Cloning,subcellular localization and expression analysis of gene BoRACK1 in Brassica oleracea

ObjectiveGene(BoRACK1)sequence of tyrosine kinase receptorC1(RACK1)from Brassica oleracea was cloned;subcellular localization and expression analysis were also conducted,in order to provide theoretical basis for research on the regulation mechanism of gene RACK1 in the process of plant growth and development.MethodGene BoRACK1 was amplified by PCR and expression vector of subcellular localization was constructed.Onion epidermal cells were transformed by the particle bombardment method.The distribution of green fluorescent protein(GEP)was observed under a laser scanning confocal microscope.The fluorescent quantitative PCR(qRT-PCR)method was used to detect the expression of gene BoRACK1 in different tissues and seedlings under abiotic stress(200 mmol/L NaCl,100 μmol/L ABA and 1 mmol/L H2O2solution).ResultThe open reading frame(ORF)sequence of gene BoRACK1 was acquired by PCR amplification.cDNA was 980 bp in length,encoding 326 amino acids.Amino acid sequence hadhigh homology with the RACK1 of other plants,which was over 65%.In particular,the homology with Arabidopsis thaliana reached 93%.Gene BoRACK1 expressed in roots,stems,leaves,flowers and seeds of B.oleracea.The expressions in roots,leaves and seeds were high while those in flowers,seedlings and stems were low.The overall expression of gene BoRACK1 was down-re-gulated under abiotic stress of ABA,H2O2and NaCl. The expression level reached the lowest point at 6 h under NaCl stress,and the expression level reached the lowest point at 4 h under other stresses.BoRACK1 was localized in the cyto-plasm,cell nuclear and plasma membrane.ConclusionThe expression of gene BoRACK1 has no tissue specificity.The gene has different degrees of correlations with development of flowers,fruits and vegetative organof B.oleracea;and the gene may also be involved in the regulation process of plant stress resistance.