Cloing And Preliminary Functional Analysis Of Calcineurin B-like Gene Of Mesembryanthemum Crystallinum L.

Mesembryanthemum crystallinum L.is a halophyte with high resistance to salt stress.Up to0.5 mol·L^-1NaCl is tolerated well and accelerates reproductive development for M.crystallinum.Thus,it has become synonymous with salt stress responses modelled at the molecular level.So it is important that study of salt-tolerance mechanism and cloning of salt tolerance genes of M.crystallinum for reveal salt resistance molecular mechanism of plant.Calcineurin B-like?CBL?protein,a Ca²⁺sensor,relay to convert Ca²⁺signals into salt stress responses.In Arabidopsis thaliana,the loss-of-function mutant of AtCBL4 shows hyper-sensitivity to salt stress.In the present study,based on the EST,McCBL1 gene was isolated from M.crystallinum by RACE-PCR.To analyze the the preliminary function of of McCBL1,tissue-specific expression,subcellular localization and over-expression in rice have been performed.It provides a basis for studying the salt tolerance mechanism of this gene.The main results are as follow:1.Based on the EST,the conserved primers were designed.The full-length cDNA sequence of McCBL1 gene was successfully obtained by RACE-PCR using the cDNA of leaf from M.crystallinum.McCBL1 gene has 1118 bp,encodes a 226 amino acides peptide,which molecular weight is 26.4 kDa and the isoelectric point is 5.0.McCBL1 protein contain two EF-hand calcium combined domain which were similar to CBL families members.The phylogenetic tree analysis of McCBL1 shows that McCBL1 gene is most closely related to AtCBL6.2.The total RNA of root,shoot and leave have been extracted in M.crystallinum.And the expression of McCBL1 in each organ was detected by fluorescence quantitative PCR.The results show the relative expression of McCBL1 was highest in leave.3.To determine the subcellular localization of McCBL1,the constructs,p163-McCBL1-GFP were transformed into rice protoplast and observed the results by confocal microscope.The result shows green flurescence signals were observer exclusively in the chloroplast of cells transformed with p163-McCBL1-GFP.4.The construct,35S-McCBL1-3×Flag has been transformed into calli of Nipponbare by Agrobacterium tumefaciens.There were 46 transgenic rice plants for 4 months.And the results of PCR shows that 37 positive transgenic rice plants were obtained.5.T1 transgenic rice plant and WT with different NaCl noncentrations?0.0%?0.4%?0.8%?1.0%and 1.2%?were selected to study the rice germination rate and germination potential,measuring the length of seedings and the root length.The results show over-expression McCBL1 can increase plant salt tolerance.