Functional Characterization Of Selected Putative Pi Regulated Transcriptional Factor Genes Of Arabidopsis

The ultimate approach of improving Pi utilization of crops and agricultural sustainable development is to understand clearly gene regulation system of Pi metabolism in plant,to discover novel genes involved in high-efficient utilization of Pi,and then to carry out genetic improvement of crops.In this article,overexpression constructs of 5 transcription factor genes that are proved to involve in Pi regulation are created.And primary function analysis of these 5 transcription factor genes has also been carried out in order to provide identifiable ground for screening novel genes involved in high-efficient utilization of Pi.The main findings are listed below:(1)The expression verification of candidates for phosphorus regulation transcript ion factor genes.We have analyzed expression of 5candidates,at5g55690,at3g03260,atlg30500,atlg66370,at4g01060 using qRT-PCR.The result indicates that the express-ion of these 5 genes increased under Pi deficiency,especially the expression of at3g03260 in overground part and the expression of the rest 4 genes in subter-ranean part.This may indicate the difference between the path-ways of the regulation of these genes.(2)The establishment of over-expression constructs of transcription factor genes.The full length DNA of 4 candidate genes have been cloned in this study.And then we created over-expression constructs of at1g30500?at1g66370?at4g01060 by using plant expression vector p3301,and the over-expression construct of at3g03260 by using p1300 in which strong promoter is added.Then these recombinant vectors were transformed into Arabidopsis mediated by Agrobacterium tume faciens.Using resistance screening and PCR amplification,we got positive mutants.(3)The analysis of mutantsThe physiologic and biochemical index of mutants of candidate genes and corresponding T-DNA insertion mutants has been studied and analyzed.The root-shoot ratio,root length,and the content of acid phosphatase and anthocyanidin have been measured.The results demonstrate that the root-shoot ratio and the activity of acid phosphatase of the over-expression construct of at4g01060 are higher than that in wild type,but the content of anthocyanidin is clearly lower under Pi deficiency.However,the salk is on the opposite.These indicate that at4g01060 can promote the synthesis of acid phosphatase under Pi deficiency,and the development of lateral root and root hair.The content of anthocyanidin in the over-expression construct of atlg66370 increased significantly under both Pi sufficiency and deficiency,but the content of anthocyanidin in salk decreased significantly under both Pi sufficiency and deficiency.This shows that atlg66370 can promote the synthesis of anthocyanidin.Although the transcription level of the rest 3 targeted genes changed,the hysiological phenotype did not change much.There may be some replenishment pathways.The specific mechanism needs further study.(4)Prokaryotic expression analysis of candidate genesIn order to search for the targeted genes of candidate genes,we have established prokaryotic expression vectors and have got the proteins of at5g55690?at3g03260?at1g30500?at1g66370?at4g01060,which lays the foundation for further research to determine their targeted genes.The mutants created in this study lay the material foundation for gene function study.And we got mutants of atlg66370 and at4g01060 with clear hysiological phenotypes.which worths further study.