Identification And Analysis The Roles Of GA Oxidase And MdGAMYB In Flower Bud Induction In Apple

The chronic phenomenon of flower induction and alternate bearing is common in'Fuji'variety,which is cultivated around 70%of total apple production in China.Gibberellic acid?GA?inhibits flowering in woody plants such as apples.Flower induction and alternate bearing motif are closely related to the GA of young fruit seeds but the specific regulation mechanism is still unclear.In this study,we identified and analyzed the GA oxidase genes GA2ox,GA3ox and GA20ox in the GA synthesis pathway in apple,investigated expression levels,cloned the sequences of candidate genes involved in flowering.Additionally,we analyzed expression levels,gene and promoter,the function and interaction protein of MdGAMYB gene in GA signal transduction pathway.The main findings are as following:1.Genome-wide identification?expression analysis and cloning of GA oxidase in apple.?1?A total of 41 gibberellin oxidase genes were identified from the apple genome,including 20 GA2ox,14 GA3ox and 7 GA20ox.Tissue-specific analysis by online database showed that the three types of genes were differentially expressed in different varieties and tissues,and their expression levels were relatively high in flowers and fruits.?2?Eight candidate genes MdGA2ox4/6/9/12,MdGA3ox5/12 and MdGA20ox1/5were selected for RT-qPCR through the transcriptome data.The expression of GA20ox was down-regulated,while GA2ox was increased during flowering induction period after GA₃treatment,the expression of GA3ox showed different trends in different stages.The expression of these genes followed to the negative feedback adjustment mechanism.However,the expression of MdGA2ox,MdGA3ox and MdGA20ox genes were down-regulated after PAC treatment in general.Most of the genes did not conform to the negative feedback regulation mechanism.?3?The MdGA2ox9,MdGA3ox12 and MdGA20ox5 genes were cloned in‘Nagafu 2'and‘Yanfu 6'.The sequence alignment of MdGA2ox9 and MdGA20ox5 were identical in the two varieties,while there are two different sequences were obtained of MdGA3ox12gene in‘Yanfu 6',which has two amino acid mutations:The 66th amino acid was mutated from valine?V?to leucine?L?,and the 137th amino acid was mutated from arginine?R?to glycine?G?,which belonged to the conserved domain region.?4?Further analysis of the expression of three genes MdGA2ox9,MdGA3ox12 and MdGA20ox5,showed that they were expressed in different tissues and MdGA2ox9 and MdGA20ox5 were high in flowers and buds relatively;MdGA20ox5 and MdGA3ox12 were higher in‘Nagafu 2'than‘Yanfu 6',MdGA2ox9 was lower reversely;MdGA20ox5 and MdGA3ox12 were higher in‘ON-year'flower buds than‘OFF-year'flower buds,and MdGA2ox9 was opposite.These results suggest that MdGA2ox9,MdGA3ox12,and MdGA20ox5 may be related to the flower induction and alternate bearing in apple.2.MdGAMYB gene coding region and promoter sequence cloning,expression characteristics and functional analysis.?1?We cloned MdGAMYB of 6 cultivars with different flowering characteristics and2 rootstocks.The results showed that sequences were different between 8 materials.However,there were little differences in conserved regions.Cloning the MdGAMYB promoter in three varieties from different flowering characteristics,the results showed that there were large differences between‘Qinguan'and‘Fuji'?‘Yanfu 6'and‘Yuhuazaofu'?.Moreover,cis-acting elements analysis revealed that there are some differences between the hormone response element and the photoresponsive element in different varieties.?2?MdGAMYB expression pattern analysis showed that MdGAMYB expressed highly in leaves,flowers and buds;the expression of MdGAMYB was significantly inhibited after GA₃ treatment,and showed the opposite expression trend after PAC treatment;The expression of‘Nagafu 2'was significantly lower than‘Yanfu 6';There was no obvious trend‘ON-year'and‘OFF-year'flower buds.These results indicate that MdGAMYB was regulated negatively by GA,and may be involved in apple flower induction.?3?Heterologous overexpression of MdGAMYB in tomato showed that the flowering time of transgenic plants was earlier than WT,and the expression of flowering genes was also significantly affected.The parthenocarpy treatment of apples showed that the expression levels of MdGAMYB were lower than the pollination control within 15 days after GA₄₊₇ treatment.While it was significantly higher in the young fruits treated with 120days.3.There were 39 candidate interacting proteins of MdGAMYB were preliminarily obtained in apple by using gold yeast two-hybrid system,including three hormone-related genes,three genes that play important roles in plant growth and development,nine functional enzyme proteins.