A number of genes that function in the terpenoid indole alkaloids (TIAs) biosynthesis pathway have been identified in Catharanthus roseus . Except for the geraniol 10-hydroxylase (G10H) gene, which encodes a cytochrome P450 monooxygenase, several of these genes are up-regulated by ORCA3, a jasmonate-responsive APETALA2 (AP2)-domain transcript factor. In this study, the G10H gene was transformed independently, or co-transformed with ORCA3 into C. roseus , using Agrobacterium rhizogenes MSU440. Hairy root clones expressing the G10H gene alone, or both the G10H and ORCA3 genes, were obtained. Alkaloid accumulation level analyses showed that all transgenic clones accumulated more catharanthine, with the highest accumulation level in the transgenic clone OG12 (6.5-fold higher than that of the non-expression clone). Following treatment with ABA, accumulation of catharanthine reached 1.96 mg/g DW in the transgenic clone OG12. The expression levels of TIAs biosynthesis genes in transgenic and non-transgenic clones were also investigated.
Shi Min,Luo Xiuqin,Ju Guanhua,Yu Xiaohong,Hao Xiaolong,Huang Qiang,Xiao Jianbo,Cui Lijie,Kai Guoyin
Tanshinone is widely used for treatment of cardio-cerebrovascular diseases with increasing demand. Herein, key enzyme genes SmHMGR (3-hydroxy-3-methylglutaryl CoA reductase) and SmDXR (1-deoxy- d -xylulose 5-phosphate reductoisomerase) involved in the tanshinone biosynthetic pathway were introduced into Salvia miltiorrhiza ( Sm ) hairy roots to enhance tanshinone production. Over-expression of SmHMGR or SmDXR in hairy root lines can significantly enhance the yield of tanshinone. Transgenic hairy root lines co-expressing HMGR and DXR (HD lines) produced evidently higher levels of total tanshinone (TT) compared with the control and single gene transformed lines. The highest tanshinone production was observed in HD42 with the concentration of 3.25 mg g −1 DW. Furthermore, the transgenic hairy roots showed higher antioxidant activity than control. In addition, transgenic hairy root harboring HMGR and DXR (HD42) exhibited higher tanshinone content after elicitation by yeast extract and/or Ag + than before. Tanshinone can be significantly enhanced to 5.858, 6.716, and 4.426 mg g −1 DW by YE, Ag + , and YE-Ag + treatment compared with non-induced HD42, respectively. The content of cryptotanshinone and dihydrotanshinone was effectively elevated upon elicitor treatments, whereas there was no obvious promotion effect for the other two compounds tanshinone I and tanshinone IIA. Our results provide a useful strategy to improve tanshinone content as well as other natural active products by combination of genetic engineering with elicitors.
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