药学学报, 2021, 56(6): 1719-1726
引用本文:
张智新, 汪逗逗, 杨林, 田少凯, 肖瑶, 刘颖. 基于基因过表达及基因沉默解析F5H基因对甘草酸生物合成的调控研究[J]. 药学学报, 2021, 56(6): 1719-1726.
ZHANG Zhi-xin, WANG Dou-dou, YANG Lin, TIAN Shao-kai, XIAO Yao, LIU Ying. Ferulate 5-hydroxylase gene (F5H) regulation of glycyrrhizic acid biosynthesis determined by gene overexpression and knockout[J]. Acta Pharmaceutica Sinica, 2021, 56(6): 1719-1726.

基于基因过表达及基因沉默解析F5H基因对甘草酸生物合成的调控研究
张智新1, 汪逗逗1, 杨林1, 田少凯1, 肖瑶2*, 刘颖1*
1. 北京中医药大学生命科学学院, 北京 102488;
2. 北京中医药大学中药学院, 北京 102488
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Bsa Ⅰ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。
关键词:    F5H      甘草毛状根      甘草酸      基因过表达      基因沉默      CRISPR/Cas9     
Ferulate 5-hydroxylase gene (F5H) regulation of glycyrrhizic acid biosynthesis determined by gene overexpression and knockout
ZHANG Zhi-xin1, WANG Dou-dou1, YANG Lin1, TIAN Shao-kai1, XIAO Yao2*, LIU Ying1*
1. School of Life Sciences, Beijing University of Chinese Medicine, Beijing 102488, China;
2. School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 102488, China
Abstract:
Ferulate 5-hydroxylase (F5H) is a key enzyme involved in the phenylpropane metabolism pathway. Based on our previous transcriptome sequencing study, F5H played a negative regulatory role in glycyrrhizic acid (GA) biosynthesis. Therefore, in this study we cloned the F5H gene and investigated its regulatory effect on GA accumulation through gene overexpression and knockout. F5H was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MK882511). A plant binary expression vector pCA-F5H was constructed by inserting F5H into pCAMBIA1305.1 at Spe I and Bgl II sites. The sgRNA sequences were designed based on the first exon of F5H. The CRISPR/Cas9 gene editing vector pHSE-F5H was constructed by inserting F5H sgRNA into pHSE401 at two Bsa Ⅰ sites. PCA-F5H and pHSE-F5H were transfected into Agrobacterium tumefaciens ATCC15834, which was used to induce hairy root overexpressing or knocking out F5H 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 F5H was significantly higher, whereas in hairy root lines overexpressing F5H 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 F5H 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.
Key words:    F5H    licorice hairy root    glycyrrhizic acid    gene overexpression    gene knockout    CRISPR/Cas9   
收稿日期: 2021-02-05
DOI: 10.16438/j.0513-4870.2021-0196
通讯作者: 刘颖,Tel:86-10-53912163,E-mail:liuyliwd@bucm.edu.cn;肖瑶,Tel:86-10-53912136,E-mail:xiaoyao9510@126.com
Email: liuyliwd@bucm.edu.cn;xiaoyao9510@126.com
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