Cloning And Functional Analysis Of BHLH Family Genes Related To Flavonoid Biosynthesis In Fagopyrum Tataricum

Fagopyrum tataricum is an annual herb of buckwheat family.Because it contains flavonoids,it has many functions,such as anti-oxidation,anti-inflammatory,hypoglycemic,hypolipidemic,blood pressure lowering,anti-tumor and so on.In recent years,there are many studies on the chemical constituents of tartary buckwheat.However,there are few reports on the biosynthesis pathway of flavonoids in tartary buckwheat,which is the main substance of medicinal value of tartary buckwheat.The regulation of flavonoid biosynthesis is influenced by many factors.This study focuses on the effects of Tartary Buckwheat bHLH transcription factors on flavonoid biosynthesis pathway in tartary buckwheat.The purpose of this study is to explore the regulatory mechanism of the flavonoid biosynthesis pathway in Tartary Buckwheat by analyzing the bHLH family and the cloning and functional analysis of the important genes in the flavonoid biosynthesis pathway.The specific contents are as follows:1.In this study,164 families of bitter buckwheat bHLH family were annotated by using the information of tartary buckwheat protein group in the transcription factor database.The structural characteristics of Tartary Buckwheat bHLH family were analyzed by MEME online software.MEGA7.0 was used to construct the Neighbo-Joining(NJ tree)family,and the 164 bHLH family sequences were divided into 26 subfamilies and 8 groups.MeV thermography software is used to visualized their expression in tissues and organs.2.Homologous cloning method was used to obtain 3 homologous genes from bHLH family genes of tartary buckwheat,named FtbHLH001,FtbHLH003 and FtbHLH004 respectively.The 4 nucleotide sequences contain the open reading frames of 2199bp,1761bp and 1887bp,respectively.The encoded amino acids are 732,586 and 628 respectively.5 promoters in the flavonoid synthesis pathway were cloned by genomic information,which were LAR,F3 'H,CHI,F3H and CHS,and the corresponding lengths were 1626bp,1784bp,1827bp,1822bp and 1550bp,respectively.3.Three genes of FtbHLH001 FtbHLH003 and FtbHLH004 were constructed into AD and BD vectors of yeast two hybrid system by double enzyme digestion.Gateway technology was used to construct three target genes,namely,over expression vector(pK7WG2D)and gene silencing vector(pK7GWIWGIID).4.The construction of 3 overexpressed plasmids was introduced into the Agrobacterium tumefaciens ACCC10060,and 3 genetic engineering bacteria(FtbHLH001-pK7WG2D-ACCC10060,FtbHLH003-pK7WG2D-ACCC 10060 and FtbHLH004-pK7WG2D-ACCC 10060)were successfully constructed.Transgenic materials were obtained from infected buckwheat tissue by genetic engineering bacteria,and 5,15 and 20 transgenic hairy root materials were obtained respectively.