Cloning And Functional Analysis Of FmSnRK2.6/FmSnRK2.10 Genes

Fraxinus mandshurica has high economic value,and is ranked into the second degree of national protected plants.Domestically and abroad physiological characteristic,crossbreeding,carbon and nitrogen metabolism,tissue culture and other aspects are outspread,however,studies on molecular biology and genetic engineering of ABA responsive genes related to drought resistance have not been reported.This thesis aims to analyze the function of transcription factor genes of FmSnRK2.6 and FmSnRK2.10 through homology-based cloning,bioinformatics analysis,expression vectors construction and experiment on physiological and biochemical indexs of overexpression materials under stress treatment of FmSnRK2.6 and FmSnRK2.10 genes responsed adversity stress,to further understand the SnRK2 gene characteristics,functions and provide the theory foundation for breeding the drought tolerant cultivars of Fraxinus mandshurica by genetic engineering.1.Two SnRK2 transcription factor gene were found and cloned in drought stress response transcriptome of Fraxinus mandshurica,and named FmSnRK2.6 and FmSnRK2.10.The identified genes are belong to the SnRK2 family by multiple sequence alignment.FmSnRK2.6 gene contains 1089bp open reading frames,and coding 362 amino acids.The encoded protein's molecular weight is 41015.5Da,and its isoelectric point is 4.83.FmSnRK2.10 gene contains 1074bp open reading frames,and coding 357 amino acids.The encoded protein's molecular weight is 41.01 kDa,and its isoelectric point is 5.43.Both of the FmSnRK2.6 and FmSnRK2.10 contain ATP binding site and the specific conserved domain of the protein kinase(Ser/Thr enzyme slipknot structure domain),and they are hydrophilic proteins.2.According to the sequences of the target genes,specific primers were designed to get the target genes FmSnRK2.6 and FmSnRK2.10 into the expression vector pROKII by Infusion clone,the recombinant vectors were introduced into Agrobacterium by triparental mating and screened the transformation of Agrobacterium.According to the Agrobacterium mediated genetic transformation technologies,we conducted a preliminary exploration on the genetic transformation conditions of Fraxinus mandshurica.The Manchurian ash explants were placed on culture medium of callus inducing containing kanamycin of different concentrations,callus induction rate was 0 when the Kan's concentration was 80mg/L;leaves,petioles,stems and roots of Fraxinus mandshurica were used as explants,in different co-culture time and delay treatment conditions for genetic transformation respectively.The results showed that when the root explants in two days of co-culture and without delay in processing conditions of genetic transformation have the highest transformation efficiency.3.The overexpression of FmSnRK2.6 and FmSnRK2.10 gene of Fraxinus mandshurica callus as test material,the Fraxinus mandshurica callus without genetic transformation for control were treated with 20%PEG and 20%PEG+300 ?mol/L ABA.Compared with the control group,the expression of SnRK2.6,SnRK2.10,ABA signal transduction pathway related gene PYR1,defense enzyme genes POD1 and SOD were significantly up-regulated in the overexpression materials,content of proline and activities of antioxidant enzymes of POD and SOD were significantly increased,level of MDA(the products of membrane lipid peroxidation)was decreased,it indicated that the overexpression of FmSnRK2.6 and FmSnRK2.10 gene enhanced drought tolerance in transgenic Fraxinus mandshurica callus.