Functional And Molecular Mechanisms Of MdTCP14/15 In Regulation Of Floral Induction In Apple (Malus Domestica)

The main cultivar’Fuji’,which accounts for more than 70%of the apple area in China,often has problems in production,such as the difficulty in forming flower buds on young trees and the obvious phenomenon of biennial bearing on mature trees,which has become one of the bottleneck problems restricting the high-quality development of apple industry in China.Studies in model plants have shown that TCP transcription factor can interact with other proteins to regulate the transcription level of downstream flowering genes,and then participate in the regulation of flower formation.However,the regulation mechanism of flowering in perennial woody plants such as apple remains to be further elucidated.In this study,’Nagafu No.2’was used as the experimental material,combined with exogenous hormone treatment,analyzed the expression characteristics of MdTCP14/15,cloned the MdTCP14 gene,analyzed its function of regulating flower formation,screened its interaction proteins,and performed the interaction mode verification analysis.It is expected to provide theoretical guidance for the regulation of flowering and the genetic improvement of flowering traits in the cultivation of high-quality,high-yield and high-efficiency apples.The main results are as follows:1.Bioinformatics analysis,expression characteristics and functional analysis of MdTCP 14/15 in apple.（1）By phylogenetic tree construction and homologous protein sequence alignment of TCP14/15 among 11 different species,it was found that TCP14/15 was conserved in the evolutionary process,and MdTCP14/15 in Apple and At TCP14/15 in Arabidopsis had high homology,indicating that they may play the same biological function in different species.（2）Expression characteristics analysis showed that MdTCP14 was highly expressed in the stem,and MdTCP15 has the highest expression level in buds.After GA₃treatment,the expression of both were significantly inhibited at all stages of flower bud differentiation.After 6-BA treatment,the expressions of the two were significantly down-regulated at 40days after the full blooming stage,and there was no significant difference in other periods compared with the control.After ABA treatment,the expression level of MdTCP14 was inhibited and down-regulated at 50 d and 60 d after flowering,and was significantly up-regulated than the control at 70 d after flowering;while the expression level of MdTCP15was significantly up-regulated at 30 d and 70 d after flowering.In different load treatments,the expression level of MdTCP14 in the trees with low fruit load was higher in the early stage of flower bud differentiation than in the trees with heavy fruit load,while the expression level of MdTCP14 in the trees with heavy fruit load was higher than that in the trees with low fruit load at the late stage of flower bud differentiation;at 70 days after flowering,the expression of MdTCP15 in the trees with heavy fruit load was significantly higher than that in the trees with low fruit load.Heterooverexpression of MdTCP14 in Arabidopsis indicated that the transgenic plants had fewer rosette leaves,higher inflorescence height,and fewer branches compared with the wild type.2.Screening,validation and regulatory mechanism analysis of MdTCP14 interacting proteins in apple.（1）MdTCP14 protein as a bait,In the apical bud c DNA library of’Nagafu No.2’in the physiological differentiation period,using yeast double hybrid technology for protein screening,get 104 interactive proteins,which were respectively related to flowering regulation,ethylene signal transduction pathways,auxin polar transport,abscisic acid signal transduction,stress response and the ubiquitin-proteasome proteolytic digestion process.（2）The results of yeast two-hybrid regression test showed that MdTCP14/15 could interact with Md WRKY6a,Md WRKY6b,Md ERF,MdTCP2 and Md HSP20.Subcellular localization of Md ERF and Md HSP20 showed that Md ERF was mainly localized in the nucleus,while Md HSP20 was localized in the cytoplasm.The expression pattern analysis showed that Md ERF was highly expressed in flowers,MdTCP2 was mainly expressed in leaves and flowers,and Md HSP20 was concentrated in buds and stems.（3）The yeast single hybridization test showed that MdTCP14 could bind to the 3’TFL1-1q and 5’TFL1-2q promoter fragments.It is speculated that MdTCP14 may regulate flower bud differentiation by regulating the transcription of MdTFL1.In conclusion,MdTCP14/15 is involved in regulating various growth and development processes of apple,and its expression level is regulated by various hormones.Interaction regulatory analysis showed that MdTCP14/15 could interact with Md WRKY6A,Md WRKY6B,Md ERF,MdTCP2 and Md HSP20,and transcriptional regulatory analysis showed that MdTCP14 could bind to the promoter fragments of MdTFL1-1 and MdTFL1-2.In apples,MdTCP14/15 may be involved in the regulation mechanism of flower bud induction through the transcriptional regulation of MdTFL1 by interacting with some proteins.