摘要
阿魏酸5-羟化酶(ferulate 5-hydroxylase,F5H)是苯丙烷代谢途径上的关键酶。本课题组前期转录组测序研究显示F5H基因在甘草酸生物合成过程中起负调控作用,因此本文拟克隆该基因,并从基因过表达和沉默两个方面对这一调控作用进行深入研究。本文克隆得到甘草F5H基因(GenBank注册号:MK882511);以pCAMBIA1305.1为载体骨架,以Spe I和Bgl II为酶切位点,利用基因融合法构建过表达载体pCA-F5H;根据F5H第一外显子设计sgRNA序列,以pHSE401为载体骨架,以BsaⅠ为酶切位点,构建CRISPR/Cas9基因编辑载体pHSE-F5H;使用电转法将pCA-F5H和pHSE-F5H分别转入发根农杆菌ATCC15834感受态细胞;以甘草胚轴为外植体材料,利用发根农杆菌介导法分别诱导F5H过表达及沉默甘草毛状根系。同时,构建野生型甘草毛状根以及携带空质粒的阴性对照甘草毛状根。利用UPLC法测定各甘草毛状根系中甘草酸的含量,结果显示F5H基因沉默甘草毛状根体系中甘草酸含量显著高于野生型和阴性对照组,而过表达F5H基因甘草毛状根系中甘草酸含量则显著低于野生型和阴性对照组。本文通过逆向遗传学策略,从基因过表达和沉默两个方面证实了F5H基因对甘草酸生物合成的负调控作用,为进一步构建甘草酸次生代谢分子调控网络奠定了基础。
Ferulate 5-hydroxylase(F5 H)is a key enzyme involved in the phenylpropane metabolism pathway.Based on our previous transcriptome sequencing study,F5 H played a negative regulatory role in glycyrrhizic acid(GA)biosynthesis.Therefore,in this study we cloned the F5 H gene and investigated its regulatory effect on GA accumulation through gene overexpression and knockout.F5 H was cloned from Glycyrrhiza glabra L.(GenBank Accession No.MK882511).A plant binary expression vector pCA-F5 H was constructed by inserting F5 H into pCAMBIA1305.1 at Spe I and Bgl II sites.The sgRNA sequences were designed based on the first exon of F5 H.The CRISPR/Cas9 gene editing vector pHSE-F5 H was constructed by inserting F5 H sgRNA into pHSE401 at two BsaⅠsites.PCA-F5 H and pHSE-F5 H were transfected into Agrobacterium tumefaciens ATCC15834,which was used to induce hairy root overexpressing or knocking out F5 H with licorice hypocotyl as explants.At the same time,wild type and negative control hairy roots were also generated.UPLC was used to assay the GA content in different hairy root lines,and results showed that the GA content in hairy root lines knocking out F5 H was significantly higher,whereas in hairy root lines overexpressing F5 H GA content was lower than that in the wild-type and negative control.In this work,through a reverse genetics strategy,the negative regulatory effect of F5 H on GA biosynthesis was confirmed through gene overexpression and knockout.This work will lay a foundation for further elucidation of the molecular regulatory network of GA biosynthesis.
作者
张智新
汪逗逗
杨林
田少凯
肖瑶
刘颖
ZHANG Zhi-xin;WANG Dou-dou;YANG Lin;TIAN Shao-kai;XIAO Yao;LIU Ying(School of Life Sciences,Beijing University of Chinese Medicine,Beijing 102488,China;School of Chinese Pharmacy,Beijing University of Chinese Medicine,Beijing 102488,China)
出处
《药学学报》
CAS
CSCD
北大核心
2021年第6期1719-1726,共8页
Acta Pharmaceutica Sinica
作者简介
通讯作者:刘颖,Tel:86-10-53912163,E-mail:liuyliwd@bucm.edu.cn;通讯作者:肖瑶,Tel:86-10-53912136,E-mail:xiaoyao9510@126.com。
