Maize Transformation With Betaine Aldehyde Dehydrogenase Gene(AnBADH) From Ammopiptanthus Nanus

Drought is one of the major abiotic stresses constraining the normal growth and development of maize.The development of drought tolerant varieties is the most economical and effective measure to overcome the drought threat.However,maize is a species sensitive to water deficiency.Drought-tolerant germplasm resources are seldom.Conventional breeding methods has not made much progress in improving drought tolerance,and it is difficult to meet the demand of agricultural production.Transgenic technology can be used to overcome reproductive barriers between species,transform drought-tolerant genes from other species,and provides a new way for the development of drought-tolerant maize varieties.Ammopiptanthus nanus,a xerophyte inhabiting arid desert region,is highly tolerant to abiotic stresses such as drought,salinity,and extreme temperatures.In our previous study,the betaine aldehyde dehydrogenase?BADH?gene AnBADH was cloned from A.nanus,and validated for its resistant function by transformation into E.Coli and Arabidopsis mutants.Based on subcellular localization and safety prediction,this study was attempted to construct monocotyledonous expression vector of the AnBADH gene,transform embryonic calli of maize by Agrobacterium mediation,regenerate plants after screening,identify positive plant lines integrated by the exogenous gene by PCR detection,confirm the expression of the AnBADH gene by semi-quantitative PCR,and phenotype drought tolerance by pot experiment,in order to lay the foundation for further drought tolerance identification and cultivation of drought-tolerant cultivars.The main results are as follows:?1?The expression vector pCAMBIA2300-35S-AnBADH-eGFP for dicotyledon plants and The constitutive expression vector pTF101.1-Ubi-AnBADH-T-nos for monocotyledonous plants was constructed by the insetion of the betaine aldehyde dehydrogenase gene AnBADH into the pCAMBIA2300-35S-eGFP and pTF101.1-Ubi-T-nos plasmid,and identified by enzyme digestion and sequencing.?2?Subcellular localization showed that the AnBADH protein functionsin the cytoplasm,and may play its tolerant effect through osmoregulation and activity protection of the cytoplasmic enzymes.?3?AnBADH protein was not homoloogus to any toxic proteins,allergens and anti-nutritional factors in the database,suggesting that it's not harmful to human halth.?4?The embryonic calli of maize inbred line“18-599”was transformed by Agrobacterium mediation.Six T₁ lines were screened,differentiated and regenerated on resistant medium and identified by specific PCR detection.?5?Semi-quantitative PCR showed that the AnBADH gene expressed in the T₂ lines.?6?Under drought stress conditions,the wilting degree of the T₂ seedlings was obviously less than the untransformed inbred line“18-599”,suggesting that the transgenic lines possessed higher drought tolerance.The above results demonstrated that the heterologous expression of the AnBADH gene in the transgenic lines significantly improves drought tolerance of maize.After further homozygous screening,phenotyping and safety evaluation,the transgenic lines are hopeful to be applied to improvement of maize varietis for drought tolerance.