胀果甘草查尔酮异构酶基因的克隆及序列分析

摘要

目的:对胀果甘草甘草苷生物合成途径的关键酶查尔酮异构酶(CHI)进行基因克隆及序列分析.方法:根据乌拉尔甘草CHI基因cDNA序列设计引物,以胀果甘草主根总RNA为模板RT-PCR扩增CHI基因cDNA序列,并对其进行分析.结果:克隆得到20条胀果甘草CHI基因eDNA序列,开放读框全长690 bp,编码229个氨基酸残基.20条胀果甘草CHI基因的cDNA序列存在22个变异位点,一致性为99.54％,可分为6种单倍型;其编码的氨基酸序列存在14个变异位点,一致性为98.98％.胀果甘草CHI基因编码蛋白为稳定性亲水蛋白,相对分子质量为24.5× 103,等电点为5.3～6.2,不合信号肽,无跨膜区,二级结构以α螺旋和无规则卷曲为主,保守结构域包含一个查耳酮超家族结构域.同源性分析显示,不同物种间的CHI基因同源性较低.结论:克隆了胀果甘草CHI基因cDNA序列,为进一步研究不同基原甘草中甘草苷生物合成的分子调控机制奠定了基础,并为优质甘草的分子选育提供了理论依据.%Objective:To clone and analyze the chalcone isomerase(CHI) gene from Glycyrrhiza inflata Bat..Methods:Specific primers were designed based on reported CHI cDNA sequence of G.uralensis,RT-PCR technique was used to amplify CHI cDNA sequences of G.inflata,and which were analyzed by bioinformatics.Results:Twenty CHI cDNA sequences of G.inflata with the length of 690 bp were obtained encoding 229 amino acid residues.Twenty-two variable sites were found in these 20 cDNA sequences and 6 haplotypes werc determined,with a similarity of 99.54％.Physicochemical property ananlysis shows that these CHI coding protiens are all stably hydrophilic protein,with a molecular weigh of 24.5 kD and a isoelectric point between 5.3～6.2.It has no signal peptides or transmembrane domains.Its secondary structure mainly consists of alpha helix and random coil.Also chalcone super family domain is included in the conserved domain.Homology analysis indicates that the homology of CHI among different species is low.Conclusion:CHI cDNA sequeces were successfully cloned from G.inflata.We hope this work will lay a foundation for further researches on the molecular mechanisms of flavonoid biosynthesis in different licorice origins and licorice molecular breeding.