Function And Regulation Mechanism Of MbACO2 Gene In Banana Postharvest Ripening

Controlling the ripening process can prolong the shelf life of banana fruit and reduce the economic loss.Ethylene synthesis and its effect are the key factors determining banana fruit ripening.Banana cultivars are mainly composed of A genome or A / B genomes.Previous studies focused on the regulation mechanism of ethylene synthesis in banana A genome,and considered that B genome mainly affected the stress resistance of banana.At present,the function and regulation mechanism of ethylene synthesis key enzyme genes in B genome have not been revealed,which greatly limits the fruit storage genetic improvement of banana germplasm containing B genome.In this study,we used yeast one hybrid,yeast two hybrid,dual luciferase and genetic transformation techniques to analyze the function and transcriptional regulation mechanism of MbACO2 during fruit ripening1.A 918 bp c DNA encoding 305 amino acids was cloned from Musa ABB group,cv Pisang Awak,FJ.Its amino acid sequence has a conserved domain PLN02299,which is most close to Musa acuminata AAA group ACO protein.2.MbACO2 gene was expressed in root,stem,leaf and fruit of FJ,which increased rapidly with ripening and was induced by osmotic,low temperature and high salt stress.3.Overexpression of MbACO2 in tomato could significantly accelerate the process of fruit ripening,increase the ethylene release,and boosting the appearance of ethylene peak,and promoted the increase of vitamin C,glucose,fructose,carotenoid content and chlorophyll degradation.4.Based on the transcriptome data of FJ,eight NAC and ERF transcription factors were identified.Y1 H and D-Luc experiments confirmed that Ma NAC29 、 Mb ERF71 and Mb ERF113 could interact with the promoter of MbACO2 and activate it’s expression.Further analysis by Y1 H with promoter fragment and core element mutation showed that Ma NAC29 interacted with CGTC element(-723 bp to-726bp)of MbACO2 promoter,and Mb ERF71/Mb ERF113 interacted with GCC box(-653 bp to-658bp)of MbACO2 promoter.5.The interaction between Ma NAC29 and Mb ERF113 at protein level was confirmed by Y2 H,Bi FC and D-Luc experiments,and the interaction between Ma NAC29 and Mb ERF113 could enhance the transcriptional activation of MbACO2.6.Based on the transcriptome data of FJ,thirteen MAPK and CDPK protein kinases were identified.The interaction between Ma MAPK1 and Ma NAC29 at protein level was confirmed by Y2 H and Bi FC experiments.7.The D-Luc experiment revealed that Ma MAPK1,Ma NAC29 and Mb ERF113 could coactivate the expression of MbACO2.Subcellular localization analysis showed that the activation of MbACO2 by the three interactions might occur in the nucleus.In conclusion,this study revealed that the interaction between Ma MAPK1 and Ma NAC29 can activate the expression of MbACO2 by binding to the CGTC element of MbACO2 promoter.Meanwhile,the interaction between Ma NAC29 and Mb ERF113 can further enhance the transcriptional activation of MbACO2,thereby promoting ethylene synthesis and fruit ripening.On the one hand,the results can deepen the understanding of the regulation mechanism of ethylene synthesis mediated by key genes in banana B genome,and on the other hand,provide theoretical basis for controlling banana fruit ripening process and prolonging banana storage period.