Identification And Functional Characterization Of The LEA Gene Family Members From Caragana Korshinskii

Caragana korshinskii belonging to the Caragana Fabr.,Leguminosae,is a perennial shrub with strong abiotic resistance and broad adaptability in desert and semi-desert area of northwest China.C.korshinskii has been proven to be an ideal material for studying the mechanisms of stress tolerance of shrubs.Previous studies have shown that late embryogenesis abundant?LEA?proteins play important roles in plant adaptation to abiotic stresses.However,reports that demonstrate their biological functions,especially in C.korshinskii,are still very limited.In this study,genes encoding LEA proteins were isolated from C.korshinskii by RACE and homology clonging based on a dehydration-induced suppressive subtractive hybridizations?SSH?cDNA library of C.korshinskii and transcriptome sequencing data of C.intermedia,and the functions of several CkLEAs in abiotic stress tolerance were further investigated.Here below are the detailed results of this study:1.A total of 26 CkLEAs were identified,including 3 of LEA₁ 8 of LEA₂,3 of LEA₃,2 of LEA₄,1 of LEA₆,2 of SMP and 7 of dehydrins.The gene structures,physico-chemical characteristics and phylogenetic relationships were analyzed.2.Quantitative real-time PCR analysis revealed that the expression levels of majority of CkLEAs showed tissue-specific expression in five different organs including roots,shoots,leaves,flowers and seeds.Moreover,the transcription of most CkLEAs could also be induced by drought,NaCl,cold,heat,high pH and abscisic acid?ABA?treatments.3.Three full-length cDNA fragments encoding LEA proteins were amplified using RACE?rapid amplification of cDNA ends?,denoted as CkLEA2-2,CkLEA2-3 and CkLEA4-2.The putative open reading frame of these genes encodes a polypeptide of 152,152 and 320 amino acid residues,respectively.4.Three expression vectors CkLEA2-3-GFP,CkLEA2-2-GFP and CkLEA4-2-GFP were constructed to investigate the protein subcellular localization.Confocal laser fluorescence microscopy showed that CkLEA4-2 localized in the mitochondria,and the CkLEA2-2 and CkLEA2-3 were distributed throughout the cytoplasm,nuclei and plasma membrane.5.The recombinant vector pE2-CkLEA2-2 was constructed and transformed into E.coli strain BL21?DE3?.The results suggest that CkLEA2-2 confers salt and osmotic tolerance to the transformed E.coli.6.Four expression vectors pCanG-CkLEA2-2,pCanG-CkLEA2-3,pCanG-CkLEA3-1,pCanG-CkLEA4-2,under the control of the CaMV 35S promoter were constructed and transformed into the wild-type Arabidopsis.Transgenic Arabidopsis overexpressing CkLEAs were generated for further analyzed.Overexpression of CkLEA2-2,CkLEA2-3,CkLEA3-1 and CkLEA4-2 in Arabidopsis resulted in enhanced tolerance to ABA treatment,osmotic and salt stresses during seed germination.CkLEA2-2 overexpression lines also showed better growth performance under salt or ABA treatment with longer primary roots.While the CkLEA2-3,CkLEA3-1 and CkLEA4-2 overexpression lines exhibited resistance to drought stress during seedling development.What's more,several genes involved in ABA signaling and other stress-responsive genes were up-regulated in CkLEAs transgenic plants.