Functional And Transcriptional Analysis Of The Thermosensitive Genic Male-sterile Gene OsTMS5

As one of the most important food crops,rice feeds more than half of the world’s population.With the population explosion in the world,how to increase rice yield becomes one of the most serous to scientists and breeders.Because hybrid rice yields about 10-20%more than conventional rice,it is widely cultivated and has made a great contribution to food security.Hybrid rice can be divided into three-line hybrid rice and two-line hybrid rice.Compared with three-line hybrid rice,two-line hybrid rice can save more time and energy and has higher application potential.Although the thermosensitive genic male-sterile gene OsTMS5 is widely adopted in rice breeding,the regulatory network of OsTMS5 remains unclear and requires further investigation.Based on the previous results of our lab,we demonstrated the function of the new multiple gene Ostms5-2 in the regulatory network of thermosensitive genic male sterile（TGMS）lines.Furthermore,we screened a yeast one-hybrid c DNA library and obtained two transcription factors Osb HLH138 and OsAlfin-Like 5 in transcription control of OsTMS5.According to our results,the transcription activator Osb HLH138could increase the expression level of OsTMS5 while the transcription repressor OsAlfin-Like 5 has opposite function.In addition,these two transcription factors could bind to the promoter region of OsTMS5 simultaneously but they did not interact with each other.The main results are summarized as following1.We transformed the genomic fragment of OsTMS5 gene including 2200-bp promoter,1900-bp gene body and 1200-bp downstream region into the 93-11s mutant.After analyzing the phenotype of T₀ generation,we found that the fertility of the transgenic lines was restored.Then we predicted the structure of OsTMS5 and Ostms5-2 using SWISS-MODEL online tool.According to the predicted structure,we found that the deletion of two amino acids in ostms5-2 changed its structure of and lost its processing function to Ub _L40-1,Ub _L40-2,Ub _L40-4 m RNA.We screened a yeast one-hybrid c DNA library derived from 93-11 young inflorescence with OsTMS5promoter as a bait and obtained 19 candidate proteins.Based on bioinformatic analysis,we chose the transcription factors Osb HLH138 and OsAlfin-Like 5 for further research.2.The bHLH gene family containes 167 members,which participate in multiple biological processes.The Osb HLH138 encodes a transcription factor and localized in the nucleous.Through yeast one-hybrid assay and Electrophoretic Mobility Shift Assay assay,we proved that Osb HLH138^360-424 region could specially bind to the promoter region.Then according to dual-luciferase assay and transcription activity test,we proved that the Osb HLH138^217-364 region alone could increase the expression level of OsTMS5.Finally,the Osb HLH138 knock out lines show the thermosensitive genic male sterile under high temperature.3.The family of Alfin-Like has been proved to participate in multiple biological processes such as the growth and development of roots,and the resistance to abiotic stresses.Furthermore,all of the AL family members function as transcription repressors in Arabidopsis thaliana.In this work,the transcription repressor OsAlfin-Like 5 could bind to the G rich domain of OsTMS5 promoter.It is important that both OsAlfin-Like 5 and Osb HLH138 could regulate the epression level of OsTMS5.However,we found there was no interaction between OsAlfin-Like 5 and Osb HLH138 by yeast two-hybrid（Y2H）assay,pull-down or Bi FC.In this study,we proved the function of a new allele,Ostms5-2,in the regulatory network of thermosensitive genic male sterile.In addition,we screened and verified two transcription factors,Osb HLH138 and OsAlfin-Like 5 in this network.The relationship between Osb HLH138,OsAlfin-Like 5 and OsTMS5 is important to understand the mechanism of thermosensitive genic male sterile.Further,the identification of Osb HLH138 and OsAlfin-Like 5 provids breeders a new strategy for developing TGMS rice lines.