Isolation And Function Characterization Of Genes Associated With Fruit Ripening And Softening In 'Taishanzaoxia' Apple(Malus Domestica Borkh.)

China is the largest production country in the world.The structure of apple cultivars needs to be imporved urgently as the proportion of late-maturing cultivar 'Fuji'is over 70% and the early-maturing cultivars hold a very small proportion.However,it becomes the limiting factor of improving structure of cultivars to be lack of high-quality early-maturing apple cultivars.'Taishanzaoxia‘,an early-maturing apple cultivar bred by our group,has excellent fruit appearance and quality characteristics.However,fruit softening,the common problem of early-maturing apple cultivars still persists.Therefore,it is not only important for the regulation of fruit quality and cultivar promotion,but also contributes to the formation mechanism of fruit ripening and softening and provides scientific theory basis for cultivar improvement and genetic breeding to study the mechanism of fruit ripening and softening in 'Taishanzaoxia‘.This study optimized the method of evalution for fruit texture quality with Texture analyzer.Two SSH libraries were generated from 'Taishanzaoxia'apple around the ethylene climacteric to isolate fruit ripening and softening-related genes.Fruit firmness,ethylene production,enzyme activity and expression of genes associated with fruit ripening and softening during fruit development period and after 1-MCP treatment were measured.Four genes related with cell wall modification were cloned and overexpressed in tomatoes to explore the effect on the expression of genes associated with ethylene biosynthesis,signal transduction and fruit softening.The main results were as follows:1.After optimizing fruit peel,probes,penetration depth and test speed,determined the optimum test condition for fruit texture evaluation by Texture analyzer was: P/2 probe with fruit peel,penetration depth 10 mm and test speed 1 mm·s-1.2.Forward and reverse SSH libraries were generated from fruits 60 DAFB and 70 DAFB of 'Taishanzaoxia'apple,and 648 unigenes differentially expressed during fruit ripening and softening were isolated from the libraries,of which 87 genes were closely associated with fruit ripening and softening,involved in cell wall modification,anthocyanin synthesis,aroma production,stress response,metabolism,transport,transcription and not-annotated function.The qRT-PCR results and correlation analysis suggested fruit texture,anthocyanin synthesis and aroma production in 'Taishanzaoxia'apple were all regulated by ethylene.3.The ACS1 genotype of 'Taishanzaoxia'was ACS1-1/-1 homozygotes while it was ACS1-2/-2 homozygotes and ACS1-1/-2 heterozygotes in 'Fuji'and 'Golden Delicious'respectively.The insert of SINE into the promotor region of ACS1-2 allele resulted in the sharp decrease of ACS1-2 transcriptional activity and ethylene production.The burst of ethylene production during fruit development period in 'Taishanzaoxia'apple may be primarily resulted from the higher transcriptional activity of ACS1 and ACO1.4.Fruit softening in 'Taishanzaoxia'apple was a result of cooperation of multiple genes including PG,?-Gal,?-Glu,XTH2,XTH10 and AM et al,which was regulated by ethylene.(1)1-MCP could delay the burst of ethylene production and fruit softening through suppression the up-regulation of ethylene biosynthesis,signal transduction gene(ACS1,ACO1 and ERF2)and fruit softening-related genes(?-Gal,?-Glu,XTH2 and XTH10).(2)Overexpression of ?-Gal,?-Glu,XTH2 and XTH10 in tomato all resulted in the elevated expression of fruit softening-related gene(e.g.PG),ethylene biosynthesis and signal transduction genes(ACS1,ACO1 and ERF2).