Functional Analysis Of AP2/EREBP Transcription Factor OsSub1C In Rice (Oryza Sativa L.) Under Low Temperature Stress

Rice is a warmth-likeing crop.Its growth and development are sensitive to low temperature,which limits rice production.Therefore exploring the low temperature stress related genes and revealing their functions can provide molecular basis for rice molecular breeding of cold tolerance.In this study,AP2/EREBP transcription factor OsSub1 C was used to study the function of OsSub1 C in Rice Seedling under low temperature stress by bioinformatics,biological function,and molecular function analysis.The results are as follows: 1.Bioinformatics analysis of OsSub1COsSub1C transcription factor belongs to the ERF subfamily of AP2/EREBP family,and contains nuclear localization sequence,KKKKR,at the N-terminal of its protein sequence.Promoter sequence analysis showed that the gene contained several abiotic stress and hormone response elements such as low temperature,abscisic acid etc.2.OsSub1 C induction and tissue specific expression analysisOsSub1C was induced by abscisic acid,6-benzylaminopurine,gibberellin,auxin,jasmonic acid and low temperature.Except 6-benzylaminopurine,the expression of OsSub1 C in other treatments increased rapidly at 6 h.3.Subcellular localization and self-activationOsSub1C-GFP transient expression vector was transferred into protoplasts of Arabidopsis thaliana.The results of fluorescence inversion microscopy showed that OsSub1 C protein was expressed in the nucleus.OsSub1 C had the characteristics of transcriptional self-activation.4.Functional analysis of OsSub1 C under low temperature stressThe survival rates of Sub1C-VP64-1 and Sub1C-VP64-2 were 40.0% and 43.3% respectively,slightly higher than those of wild type,Kitaake(37.6%),while the survival rates of Sub1C-EAR-1 and Sub1C-EAR-2 were 13.3% and 10.0% respectively,which were significantly lower than those of wild type under low temperature treatment.At 0,6 and 12 h of low temperature stress,the relative expression of 12 low temperature stress related genes were different from that of wild type in transcription-activated line and transcription-inhibited line,which indicated that OsSub1 C gene expression affected the expression of low temperature stress related genes.The expression levels of cold-sensitive genes OsWRKY45,OsRAN2,OsbZIP52 and ZFP177 in Sub1C-EAR-1 line were significantly higher than those in wild type and Sub1C-VP64-1 line.The expression patterns were consistent with phenotypic identification results.5.Screening and validation of OsSub1 C interacting proteinA NAD-dependent differential isomerase(OsNAD3)protein was screened by yeast two-hybrid analysis,and the interaction between OsSub1 C and OsNAD3 was verified by bimolecular fluorescence complementarity test(BiFC).