Cloning And Expression Of ZmPIFs And Study On The Drought Resistant Function Of AtPIFs

PIFs(phytochrome interacting factors)or PILs(phytochrome interacting factor-like),belong to the fifteenth subfamily of the bHLH transcription factors family,playing an important role in plant signaling network.In recent years,the PIFs protein family has been considered as a"hub" of environmental signals and hormonal signals at the molecular level.However,the research on PIFs is mainly dependent on the model plant--rabidopsis thaliana,instead of in other crops(such as rice,wheat,maize).In this study,according to the sequence of PIFs in Arabidopsis,we identified six PIFs from 308 bHLH transcription factor in maize on the basis of the PlantTFDB database through sequence alignment,named ZmPIF1,ZmPIF2,ZmPIF3,ZmPIF4,ZmPIF5 and ZmPIF6.ZmPIFs were evaluated by conserved domain analyses and phylogenetic analyses,sequence comparison showed that these six ZmPIFs all have bHLH domain and APB domain,three of which include ZmPIF1,ZmPIF2 and ZmPIF3 also have an APA domain.Phylogenetic analysis showed genetic relationships are close among ZmPIFl,ZmPIF2,ZmPIF3,AtPIF1,AtPIF3,OsPIL15 and OsPIL16,coincidentally,both of them have APA domain.We designed experiment to test whether ZmPIFs interact with phytochrome in yeast two hybrid system,the results showed that ZmPIF1,ZmPIF2,ZmPIF3,ZmPIF4,ZmPIF5 and ZmPIF6 interact with ZmPhyB1,in which ZmPIF1,ZmPIF2 and ZmPIF3 also can interact with ZmPhyAl.It is indicated that ZmPIFs indeed act as a phytochrome interacting factor.We used qRT-PCR to research the expression characteristics of these six PIFs in Maize at different developmental stages,the results showed that these genes all expressed in roots,stems,leaves,stamen,pistil and seed with tissue-specific expression patterns.Then we explored their expression condition at different time points in a day,qRT-PCR analysis showed that the expression pattern of ZmPIFs has rhythm.The expression of ZmPIFs was strongly induced by dehydration,high salt,cold and heat in five-leaf stage maize seedlings.qRT-PCR analyses showed that different ZmPIFs have different responses to abiotic stress,suggesting that they might participate in the process of enhancing tolerance to abiotic stress in maize.In order to further explore the signal path they may participate in,we treated the maize with plant hormone including ABA,GA,JA,BR,IAA and ETH,the results showed that the expressions of ZmPIFs are changed,suggesting that ZmPIFs is associated with GAs,ABA,BRs,JA,auxin and ethylene signal,and PIFs may be the key node to integrate external signals and internal signals in plant growth and development.In the present study,the expression of ZmPIFs were induced by PEG treatment.In order to verify the function of PIFs,we constructed the expression vector of ZmPIFl,ZmPIF3,AtPIFl,AtPIF3,and transformed them into Arabidopsis by dipping flower infection.After resistance screening,the T3 generation pure lines were obtained.PCR and qRT-PCR results showed that the genes had been integrated into the genome of Arabidopsis and expressed at the transcriptional level.The wild type Arabidopsis,PIF1 mutant and PIF3 mutant were obtained by the method of purchase,and the double mutation of PIF1 and PIF3 was obtained by hybridization.The results showed that the transpiration rate of Arabidopsis mutants was lower than that of wild type,while the transpiration rate of PIF1 or PIF3 over expression material was higher than that of wild type.The same results were also found in ZmPIFl and ZmPIF3 transgenic Arabidopsis.In order to further verify the difference of transpiration rate,we compared their blade water loss rate.The results showed that the water loss rate of over expressing lines was lower than that of wild type,and mutant was higher than wild type.Therefore,PIFs can To achieve the purpose of water-saving drought-tolerance by reducing transpiration.The above results reveal the role of PIFs in abiotic stress response,and lay the foundation for further establishment of PIFs model for drought tolerance.