Functional Study Of Variable Splicing Isoforms Of Multi-Season Flowering Candidate Genes Of Osmanthus Fragrans

Osmanthus fragrans Lour.belongs to the genus Osmanthus.O.fragrans has been listed as one of the top ten traditional flowers because of its unique ornamental value since ancient times.O.fragrans is divided into two categories: ‘Qiugui’ and ‘Sijigui’.O.fragrans showcases a rich diversity,and different variety groups have different flower bud differentiation and flowering mechanisms,which provides excellent experimental materials for studying the multi-season flowering mechanism of higher plants.In our previous studies,we found that the candidate genes OfAP1(with homologous genes OfAP1-a and OfAP1-b)and Of TFL1 had Alternative Splicing(AS)events in ‘Sijigui’ and ‘Qiugui’,respectively.Named OfAP1-a1/OfAP1-a2,OfAP1-b1/OfAP1-b2,Of TFL1-1/Of TFL1-2.On this basis,we conducted tissue-specific expression analysis,subcellular localization,construction of overexpressed Arabidopsis transgenic lines,fluorescence quantification,protein interaction and other methods to explore the functions of different variable splicing isomers in the flower formation process,and explored the mechanism of multi-season flower formation in O.fragrans from a new perspective of variable splicing.The results are as follows:1.Tissue-specific expression analysis showed that OfAP1-a1,OfAP1-a2,OfAP1-b1,OfAP1-b2,Of TFL1-2 were expressed similarly in ‘Qiugui’ and ‘Sijigui’,and their expression levels were higher in flowers,while Of TFL1-1 was highly expressed in the stems of ‘Qiugui’and in the roots of ‘Sijigui’.These results suggested that OfAP1-a1,OfAP1-a2,OfAP1-b1,OfAP1-b2,Of TFL1-2 might play a role in the development of the flowers of ‘Qiugui’ and‘Sijigui’,while Of TFL1-1 played a role in the stem of ‘Qiugui’ and in the root of ‘Sijigui’.1.Tissue-specific expression analysis showed that OfAP1-a1,OfAP1-a2,OfAP1-b1,OfAP1-b2,Of TFL1-2 were expressed similarly in ‘Qiugui’ and ‘Sijigui’,and their expression levels were higher in flowers,while Of TFL1-1 was highly expressed in the stems of ‘Qiugui’ and in the roots of ‘Sijigui’.These results suggest that OfAP1-a1,OfAP1-a2,OfAP1-b1,OfAP1-b2,Of TFL1-2 may play a role in the development of the flowers of ‘Qiugui’ and ‘Sijigui’,while Of TFL1-1 plays a role in the stem of ‘Qiugui’ and in the root of ‘Sijigui’.2.Transient overexpression was transferred into Nicotiana benthamiana and subcellular localization of isoforms was observed by laser confocal microscopy.It was found that OfAP1-b1,OfAP1-b2 and Of TFL1-2 were located in the nucleus,and Of TFL1-1 was located in the cytoplasm,which was consistent with the predicted results.Previous studies have shown that OfAP1-a1 and OfAP1-a2 are localized in the nucleus.3.Phenotypic observation of T3 homozygous overexpressed Arabidopsis thaliana lines:there was no significant difference in flowering time between OfAP1-a1 and OfAP1-a2 transgenic plants and wild-type plants;OfAP1-b1 transgenic plants blossomed 7 days earlier than wild type plants,and OfAP1-b2 transgenic plants blossomed 7 days later than wild type plants.Of TFL1-1 transgenic plants blossomed 7 days earlier than wild-type plants,and Of TFL1-2 transgenic plants blossomed 7 days later than wild-type plants.Different isomers had different effects on flowering time,and the functions of the isoforms were differentiated.4.The expression levels of flowering related endogenous genes in A.thaliana transgenic lines were analyzed.The results showed that the expression levels of At AP2,At SVP and At LFY genes were slightly up-regulated in OfAP1-a1 transgenic lines.The expressions of At AP2,At SVP,At LFY and At14-3-3 genes were slightly up-regulated in OfAP1-a2 transgenic lines.In OfAP1-b1 transgenic lines,the expressions of At AP1,At AGL24,At SOC1,At LFY,At SVP,At AP2 and At14-3-3 were significantly up-regulated,while the expressions of At FT,At TFL1 and At FD genes were significantly down-regulated.In OfAP1-b2 transgenic lines,the expressions of At FT,At TFL1 and At FD genes were significantly up-regulated,while the expressions of At AP1,At AGL24 and At SOC1 genes were significantly down-regulated.In Of TFL1-1 transgenic lines,the expressions of At AP1,At LFY,At SVP and At AP2 were significantly up-regulated,while the expressions of At FT,At TFL1 and At FD were significantly down-regulated.In Of TFL1-2 transgenic lines,the expressions of At14-3-3,At TFL1 and At FD were significantly up-regulated,while the expressions of At AP1,At AGL24,At SOC1 and At SVP were significantly down-regulated.The over-expression of different isoforms into A.thaliana affected the expression of endogenous genes in A.thaliana,which may be the potential cause of affecting the flowering changes.5.Interaction protein analysis: OfAGL24 and Of FD genes were cloned from the flower of O.fragrans.Sequence analysis showed that OfAGL24 contained MADS-box conserved domain,Of FD contained b ZIP conserved domain,and both OfAGL24 and Of FD were located in the nucleus.OfAP1-a1,OfAP1-a2,OfAP1-b1,OfAP1-b2 can interact with OfAGL24 in the nucleus of Y2 H and Bi FC experiments.Of TFL1-1 can interact with Of FD in cytoplasm,and Of TFL1-2 can interact with Of FD in nucleus.