Evaluation And Creation Of Germplasm And Mechanism Mediated By LcSRA13 To Response Salt Stress In Lotus Corniculatus

Lotus corniculatus is one of leguminous forage with rich nutrition,high quality and widely ecological adaptability.It is widely used for Livestock breeding,ornamental and ecological restoration.Soil salinization seriously threatens the development of agriculture and animal husbandry.Breed/create new salt-tolerant plant is crucial to solve this worldwide ecological problems.AP2/ERF transcription factors,which is gene family mainly involved in the metabolic pathways of plant growth and environmental stress.Here,we evaluated the germplasm and analyzed the phenotypes of hundreds of ecotypes of Lotus corniculatus.Phylogenetic analysis was performed to investigate their evolutionary relationship.We further created a new germplasm with enhanced drought resistance and salt tolerance using genetic engineering technology.Moreover,we exmained the molecular mechanism of regulation role of AP2/ERF family gene of LcSRA13 on plant growth and salt stress.The main results of the study are as follows:1.In this study,agronomic traits of 215 germplasm lines of Lotus corniculatus from all over the world were evaluated.Genetic diversity analysis and phylogenetic relationship among them were studied as well.The phylogenetic analysis was based on ITS sequences from all the samples.Base on the phylogenetic result,we proposed an evolution and transmission route of germplasm resources of Lotus corniculatus.2.The two genes PeDREB2 a and KcERF were co-overexpressed in Lotus corniculatus using Agrobacterium-mediated method.In the transgenic lines,the physiological and biochemical parameter were examined,and expression of many stress-response related genes were investigated under drought and salt stress conditions.Our results demonstrated that co-overexpression of the two transcription factors PeDREB2 a and Kc ERF improved drought and salt tolerance in Lotus corniculatus.3.We obtained LcSRA13 overexpression(LcSRA13-OE)plants and LcSRA13 interference expression(LcSRA13-RNAi)plants,respectively.The results showed that LcSRA13 affected plantbranch number and plant height.Compared with the wild type,the number of branches was increased and the height of plant was decreased in the LcSRA13 overexpressed plants.The number of branches was decreased and the height of plant was increased in the LcSRA13 interference expression plants.The results of salt stress treatment showed that the LcSRA13 overexpression improved salt tolerance,and LcSRA13 interference reduced salt tolerance in Lotus corniculatus.4.Based on RNA-Seq of LcSRA13 overexpression and Lc SRA13 interference expression plants,the differentially expressed genes mainly distributed on 14 metabolic pathways,such as transcription,secondary production biosynthesis,signal transduction,carbohydrate metabolism,environmental adaptive substances,amino acid biosynthesis and so on.Further analysis revealed that 26 differentially regulated genes were related to plant growth and salt stress response.4candidate genes,Lj1g3v3330050,Lj0g3v0070989,Lj0g3v0242029 and Lj0g3v0342769,regulated by LcSRA13 were screened out.5.The analysis of bioinformatics,together with yeast one hybrid(Y1H)and EMSA results demonstrated that Lc SRA13 binds to GCC-box in the promoter of Lj0g3v0242029.The yeast two hybrid(Y2H)and bimolecular fluorescence complementation(BiFC)analyses demonstrated that LcSRA13 and LcMED25 interacted with each other.In conclusion,LcSRA13 regulating plant branching and salt stress response by regulates downstream related genes through different pathways.