苦荞查尔酮合成酶多克隆抗体制备及组织表达分析

Preparation of Polyclonal Antibody and Tissue Expression Analysis of Chalcone Synthase from Tartary Buckwheat

苦荞查尔酮合成酶(FtCHS)是黄酮类化合物合成过程中的限速酶之一,在苦荞黄酮类次生代谢物合成中起到重要作用。以苦荞种子灌浆期cDNA为模板,采用RT-PCR方法克隆获得FtCHS基因,通过生物信息学分析确定FtCHS的截短抗原基因序列(TrCHS)。再通过原核表达、亲和层析等方法制备并纯化TrCHS抗原,以纯化的抗原免疫大白兔获得TrCHS的多克隆抗体,利用半定量RT-PCR、Western blotting方法分析苦荞不同器官FtCHS的表达情况。结果表明,成功克隆FtCHS基因开放阅读框(ORF)大小为1182 bp,共编码393个氨基酸,通过原核表达获得TrCHS抗原的分子质量约34.71 ku,所制备的多克隆抗体具有较高的特异性,FtCHS基因在苦荞叶子、种子的表达量明显最高。

Chalcone synthase is one of the rate-limiting enzymes in the biosynthsis of flavonoids synthesis,and plays an important role in secondary metabolites synthesis path of Tartary buckwheat.The full-length cDNA of FtCHS was cloned from tartary buckwheat seed and truncated coding region of Chalcone synthase(TrCHS)was identified as antigen fragments by bioinformatics comparative analysis.TrCHS was cloned and inserted to a prokaryotic expression vector pET-28a(+).The recombinant vector was introduced into Escherichia coli BL21(DE3)to induce expression.The Newland White rabbits were immunized with the recombinant protein purified by Ni-NTA Resin,and the antibody specificity was determined by western blot.The expression pattern of FtCHS mRNA and protein in different tissues were detected by semi-quantitative RT-PCR and the FtCHS protein expression in various tissues was analyzed with this newly prepared antibody by western blotting.The results showed that FtCHS gene contained a 1182 bp-sized open reading frame(ORF),of which 393 amino acids were encoded,and SDS-PAGE analysis showed that the TrCHS protein had molecular mass of about 34.71 ku,which was obtained by prokaryotic expression.Western blot analysis showed that the polyclonal antibody of FtCHS protein was obtained and the expression of FtCHS in different tissues from buckwheat was explicit.The analysis of the expression pattern of FtCHS gene by semi-quantitative RT-PCR and western blotting showed that FtCHS could express in seeds,leaves,flowers and stems,and the expression level was higher in seeds and leaves.The results of this study will provide reference for further research on the function of FtCHS in flavonoids biosynthetic pathway and molecular breeding of buckwheat.