PJB-2018-1509
Enhancing glycyrrhizic acid accumulation by root-specifically over-expressing 3-hydroxy-3-methylglutary coa reductase (HMGR), squalene synthase1 (SQS1), and β-amyrin synthase (β-AS) genes in Glycyrrhiza uralensis
Bo-Chuan Yuan, Rui Yang, Yong-Sheng Ma, Shan Zhou, Wen-Dong Li and Ying Liu
Abstract
Glycyrrhizia uralensis is one of the most widely-used Chinese herbal medicines, and glycyrrhizic acid (GA) is its marker component. However, the GA content in G. uralensis cultivars is generally low. We selected three functional genes involved in GA biosynthetic pathway in G. uralensis, 3-hydroxy-3-methylglutary CoA reductase gene (GuHMGR), squalene synthase1 gene (GuSQS1), and β-amyrin synthase gene (GuBAS), and constructed three root-specific expression vectors by gene fusion, then transformed them into disarmed Agrobacterium tumefaciens EHA105 cells using CaCl2 freeze-thaw method, which was used to infect G. uralensis explants. There were about 13% explants survived in infection and then dedifferentiated to resistant callus. The shoots differentiation rate was only about 7%, but they all successfully took root and finally, about 60% regenerated G. uralensis plantlets survived in transplantation. qRT-PCR was used to analyze the expression level and calculate the copy number of GuHMGR, GuSQS1, and GuBAS genes in the regenerated transgenic G. uralensis plants. Root-specific over-expression of GuHMGR, GuSQS1, and GuBAS genes were respectively observed in the regenerated transgenic G. uralensis plants. Thirteen GuHMGR transgenic G. uralensis plants were obtained with the gene copy number of 2, 3, 4, 5, or 6, respectively, six GuSQS1 transgenic G. uralensis plants were obtained with the gene copy number of 2, 3, 4, or 6, respectively, and five GuBAS transgenic G. uralensis plants were obtained with the gene copy number of 3 and 5, respectively. HPLC was used to detect the contents of GA and isoglycyrrhizic acid (IGA) in regenerated G. uralensis plants. It was found that GA and IGA contents were both higher in transgenic plants than that in blank control, and with the increase of copy number of GuHMGR, GuSQS1, and GuBAS both contents were increased. Root-specific over-expression of GuHMGR, GuSQS1, and GuBAS genes enhances the accumulation of GA and IGA
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