Expression Vector Construction Of Tomato High Affinity Phosphorous Transporter Gene SlPT1 And Functional Analysis

Phosphorus plays a key role in the growth and development of plants,and the lack of phosphorus in a large number of nutrients will affect every link of the plant.Phosphorus content in soil is much,but it can be used very little.In order to deal with these environmental constraints,plants have evolved their own phosphorus utilization system.Phosphorus transporter PHT family plays a key role in the absorption and utilization of phosphorus.Under phosphorus stress,most of the phosphorus transporter genes have been expressed in large quantities to improve the content of phosphorus in cells.Tomato is a main vegetable cultivation crop.Phosphorus deficiency can cause tomato yield and quality decline.Studying the function of tomato high affinity phosphate transporter SlPT1 gene can help solve tomato’s utilization of phosphorus.In this project,we use the forward genetics method to construct the over expression vector of SlPT1 gene and transform it into Arabidopsis mutants,and verify its function in Arabidopsis mutants.the initiation of SlPT1gene was cloned and its characteristics were preliminarily studied.the experimental results are as follows:1.The tomato gene database online by NCBI,got the sequence of tomato SlPT1 gene,analyzed the location open reading frame and designed primers,and amplified a DNA fragment with 1654bp length by PCR amplification.the blast gene sequence alignment and amino acid sequence alignment analysis showed that the full length of tomato phosphorus transporter gene SlPT1 was 2002bp（GenBank ACCESSION NM₀01247432）,the open reading frame was 1617bp,encoding a 538 amino acid protein.the molecular formula of the phosphor transporter encoded by tomato SlPT1 gene is C₂₇₁₆H₄₁₃₇N₆₇₃O₇₂₂S₂₉.the composition of amino acids is 2.6,the molecular weight is 58699.54D,the isoelectric point is 8.51,the average hydrophilic coefficient is 0.401,and the instability coefficient is 29.20.tomato SlPT1 has high homology with AtPT4 of Arabidopsis.2.The amplified cloned SlPT1 gene was connected to the T-Vector pMD 19 vector and was sequenced.the correctly extracted plasmid,SlPT1-T plasmid and pCAMBIA3301/luc vector plasmid were processed by Xba I and Xma I restriction enzyme respectively,then connected and transformed,and the expression vector was constructed and named as SlPT1-pCAMBIA3301/Luc.3.The agrobacterium mediated dipping method was applied To genetic transformation of Arabidopsis thaliana mutants.the transgenic plants were screened and obtained,and the seeds of T1 generation were obtained.then the phenotype of T1generation was observed.compared with the control plant,the difference of phosphorus deficiency symptoms was not obvious but the root morphologic changes were obvious in the normal culture conditions.Transgenic plants increased the uptake of phosphorus,which was higher than that of the control under normal culture conditions.4.The amplification obtained a 903bp sized promoter sequence,on line sequence analysis and cis acting element prediction.The cloned fragments were sequenced by T-Vector pMD 19 vector,and SlPT1-Pro-T plasmid and pBI121 vector were digested by enzyme cut site Hind III to Xma I for double enzyme digestion.The linkage transformation and validation were completed,and the expression vector of CaMV35S promoter was constructed.through the transient expression of Arabidopsis thaliana,according to the comparison and dyeing effect analysis,the promoter activity of SlPT1 gene was obvious in root.In conclusion,the expression vector SlPT1-pCAMBIA3301/Luc was constructed,and the mutant of Arabidopsis thaliana was genetically transformed with Agrobacterium tumefaciens dipping method,and the transgenic plants were screened and obtained.Then the phenotype of T1 generation was observed.Compared with the control plant,the difference of phosphorus deficiency symptoms was not obvious but the root morphologic changes were obvious in the normal culture conditions.the transformation of SlPT1 gene promoter into Arabidopsis thaliana was preliminarily studied,and the promoter activity of SlPT1 gene was obvious at root.