Study On Mechanism Of Cloning,Expression And Functional Analysis Of Panax Ginseng PgCyP Gene In Arabidopsis Thaliana

Objective:To study the role of ginseng cyclophilin pgCyP gene in resistance to ginseng blight and high salt stress,and provide more choices for ginseng resistance to disease and stress.To study the role of ginseng cyclophilin pg Cy P gene in anti-cancer,and provide more choices for anti-cancer.Method:Ginseng RNA is obtained by TRizol method,CyP target gene is increased by RT-PCR,TA cloning,sequencing gene and construction of eukaryotic expression vector.Agrobacterium freeze-thaw transformation,Agrobacterium-mediated flower soaking transformation of Arabidopsis thaliana;Screening hygromycin and molecular identification to obtain transgenic positive plants;Wild Arabidopsis thaliana was used as the control group to isolate the whole plant and analyze the disease resistance of leaves and protein extracts.RT-PCR Analysis of Genes Related to Arabidopsis Disease Resistance before and after Stress.The plants were treated with high salt in culture medium and soil and the related physiological indexes were detected.CCK8 assay was used to detect the cytotoxicity of three kinds of cancer cells.Result:First,ginseng CyP target gene was successfully amplified,with 522 bp.The plant expression vector p CAMBIA1303-Cy P was successfully constructed.Secondly,PCR detection shows that the target gene has been integrated into Arabidopsis thaliana genome and transcribed.Western Bolt experiment showed that the target gene Cy P protein was successfully translated.Thirdly,the expression of Cy P protein in transgenic phytophthora infestans before and after stress was higher than that in wild type(p < 0.05).The growth state of transgenic Arabidopsis plants is better than that of wild type after disease stress.In vitro leaves of transgenic plants have better resistance to Phytophthora ginseng.Protein extracts from transgenic plants can also slow down the growth of Phytophthora infestans spores.Fourth,RT-PCR results showed that PRs of disease-resistant genes before Phytophthora blight stress was lower and the contents of positive plants and wild-type plants were not significantly different.PRs content of transgenic Arabidopsis thaliana was higher than that of wild type(P<0.05).Fifthly,the results of salt tolerance experiments show that the relative chlorophyll content of transgenic Arabidopsis thaliana is greatly increased,the water loss rate is lower,and the electrolyte rate is lower than that of wild Arabidopsis thaliana under high salt conditions.The expression of proline per unit fresh weight increased.Sixthly,CCK8 assay showed that the transgenic Arabidopsis thaliana protein had inhibitory effects on three kinds of cancer cells.Conclusion:Ginseng CyP gene has some disease resistance,stress resistance and anti-tumor effects.