Cloning And Expression Of Phosphate Transporter Gene SpPHT1 From Spirodela Polyrhiza

Phosphorus(Pi)plays an important role in plant metabolism.Phosphorus transporter is an essential protein for plant transport,absorption and utilization of phosphorus.Spirodela polyrrhiza is very common in the waters of various places and has extremely strong vitality.It can absorb calcium,nitrogen,phosphorus and other elements in the water,as well as copper,iron,cadmium and other heavy metal elements.Therefore,the cloning,development and rational use of the phosphorus transporter gene of Spirodela polyrrhiza can provide an important theoretical basis for studying the mechanism of Spirodela polyrrhiza purification of water,analyzing the molecular mechanism of it's efficient phosphorus absorption and using it to improve the absorption of phosphorus by crops Economic crops of excellent nature provide genetic resources.In this paper,the Spirodela polyrrhiza was used as a material to clone the phosphorus transporter gene and predict the physical and chemical properties,and to study the expression pattern of the gene in different external environments.The upstream promoter sequence of the gene was analyzed to predict the presence of cis-acting elements.Four 5' promoter deleted fragments were cloned and introduced into transgenic tobacco for stable expression.The main conclusions are:1.Clone the purple transporter gene and name Sp PHT1.Analysis using biological information software showed that the Sp PHT1 gene sequence has 1620 bp,no intron region,and encodes 539 amino acids.Sp PHT1 protein has a molecular weight of59.19 k D,an isoelectric point of 8.51,and 11 transmembrane regions.It is a stable hydrophobic protein and contains domains similar to the MFS superfamily and sugar transporter family,and the amino acid sequence of Sp PHT1 protein is similar to Zea mays,Sorghum bicolor,Camellia oleifera,Camellia sinensiscorn and so on 15 plant PHT1 amino acid sequences have more than 80% similarity,Speculates that the protein encoded by Sp PHT1 belongs to a member of the PHT1 family.2.To study the expression pattern of Sp PHT1 gene in different environments by fluorescence quantitative PCR.The gene under three conditions of low phosphorus,protophosphorus and high phosphorus showed that: low phosphorus promoted geneexpression,and the relative expression reached the peak when the root and leaf growth of P.sylvestris were at 48h;high phosphorus conditions inhibited gene expression;Under different phosphorus concentrations,the genes were significantly more expressed in the leaves of Spirodela polyrrhiza than in the roots.The expression of genes in circadian rhythm showed that: the expression trend of genes in roots and leaves of Spirodela polyrrhiza.Increased first then decreased and then increased,but the expression reached the peak in dark,and the same gene expression in leaves of Spirodela polyrrhiza was still more than in roots.It showed that the Sp PHT1 gene was expressed in the roots of P.chinensis and was affected by the negative regulation of outside phosphorus and light.3.Clone the 5' promoter sequence(1435 bp)upstream of the Sp PHT11 gene of Spirodela polyrrhiza.And name it Sp PHT1 p.Using bioinformatics software to analyze the sequence contains cis-acting components: core components(CAAT-box,TATA-box),optical response components(Box 4,G-Box,G-box,TCCC-motif),hormone response components(ABRE,TGACG-motif,ERE),stress control elements(ARE),meristem specific elements(CCGTCC-box),other functional elements(AAGAA-motif,Unnamed1).Four expression vectors with 5' deleted fragment promoters were constructed and successfully transferred into tobacco for stable expression to obtain four corresponding transgenic tobaccos ZF1,ZF2,ZF3 and ZF4.The roots,stems and leaves of the control group and the transgenic tobacco were subjected to GUS staining and GUS enzyme activity tests.The results consistently showed that the promoter activity of this gene increased with the growth of the promoter fragment,but the root,stem,and leaf of the plant with the longest promoter fragment had no GUS activity.It is speculated that the Sp PHT1 p sequence has a function of negatively regulating downstream gene expression The element;the promoter only has GUS active expression in the roots and stems of the plant,and the expression amount in the roots of the plant is obviously redundant.It is speculated that there are functional elements of root and stem tissue-specific expression elements in the Sp PHT1 p sequence.