Cloning And Analysis Of Phosphate Transporte Protein Gene (PT) In Maize

Phosphorus is one of essential minerals for plant growth and development in natural ecosystems.Supplying adequate phosphate fertilizer is the important path for raising crops production.Howere,low available phosphate is the most common characterization of soils. From 1960s,the global phosphorite reserves has been in danger of disappear,which makes the plant phosphorite supply an increasingly serous problem.Most of the soil in southwest of our country is red soil,the leanness and the phosphorite deficiency act as the key non-biotic stress to confine the maize yield improvement.Therefore,study the mechanism of maize phosphorite transfer it an effective method to cultivate new maize variety which can effectively absorb the soil phosphorite.Homologous Clone is a new method for finding Phosphate Transporte protein(PT)genes from plants.The procedures are as follows,first the polymerase chain reaction(PCR)for PTs were carried out by using the degenerate primer that designed according to the conserved domains of the cloned PT genes and the plant genomic DNA or cDNAs as template;second the PTs were sequenced and homologous and phylogenetic relations among the PTs and the cloned PTs genes were analyzed;third some of PTs were selected as the candidate Phosphate Transporte protein genes;and finally a new plant PT genes were discovered from the candidate.Compare with other methods of clonging genes,this method has the strongpoint of convenience,saving labor and time.Up to now,some PTs from different plants had been isolated.In this research,expression of PT genes in the root and leaf of low phosphate-proof "178" and sensitive low phosphate "9782" were studied.Meanwhile,the functional domains and homology of PT genes from this two cultivars were analyzed by using the methods of Bioinformatics.1 Used cultivars low phosphate-proof "178" and sensitive low phosphate "9782" genomic cDNA as the templates,according to the conservative regional of PT gene to design primers for PCR amplification,we found 6 amplificated fragments,which were about 700 bp and 2 amplificated fragments,which were about 1000 bp.2 The expressional difference of PT genes during root and leaf of "178" and "9782" in 7 time slots(0 h,6 h,12 h,24 h,48 h,72 h,96 h),the results indicated that:the expression level of Zea_Pts were different among these time slots,meanwhile,the expression level of Zea_Pts(Zea_Pt3,Zea_Pt7,Zea_Pt3andZea_Pt9)were significant,moreover,the electrophoretic detection indicated that the 4 Zea_Pts expression level in "178" were higher than that in "9782",the expression of PT genes were up-regulated with the extention of low-phosphate treatment.3 Submitted the 8 different sequences to Genbank in a Blast comparison,database searches indicated that the gene sequences of PTs were 98%,95%,93%,87%identical to that of maize PTs.We found that PTs were known function of the gene,these genes belong to a type of H₂PO₄^-/H⁺ symporters involved in the uptake of Pi from the soil solution and redistribution of Pi within the plant.4 Cloned sequenced and analysised the differert fragments,the results showed that 8 cloned fragments had conservative structure of PT gene.Deduced and analysised amino acid sequence of the 8 PTs show that:This 8 sequences had high homology and similarity in the functional and transmembrane domain,there exist Protein kinase C phosphorylation site,Casein kinaseâ…¡phosphorylation site,Tyrosine kinase phosphorylation site.These analysis were mutual corroboration with the biochemical structural feature of Pht1 family.