Study On Regulatory Roles Of MicroRNA166s And Its Target Genes During GA-induced Grape Seedless Berry Development

The fruit nucleus is an important part of the fruit such as grapes,peach,apricot,cherry,plum,hawthorn and jujube.However,with the development of variation,natural selection and production demand,a large number of nuclear-free fruits with degenerate or disappeared kernels gradually appeared in production.However,lackness of the synthesis of the hard shell composed of lignin was an important factor that led to the degradation of the fruit kernel,thus producing the fruits with nuclear softness,nuclear thinness and isappearance of nuclear degradation.Through the research and analysis of the synthetic pathways of lignin and its dynamic distribution during synthesis,related enzymes and hormones,the metabolic status of sugars and phenols affecting the lignin synthesis substances,and the gene regulation related to the development of cores were observed.Nucleus development and other aspects were studied to illustrate the mechanism of fruitless seedling and understand the effect of lignin on the development of the fruit core,and thus providing a theoretical basis for the fruitless breeding of fruit trees.The Vv-miR166s family is an important miRNA family in response to GA induction that can target the HD-ZIP family of VvHOX32,VvREV,and VvATHB15,and these three members regulate lignin accumulation;on the other hand,GA inhibits lignin synthesis,can induce seedless grape fruit,but its role in this process so far has not been reported.In addition,the mechanisms of the lignin synthesis related genes VvPAL,Vv4CL,and VvCAD in regulating the development of seedless grapes were studied,to understand the regulatory roles of mcroRNAs and genes related to lignin synthesis during GA-induced grape seedless berry development.The findings are as follow:1.We identified that the complementary degree of grape Vv-miR166s:their target genes(VvHOX32,VvREV and VvATHB15)pairs possessed some discrepancy,implying their regulatory roles might diverge,while the promoters of Vv-miR166s and the target genes both had some similar motif types of GA related factors,suggesting they might function in responding GA.Vv-miR166s-mediated VvHOX32,VvREV and VvATHB15 were verified in grape berries by both RLM-RACE and PPM-RACE.Subsequently,the expression of Vv-MIR166s and target genes in the three different periods of the grape flesh,peel and seed tissues showed the character of spatio-temporal differential expression,and Vv-miR166s on their targets had the obvious negative correlation at some special stages,especially at the stone hardening stages,indicating they might significantly function in this stage.Furthermore,the modes responsive to GA of Vv-MIR166s and their targets also appeared the seeds-specific traits.Our findings could provide the significant information for recognizing the roles of Vv-MIR166s responsive to GA in the involvement of modulating grape seedless berry development.2.To investigate the potential role of VvPAL,Vv4CL and VvCAD genes in the development of fruit,especially fruit seed development in Vitis vinifera cv.'Rosario Bianco',the components of VvPAL,Vv4CL and VvCAD genes were analyzed by bioinformatics and gene chip analysis,qRT-PCR technique.Localize the position of grape VvPAL,Vv4CL,VvCAD,whose structure and promoter motif elements were analyzed.The effects of protein coding region,structure and interacting protein were further studied.The expression pattern of GA in different tissues of 'Rosario Bianco' fruit was identified.The homology of grape VvPAL,Vv4CL,VvCAD and the ones in other species showed highly and strong conservation.Gene promoter analysis showed that they all contained tissue-specific components of endosperm development,and Vv4CL gene contained GA responsive elements;The prediction of subcellular localization of VvPAL,Vv4CL and VvCAD genes in grapes showed that they are mainly expressed in cytoplasm and cell membranes;their interaction proteins are mainly key enzymes in the synthesis of lignin such as C4H and OMT;the electronic expression profile of grape chips found that VvPAL and Vv4CL genes are highly expressed in tissues with relatively high lignification,such as roots and stems,whereas VvCAD genes are only relatively highly expressed in seeds.The results of qRT-PCR analysis in this study show that GA significantly down-regulated the expression of VvPAL,Vv4CL and VvCAD genes in seeds in certain periods.VvPAL,Vv4CL and VvCAD genes were involved in the regulation of fruit development of 'Rosario Bianco' grape,and GA may inhibit the development of 'Rosario Bianco' grape seed by down-regulating the expression of VvPAL,Vv4CL and VvCAD genes.