| Persimmon(Diospyros kaki L.),belongs to the genus Diospyros in the family Ebenaceae,is an important commercial and deciduous fruit tree.China is one of the country of origin of persimmon and also the world's largest producer of persimmon.Although persimmon is grown in various countries and is increasingly appreciated for its nutritional value,health benefits and rich flavor,surprisingly little research has been conducted to uncover flavor,particularly genes responsible for aroma volatile,which consider as one of the important characteristics determined fruit quality and consumer acceptance.Evaluation of volatiles at genes level is useful tools aimed to improvement of fruit quality,via effect on fruit aroma.Recent progress in omics technologies resulted in the isolation of genes encoding enzymes responsible for the biosynthesis of many volatiles and contributed to our understanding of regulatory mechanisms involved in volatile compound formation.In this research persimmon fruits were used as plant materials to evaluate the main fruit quality characteristics of 34 different cultivars and to study the effect of ethylene and salicylic acid in main quality characteristic of two persimmon cultivars,namely 'Xiaofangshi' and 'Naiyoushi',during different ripening stages.On the other hand,the aroma volatile profiles of persimmon fruits were determined in both different cultivars and during different ripening stages.Moreover,alcohol acyltransferase was isolated and characterized and the effects of ethylene and salicylic acid on expression pattern of 'Xiaofangshi' and 'Naiyoushi' during different ripening stages were also evaluated.Furthermore,the transcriptome analysis was performed to create a transcript sequence database of persimmon and to identify candidate genes involved in volatile aroma biosynthesis.The main results were as follows:1.The average weight of fruit of non-astringent persimmon cultivars was heavier compared to astringent persimmon,while the differences in fruit shapes index among all the astringent persimmon cultivars was larger than that of non-astringent persimmon cultivars.Moreover,the average total soluble solids of the astringent persimmon cultivars was higher than that of the non-astringent persimmons,while titratable acidity was basically consistent in both non-astringent and astringent persimmon cultivars.On the other hand,results showed that there were no significant differences in main fruit quality including fruit weight,diameter and fruit shape index in two different cultivars during different ripening stages.While,both fruit firmness and total soluble solids showed significant differences in both two cultivars,result revealed that fruit treated with ethylene had the lowest firmness and the highest total soluble solids followed by control fruits,while fruit treated with salicylic acid had the highest firmness and the lowest total soluble solids.2.A comparison of fruit color between different persimmon cultivars showed that both the pulp and the peel of astringent persimmon had deeper orange red color compared to non-astrignant cultivar.Moreover,hue angle(H°)of both 'Xiaofangshi' and 'Naiyoushi' was higher at harvest then decreased in different treatment during different ripening stages reflected the development in the color of fruit to deep orange.3.Many different volatile compounds were identified in the headspace of the pulp of 8 different non-astringent and 26 astringent cultivars of persimmon fruit and 48 and 61 of them were quantified,respectively.Generally,the volatile compounds varied between different cultivars.Benzaldehyde,cyclopropylmethanol,2-ethyl-1-hexanol,nonanone,?-ionone and bornyl acetate were the most dominant volatiles in non-astringent cultivars.While,decanal,2-ethyl-1-hexanol,?-ionone,nonanone,benzyl acetate and bornyl acetate were the most dominant aldehydes produced in astringent cultivars.On the other hand,hexanal,cyclopropylmethanol,6-methyl-hept-5-en-2-one and ethyl acetate;2-hexenal,cyclopropylmethanol,nonanone,ethyl acetate and butyl acetate was the most abundant volatiles in non-astringent and astringent cultivars,respectively.Moreover,42 and 34 different volatile compounds were identified in the headspace of the fruit pulp of'Xiaofangshi' and'Naiyougshi' during different ripening stages.Nonanal,(Z)-9-hexadecen-1-ol,3-tridecanol,3-hydroxy-2-butanone and benzylacetate were the most dominant volatiles produced during different ripening stages.While in 'Naiyougshi',acetaldehyde,nonanal,(Z)-9-hexadecen-1-ol,linalool,2-ethyl-1-hexanol and 1-octanol were the most dominant volatiles produced in fruits during different ripening stages,while 3-hydroxy-2-butanone was the only one ketones produced in fruits during different ripening stages.4.Among the esters quantified,results showed that benzylacetate was the most dominant esters produced during different ripening stages of 'Xiaofangshi',while ethyl acetate and methylphenyl acetate were the most dominant esters produced during different ripening stages of'Naiyoushi'.Moreover,both butyl acetate and ethyl acetate were the most abundant ester produced in the two cultivars.Generally,'Xiaofangshi' fruits treated with salicylic acid had the highest total aldehydes,ketones,esters and acids at day 9,15,15 and 12 compared to other treatments,and control fruits had the highest alcohols at day 15.On the other hand'Naiyoushi' fruits treated with salicylic acid had the highest total aldehyde,alcohols,ketones and acids,while the highest total esters was produced in fruits treated with ethylene.5.AAT activity of DkAAT protein was quantified by its ability to convert butanol and acyl-CoAs into the corresponding ester.Results showed different trend during different ripening stages of persimmon fruits,however,in both 'Xiaofangshi' and 'Naiyoushi' fruits treated with salicylic acid had the highest activity at day 12 and 15,respectively.6.The cloning of DkAAT1 was achieved by application of 3' and 5' RACE strategies with specific primers.The spliced results of 3,and 5' cDNA ends showed that the full-length of DkAAT1 cDNA was 1637 bp,contained an ATG initiation codon,an open reading frame(ORF)of 1380 bp,a TAA stop codon,a 5'-untranslated region(5'-UTR)of 75 bp,a 3'-UTR of 182 bp and a poly(A)tail which encoding 459 amino acids.Additionally,DkAAT1 had been submitted in GenBank with accession number KP271995.Homology analysis using BLAST of GenBank revealed that the deduced amino acid sequence shared high similarities with sequences encoding AAT from other plants.On the other hand,in order to analyze the complexity of persimmon's AAT family,genomic analysis was performed using DNA gel blots.Results suggested the existence of one copy of the DkAAT gene with all the restriction enzymes tested.7.The expression of DkAATl was analyzed by RT-qPCR in different vegetative tissues,as well as in mature fruit of two different cultivars.Results showed significant differences between different tissues,among tissue analyzed the petal showed the highest transcript accumulation followed by the leaf,while the fruit showed the lowest transcript accumulation.Generally,DkAAT1 gene was highly expressed in cultivar 'Xiaofangshi' compared to'Naiyoushi'.Moreover,the peel of the two cultivars showed higher expression level compared to the pulp.On the other hand,the effect of ethylene and salicylic acid on the expression level was studied in two different cultivars of persimmon fruits during different ripening stages.Results showed that transcript accumulation was observed in all fruits tested during different ripening stages.However,the expression level showed significant differences between different treatments during different ripening stages.Generally,In'Xiaofangshi' a notorious increment in the level of DkAATl transcripts was observed during ripening,reaching the highest level of 53.99 at day 9 in fruits treated with ethylene and 40.76 in fruits treated with SA at day 12.While in 'Naiyoushi' the highest transcripts level was 31.73 and 5.08 at day 12 and 9 in fruits treated with ethylene and fruits treated with salicylic acid respectively.8.The de novo transcriptome sequencing analysis of persimmon fruit using the Illumina HiseqTM 2000 platform revealed that a total of 84759380 raw reads libraries were generated from the pulp cDNA and 81973802 total high quality clean reads were obtained,which were assembled into 71194 unigenes with an average length of 642 nt.Eukaryotic Orthologous Groups(KOG)clustering analysis revealed 26 different functional classifications,among them,the largest group was the predicted general functions followed by the group with transcriptional functions.For Gene ontology(GO)classification,the three largest percentages of genes identified within these functional categories were cellular process,binding and metabolism.Subsequently,analysed in the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway showed that translation pathways demonstrated the largest number of unigenes followed by carbohydate metabolism,while membrane transport pathways demonstrated the lowest number of unigenes.Moreover,transcriptome analysis create a transcript sequence database of persimmon to identify candidate genes involved in volatile aroma biosynthesis particulary,alcoholtransferase(AATs),fatty acid hydroperoxide lyase(HLP),3-ketoacyl-COA thiolase(THMF)and carotenoid cleavage dioxygenase(CCDs). |
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