红花查尔酮合成酶基因的克隆、生物信息学分析及表达

目的克隆红花查尔酮合成酶基因(chalcone synthase,CHS),运用生物信息学分析CHS,比较花期中每天CHS的表达,为红花有效成分合成及调控机制研究提供基础。方法从新鲜红花花冠中提取RNA,反转录为cDNA,设计特异性引物,克隆获得CHS。通过生物信息学对该基因蛋白的特征进行分析,使用MEGA5.1构建CHS与相关物种CHS的系统进化树,利用real-time PCR分析花期中每天红花CHS的表达量,并进行分析和比较。结果克隆获得的红花CHS,序列全长为1 149 bp,具有1 041 bp的完整开放阅读框,编码346个氨基酸。将该蛋白通过NCBI上的Blastp比对发现,该蛋白属于CHS家族,比对结果显示红花CHS与100余种NCBI上登录的植物有相似性,其中与水飞蓟、翠菊、菊花、红凤菜的相似性分别达95%、95%、94%、94%。将相似性在90%以上的物种用MEGA5.1构建进化树,结果显示红花CHS与水飞蓟CHS亲缘关系最近。通过ProtParam预测红花CHS蛋白分子式为C1678H2693N451O493S20,相对分子质量为37 700,等电点为6.10,负电荷的氨基酸残基数(Asp+Glu)为42,正电荷的氨基酸残基数(Arg+Lys)为38。基因表达分析结果表明红花CHS在花期第3天的相对表达量最高,远远高于花期其余时间。结论成功克隆、分析并表达了红花CHS,为红花有效成分合成及调控机制研究提供基础。

Cloning, bioinformatic analysis, and expression of chalcone synthase gene in Carthamus tinctorius

Objective To clone the chalcone synthase (CHS) gene in Carthamus tinctorius, to analyze the bioinformation of CHS, to compare the expression of CHS during the florescence, and to provide the foundation for composition and regulation mechanism of the active ingredients in C. tinctorius. Methods RNA was obtained from fresh safflower corolla, cDNA was reversely transcriped, specific primers were designed, and then CHS was cloned. The protein characteristics was analyzed using bioinformatics, the phylogenetic tree of CHS was constructed using MEGA5.1, the expression of CHS during the florescence was analyzed using real time-PCR. Results The 1 149 bp CHS sequence in C. tinctorius was obtained, which has a 1 041 bp ORF, encoding 346 amino acids. This protein belongs to the CHS family according to Blastp in NCBI. The CHS in safflower was similar to that in above 100 plants, and the similarities to Silybum marianum, Callistephus chinensis, Chrysanthemum x morifolium, and Gynura bicolor were respectively reaching 95%, 95%, 94%, and 94%. It has the closest relationship to S. marianum according to the phylogenetic tree using MEGA5.1. The CHS formula was C₁₆₇₈H₂₆₉₃N₄₅₁O₄₉₃S₂₀ and the molecular weight was 37 700, with the isoelectric point of 6.10. The number of negatively charged amino acid residues (Asp + Glu) was 42, and the number of positively charged amino acid residues (Arg + Lys) was 38. The gene expression analysis showed that the highest expression of CHS in safflower was on day 3 of florescence, much higher than that on the other days. Conclusion The CHS in safflower is successfully cloned, analyzed, and expressed, which provides the foundation for composite and regulation mechanism of the active ingredients in C. tinctorius.