Molecular Cloning And Functional Analysis Of CHIL And F3H In Flavonoid Biosynthesis Pathway In Mulberry

Mulberry(Morus alba)is an important economic crop,and its mature fruit is rich in flavonoids and anthocyanins,which are widely used in the fields of food and cosmetics.Chalcone isomerase-like protein(CHIL)belongs to the fourth class of CHI,which does not have CHI catalytic activity,but still affects the accumulation of plant flavonoids,and is called flavonoid biosynthesis enhancer.Flavanone 3-hydroxylase(F3H)catalyzes the conversion of naringenin to dihydroflavonols and is involved in the biosynthesis of flavonols and anthocyanins.More and more studies have shown that genes upstream of the flavonoid biosynthetic pathway may affect the synthetic yield of flavonoids.In this study,the CHIL and F3 H genes were cloned in mulberry,and the functional analysis of these two genes was carried out,which preliminarily revealed the roles of CHIL and F3 H in the flavonoid biosynthesis pathway in mulberry,and by regulating the expression levels of these two genes Effects on flavonoid and anthocyanin content in mulberry.The main results are as follows:1.The published CHIL and F3 H sequences of different plants were obtained by online analysis with blast software of NCBI website and submitted in Morus DB database.The homologous sequences were found by HMMER search,and primers were designed to obtain CHIL and F3 H genes from mulberry.The open reading frame length of CHIL gene is627 bp and encodes 209 amino acids.The open reading frame length of F3 H gene is 1098 bp,encoding 365 amino acids.The sequence is stored in Gen Bank(accession number:ON055162)and named Mazs F3 H.Phylogenetic analysis of CHIL and F3 H showed that CHIL gene belongs to Type IV CHI gene.Mazs F3 H clustered with F3 H of other species and could be distinguished from FLS and ANS genes belonging to a 2-ketoglutarate dependent dioxygenase(2-ODD)superfamily;In vitro enzyme activity analysis showed that Mazs F3 H could catalyze naringin to produce dihydrokaempferol.2.q RT-PCR was used to analyze the expression differences in different organs including leaves,buds,stems and fruits at different stages of mulberry.In this study,the expression level of CHIL in mulberry leaves is very low and almost not detected.F3 H showed low expression level in mulberry buds,and medium expression levels of F3 H were observed in stems,ovaries and stigmas.3.In order to explore the effects of CHIL and F3 H on flavonoid biosynthesis pathway,knockdown and transient overexpression of the two genes were carried out respectively.Knockdown of CHIL in mulberry and transient overexpression of CHIL in tobacco showed a positive correlation between CHIL gene expression level and total flavonoid content.In mulberry trees with successful knockdown of F3 H,different knockdown levels had different effects on anthocyanin and flavonoid contents.The results showed that only when the expression level of F3 H gene was down-regulated by more than 70%,the anthocyanin content could be significantly reduced.In tobacco with transient overexpression of F3 H,anthocyanin content was significantly decreased,and total flavonoid content was almost not significantly changed.4.To study the effect of F3 H expression level on other anthocyanin biosynthesis related genes,and determine the expression level of each gene in anthocyanin biosynthesis pathway when F3 H was knocked down.CHS,CHI2,CHIL and other upstream F3 H genes showed different changes.When the F3 H expression decreased by more than 70%,the expression of CHS as the upstream genes increased in the plants,while CHI2 expression decreased and CHIL expression level showed no significant changes which was reported to be CHS interaction protein and play important roles in enhancing flavonoid biosynthesis.The downstream structural genes involved in anthocyanin biosynthesis(including F3’H and ANS)were significantly decreased,and the expression level of FLS responsible for flavonol biosynthesis was significantly increased in F3 H down-regulated mulberry trees.Therefore,the results showed that F3’H and ANS were co-expressed with F3 H and responsible for anthocyanin accumulation,while the upstream genes of flavonoid biosynthesis pathways had different responses to the interference of F3 H expression level.