珠子参中HMGR基因对皂苷生物合成的影响

该研究以珠子参愈伤组织为材料,通过同源克隆方法获得了珠子参3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)基因(PjHMGR,登录号:MG712296)的cDNA序列,并对其进行生物信息学分析。基于PjHMGR序列构建了珠子参过表达载体pCAMBIA2300s-PjHMGR,通过根瘤农杆菌转化法将其转化到珠子参细胞中,成功获得7株阳性转PjHMGR基因细胞系;通过实时荧光定量PCR、比色法及皂化法等技术测定阳性细胞系PjHMGR基因的相对表达量、HMGR酶活、皂苷和植物甾醇含量变化。结果显示:(1)与野生型珠子参细胞系相比,转PjHMGR基因珠子参阳性细胞系中,PjHMGR基因的相对表达量、HMGR酶活和皂苷含量均有不同程度的提高;在效果最好的阳性细胞中,PjHMGR基因的相对表达量、HMGR酶活和皂苷含量分别为对照的7.15、6.14和3.50倍。(2)在研究过程中,发现转基因细胞系的植物甾醇含量也有所升高。研究认为,将珠子参关键酶基因PjHMGR过表达载体导入珠子参愈伤组织细胞中,可引起相关关键酶基因相对表达量和PjHMGR酶活性的增加,从而调控珠子参总皂苷的合成,对皂苷合成途径中关键酶基因进行调控将可能实现对皂苷生物合成的调节。

Effect of HMGR Gene on the Biosynthesis of Saponins in Panax japonicus

The cDNA sequence of PjHMGR was cloned from Panax japonicus callus and bioinformatics analysis of it was conducted in the study. Overexpression vector pCAMBIA2300s PjHMGR was constructed and then transferred into the P. japonicus callus to obtain the transgenic cell lines successfully by the method of Agrobacterium tumefaciens transformation. The relative expression level of PjHMGR, the enzyme activity of PjHMGR, the contents of PJS and phytosterols in transgenic cell lines were determined by the fluorescence quantitative PCR, colorimetric method and saponification. The results showed that: (1) all of them in PjHMGR transgenic cells achieved enhancements to some extent compared with the control. Especially, the expression level of PjHMGR, the enzyme activity and PJS content of the best performing positive cell line were 7.15 times, 6.14 times and 3.50 times of those in control, respectively. Meanwhile, the content of phytosterol in transgenic cell lines was also found to be enhanced. (2) The study considers that if the key enzyme genes (For example: the over expressing vector of PjHMGR in the biosynthesis pathway, was transformed into Panax japonicus callus, which will increase the relative expression level of the key enzyme genes and the activity of the PjHMGR enzyme, thereby regulating the synthesis of the total saponins of Panax japonicus.) take part in the biosynthesis of saponin were regulated. The regulation of the synthetic pathway of saponin will be realized.