| Global drought is growing fast these years, drought and salinization of land become a worldwide problem for humanity, especially the arid and semi-arid region in northwest of our country, drought and salinization of land become the major limiting factor for the development of the agricultural economy because of the shortage of water resource. The plants metabolize slowly and growing is inhibited under salt stress, it has seriously affected the production and land utilization.The production of corn just lower than rice in our country as the major grain crops.Corn is sensitive to warter, so drought and salinization become a important abiotic factor affecting the growing of corn.With the rapid development of modern molecular biology,to cultivate high-yielding and drought tolerant varieties by transgenic technology is one of the most effective way to resist drought.Plants resist drought mainly by the synergy of vacuolar membrane Na+/H+-antiporter protein and proton pyrophosphatase (H+-PPase).This study has integrated the gene of Na+/H+antiporter protein and H+-PPase into plant expression vector pCAMBIA3301,and transformed embryogenic callus of maize inbred lines18-599R by Agrobacterium-mediated method. At the same time,we have optimized the system of callus induction and genetic transformation mediated by Agrobacterium,and obtained the transgenic plants.The main results are as follows:1.Construction of the plant expression vector pCAMBIA3301-ZxNHX-ZxVP1-1.We digested gene ZxNHX and ZxVP1-1including promoter by restriction enzymes EcoR I and BstEⅡ from the expression vector pCAMBIA1302and integrated it into pCAMBIA3301.Through this method,we obtained a new binary vector which use bar gene as the selectable marker and proved the correctness of the results by restriction enzyme digestion.2.Optimize the callus induction systerm of maize18-599R.We found the optimal concentration of2,4-D is2mg/L though comparison of experimental results, and we gained the highest induction rate when the size of immature embryo is between1.0-1.5mm,the induced callus is right yellow color and dense stucture.When the size of immature embryo is less than1.0mm,the moisture content of callus is too high to continue growing and regeneration though it has a high induced rate and not easy to sprouting. When the size of immature embryo is greater than1.5mm,the induced rate is low because the growing of root and shoot and forming a hard shell easily.This study also show that callus embryogenic rate declined slightly with the increased time of subculture when subculture less than three times.The callus subcultured two times growing significantly while callus subcultured three times with the best quality is most suitable for genetic transformation.When subcultured more than three times,the callus differentiated seriously and embryogenic rate declined obviously.3.Optimize the genetic transformation systerm mediated by Agrobacterium.In this experiment,we investigated the influence of the concentration of bacteria, infection time,co-culture temperature and co-culture time for infection efficiency. We concluded that the best concentration of bacteria and infection time is infecting ten minutes at OD600=0.6.Increasing the infection time can improve transformation efficiency when OD600=0.4.The best condition for co-culture is culture two days at27℃, followed by two days at23℃and three days at18℃,it show that different co-culture temperature require different time.4.The binary vector transformed maize callus and the preliminary identification of transgenic plants.We obtained142resistant plants after transformation and selection,8of which are transgenic plants which can detected specific fragment of gene ZxNHX and ZxVP1-1by PCR identification.It proved that the binary vector pCAMBIA3301-ZxNHX-ZxVP1-1had successfully integrated into the genome of maize18-599R. |
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