Studies On The Screening Of MaEBF1 Interacting Proteins And Expression Of Key Genes In Ripening Disorder Of Dwarf Banana Fruit Caused By Low Temperature Stress

The dwarf banana?Musa ABB group,Pisang Awak?belongs to banana hybrid cultivar of banana species.Dwarf banana has good quality and flovour,high nutritional value and economic value,but it usually harvested with higher maturity,so it is difficult to preserve.Low-temperature storage is commonly used to prolong the preservation time of banana fruit.However,unsuitable temperature and storage time,and the fruit harvested in winter will cause cold injury,and even lead to fruit ripening disorders.As a result,the fruit can not soften and turn yellow normally,and the starch degradation is inhibited.In this study,we analyzed the effects of low temperature stress?7??storage on the physiology and expression of starch degradation enzymes of dwarf banana during storage and shelf-life period,in comparison with 11?storage as the control.A cDNA library of dwarf banana fruit under low temperature stress was constructed.The ethylene signal transduction component EBF1 was used as a bait protein to screen the interacting proteins.Several identified interacting protein code genes related to fruit ripening and cold resistance were selected for RT-qPCR expression validation to further study the mechanism of fruit ripening disorder caused by low temperature stress.The main results were listed as follows:1.The fruits stored at 7?and 11?were still with a green state on 12^thd..The fruit suffered cold injury after storage at 7?for 6 d,and cold injury symptom even more severe at 12 d,with the chilling injury index about 2.2.On the 3rd,6th and 12th day of storage,the fruit were treated with ethephon and moved to room temperature to study the effects of low temperature stress on fruit ripening.Fruit ripening after being stored at 7?and 11?for 3 days had little difference in phenotypes compared with control,and the ripening process was almost the same with the control fruit.Fruit stored at 7?for 6 days,the full ripening time of the fruit was delayed by 2 days than that of control?11??fruits.Fruit ripening after 7?storage for 12 days was obviously suppressed,fruit showed severe cold injury symptoms,and could not be softened and ripening normally.2.The expression of 9 genes encoding starch degradation enzymes?PWD1,GWD1,AMY2B/3,BAM4/7,SEX4,LSF1,ISA3?under different temperatures during the storage were investigated.Expression analysis revealed that the expression of these genes increased significantly during banana fruit ripening at the room temperature,and their expression were significantly inhibited by cold storage.The expression of PWD1,GWD1,AMY3,BAM4/7,SEX4 and ISA3 were inhibited during shelf-life ripening after storage at7?,and longer duration of chilling temperature storage suffered,more severely inhibition observed.However,no significant inhibition of the expression of AMY2B and LSF1 were observed.3.The invitrogen system was used to construct a cDNA library of the dwarf banana fruit under low temperature stress through gateway recombination cloning technology.MaEBF1 was used as a bait to screen the interacted proteins by yeast two-hybrid from the cDNA library and 33 positive interaction proteins were obtained.Among these proteins,four of which contain the zinc finger domains,which play an important role in the stress resistance of plants.Two genes are vacuolar sorting proteins related genes,which play an important role in the intracellular vesicle trafficking and maintenance of cell polarity.Three proteins are related to hormone?ethylene,auxin,abscisic acid?signal transduction.Six proteins encoding genes are related to plant resistance,among which CHIT-like,CHIT,NAC67-like,HSA32 are related to plant cold resistance.Two proteins are related to the transport of substances.Three proteins are involved in protein degradation and other five proteins are fuction unknown.4.Several genes encoding the screened proteins with high frequency n the results and cold-resistance related were selected for the expression alalysis,which included VPS18/36,P21-like,BEL1-like,ZFN33-like1,ZFN33-like2,CHIT-like,CHIT,NAC67-like,HSA32 and unknown1.The result showed that low temperature induced the expression of ZFN33-like1,ZFN33-like2 and unknown 1 genes,and inhibited the expression of VPS18,P21-like and BEL1-like genes,but no significant effects on the expression of CHIT-like,CHIT,HSA32and VPS36 genes.After ethephon ripening treatment,the expression levels of VPS18,P21-like,BEL1-like and NAC67-like genes were significantly inhibited in fruits with chilling injury,and the expression of CHIT-like,CHIT,HSA32 and VPS36 was not affected by chilling injury.Genes including ZFN33-Like1,ZFN33-like2 and Unknown-1 showed low expression at room temperature.