Isolation and expression analysis of MADS-box gene related to flowering regulation in sweet cherry

Abstract:【Objective】It has been known that MADS-box gene family members participate in floweringregulating pathway of higher plants.However, there are very few reports and evidence of MADS-box family genes functioning on cherry flowering.This study aims to clone and characterize MADS-box family genes of sweet cherry (Prunus avium) , and analyze the spatial-temporal gene expression of them in different cherry tissues.This research will provide important evidences for further understanding the effects of MADS-box family transcription factors in cherry flowering.【Methods】Here we first used sweet cherry'Summit'as material, referring to peach genome information as sweet cherry genome is still unknown, we cloned and characterized 14 MADS-box genes that might be related to flowering regulation, named PaSOC1, PaAG, PaAP1, PaAP1-2, PaAP3, PaPI, PaSVP, PaAGL24, PaSEP1, PaSEP2, PaSEP3, PaSEP4, PaSEP5 and PaFLC.Besides, the bioinformatics software of DNAMAN, SMART and protein BLAST were used to analyze the features of the 14 MADS genes.Additionally, MEGA5 was also used for a cluster analysis of these 14 MADS-box genes and other 132 MADS-box genes.And the spatial-temporal RT-PCRgene expression analysis of these 14 genes were conducted in the tissues of root, leaf bud, leaf, flower bud, flower and phloem in sweet cherry.【Results】14 full-length MADS-box genes (PaSOC1, PaAG, PaAP1, PaAP1-2, PaAP3, PaPI, PaSVP, PaAGL24, PaSEP1, PaSEP2, PaSEP3, PaSEP4, PaSEP5 and PaFLC) were 690, 732, 753, 768, 615, 633, 678, 696, 735, 723, 723, 735, 756 and 648 bp, respectively, and the molecular weight were 26, 28, 29, 29, 24, 24, 26, 26, 28, 28, 28, 28, 29 and 24 ku, respectively.Except the isoionic point of PaAGL24 was acidic, the other 13 genes that PaSOC1, PaAG, PaAP1, PaAP1-2, PaAP3, PaPI, PaSVP, PaSEP1, PaSEP2, PaSEP3, PaSEP4, PaSEP5 and PaFLC were alkaline.The structural analysis results showed that all these 14 genes contained MADS-box domain and K-box domain.Among them, PaAG, PaAGL24, PaAP3, PaPI, PaSVP, PaFLC contained 6 introns, PaSOC1, PaAP1, PaAP1-2, PaSEP1, PaSEP2, PaSEP4, PaSEP5 contained 7 introns and PaSEP3 contained 8 introns.In addition, it was interesting that the length and distribution of introns of those genes presented evolutional regularity, that is, the genes with high homology showed the same distribution of introns.Nevertheless, the result of phylogenetic analysis revealed that the 14 cherry MADS-box genes could be manually divided into 7 subclasses.The details were descripted as following:PaSEP1, PaSEP2, PaSEP3, PaSEP4 and PaSEP5 belonged to SEP subclass.PaAP1 and PaAP1-2 belonged to AP1 subclass.PaAG belonged to AG subclass.PaSOC1 belonged to SOC1 subclass.PaAP3 and PaPI belonged to AP3/PI subclass.PaSVP belonged to SVP subclass.PaAGL24 belonged to AGL24 subclass.While PaFLC was extremely special, because that subclass with FLC of woody plant pear neither belonged to a subclass with FLC of herbaceous plant Arabidopsis nor belonged to a subclass with FLC of vine plant Vitis vinifera.The results allowed us to speculate that except for the difference in verbalization and dormancy there were also some differences in the function of FLC genes in different plant species.Comprehensively, RT-PCR results confirmed that all MAD-box family genes performed their own expression pattern in all tissues.The detailed were listed as follows.PaSOC1 was expressed in the tissues of cherry root, leaf bud, leaf, flower bud and phloem.PaAG was expressed in cherry leaf, flower bud, flower and phloem.Furthermore, PaAP1 was mainly expressed in cherry flower bud, and were weakly expressed in leaf, flower and phloem, while PaAP1-2 was expressed in various cherry tissues.PaAP3 was expressed in cherry root and were weakly expressed in flower.PaPI was expressed in cherry leaf bud, leaf, flower bud flower and phloem.PaSVP was expressed in cherry root, leaf bud, leaf, flower bud and phloem.PaAGL24 was expressed in cherry leaf bud, leaf, flower bud and phloem.PaSEP1 was expressed in cherry flower bud and flower.PaSEP2 was expressed in cherry leaf bud, flower bud, flower and phloem.PaSEP3 was expressed in cherry leaf bud, flower bud, flower and phloem.PaSEP4 was expressed in cherry flower bud and flower.PaSEP5 was expressed in cherry flower bud and flower.PaFLC was expressed in cherry leaf, flower bud and phloem.Except for PaAP3, PaSEP1, PaSEP4 and PaSEP5, the rest genes were also expressed in phloem.These results had been never reported in previous researches, which allowed us to speculate that the specify and universality of phloem-derived gene expression pattern might be a unique character of woody plants.In addition, these results suggested that MADS-box genes were widely involved in sweet cherry growth and development process.【Conclusion】14 PaMADS-box family genes obtained in sweet cherry were highly and structurally conserved.They were involved in regulation of flowering and flower development processes in sweet cherry.