Functional Analysis Of Chalcone Isomerase (CsCHIs) In Tea Plants

Flavonoid compounds represent a larger group of plant secondary metabolism with many important physiological functions.Chalcone isomerase isomerize 4’,2’,4’,6’-tetrahydroxychalcone(naringenin chalcone)to form 5-hydroxyflavanone,namely(2S)-naringenin.As a direct catalytic product of CHI,flavanones are one of the important substrates to produce other flavonoid substances.Therefore,CHI plays a crucial role in the accumulation of flavonoids in tea plants.In this study,‘shu cha zao’was used as the experimental material to analyze the function of chalcone isomerase in tea plant.The main finding are as follows:1.In this study,four genes of CHI family were cloned and were named as CsCHIa,CsCHIb,CsCHIc and CsCHId.Amino acid sequence alignment and phylogenetic tree analysis were carried out using DNAMAN and MEGA method.The phylogenetic analysis showed that they were belonged to Type Ⅰ,Type Ⅳ and Type Ⅲ of CHI family,respectively.2.The transcriptional profile of the CHI family was obtained by qRT-PCR technology,and the results presented a tissue-specific and inducible pattern.3.The functional verification of CHI family in vitro was carried out based on construction of heterologous expression vector,prokaryotic expression,protein purification.The results in vitro showed that CsCHIa could catalyze naringenin chalcone while the other three could not.4.The functional verification of CsCHI family in vivo was carried out based on genetic transformation to tobacco and genetic transformation to Arabidopsis thaliana.The genetic transformation experiment showed that CsCHIa gene could improve the content of PAs and flavonol glycosides in pod of transgenetic tt5 mutants and recovered of mutant coat color to brown.What’s more,CsCHIa and CsCHId increased the content of proanthocyanidins and flavonol glycosides in the pod of wild arabidopsis thaliana,and CsCHIb also increased the content of flavonol glycosides in Arabidopsis thaliana.All the four CsCHIs increased anthocyanin content in transgenic tobacco flowers,especially in CsCHIa transgenic tobacco.The content of flavonol glycosides in transgenic tobacco was increased by CsCHIb and CsCHIc.Through the analysis of four chalcone isomerase,it was found that CsCHIa had catalytic enzyme activity in vitro and increased the accumulation of anthocyanins PAs and flavonols in transgenic Arabidopsis thaliana and tobacco.CsCHIb can increase the accumulation of anthocyanins and flavonoids in transgenic Arabidopsis thaliana and tobacco.CsCHIc can increase the accumulation of anthocyanins and flavonoids in transgenic tobacco.CsCHId increased the accumulation of flavonoids in transgenic Arabidopsis thaliana.