Preliminary Functional Analysis Of Transcription Factor OsbHLH153 And Its Homologous Factors In Rice

There are many members in the bHLH transcription factor gene family and they are indispensable in the processes of plant growth and development.They play important roles in the aspects of plant morphogenesis,physiological metabolism,cell and tissue formation,stress response and other biological processes.At present,the function of OsbHLH153 have not been identified in rice.The homologous analysis of bHLH gene family found that there are two transcription factors that are highly homologous to OsbHLH153 in rice,namely,OsbHLH172 and OsbHLH174.In this preliminary study,the function of the transcription factor OsbHLH153 and its two close homologs OsbHLH172 and OsbHLH174 were studied by using CRISPR/Cas9 gene-editing technology.The results obtained are as the follows:(1)Using the CRISPR/Cas9 gene-editing technology,we obtained the triple mutants of osbhlh153/osbhlh172/osbhlh174,the double mutants of osbhlh153/osbhlh172 and osbhlh153/osbhlh174 and the single mutants of osbhlh153,osbhlh172 and osbhlh174.The phenotype of the triple mutants showed shorter plant heights and the decreased seed setting rates,while the double and single mutants had no obvious defective phenotypes.(2)Overexpression of any of the three genes resulted in obvious and similar phenotypes at the tissue culture stage,Most calli kept proliferating with no stems and leaves formed.Deformed stems and leaves emerged ony from a small number of calli.(3)Analysis of the expression patterns of the three genes with data from databases RiceXPro and HZAU CREP showed that all the three genes were highly expressed in calli,roots,stems,leaves and endosperms of rice.At present,expression constructs PLV4/Promoter:3xGFP and PLV15/Promoter:GUS of the three genes have been made for further analysis of the functions of the three genes in rice.(4)By using yeast two-hybrid technique,we preliminary determined that transcription factors OsbHLH175 and OsbHLH176 interacted with OsbHLH153,OsbHLH172,and OsbHLH174,respectively.OsbHLH153 interacted with OsbHLH172 and so is OsbHLH172 with OsbHLH174.The preliminarily results above showed that the three genes OsbHLH153,OsbHLH172 and OsbHLH174 had strong functional redundancy and they played silimar roles in rice in the development of roots,stems and leaves.The three transcription factors could form heterodimers with others to regulate growth and development of rice.