Clonning And Function Analysis Of CkLEA4-3 From Caragana Korshinskii Kom.

Throughout the entire life of plants,drought,soil alkalization and freezing injury become critical adverse stress factors restricting the growth and development of plants.During the long evolution history,plants have developed various physiological and biochemical mechanisms to adapt to the different environment.The late embryogenesis abundant protein(LEA)is a series of functional protein,which closely relates to the stress resistance function of plants.Caragana korshinskii,which belongs to the family of leguminosae,has the characteristics of drought,cold,saline-alkali tolerance and infertility bearing.It is an important bush species used for afforestation in arid desert in northwest China.In this study,an LEA encoding gene has been cloned from the C.korshinskii,and its function under stress has been analyzed.The results are as follows:1.The cDNA length of the cloned LEA is 971 bp,with an open reading frame of 636 bp.It encods 211 amino acids,and the speculating molecular weight is 22.44 kD,the isoelectric point is 5.76.The gene has 31 phosphorylation sites and 6 glycosylation sites.Through sequence alignment and phylogenetic analysis,it shows that the protein has no specific domain,and has the closest homology with Arabidopsis LEA4(AT5G44310),so the gene is named CkLEA4-3.2.The expression pattern of CkLEA4-3 has been detected by real-time quantitative PCR analysis revealed that the expression of CkLEA4-3 has certain tissue specificity in root,stem,leaf.Moreove,CkLEA4-3 could be induced by drought,dehydration,ABA,cold,heat and high salinity stress,which indicates that it may be related to stress.3.The over-expression vector CkLEA4-3-HA has been constructed and transformed into wild-type Arabidopsis.All the obtained transgenic homozygous lines showed CkLEA4-3 overexpression through real-time fluorescent quantitative RT-PCR detected that shows strains have varying degrees of expression.4.The germination rate of CkLEA4-3 over-expressed Arabidopsis under treatment of400 mM mannitol and 150 mM NaCl was significantly higher than that wild-type,and the primary root length of the CkLEA4-3 over-expressed line is significantly longer than that of the wild-type.Also,the transgenic seedlings are slightly larger than the wild ones under mannitol treatment.5.Under the drought treatment,the 4-week-old transgenic Arabidopsis showed resistance to drought,and had significantly higher survival rate than thewild-type;The MDA contents in over-expressed plants was significantly lower than that of wild-type ones;meanwhile the chlorophyll content of over-expressed plants is higher than that of wild-type.