Identifying KLF4 Gene Transcription Initiation Site by Optimized 5' RACE Technique in Bos taurus Adipose Tissue
HAO Rui-Jie1, ZHANG Yan-Ling1, XU Shu-Yu1, LI Min1, ZHANG Ming-Ming1, LI Xing-Yu1, CHEN Ya-Zhen1, GUO Yun1, ZAN Lin-Sen2*
1 College of Life Science, Xinyang Normal University, Xinyang 464000, China; 2 College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
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.
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