Improvement Of Salt Tolerance Of Lettuce Seedlings By Introduction Of AcdS Gene

Nearly 10% of the world's cultivated land belongs to saline-alkali soil.Salinization of soil is one of the significance factors affecting agricultural production and limiting plant yield,which severely limits the sustainable use of cultivated land and affects the sustainable development of agriculture.Lettuce(Lactuca)has become the favorite vegetable crops because of its high nutrition value,low calorie and can be eaten directly.Lettuce is often suffer from drought,salinity,high temperature and other adverse stresses in the production process,leading to lower quality and yield.The use of genetic engineering for germplasm innovation and variety improvement has become a fast and effective method to improve the salt tolerance of crops.Establish a highly efficient and stable lettuce regeneration system,cultivate new varieties of salt-tolerant lettuce can provide technical support and theoretical basis for the cultivation of lettuce in saline-alkali soil.ACC deaminase catalyzes metabolism of ethylene precursor ACC(1-aminocyclopropane-1 carboxylic acid)into ?-ketobutyric acid and ammonia,thereby reducing the amount of ethylene produced in plants to improve plant resistance.In our experiment,the acd S gene of Bacillus cereus was cloned,the plant expression vector 35S::acd S-GFP was constructed and transformed into Hong Kong glass lettuce(WT)by leaf disk method using agrobacterium.In this experiment,the genetic transformation system of Hong Kong glass lettuce was optimized.The leaf-explants of lettuce were preincubated on differentiation medium for 2 days,the differentiation medium was MS+6-BA0.5mg/L+NAA0.1mg/L,and then transformed into Agrobacterium tumefaciens with OD600 for 8 min.After co-culture for 3 days,the explants were transferred to the differentiation culture medium,of which Hyg10mg/L as screening antibiotic,Cef300mg/L as antibacterial antibiotic.The rooting medium was selected 1/2MS + 0.05mg/L NAA + Hyg10mg/L.The transgenic lettuces were identified by PCR analysis of the fusion gene,and western blot analysis demonstrated the acd S protein was expressed in transgeniclettuce.And the transgenic lettuce seedling lines stably expressing the acd S gene were obtained.The fusion gene was located on the cell membrane or cell wall of the root cell.The introduction of exogenous acd S gene resulted in significant ACC deaminase activity in transgenic lettuce.The results of salt stress experiment showed that the growth of transgenic lines under salt stress was better than that of wild type.The content of proline in transgenic lettuce reached 802.02?g/g at 300mmol/L Na Cl and 1.36 times that of wild type.In300mmol/L Na Cl treatment,the soluble sugar content in transgenic lettuce was43033.80?g/g,which was 2.09 times of that of wild type.And the activity of SOD in the wild type lettuce was 2.14 times when the Na Cl concentration was 300mmol/L,the activity of SOD in the transgenic lettuce was 3.47 times higher than that in the wild type plant.When the concentration of Na Cl was 200mmol/L,the chlorophyll content of transgenic lettuce was 0.16mg/g while the control was 0.09mg/g.When treated with 300mmol/L Na Cl,the relative water content of transgenic lettuce was70.95%,while the wild type was 60.95%.In 300mmol/L Na Cl treatment,the content of MDA in the wild type plants was 4.80×10-3?mol/g and the content of MDA in the transgenic lettuce was 4.00×10-3?mol/g,which was significantly lower than that of WT plants(P <0.05).In summary,the introduction of acd S gene can effectively eliminate salt damage to lettuce and improve the salt tolerance of transgenic lettuce.In this study,the ACC deaminase gene of Bacillus cereus was introduced into lettuce and the transgenic lettuce lines with significant increase of ACC deaminase level were obtained.The effects of the introduction of acd S gene of transgenic lettuce on the salt tolerance were studied by simulating salt stress,which laid a foundation for cultivating salt tolerant lettuce.