管花肉苁蓉花中查耳酮合酶的基因克隆、功能鉴定与表达分析

Cloning,functional identification and expression analysis of chalcone synthase from Cistanche tubulosa

目的 研究管花肉苁蓉Cistanche tubulosa花中查耳酮合酶(chalcone synthase)Ct CHS蛋白的功能和Ct CHS基因在白、红、紫3种颜色花中的表达模式。方法 利用RT-PCR克隆Ct CHS基因并进行生物信息学分析。将p ET-28a-CtCHS质粒转入大肠杆菌通过IPTG诱导蛋白表达。将Ct CHS重组蛋白与底物4-香豆酰辅酶A、丙二酰辅酶A孵育并利用HPLC和MS检测产物。将p CAMBIA1300-35S-GFP-CtCHS质粒转入拟南芥原生质体观察CtCHS蛋白亚细胞定位。利用q RT-PCR检测Ct CHS基因在3种颜色管花肉苁蓉花中的表达模式。结果 克隆出1条Ct CHS基因,开放阅读框(open reading frame,ORF)长度为1173 bp,编码390个氨基酸,蛋白相对分子质量为42 800,具有查耳酮合酶保守的催化残基Cys164、His303、Asn336。利用大肠杆菌外源表达Ct CHS蛋白并纯化获得重组蛋白。Ct CHS蛋白能够催化4-香豆酰辅酶A与丙二酰辅酶A生成柚皮素查耳酮。Ct CHS蛋白主要定位于细胞质。Ct CHS基因在紫、红色花中高表达,相对表达量分别为白色花中的8.44、3.21倍。结论从管花肉苁蓉花中克隆出1条Ct CHS基因并外源表达蛋白,CtCHS蛋白具有查耳酮合酶功能,Ct CHS基因在颜色更深的花中表达量更高,表明Ct CHS基因可能调控管花肉苁蓉花色,为进一步研究管花肉苁蓉花色调控机制奠定基础。

Objective To study the function of chalcone synthase from Guanhuaroucongrong(Cistanche tubulosa)and the expression pattern of CtCHS gene in white,red and purple flowers.Methods The CtCHS gene was cloned by RT-PCR and bioinformatics analysis was carried out.The pET-28a-CtCHS plasmid was transferred into the Escherichia coli to induce protein expression by IPTG.The CtCHS recombinant protein was incubated with the substrates p-coumaroyl CoA and malonyl CoA,and the products were detected by HPLC and MS.The pCAMBIA1300-35S-GFP-CtCHS plasmid was transferred into the protoplast of Arabidopsis thaliana to observe the subcellular localization of the CtCHS protein.The expression pattern of CtCHS gene in the flowers of C.tubulosa with three colors was detected by qRT-PCR.Results A CtCHS gene was cloned.The length of the open reading frame(ORF)was 1173 bp,encoding 390 amino acids,and the molecular weight of the protein was 428000.CtCHS contained the catalytic residues Cys164,His303,Asn336,which were conserved in chalcone synthase.The CtCHS protein was expressed in E.coli and purified to obtain the recombinant protein.CtCHS protein could catalyze p-coumaroyl CoA and malonyl CoA to produce naringenin chalcone.CtCHS protein was mainly localized in the cytoplasm.CtCHS gene was highly expressed in purple and red flowers,and the relative expression levels were 8.44 and 3.21 times higher than that in w hite flowers,respectively.Conclusion A CtCHS gene was cloned from the flower of C.tubulosa and protein was expressed.CtCHS protein has chalcone synthase function,and the expression of CtCHS gene is higher in darker flowers,indicating that CtCHS gene may regulate the flower color of C.tubulosa,which lays a foundation for further research on the regulation mechanism of the flower color of C.tubulosa.