| As an important crop for food and oil,soybean(Glymine max(L)Merr.)has a long cross-time between vegetative and reproductive growth,which induced two energycompetition processes including nodulation and flowering.The regulation balance between flowering and nodulation process is a great significance to explicit the developmental pattern of soybean,improve the soybean production and guide the soybean breeding.GmNMHC5 is a MADS-box gene originally cloned from soybean nodules,it can significantly promote the lateral roots development and nodulation and might involve in flowering process.The regulatory and molecular mechanism involved in this bifunctional regulation of GmNMHC5 plays a key role to clarify the regulation between nodulation and flowering.In this study,the function of the promoter of GmNMHC5 was analyzed to explore the regulatory mechanism of GmNMHC5 in transcriptional level.Sequence analysis revealed that GmNMHC5 promoter contains a large scale of cis-elements.The GmNMHC5 promoter was introduced into soybean hairy roots and Arabidopsis thaliana to analyze the promoter activity.As shown the GUS expression pattern,the GmNMHC5 promoter is a vascular-preferential promoter.In order to identify its core fragment,three 5’-deletion fragments were amplificated by PCR and the vascular specificity was disappeared in three 5’-deletion fragments.At the same time,sucrose could highly induce the promoter activity,while it did not work to 5’-deletion fragments which don’t contain sucrose response element.It demonstrated that its core fragment for vascular-preferential expression might lie between-2051 bp and-1418 bp and the sucrose response element might be one of the core regulatory elements of GmNMHC5 promoter.Using yeast one-hybrid method,a S-bZIP subfamily transcriptional factor GmbZIP33 was screened from soybean cDNA which may bind to GmNMHC5 promoter sucrose-response element.This result revealed that the transcription of GmNMHC5 was regulated by S-bZIP transcriptional factor.In order to identify the function of GmNMHC5 in flowering,GmNMHC5 was ectopic expressed in Arabidopsis.It was showed that the overexpression of GmNMHC5 was able to trigger the flowering process both in long-day and short-day,ahead of 27-30.1% and 29.5-35.7%,respectively.As detected by qRT-PCR and RNA-seq,the transcription levels of the flowering time inducible genes FT,SOC1,AP1 and LFY were promoted significantly in the transgenic lines.Therefore,GmNMHC5 acts as a positive regulator in promoting the flowering time.All the results provided an important base to explore the site of GmNMHC5 in flowering regulated network.An early flowering phenotype was observed in GmNMHC5-overexpression soybean,it confirmed that GmNMHC5 can promote flowering time in soybean.In conclusion,GmNMHC5 is regulated by GmbZIP33 transcription factor and sucrose in transcriptional level,in reverse,GmNMHC5 might was involved in the metabolism and transportation of sucrose in plants due to its phloem specific expression.The regulated association between GmNMHC5 and sucrose could be an important foundation to analysis the molecular mechanism of GmNMHC5 involved in both flowering and noduation processes. |
