Construction Of Bivalent Gene Vector For Resistancing Glyphosate And Increasing Phosphorus Uptake And Its Genetic Transformation In Brassica Napus L.

Brassica naups L.belongs to Cruciferous Brassica from the plant taxonomy,it is an extremely important oil and feed crop in China.With the continuous growth of the global population and the rapid economic development,people’s demand for vegetable oil has increased dramatically.This poses new challenges to the production of B.naups.Due to the limited availability of phosphorus and grass damage during its growth,its development has been greatly limited.Therefore,the plant recombinant expression vector pC-NLTA containing rape chloroplast peptide eCTP,glyphosate resistance gene EPSPS,root specific promoter Pht1;2 and phytase gene phyA was constructed for obtaining a new B.napus germplasm which with high-efficiency phosphorus absorption and use in this study.And we adopted the Agrobacterium-mediated transformation of three varieties of B.napus,‘Long Shang Dong’,’1831R’,and ‘Xinjiang 21109’.It has a certain theoretical significance for definiting the role of phytase gene phyA and the glyphosate resistance gene EPSPS to the absorption and utilization of phosphorus and glyphosate resistance in the above three B.napus species.At the same time,it also provides theoretical basis and reference for obtaining the new rapeseed germplasm resources which both have high-efficiency phosphorus absorption and utilization and glyphosate resistance.The current research has achieved the following results:1.Bioinformatics software: TargetP 1.1 Server and ChloroP 1.1 Server were used to predict the subcellular localization of rape rbcS gene,its transit peptide and cleavage site in the encoded amino acid sequence were also be predicted.Analyzed the results and choose the ideal sequence and named as eCTP;2.We have obtained the EPSPS gene by subcloning the plasmid pTOPO-EPSPS;We have obtained the phyA gene by subcloning the plasmid PGA1611-E-PhpAO;We have obtained the nos gene by subcloning the plasmid pMD-nos;We have obtained the root-specific promoter Pht1;2 gene by cloning the wild-type Arabidopsis DNA;We have obtained the chloroplast leader peptide eCTP by cloning the Brassica napus RG2 cDNA;3.The overlap PCR method was used to fuse the chloroplast leader peptide eCTP and EPSPS gene,and the fusion fragment L was obtained;4.The nos terminator,the fusion fragment L,the root-specific promoter Pht1;2and the phytase gene phyA were inserted successively into the basic vector pCEPSP using the method of enzymatic ligation.Finally,the plant recombinant expression vector pC-NLTA was constructed.And it was integrated into Agrobacterium LBA4404 using freeze-thaw transformation;5.The three varieties of B.napus(‘Long Shang Dong’,’1831R’,and ‘Xinjiang21109’)were transformed by Agrobacterium-mediated,after glyphosate screening and PCR detection,we obtained two ‘Xinjiang 21109’ positive transgenic plants,two‘1831R’ positive transgenic plants and one ‘Longshangdong’ positive transgenic plant;6.The expression of each gene in the positive plants was detected by RT-qPCR.The results showed that the phytase gene phyA and EPSPS gene expression levels in positive transgenic plants were significantly higher than the control plants;...