The Mechanism Of WRKY Transcription Factors Involved In Regulating ABA-induced Cold Tolerance In Banana Fruit

Chilling injury happened to banana fruit easily and affect its commodity value when the storage temperature is below 13 ℃.Therefore,the research of cold tolerance molecular mechanism has important scientific significance in postharvest banana fruit.WRKY is a peculiar kind of plant transcription factors involved in the regulation of plant growth,aging,hormone signal transduction,and stress response and a series of biological processes.Abscisic acid(ABA)is one of the main hormones in plants playing a important role in stress response.However,whether WRKY transcription factors involved in ABA-induced cold tolerance and what’s its specific mechanism in banana fruit are not clear now.therefore,This article is based on RNA-seq expression spectrum related to the cold stress response of banana fruit,select the WRKY transcription factors involved in cold stress response,analysis the expression of genes,subcellular localization and transcription activity etc.,selective analysis the transcription regulation of WRKY to 9-cis-epoxy carotenoids two-plus oxygen(NCED),the key gene of ABA biosynthesis,in order to clarify the specific mechanism of WRKY transcription factors regulating ABA-induced cold tolerance in banana fruit,the main results were as follows:1.100 μM exogenous ABA treatment can reduce chilling injury and enhance cold resistance of banana fruit;this paper pick out four cold stress response WRKY transcription factors from RNA-seq expression spectrum,and name them MaWRKY31,MaWRKY33,MaWRKY60 and MaWRKY71.Further real-time fluorescence quantitative analysis show that cold stress induce the expression of the MaWRKY31/33/60/71,and the expression of MaWRKY31/33/60/71 become stronger after exogenous ABA treatment.2.This experiment has cloned the total gene sequence length of MaWRKY31,MaWRKY33,MaWRKY60 and Ma WRKY71.Sequence alignment and evolutionary tree bioinformatics analysis show that MaWRKY33 contains two WRKY domain structure belong to the first class I of WRKY family,MaWRKY31,MaWRKY60 and MaWRKY71 contain a WRKY domain structure,respectively belong to the WRKY family type IIb,IIa and IIc class.Subcellular localization show that MaWRKY31 MaWRKY33,MaWRKY60 and MaWRKY71 are nucleoprotein located in nucleus;Transcriptional activity analysis show that MaWRKY31 MaWRKY33,MaWRKY60 and MaWRKY71 have transcriptional activation activity,they are transcriptional activator.3.This experiment has analysised the expression characteristics of five NCED genes(MaNCED1-MaNCED5)in the banana genome.Cold stress induce the expression of MaNCED1/2/4/5 except MaNCED3,the expression is stronger after exogenous ABA treatment,especially the MaNCED1 and MaNCED2.4.This experiment has cloned the promoter of MaNCED1 and Ma NCED2,the analysis show that the promoter contains a WRKY transcription factors binding element W-box(TTGAC).Further Gel block analysis(EMSA)and Dual luciferase analysis(Dual-Luc)show that MaWRKY31,MaWRKY33,MaWRKY60 and MaWRKY71 can combine the promoter of MaNCED1 and MaNCED2 directly,and activate their activity.The results show that WRKY transcription factor,though directly regulate the expression of NCEDs which are the key genes of ABA synthesis,participate in the response of cold stress and the ABA-induced cold tolerance in banana fruit.