophila mRNAs using RNA ligase me-diated amplification of cDNA ends (RLM-RACE)[J]. Nucleic Acids Research, 21(21): 4954-4960.
[32] Machida R J, Lin Y Y, Cees O. 2014. Four methods of prepar-ing mRNA 5' end libraries using the Illumina sequenc-ing platform[J]. PLOS ONE, 9(7): E101812.
[33] Pinto F L, Lindblad P. 2010. A guide for in-house design of template-switch-based 5' rapid amplification of cDNA ends systems[J]. Analytical Biochemistry, 397(2): 227-232.
[34] Potenza C, Aleman L, Sengupta-Gopalan C. 2004. Targeting transgene expression in research, agricultural, and envi-ronmental applications: Promoters used in plant transfor-mation[J]. In Vitro Cellular & Developmental Biology-Plant, 40(1): 1-22.
[35] Potter J, Zheng W, Lee J. 2003. Thermal stability and cDNA synthesis capability of SuperScript III reverse transcrip-tase[J]. Focus (San Francisco, Calif. ), 25(1): 19-24.
[36] Rosenberg M, Court D. 1979. Regulatory sequences involved in the promotion and termination of RNA transcription[J]. Annual Review of Genetics, 13(1): 319-353.
[37] Sandelin A, Carninci P, Lenhard B, et al. 2007. Mammalian RNA polymerase Ⅱ core promoters: Insights from ge-nome-wide studies[J]. Nature Reviews Genetics, 8(6): 424-436.
[38] Schmidt W M, Mueller M W. 1999. Cap Select: A highly sen-sitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs[J]. Nucleic Acids Research, 27(21): E31(1-4).
[39] Schramm G, Bruchhaus I, Roeder T. 2000. A simple and reli-able 5'-RACE approach[J]. Nucleic Acids Research, 28 (22): E96(1-4).
[40] Scotto-Lavino E, Du G, Frohman M A. 2006. 5' end cDNA amplification using classic RACE[J]. Nature Protocols, 1(6): 2555-2562.
[41] Sharma C M, Hoffmann S, Darfeuille F, et al. 2010. The pri-mary transcriptome of the major human pathogen Helico-bacter pylori[J]. Nature, 464(7286): 250-255.
[42] Shell S S, Chase M R, Ioerger T R, et al. 2015. RNA sequenc-ing for transcript 5'-end mapping in Mycobacteria[J]. Methods in Molecular Biology, 1285: 31-45.
[43] Vitale L, Caracausi M, Casadei R, et al. 2017. Difficulty in ob-taining the complete mRNA coding sequence at 5' region (5' end mRNA artifact): Causes, consequences in biolo-gy and medicine and possible solutions for obtaining the actual amino acid sequence of proteins (Review)[J]. In-ternational Journal of Molecular Medicine, 39(5): 1063-1071.
[44] Yeku O, Frohman M A. 2003. Rapid amplification of cDNA ends (RACE)[J]. Methods in Molecular Biology, 226: 105-116.