Functional Characterization Of The Promoter Of Flowering Gene ZmCOL3 In Maize

Photoperiod sensitivity is an important factor for florescence regulation in maize.The existence of photoperiod sensitivity severely limits breeding application of maize germplasm from tropical and subtropical regions into temperate region.Previous studies revealed that ZmCOL3 is one of the crucial genes functioning in photoperiod regulation network of maize flowering stage.It was noticed that there was no difference in the coding region of ZmCOL3 gene except two completely different alleles in the promoter region,implying that the promoter is likely a functional differentiation point of ZmCOL3.To this end,we cloned the ZmCOL3 promoter of the tropical maize CIMBL119 and the temperate maize B73 respectively,and designated ZmCOL3_pro217ro217 and ZmCOL3_pro551ro551 accordingly.Assessments of bioinformatics,molecular biology and histochemistry proved that the promoter region is one of the important targets of ZmCOL3 genetic variation.The results are as follows:1.Pairwise alignment of the two promoters revealed that there are base substitutions of 217 bp for ZmCOL3_pro217ro217 and 551 bp for ZmCOL3_pro551ro551 respectively.Bioinformatics analysis indicated that ZmCOL3 promoter contains several cis regulation elements of meristem-specific genes,photo-responsive genes and stress responsive genes etc,suggesting that ZmCOL3 promoter is likely to be a tissue specific one involving in maize flowering regulation in response to light and stress responses.2.To verify the functionality of the two promoters,we assessed ZmCOL3 gene expression in leaf of 18 inbred lines including 9 from tropical and 9 from temperate.The results showed that both ZmCOL3_pro217ro217 and ZmCOL3_pro551ro551 possess promoter activity,but ZmCOL3_pro217ro217 exhibited in average a 1.1-fold higher capability in driving ZmCOL3 expression.3.The tissue specificity and photoperiodic rhythm of the two promoters were analysised by gus gene reporter system.The results showed that:?1?there was a significant difference in photoperiod induced expression between the two promoters.ZmCOL3_pro217ro217 showed higher expression than ZmCOL3_pro551ro551 by long day photoperiod.?2?it was obviously different in the tissue specificity of the reporter gene expression driven by the two promoters.ZmCOL3_pro217ro217 was expressed in both root and leaf,while ZmCOL3_pro551ro551 was expressed in root?3?ZmCOL3_pro217ro217 exhibited a stronger photoinduced rhythm under long sunshine condition than ZmCOL3_pro551.In summary,our research confirms that there are significant differences in expression levels,tissue specificity and photoperiodic rhythm between ZmCOL3_pro551and ZmCOL3_pro217.The results together suggested that the large fragment variation of ZmCOL3 promoter leads apparently to different biological functions in flowering-time regulation.This genetic variations appear to play crucial roles as maize spread geographically from its tropical origin to higher latitudes.