Gene Clone And Functional Analysis Of ZmPIFs In Maize

The growth of plants can't be independent with light in nature environment,the plants get energy from light and accumulate the photosynthate;on the other hand,light also plays a role of signaling molecule and the plants adjust development by sensing the change of intensity of light.Phytochrome(PHY)is F/FR photoreceptor of plant,and modulates growth by interacting with phytochrome-interacting factors(PIFs).The accumulation of PIFs results long hypocotyls,appressed cotyledons,inhibition of photomorphism.In conclusion,PIFs are crucial to the development of plants.The knowledge of PIFs are still unknown in higher crop,especially in maize,due to most researches focus on model plant.So the study in ZmPIFs will be significatly.In this study,we identified 10 ZmPIFs genes from maize in the database MaizeGDB,researched the expression pattern and cloned part members of this gene family.We generated4 kinds of transgenic Arabidopsis thaliana plants,and found that bHLH44 gene can inhibit the photomorphosis and affect the gravitropism in plant.The major results are as follows:(1)According to BLAST analysis on MaizeGDB,17 ZmPIFs genes were identified as homologous genes compared with the sequence of Arabidopsis thaliana AtPIFs gene family which were found on NCBI.We investigated the structure and function of ZmPIFs by phylogenetic relationships,genes sequence analysis,amino acid sequence analysis and protein structure domain prediction.Finally,10 maize ZmPIFs genes were regard to play the main role as Arabidopsis thaliana AtPIFq: ZmbHLH12,ZmbHLH30,ZmbHLH37,ZmbHLH40,ZmbHLH44,ZmbHLH51,ZmbHLH74,ZmbHLH88,ZmbHLH113 and ZmbHLH180.We used serial number of bHLH gene family when named the maize ZmPIFs genes,due to PIFs belonging to bHLH gene family.According to analysis by qRT-PCR and high-through put RNA sequencing,we found the maize ZmPIFs genes expression patterns are specificity in spatial,temporal and shady environment,and also verified the circadian rhythmicity of bHLH24 and bHLH74 under short day cycles.(2)10 ZmPIFs were cloned: bHLH1,bHLH12,bHLH24,bHLH30,bHLH40,bHLH44,bHLH51,bHLH113,bHLH130 and bHLH180.In view of the accuracy of gene sequence,the integrity of protein domains and whether played the main role in maize,we transferred 4 of ZmPIFs into wild-type Col and mutant pifq.Furthermore,bHLH44 gene showed the most conspicuous function in repressing photomorp-hogenesis among all ZmPIFs.It promoted the hypocotyl length of 35S:bHLH44-3FLAG/pifq and 35S:bHLH44-3FLAG/Col,and changed the angle of cotyledons in dark.The function of repressing photomorphogenesis between bHLH44 and AtPIFs were similar.(3)bHLH44 gene affected the gravitropism in plant: there was no hypocotyl negative gravitropism in mutant pifq,but transgenic 35S:bHLH44-3FLAG/pifq and wild-type Col showed the gravimetric response in dark environment.In consist with Arabidopsis thaliana AtPIF1,bHLH44 inhibited the conversion of endodermal amyloplast to etioplasts in plant.We provided the evidence by using I2-KI staining experiments to detect the starch synthesis in plant.