Studies On Transformation Of Phosphonate Dehydrogenase Gene PtxD In Brassica Napus

At present,the abuse of phosphorus fertilizer and herbicides to make rape high yield and quality has become the norm.This will not only increase the production cost of rapeseed oil,but also make weeds resistant,which will cause a series of ecological and environmental problems.How to create methods that can not only pursue economic benefits,but also protect ecological benefits to promote high yield and quality of rape has become an urgent problem to be solved.The phosphate fertilizer used in agriculture is non renewable orthophosphate(PO₄^3-or Pi)with limited output,while the phosphite(PO₃^3-or Phi)which can not be used by plants has a large stock.In this study,the phosphonate dehydrogenase gene ptxD was transformed into Brassica napus,to obtain transgenic rape which can use phosphite as weed control system and source of phosphate fertilizer.To solve a series of problems caused by overuse of phosphate fertilizer and herbicide,so as to find new methods for the application of phosphite in China to improve the sustainable development of agriculture.The main results are as follows:（1）According to the phosphonate dehydrogenase gene ptxD（Gen Bank accession number:WP＿003118429.1）,without changing the amino acid sequence,the codon was optimized according to the codon preference of Brassica napus.Increase the expression of genes in Brassica napus.（2）After optimizing the gene sequence of codon,using DA-PCR+OE-PCR two steps method synthesis of the phosphonate dehydrogenase gene ptxD,and the expression vector pcambia1303（ptxD）was successfully constructed.（3）By tissue culture,the plant expression vector pcambia1303（ptxD）was successfully transformed into Brassica napus Westar by Agrobacterium-mediated method.15 transgenic rapes were identified by PCR,and the positive transformation rate was 21%.（4）Using the transient expression activity of GUS gene to detect the effect of co culture time on the transformation and regeneration frequency of explants after being immersed in Agrobacterium.The experimental results show that the transient expression frequency of GUS gene was 43%,89%,93%and 83%respectively after co culture with Agrobacterium for 1-4 days,and the corresponding transformation and regeneration frequency was 10.2%,20.8%,21.1%and 12.8%respectively positive correlation.（5）Using the cDNA of the root,stem and leaf of transgenic rape as template,real-time fluorescence quantitative PCR was used for detection.The results showed that ptxd was expressed in transgenic plants.Among them,leaf expression was the highest,root was the second,stem was the lowest.Therefore,the following weeding experiments were carried out by spraying Phi on the leaves.（6）The resistance identification of transgenic rape treated with 300mM phosphite（Na₂HPO₃·5H₂O）showed that the leaves of WT plants began to be damaged from the edge,showing scorching like,and then gradually turned yellow towards the center.On the contrary,there was no damage or abnormality in the leaves of the transgenic plants.The results showed that the transgenic rape with the phosphonate dehydrogenase gene ptxD could oxidize Phi to Pi.（7）After spraying 300 mM phosphite（Na₂HPO₃·5H₂O）on the leaves of transgenic,non transgenic rape and common weeds in rape field for three times,the weeds and non transgenic rape withered and died,and the T₁transgenic rape grew normally.It was proved that the introduction of the phosphonate dehydrogenase gene ptxD and the combination of Phi could effectively control weeds.