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1 161 bp，开放阅读框长654 bp，编码217个氨基酸，理论分子质量约为23.14 ku，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 Carthamus 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.Real-time PCR results indicated that relative expression of CHI gene was highest in bud stage.【Conclusion】CHI gene was cloned successfully from Carthamus tinctorius flower,which had highest expression inCarthamus tinctorius bud stage