The Regulation Mechanism Of MdBam5 In Starch Degradation Regulation Of Postharvest Apple Fruit

Apple is an important fruit tree in northern China,and it ranks first in the world in both output and export volume.Starch degradation occurs during the ripening of apple fruit,which results in texture softening and sugars production.Although starch degradation has been well studied in starchy fruits during ripening,but in apple fruit are largely unknown.Previous studies have shown that β-amylase plays a crucial role in the degradation of apple fruit starch,but the functional proof for specificβ-amylase genes responsible for apple starch degradation is still not available.Therefore,in this study,the β-amylase genes were identified from a genome-wide perspective,combined with q RT-PCR,yeast one hybridand,GUS staining,and transient overexpression,and screened the key to the degradation of starch in apples after harvest,and to carry out corresponding research on the transcriptional regulation mechanism of this gene.The main results are as follows:1.Based on the Apple Genome Database,the Arabidopsis thaliana β-amylase genes were employed as queries determine the β-amylase gene family members in apple,21 β-amylase family members were identified.Phylogenetic analysis showed that Apple MdBam5 and the catalytically active At BAM3 of Arabidopsis β-amylase gene family members aggregated in the same branch,and it is likely to participate in the degradation of apple starch.2.Many physiological indexes were measured in different treated apple,including ethylene production,firmness,soluble sugar and starch content,and the relative expression levels of 21β-amylase genes that have been identified were determined.The results of expression analysis showed that the relative gene expression of MdBam5、MdBam8、MdBam13、MdBam15 and MdBam16 was inhibited by low temperature,and the gene expression of the MdBam5,MdBam6,MdBam8,MdBam14 and MdBam19 was rapidly induced by exogenous ethylene and inhibited by 1-MCP.Based on the gene expression abundance and overall expression trend,MdBam5 was selected as a candidate gene to study the starch degradation of apple fruit after harvest.The starch content of apple pulp with transient overexpression of MdBam5 was significantly lower than that of the control,indicating that MdBam5 could promote starch degradation.3.MdWRKY32 was identified based on a yeast one-hybrid screening using MdBam5 promoter as a bait.GUS reporter gene fluorescence detection showed that MdWRKY32 could trans-activate the promoter of MdBam5.Expression analysis indicated that MdWRKY32 was induced by ethylene and inhibited by 1-MCP and low temperature treatment,which coincides with the expression pattern of MdBam5.In addition,the MdWRKY32 transient overexpression led to starch degradation in the leaves of tobacoo,and ethylene can induced the activity of MdWRKY32 promoter.