红花查尔酮异构酶基因全长cDNA的克隆及表达分析

Cloning and expression of Chalcone isomerase gene cDNA of Carthamus tinctorius

【目的】克隆红花(Carthamus tinctorius L.)黄酮合成途径中的关键酶查尔酮异构酶(Chalcone isomerase,CHI)基因的全长序列,研究其组织表达特异性,为红花代谢调控研究提供参考。【方法】利用RT-PCR技术克隆CHI基因的cDNA全长,并对其全长基因进行生物信息学分析;构建系统发育树,研究其与相似序列的同源性;利用实时荧光定量PCR方法,分析CHI基因在红花不同开花时期的表达量。【结果】CHI基因全长1 161bp,开放阅读框长654bp,编码217个氨基酸,理论分子质量约为23.14ku,等电点为5.67,序列含有典型的加尾信号序列AATAA和Poly(A)。系统发育树表明,该基因与其他物种CHI基因具有较高的同源性,其中与青木香的同源性最高,达到82%。实时荧光定量PCR结果表明,CHI基因在红花花蕾期的表达量最高。【结论】克隆得到了红花CHI基因,其在红花花蕾期的表达量最高。

[Objective] This study cloned full-length of Chalcone isomerase（CHI）gene in flavonoids biosynthesis from Cartharnus tinctorius and investigated tissue expression specificity to provide reference for the research of Carthamus tinctorius metabolic regulation. [Method] The full-length cDNA of CHI gene from Carthamus tinctorius was cloned using RT-PCR and analyzed using the bioinformatics methods. Phylogenetic tree was constructed for studying homology with similar sequences and the expression at different flowering periods was analyzed by real-time PCR. [Result] The full-length cDNA of CHI gene was 1 161 bp with an opening reading frame （ORF） of 654 bp encoding 217 amino acids. The putative protein of CHI gene had a molecular weight of 23.14 ku and a theoretical pI of 5.67, containing typical AATAA tail signal sequence and Poly（A）. Genealogical tree showed that CHI gene had high homology with other species and the highest value was 82% with Saussurea medusa. Realsion of CHI gene was highest in bud stage. [Conclusion] mus tinctorius flower,which had highest expression in Ca time PCR results indicated that relative expresCHI gene was cloned successfully from Cartharthamus tinctorius bud stage.