过表达CHI基因提高甘草毛状根中黄酮类化合物含量的研究

查尔酮异构酶(chalcone isomerase,CHI,EC5.5.1.6)是甘草黄酮类有效成分生物合成途径中的第二个限速酶,发挥重要的调控作用。本课题组在前期研究基础上,筛选出黄酮高含量甘草特异对应的CHI基因型,通过基因融合法构建了过表达CHI基因的植物双元表达载体,并通过电转法将其转化到发根农杆菌ACCC10060中,用于侵染甘草子叶和胚轴,获得过表达CHI基因的甘草毛状根系,利用qRT-PCR法测定各甘草毛状根系中CHI基因的拷贝数,并利用UPLC法测定各甘草毛状根系中4种黄酮类化合物的含量。结果显示获得了拷贝数分别为1和5的过表达CHI基因甘草毛状根系,且其总黄酮、甘草苷、甘草素和异甘草素的含量均显著高于野生型毛状根,表明过表达CHI基因能显著提高甘草毛状根中黄酮类化合物的含量。本文为解析CHI基因的功能提供了理论依据,筛选出3个过表达CHI基因甘草毛状根系用于后续扩大培养,可为离体积累甘草黄酮类化合物奠定基础。

Overexpressing of chalcone isomerase(CHI)gene enhances flavonoid accumulation in Glycyrrhiza uralensis hairy roots

Chalcone isomerase(CHI)is the second rate-limiting enzyme involved in the biosynthetic pathway of flavonoids in Glycyrrhiza uralensis.Based on our previous studies,we selected the specific CHI haplotype(GenBank Accession No.KY115232)to maximize flavonoid accumulation.We constructed a plant binary expression vector for overexpression of this CHI gene by the gene fusion method and transfected the plasmid into Agrobacterium tumefaciens ACCC10060 by electroporation.The recombinant A.tumefaciens ACCC10060 subsequently was used to infect cotyledons and hypocotyls of G.uralensis to obtain transgenic hairy roots.A qRT-PCR method was used to determine the copy number of CHI and a UPLC method was used to assay the content of four flavonoids in different hairy root lines.The qRT-PCR results showed that the copy number of CHI in hairy roots was 1 or 5.UPLC results showed that the content of total flavonoids,liquiritin,liquiritigenin,and isoliquiritigenin in transgenic hairy root samples was significantly higher than that in wild-type samples.This study demonstrates that overexpression of CHI significantly increases the content of flavonoids in hairy roots of G.uralensis.This work provides a theoretical basis for clarifying the function of CHI.Three transgenic hairy root lines of G.uralensis were isolated which can be used to increase the accumulation of licorice flavonoids in vitro.