Functional Characterization Of Drought Response Gene NtabDOG1L In Nicotiana Tabacum

Drought is one of the major environmental stresses limiting crop growth and production.It is very important to exploit and utilize drought-tolerance genes to improve crop drought-resistance.In this study,a novel drought-responsive gene NtabDOG1L(DELAY OF GERMINATION 1 like gene)was cloned in tobacco,the expression pattern in different tissues and under different abiotic stresses,subcellular localization,transgenic over-expression analysis was systematically studied.The main findings are as follows:(1)Sequence information of NtabDOG1L gene was obtained from tobacco variety K326 by homologous cloning.Bioinformatics analysis showed that tetraploid common tobacco had two homologous sequences of NtabDOG1L,which were from diploid tobacco N.tomentosiformis(TT)and N.sylvestris(SS),named NtabDOG1L-T and NtabDOG1L-S.The length of NtabDOG1L coding region is 741 bp,encoding a protein containing 246 amino acids.Prediction of protein domain shows that the protein has no typical domain.Pfam database analysis shows that the protein belongs to the DOG1 family.Specific primers were designed to amplify the upstream promoter sequence of ATG and analyzed.It was found that the NtabDOG1Ls promoter sequence contained ABRE elements,TCA-elements,CGTCA-motif,ERE,AuxRR,TATC-box and MBS regulatory elements.(2)The expression pattern of NtabDOG1L in different tissues and organs of common tobacco K326 and under different abiotic stresses was analyzed by qPCR.It was found that NtabDOG1L was mainly expressed in roots,and its expression level was highest in lateral roots.Its expression was induced by PEG,NaCl,low temperature and ABA.The constructed vectors with target genes and GFP markers were transiently transformed into tobacco for subcellular localization.The results showed that NtabDOG1L-T was expressed in nucleus,cytoplasm and cell membrane.(3)The pWM101:NtabDOG1L-T overexpression vector was transformed into tobacco by Agrobacterium tumefaciens-mediated leaf disc transformation method.The overexpression transgenic plants were screened and identified.The roots of the overexpression NtabDOG1L plants grew vigorously.Under drought stress,the overexpression plants had lower leaf water loss rate,electrolyte leakage and malondialdehyde(MDA)content and higher antioxidant activity compared with the control plants.Enzyme activity,expression level of genes encoding key antioxidant enzymes and drought-related proteins increased significantly.Overexpression of NtabDOG1L could significantly improve the drought resistance of tobacco.