Cloning And Function Analysis Of CkLEA4 From Caragana Korshinskii Kom

Drought, high temperature, low temperature and saline are the important factors torestrict plant growth and development. Caragana korshinskii Kom. belongs to Caraganaspecies, is an important cultivated shrub with high ecological and forage value, widely distributed in Northwest China. Late embryogenesis abundant (LEA) proteins could be induced in plants under stress conditions. In this study, a CkLEA4 was cloned for the first time from Caragana korshinskii Kom. and its primary function was analyzed. The concrete conclusions are as following:1. The full length cDNA was cloned by rapid amplification of cDNA ends technique. Sequencing results showed that the full length cDNA was 870bp, with an open reading frame of 510bp. It encodes a protein composed of 169 amino acids with a calculated molecular mass of 17.03kD, and a theoretical pI of 9.3. The deduced protein is strongly hydrophilic. Sequence and phylogenetic analysis indicated that the protein belongs to the LEA4 subfamily, therefore it was designated as CkLEA4. Phylogenetic anaysis shows that CkLEA4 is more closely related to MtLEA from Medicago truncatula than other LEAs and shares 85% amino acid identities with MtLEA.2. Real-time-quantitative PCR analysis showed that ABA, NaCl and dehydration stress enhanced CkLEA4 expression at the transcriptional level, indicating that this gene may be involved in the ABA and other stress resistance molecular mechanisms of C. korshinskii Kom.3. Fusion construct p35S::CkLEA4-GFP was transformed into A. thaliana to get permanent transgenic lines. Fluorescence observation under confocal laser-scanning microscopy confirmed that the GFP tagged CkLEA4 was localized in both cytoplasm and nuclei, but we can’t determine where the exact location is.4. Binary vector p35S::CkLEA4-HA was constructed and eight homozygous transgenic Arabidopsis lines overexpressing CkLEA4 had been obtained. Under mannitol and NaCl treatment, the transgenic lines were more resistant than the WT, and were also insensitive to ABA inhibition of germination.