三七转录因子PnWRKY1对三七皂苷生物合成的影响

Effect of Transcription Factor PnWRKY1 on the Biosynthesis of Panax notoginseng Saponins

该研究以野生型三七为材料,采用同源克隆的方法,获得三七转录因子PnWRKY1基因,运用农杆菌转化法构建转基因细胞系,通过测定转基因细胞系中的总皂苷含量以及重要单体皂苷的含量,并采用qRT-PCR分析皂苷合成途径中相关基因的表达情况,为三七皂苷生物合成高效调控策略的建立提供理论依据。结果显示:(1)转录因子PnWRKY1长度为810bp,编码269个氨基酸。(2)成功构建PnWRKY1过表达载体pCAMBIA2300sPnWRKY1,经农杆菌转化获得了6株具有卡那霉素抗性的转基因细胞系(T_1~T_6),对卡那霉素基因nptⅡ进行PCR检测表明,所有细胞系均有与预期大小一致的450bp特异性条带,说明成功获得了6株PnWRKY1过表达转基因细胞系。(3)6株转基因细胞系中的PnWRKY1表达水平均极显著高于野生型细胞系,其中表达量最高的T_3细胞系比野生型增加了5.36倍。(4)过表达PnWRKY1基因细胞系的总皂苷生物合成均得到显著提高,其中T_1~T_6中总皂苷含量分别为野生型细胞系的2.46、1.98、2.67、1.74、2.54和1.98倍;6株过表达PnWRKY1细胞系中的4种单体皂苷R1、Rg1、Re、Rb1的含量与野生型细胞系相比均有不同程度的提高,且T_3细胞系中的单体皂苷Re含量最高(37.81mg/g)。(5)与野生型细胞系(WT)相比,过表达PnWRKY1细胞系中三七皂苷合成途径中的关键酶基因PnDS、PnSS和PnSE的最高表达水平分别增加3.1、4.0和4.5倍。研究表明,转录因子PnWRKY1在三七细胞中的过表达可能参与调节皂苷生物合成部分重要酶基因的表达,且PnWRKY1可能通过调控三七皂苷生物合成途径中关键酶基因的表达间接影响三七皂苷的合成。

In this study,the wild-type Panax notoginseng was used as the experimental material.The transcription factor gene PnWRKY1 was isolated from the P.notoginseng by homologous cloning,and the overexpression vector pCAMBIA2300 s-PnWRKY1 was transferred into P.notoginsengcells by Agrobacterium tumefaciens-mediated transformation system.The contents of total saponins and four important monomer saponins were detected,and the expression levels of enzyme genes in the biosynthetic pathway were analyzed by qRT-PCR.It provides a theoretical basis for the establishment of a highly efficient regu-lation strategy for the biosynthesis of P.notoginsengsaponins.The results showed that:(1)the transcription factor PnWRKY1 is 810 bp in length encoding 269 amino acids.(2)PnWRKY1 overexpression vector pCAMBIA2300 s-PnWRKY1 was successfully constructed.Six transgenic cell lines(T1-T6)with resistance to kanamycin were obtained by Agrobacteriumtransformation.PCR analysis of the kanamycin gene nptⅡshowed that all cell lines had a 450 bp specific band consistent with the expected length,indicating that six PnWRKY1 transgenic cell lines were obtained,which overexpressed PnWRKY1 successfully.(3)The expression levels of PnWRKY1 in six transgenic cell lines were very significantly higher than that in wild-type cell lines,and the T3 cell line with the highest expression was 5.36 times higher than the wildtype cell line.(4)The total saponins biosynthesis in PnWRKY1-overexpressed cell lines was significantly promoted,the contents of total saponins in T1-T6 were 2.46,1.98,2.67,1.74,2.54 and 1.98 times of the wild-type cell line,respectively.In six PnWRKY1-overexpressed cell lines,the content of four monomer saponins(R1,Rg1,Re and Rb1)were increased to some content compared with wild-type cell line,especially the T3 cell line,the content of monomer saponin Re was the highest(37.81 mg/g).(5)Compared with the wild-type cell line,the highest expression levels of key enzyme genes PnDS,PnSS and PnSEin the synthesis pathway of P.notoginsengsaponins in the overexpressed PnWRKY1 cell line were increased by 3.1,4.0 and 4.5 times,respectively.This study showed that the transcription factor PnWRKY1 may affect the synthesis of P.notoginsengsaponins indirectly by regulating the expression of key enzyme genes in the biosynthetic pathway of P.notoginsengsaponins.The results provide references for the establishment of a highly efficient regulation strategy about the biosynthesis of P.notoginseng saponins.