Screening And Functional Identification Of NAC Transcription Factors Related With Apple Fruit Ripening

Apple(Malus domestica Borkh.),is one of the most economically important fruit for fresh markets.Ethylene synthesis,increases at the onset of ripening,is the key driver of fruit softening in climacteric fruits.Genes associated with ethylene biosynthesis and perception pathways have been identified in apple,including 1-aminocyclopropane-1-carboxylate(ACC)synthetase ACS,ACC oxidase ACO,ethylene receptors(ETR1),CTR1 orthologue,ethylene insensitive 3-like(EIL)genes,and the ethylene responsive factor(ERF).Plant-specific NAC transcription factors(TFs)play important roles in regulating diverse biological processes.It participates in the process of fruit development and maturation through the pathway of ethylene biosynthesis and signal transduction.However,the molecular mechanism of NAC TRs regulating fruit developing and ripening processes of apple via ethylene synthesis and transduction pathway is still unclear.In this study,taking ?Golden Delicious‘ apple fruit as experimental materiasl,13 NAC genes were screened out from apple genom and were expressed differently at apple fruit development and ripening stages.Based on this result,further study was emphasized on the functional identification of differently expressed genes of MdNAC78 and MdNAC80.The main results were as follows:1.Thirteen MdNAC genes were differently expressed during the stages of fruit growth and ripening in the fruit of ?Golden Delicious‘ after screening apple genome.2.MdNAC2,MdNAC26,MdNAC41,MdNAC57,MdNAC80,MdNAC91,MdNAC119,and MdNAC141 were up-regulated by ethylene,the expression trends of above 8 MdNAC genes mirrored ethylene production rates during storage.The expressions of both MdNAC1 a and MdNAC78 were repressed by ethylene and induced by 1-MCP during storage,the expression trend was opposite to that of ethylene release.MdNAC1,MdNAC16 and MdNAC32 did not respond obviously after 1-MCP exposure and the trend of expression was not consistent with ethylene production.3.The full-length of MdNAC78 and MdNAC80 were cloned;MdNAC78 had 615 bp in cDNA and gDNA,and encoded 204 amino acid with one extron.MdNAC80 had 1053 bp in cDNA and encoded 350 amino acids.MdNAC80 had 1258 bp in gDNA with three extrons and two introns(188 bp~297 bp,576 bp~670 bp).4.The MdNAC78 and MdNAC80 genes were overexpressed and silenced in apple fruit by the method of Agrobacterium mediated transient expression.The results showed that ethylene production increased in MdNAC78 silenced fruit and MdNAC80 overexpressed fruit respectively,but decreased in MdNAC78 overexpressed fruit and MdNAC80 silenced fruit.It was identified that MdNAC78 is a negative regulator of ethylene production in apple fruit,and MdNAC80 is a positive regulator of ethylene production in apple fruit.5.The results of Yeast one-hybrid(Y1H)experiments showed that MdNAC80 was able to bind to the promoters of MdACS1 and MdACS3 a,while MdNAC78 was unable to bind to the promoters of MdACS1 and MdACS3 a.6.Taking pGBKT7-MdNAC78 and pGBKT7-MdNAC80 as baits to hybrid with the cDNA library of apple fruit,16 interacting proteins with MdNAC78 were screened,including the auxin-repressed protein MdAP1,and 26 interacting proteins with MdNAC80 were screened,including ethylene receptor protein MdETR1 and desiccation-related protein MdPCC.