Cloning and expression analysis of SoNCED gene in key enzyme for sugarcane ABA biosynthesis

ObjectiveThe research cloned 9-cis-epoxycarotenoid dioxygenase gene (SoNCED) from sugarcane,analyzed its biological information and expression under stress,in order to provide theoretical basis for further exploring the role of SoNCED gene in sugarcane ABA biosynthesis.MethodTaking sugarcane variety Guitang 21 as materials,when sugarcane seedlings grew 5-6 leaves,drought,low temperature,high salt and oxidation stresses were applied to them.SoNCED gene was cloned using reverse transcriptional PCR (RT-PCR) and rapid amplification of cDNA end (RACE-PCR).Bioinformatics was used to analyze amino acid sequence.Prokaryotic expression vector was established and induced expression of target protein was conducted.Real-time fluorescence quantitative PCR (qRT-PCR)method was adopted to study the expressions of SoNCED geneunderdifferent stresses.ResultThe cDNA of cloned SoNCED(GenBank accession number JQ314108) in sugarcane was 2521 bp in length with an open reading frame(ORF) of 1827 bp,encoding 608 amino acids.Multiple sequence alignment and phylogenetic tree analysis showed that amino acid sequence of SoNCED gene coding was similar to that of other plants.It showed the highest homology with Sorghum bicolor and Zea may,which reached 96%.Prokaryotic expression vector of gene pET-SoNCED was established.And 66.0 kD protein was obtained under IPTG induction,which was determined as expression protein of SoNCED gene.Results of qRT-PCR analysis showed that SoNCED gene was able to express under drought,low temperature,high salt,and oxidation stresses.Under oxidative stress,the expression level reached the peak in 6 h;and the expressions reached the peak in 12 h under other stresses.ConclusionGene SoNCED was cloned from sugarcane.Different stresses are employed to predict the role of SoNCED gene in regulation of abiotic stresses such as drought,high salt,low temperature and oxidation.