Maize Transformation With Vacuolar Hydrogen Ion Pyrophosphatase Gene From Ammopiptanthus Nanus

Abiotic stresses such as drought are the main limiting factors for maize production.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 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 distributing in arid desert region,is highly tolerant to abiotic stresses such as drought,salinity,extreme temperatures.In the previous study,the vacuolar hydrogen ion pyrophosphatase gene AnVP1 was cloned from A.nanus,and validated for its resistant function by transformation into the yeast and Arabidopsis mutants.This study was attempted to construct monocotyledonous expression vector of the AnVPl gene,transform embryonic calli by Agrobacterium mediation,regenerated plants after screening,identify positive plant lines integrated by the exogenous gene by PCR detection,confirm overexpression of the AnVP1 gene by quantitative PCR,and phenotype drought tolerance by pot experiment,in order to create novel germplasm for breeding drought-tolerant maize varieties.The main results are as follows:(1)The vacuolar hydrogen ion pyrophosphatase gene AnVPl of A.nanus,ubiqintin promoter of maize,and terminator of the nopaline synthase gene of Agrobacterium tumefaciens(T-nos)were inserted plasmid pZZ00026 constructing the monocotyledonous expression vector pZZ00026-Ubi-AnVP1-T-nos.It was confirmed by the enzyme digestion and sequencing.(2)The expression vector pZZ00026-Ubi-AnVP1-T-nos was transformed into embryonic calli of maize inbred line CO1 by Agrobacterium mediation.Forty-seven T1 lines were obtained after screening on the resistant medium,differentiation and regeneration,herbicide screening in field,and PCR detection.Among them,three lines numbered 737.760 and 765.(3)The result of real-time quantitative PCR showed that the AnVP1 gene overexpressed in the T2 lines reproduced from the 737,760 and 765 T1 lines.Under the simulated drought treatment of 16%polyethylene glycol 6000,the expression of the AnVP1 gene was up-regulated in the leaf and root of the T2 lines.The relative expression levels in leaf of line 760 and root of line 765 were significantly higher than the untreated control.However,the relative expression levels in leaf and root of the other lines were not significantly higher than the untreated control.(4)After two days of 16%polyethylene glycol 6000 treatment and two weeks of natural drought treatment,the wilting degree of the T2 lines 737,760 and 765 was obvious less than the untransformed inbred line C01.The above results indicated that showed that the AnVP1 gene had been integrated into the maize genome,and its overexpression enhanced drought tolerance of the transgenic lines.The transgenic lines is hopeful to be applied to variety improvement for drought tolerance after further screening,phenotyping and safety evaluation.