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Tanshinone is a group of active diterpenes, which are widely used in the treatment of cardiovascular disease. In this study, methyl jasmonate (MJ) and salicylic acid (SA) were used to investigate their effects on tanshinone accumulation and biosynthetic gene expression in the hairy roots of geranylgeranyl diphosphate synthase (SmGGPPS) overexpression line (G50) in Salvia miltiorrhiza. High-performance liquid chromatography analysis showed that total tanshinone content in G50 was obviously increased by 3.10-fold (11.33 mg/g) with MJ at 36 H and 1.63 times (5.95 mg/g) after SA treatment for 36 H in comparison with their mimic treatment control. Furthermore, quantitative reverse-transcription PCR analysis showed that the expression of isopentenyl-diphosphate delta-isomerase (SmIPPI), SmGGPPS, copalyl diphosphate synthase (SmCPS), and kaurene synthase-like (SmKSL) increased significantly with MJ treatment. However, the expression of SmIPPI reached the highest level at 144 H, whereas those of SmGGPPS, SmCPS, and SmKSL only increased slightly with SA treatment. The two elicitor treatments suggested that tanshinone accumulation positively correlated to the expression of key genes such as SmGGPPS, SmCPS, and SmKSL. Meanwhile, the study also indicated that it was a feasible strategy to combine elicitor treatment with transgenic technology for the enhancement of tanshinone, which paved the way for further metabolic engineering of tanshinone biosynthesis.

Hepatocellular carcinoma (HCC) is a common clinical primary malignant tumor; however, efficient drugs for the treatment of HCC are still lacking at the present time. To develop a new approach for liver cancer therapy, we designed a chimeric gene (his-HR) encoding a single-chain variable fragment of human HAb25 (hHscFv) fused to a cytotoxic ribonuclease from Rana catesbeiana (RC-RNase) and expressed the corresponding fusion protein in transgenic tobacco (Nicotiana tabacum). Eleven positive transgenic plant lines were identified from 204 regenerated tobacco plants by PCR and Southern blot analysis, and the immunocompetence of the recombinant his-HR protein was confirmed by Western blotting. The expression levels of his-HR protein ranged from 0.75 to 1.99 µg/g in the fresh tobacco leaves. To characterize the bifunction of the expressed his-HR protein in tobacco, binding specificity and cell toxicity to several cell lines were examined by the indirect immunocytochemical streptavidin-biotin complex method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Data indicated that the his-HR protein had stronger specific binding affinity to HepG2 (human liver HCC cell line) than to the other tumor cell lines and normal liver cell line, and the capacity to kill the HCC cell lines SMMC7721 and HepG2 with an half maximal inhibiting concentration of 2.0 and 2.4 nM, respectively. The results suggest that recombinant bifunctional his-HR protein derived from transgenic plants may provide a novel strategy to treat HCC in the future.

Tanshinone is a group of active diterpenes, which are widely used in the treatment of cardiovascular disease. In this study, methyl jasmonate (MJ) and salicylic acid (SA) were used to investigate their effects on tanshinone accumulation and biosynthetic gene expression in the hairy roots of geranylgeranyl diphosphate synthase (SmGGPPS) overexpression line (G50) in Salvia miltiorrhiza. High-performance liquid chromatography analysis showed that total tanshinone content in G50 was obviously increased by 3.10-fold (11.33 mg/g) with MJ at 36 H and 1.63 times (5.95 mg/g) after SA treatment for 36 H in comparison with their mimic treatment control. Furthermore, quantitative reverse-transcription PCR analysis showed that the expression of isopentenyl-diphosphate delta-isomerase (SmIPPI), SmGGPPS, copalyl diphosphate synthase (SmCPS), and kaurene synthase-like (SmKSL) increased significantly with MJ treatment. However, the expression of SmIPPI reached the highest level at 144 H, whereas those of SmGGPPS, SmCPS, and SmKSL only increased slightly with SA treatment. The two elicitor treatments suggested that tanshinone accumulation positively correlated to the expression of key genes such as SmGGPPS, SmCPS, and SmKSL. Meanwhile, the study also indicated that it was a feasible strategy to combine elicitor treatment with transgenic technology for the enhancement of tanshinone, which paved the way for further metabolic engineering of tanshinone biosynthesis.

Hepatocellular carcinoma (HCC) is a common clinical primary malignant tumor; however, efficient drugs for the treatment of HCC are still lacking at the present time. To develop a new approach for liver cancer therapy, we designed a chimeric gene (his-HR) encoding a single-chain variable fragment of human HAb25 (hHscFv) fused to a cytotoxic ribonuclease from Rana catesbeiana (RC-RNase) and expressed the corresponding fusion protein in transgenic tobacco (Nicotiana tabacum). Eleven positive transgenic plant lines were identified from 204 regenerated tobacco plants by PCR and Southern blot analysis, and the immunocompetence of the recombinant his-HR protein was confirmed by Western blotting. The expression levels of his-HR protein ranged from 0.75 to 1.99 µg/g in the fresh tobacco leaves. To characterize the bifunction of the expressed his-HR protein in tobacco, binding specificity and cell toxicity to several cell lines were examined by the indirect immunocytochemical streptavidin-biotin complex method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Data indicated that the his-HR protein had stronger specific binding affinity to HepG2 (human liver HCC cell line) than to the other tumor cell lines and normal liver cell line, and the capacity to kill the HCC cell lines SMMC7721 and HepG2 with an half maximal inhibiting concentration of 2.0 and 2.4 nM, respectively. The results suggest that recombinant bifunctional his-HR protein derived from transgenic plants may provide a novel strategy to treat HCC in the future.

Talpha1 (thymosin alpha1), an immune booster, plays an important role in the maturation, differentiation and function of T-cells. It can also activate the production of cytokines in dendritic cells. Talpha1 is one of two thymosin proteins that have potential future clinical applications. In order to express Talpha1 protein in plants, we designed and synthesized the Talpha1 gene according to the plant codon usage bias and created a novel 4 x Talpha1 concatemer (four copies of the Talpha1 gene arranged end-to-end in tandem, designated 4 x Talpha1). Subsequently, a plant binary expression vector, PG-pRD12-4 x Talpha1, was constructed and introduced into tomato via Agrobacterium tumefaciens-mediated transformation. Through selection, 54 regenerated tomato plants resistant to kanamycin were obtained, and four transgenic tomato plants were further confirmed by PCR and Southern blotting. RT-PCR (reverse transcription-PCR) analysis showed that the 4 x Talpha1 gene was transcribed specifically in tomato [Solanum lycopersicum (formerly Lycopersicon esculentum)] fruits. ELISA analysis showed that the content of the 4 x Talpha1 protein reached a maximum of 6.098 microg/g fresh weight in mature tomato fruit. Western-blot analysis further confirmed the expression of 4xTalpha1 protein in transgenic tomato fruits. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay showed the 4 x Talpha1 protein derived from transgenic tomatoes exhibited bioactivity that can stimulate the proliferation of mice splenic lymphocytes in vitro, and the specific activity of Talpha1 protein from the artificial system was higher than that from the synthetic Escherichia coli system. This study is the first to report successful expression of bioactive Talpha1 in plants, and also it will provide the basis for further development of the plant system to produce Talpha1.

Talpha1 (thymosin alpha 1) is important in treating immunodeficiency and other diseases. In order to study the feasibility of expressing Talpha1 in plants, as the first attempt, we designed and synthesized the Talpha1 gene according to the plant codon usage preference and constructed the 4xTalpha1 concatemer (four copies of a DNA sequence arranged end-to-end in tandem). The latter was inserted into Escherichia coli expression vector pQE30, resulting in a recombinant plasmid that was subsequently transformed into E. coli M15. The 4xTalpha1 concatemer protein was successfully expressed in E. coli in a soluble form. The expressed protein was purified and its bioactivity was analysed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay. Preliminary results showed that the 4xTalpha1 concatemer protein could stimulate the mice spleen lymphocyte proliferation. This is the first report on the expression of 4xTalpha1 concatemer that was synthesized according to plant codon usage preference in an E. coli expression system. The present study provides the basis for expressing the synthesized active Talpha1 gene in plants in the future.

In the present study, a plant binary expression vector PG-pRD12-hFIX (where PG is polygalacturonase) harbouring the hFIX (human coagulation Factor IX) gene was constructed and introduced into tomato (Lycopersicon esculentum) via Agrobacterium tumefaciens-mediated transformation. After kanamycin selection, 32 putative independent transgenic tomato plants were regenerated. PCR and Southern-blot analyses confirmed the transgenic status of some plants. RT (reverse transcription)-PCR analysis for the expression of the introduced gene (hFIX) demonstrated that the hFIX gene was expressed specifically in fruits of the tomato. Western-blot analysis confirmed the presence of a 56 kDa band specific to hFIX in the transformed tomatoes. ELISA results showed that the expression of hFIX protein reached a maximum of 15.84 ng/g fresh weight in mature fruit. A blood-clotting assay demonstrated the clotting activity of the expressed hFIX protein in transgenic tomato fruits. This is the first report on the expression of hFIX in plants, and our research provides potentially valuable knowledge for further development of the plant-derived therapeutic proteins.