Function Verification Of LcGR Gene In Tobacco And Application Of LcGR In Heavy Metal Resistance Of Lily

Heavy metals act as important abiotic stress factors during plant growth,threatening the normal development of plants and heavy metals disturb metabolic functions severely.In order to survive under heavy metals such as cadmium and copper,plants developed different mechanisms to reduce damages during evolution.Glutathione metabolic pathway is one of the important adversarial confrontation mechanisms in plants.Glutathione reductase plays a key role in this metabolic pathway,reducing the oxidized form of glutathione GSSG into reduced form GSH.By maintaining the high ratio of GSH/GSSG,GR enhances the ability of plant cells to eliminate ROS.Lily?Lilium longflorum L.?is a popular plant for decoration with strong ornamental qualities.Being able to grow easily and propagate fast while remains its beauty,Lily is worthy for modifications.Lilies are relatively sensitive to heavy metals,but new transgenic lines are developed by molecular level modification,targeted to enhance their ability to resist heavy metal stress.New transgenic lines also can be used to improve the ecology of heavy metal soil environments.The purpose of this study is to explore the stress-tolerant gene LcGR from Lycium chinense.In the research,the function of LcGR was verified by using model plant tobacco.And finally,using engineering method,LcGR was inserted into Lily.Results are as follows:1.The LcGR gene was cloned from Lycium chinense and over-expressed in model plant tobacco.Under heavy metal stress,we assessed growth situations and enzyme activities of transgenic tobacco.The results indicate that LcGR gene over-expression significantly improved heavy metal resistance of tobacco.Under the stress tests,the content of MDA in transformed tobacco was lower than the control significantly,and the enzyme activity of CAT and GR was higher than that in control.Moreover,GR-10was the most resistant type in all transgenic lines.2.This study aimed to improve heavy metal stress resistance of lily and to improve its surrounding soil environment.Tissue culture regeneration system of lily scales was optimized by orthogonal experiment.The optimal influencing factors of the transformation system were found,and finally the transgenic plants were obtained.The results showed that the medium with MS+2 mg/L 2,4-D+0.1 mg/L NAA+90 g/L sucrose had the best effect on scale expansion.The medium of MS+1.0 mg/L 6-BA+0.2 mg/L NAA+30 g/L sucrose was used to induce the direct differentiation of scales with the highest efficiency.The ratio of the medium MS+2.5 mg/L 2,4-D+0.4 mg/L TDZ+60 g/L sucrose was used to induce the highest efficiency of indirect differentiation of scales.In this research,Agrobacterium-mediated method was used to perform genetic transformation of LcGR on lily scales.According to the experiment results,the optimal genetic transformation system of scales is:Agrobacterium OD₆₀₀=0.6,with recipient scales pre-cultured for 3 days,and perform infestation for40 min,with 200?M concentration for AS.Using this procedure,the scale has the highest genetic transformation efficiency of 17.50%.Under the condition of Agrobacterium OD₆₀₀=0.4,recipient callus pre-cultured for 5 days,infestation for 40min and AS concentration was 200?M,the callus had the highest genetic transformation efficiency and the positive survival rate was as high as 12.60%.3.Through the optimized genetic transformation system described above,LcGR transgenic Lilies were obtained and tested under heavy metal stress.According to the results,the growth state and antioxidant enzyme activities of the transgenic Lily were significantly higher than control.The LcGR gene overexpression in Lily enhanced its resistance over the stresses of cadmium and copper.And these results lay the basis for future researches on phytoremediation.