A Preliminary Study On The Function Of Low-Phosphorus Responsive Transcription Factor ZmPHR In Maize

Phosphorus is one of 14 essential mineral nutrient elements of plant,which participates in the entire life cycle of plant.The synthesis of biomacromolecular such as nucleic acids and phospholipid needs phosphorus,and phosphorus also plays a key role in the physiological and biochemical responses such as signal transduction and energy metabolism.Plant absorbs phosphate from soil,total phosphorus content is rich in soil,and however,the reality is that the phosphorus in soil which can be absorbed by plant has a very low level.Furthermore,the most agricultural land of china and even the whole world is in low available phosphorus condition.Since phosphate fertilizer used in agriculture,the problem of lacking available phosphorus in cultivated land has solved,but using phosphate fertilizer brings another crisis.Phosphorite is the main source of phosphate fertilizer,but the global total phosphorite resource is limited and non-renewable.The result is that the phosphorite will be exhausted in the near future;moreover,using overfull phosphate fertilizer in the farmland causes environmental pollution.Recently,researchers pay more attention to solve the problem of low-phosphorus tolerance of plant in biological ways,and the focal point is the research of key genes.Key genes' research not only perfects the signal network of phosphate starvation stress,but also has important significance of germplasm resources screening and maize breeding for tolerance to low-phosphorus stress conditions.PHR transcribes a transcription factor and plays a critical role in responding phosphate starvation.In this work,two ZmPHR genes(ZmPHRl and ZmPHR2)were isolated from a maize inbred line 178 with high tolerance of phosphate deficiency by homology-based cloning strategy.By bioinformatics methods,we have analyzed the characteristics of ZmPHR1 and ZmPHR2 genes' nucleic acid and protein sequences,prediction of cis-elements of promoters in ZmPHRl and ZmPHR2,evolutionary relationships of ZmPHRl and ZmPHR2,and prediction of conserved domains in ZmPHR1 and ZmPHR2.Bacterial expressed recombinant ZmPHR1 or ZmPHR2 protein has revealed the information of ZmPHR1 and ZmPHR2.In the yeast expression system,we have revealed the transcription activity of ZmPHR1 and ZmPHR2.We also revealed the spatial expression patterns of ZmPHR1 and ZmPHR2 under phosphate starvation in seedling period of maize through qRT-PCR.Finally,transgenic Arabidopsis plants with the two overexpressed genes were obtained;these transgenic materials can be used for deeply research of ZmPHR1 and ZmPHR2.Making a summary of our works,the main research conclusions are as follows:1.ZmPHRl and ZmPHR2 were isolated from a maize inbred line 178 with high tolerance of phosphate deficiency by homology-based cloning strategy,the length of cDNA sequence of ZmPHR1 and ZmPHR2 are 1743bp and 1936bp respectively,ZmPHR1 and ZmPHR2 were located in the 1 chromosome and the 7 chromosome respectively.Through research,Myb_CC_LHEQL and Myb-like DNA-binding domains are both in ZmPHR1 and ZmPHR2.In promoters of ZmPHR1 and ZmPHR2,there are many cis-elements of hormone responses and abiotic stress responses.2.Proteins of bacterial expressed recombinant ZmPHR1 and ZmPHR2 are 66.4 KDa,and the best inducing conditions is 1 mmol/L IPTG.3.We have demonstrated ZmPHR1 and ZmPHR2 have the transcription activity function,and the N-terminal peptide with Myb-like DNA-binding domain of ZmPHR had the transcription activity.4.The qRT-PCR showed that ZmPHRl and ZmPHR2 expression are not induced by phosphate starvation in seedling period of maize.Under phosphate starvation in seedling period of maize,ZmPHR2 expression didn't have significant difference.ZmPHR1 expression had significant differences between maize inbred line 178 with high tolerance of Pi deficiency and maize inbred line 9782 with low tolerance of Pi deficiency,it was induced the expression in shoot of 178,and there was no significant difference in shoot of 9782.5.Plant overexpression vector pC AMBIA3 3 01-3 5 S-ZmPHR1 and pCAMBIA3301-35S-ZmPHR2 were built successfully,and we screened 11 transgenic Arabidopsis plants of ZmPHRl and 2 transgenic Arabidopsis plants of ZmPHR1.Finally,we cloned the genes ZmPHR1 and ZmPHR1 from maize successfully,described the molecular characteristics of ZmPHR1 and ZmPHR2,and demonstrated both ZmPHRl and ZmPHR2 have the transcription activity function,which are included in the system of phosphate starvation responses.