Evaluation Of Environmental Friendliness Of β-aminobutyric Acid And Its Transcriptional Regulation Of Priming Response In Grape Berrie

Chemical fungicides could effectively control diseases of postharvest grape fruits,but long-term application will lead to some negative effects such as pathogenic bacteria resistance,pesticide residues,environmental pollution,etc.,which will not only fail to achieve antiseptic effect,but also reduce product safety,endanger human health and affect ecological balance.Therefore,it is particularly important to find ways to replace chemical fungicides.This thesis aimed to research the inhibitory effect of different concentrations of BABA and thiophanate-methyl on B.cinerea that is a pathogen causing postharpostharst grape gray mold,discussing the the effects of BABA and thiophanate-methyl on Vero cell morphology and survival,and the effects on model organism Caenorhabditis elegans movement behavior and life span were also studied,so as to determine the environmental friendliness of BABA.Moreover,the regulation mechanism of resistance to postharvest grape fruit induced by BABA treatment was further studied from multiple perspectives such as enzyme activity,gene expression and molecular level.The results are as follows:（1）The environmental friendliness of BABA and thiophanate-methyl was studied.The result showed that 1‰thiophanate-methyl had obvious inhibition on B.cinerea and had obvious toxicity to Vero cells and Caenorhabditis elegans.In vitro experiments of BABA on B.cinerea showed that 0.1 mmol/L and 1 mmol/L BABA could not significantly inhibit the growth of B.cinerea,but when BABA concentration was equal to or greater than 10 mmol/L,the spore germination and mycelium growth of B.cinerea could be significantly inhibited.It was also found that 10 mmol/L BABA could effectively reduce the incidence of gray mold and inhibit the spread of spot diameter.Moreover,0.1-100mmol/L BABA had no significant effect on Vero cells viability,and movement behavior and life span of Caenorhabditis elegans.These results suggest that BABA has no toxic effect compared with 1‰thiophenazim methyl,and has improved fruit resistance to disease.BABA can be used as an alternative fungicide for postharvest fruit preservation,thus reducing environmental and health risks associated with chemical application.（2）The effects of BABA treatment on postharvest grape fruit disease resistance were studied.The result showed that 10 mmol/L BABA treatment effectively lowered the disease incidence and delayed the increase of lesion diameter of gray mold caused by B.cinerea in grapes.BABA,applied alone,could induce the activities of phenylpropanoid metabolic enzymes and the contents of total phenol,lignin,flavonoid and anthocyanin in grapes.BABA treatment combined with B.cinerea inoculation showed most significantly effects on the induction of H₂O₂ burst,and the activities of disease-resistance related enzymes such as CHI,GLU,PAL,C4H,4-CL,and POD as well as contents of total phenol,lignin,flavonoid and anthocyanin in the samples during the storage.These results suggested that the BABA treatment could activate the strong and rapid disease resistance upon the pathogenic infection by an induction of priming defence,by which the treatment resultantly maintained the overall quality in grapes during the postharvest storage.（3）The the mode ofβ-aminobutyric acid（BABA）-induced disease resistance and to analyze the mechanism involved in BABA-mediated accumulation of soluble sugar in basis of sorbitol metabolism and transcriptional regulation in postharvest grapes were studied.The result exhibited that The results exhibited that treatment of grapes with BABA alone could induce the defensive response at basic level;however,the transcriptional values of PRs genes（Vv NPR1,Vv PR1,Vv PR2 and Vv PR5）and content of individual phytoalexin in BABA-treated grape berries were significantly enhanced upon inoculating the berries with B.cinerea,indicating that the resistance induced by 10 mmol/L BABA can be attributed to a priming mechanism in grapes,since only the BABA-treated fruit showed an enhanced capacity to augment defense responses upon challenge with the pathogen.On the other hand,BABA treatment promoted the activities of a set of sorbitol metabolism-related enzymes,including S6PDH,NAD⁺-SDH,NADP⁺-SDH and SOX,resulting in the accelerated conversion of sorbitol into glucose and fructose.Meanwhile,Vv WRKY40 bound to the W-box motif in the promoter of sucrose-metabolizing enzyme genes and activated their transcription,by which the BABA up-regulated expression of Vv WRKY40 gene and indirectly enhanced sucrose synthesis in grapes.Therefore,BABA elicitation not only activates the priming resistance for inhibiting gray mold disease in grapes during the stroage,but also promotes the accumulation of soluble sugars through the regulation of sorbitol metabolism and transcriptional activity of Vv WRKY40,which suggests the BABA-triggered priming response exerts a balance between elevated disease resistance and maintenance of primary metabolism.（4）The regulation of TCP transcription factor on priming response of grapes was studied at molecular level.The results exhibited that BABA significantly upregulated the expression abundance of ICS and IPL genes as well as PAL and BA2H genes in grape fruits,thereby fully activating the ICS and PAL pathways and stimulating the synthesis of SA.Vv TCP7 was highly homologous to At CHE in Arabidopsis thaliana by homology analysis,and BABA could induce a significant increase in Vv TCP7 expression abundance in fruits,accompanied by an up-regulation of PRs gene transcription level and a decrease in the incidence of gray mold of grapes.On the other hand,Vv TCP7 is correlated with ICS and IPL genes（ICS pathway）,as well as PAL and BA2H genes（PAL pathway）,which are key genes in SA synthesis pathway.In addition,Y1H（in vivo）and DLR experiments confirmed that Vv TCP7 could directly activate ICS genes,and the endogenous SA content and SAR response level of arabidopsis thaliana Vv TCP7 overexpressed plants were significantly increased.These results indicate that Vv TCP7 could be involved in BABA induced SAR priming resistance by activating the expression of key enzyme genes for SA synthesis.However,BABA concentration and treatment time should be strictly controlled in actual fruit preservation to prevent excessive induction of resistance.