| Aroma is one of the most important factors for fruit quality.The aroma of most pear cultivars in china was light,which could not compete with European pears in the market,and required to carry out study on the theory and technology for improving the quality of pear fruit aroma.In this study,the fruit of 'Nanguoli' was selected as materials.On the basis of research on the physiological mechanism of exogenous calcium regulation aroma synthesis,I used RNA-seq and metabolomics technique analyzed the differentially expressed genes of 'Nanguoli' pear fruit ripening process and calcium treatment.I also cloned PuADHI gene,and then performed the verification by transgenic tomatoes,the main research results are as follows:1.The effect of calcium treatment on composition and content of 'Nanguoli' pear fiuit aroma substances were studied,the result showed that fruit total calcium and water soluble calcium content were significantly increased,and the soluble solids content of 'Nanguoli'fruit were also increased.Fifty-one volatile aromatic substances were isolated and identified in 'Nanguoli' fruit,content of aroma substance had significant difference between calcium treatment and CK.Esters,especially ethyl esters were significantly increased in'Nanguoli' with calcium treatment pear fruit.Acetaldehyde,alcohol and other substances were also significantly increased,but calcium treatment inhibited the formation of ethylene in fruit.Improvement in 'Nanguoli' pear fruit aroma content under calcium treatment was mainly achieved by the fatty acid metabolic pathway,and the mainly improved fruit aroma content is ethyl esters.2.In order to study the physiological mechanism of calcium treatment improving the aroma of pear,the 15 days before harvest calcium treatment 'Nanguoli' pear fruit was taken as the material.The results showed that pre-harvest calcium treatments significantly changed in 'Nanguoli' pear fruit epidermal waxy layer,pore and arrangement of the cells.Calcium treatment fruit peel wax thickness was less than that of the control and covered as a homogenous control in harvest;in 5 days after postharvest,calcium treatment fruit peel wax layer almost completely detached,control peel wax layer only partially exfoliated.There was a significant difference in the stomatal morphology of Ca2+ treated and control.In harvest,calcium treatment of 'Nanguoli' pear fruit peel stomatal surrounding cuticle shedding was more than control,the center void number was more than those in control,and the center void space was bigger than control.There were differences in the surface cell arrangement and control in the fruit peel of Ca2+ treated fruit.The thickness of cuticle of the calcium treated fruits was decreased,and the intercellular space increased,and the number of epidermal cells decreased.Compared with the control,contents of the epidermal cells of calcium treated fruits were increased.The main deposits of calcium include:intercellular space,cell wall,vacuole,cytoplasm,duct and so on.The epidermis wax content of calcium treatments 'Nanguoli' pear fruit decreased slightly,but was not significant.'Nanguoli' fruit epicuticular wax chemical composition is complex,including alkanes,alkenes,alkanoic acids,aldehydes,terpenes.There was no significant difference of epicuticular wax chemical composition between calcium treated and control,but waxy component content changed.Four kinds of soluble sugar,6 types of organic acid,15 forms of amino acids,32 kinds of fatty acids were separated and identified from the 'Nanguoli' pear fruits.Calcium treatment significantly increased the content of linoleic acid and linolenic acid,the linoleic acid content of 'Nanguoli' pear fruit was 223%higher than that of control in harvest,and linolenic acid content improved 740%comparing with the control;preharvest calcium treatment significantly increased the activity of ADH,PDC,LOX,especially the ADH activity improved 417.68%than control.Taken together,changes of volatile aromatic substances in 'Nanguoli' pear fruit mainly relied on improving the metabolic substrates(linoleic acid and linolenic acid)content and ADH,PDC,LOX enzyme activities.Seven bound aroma substances were isolated and identified from the 'Nanguoli' pear fruit,calcium treatment significantly decreased the total content of bound aroma compounds,bound aroma compounds in peel reduced 26.74%compared with the control,and the content of bound aroma compounds in pulp decreased 11.11%compared with the control.Calcium treatments of ?-glucosidase activity was significantly higher than control,?-glucosidase activity of calcium treated peel was 146.25%of control,the ?-glucosidase activity of pulp was 124.44%of control.Therefore,calcium treatment improved the activity of fglucosidase to promote the release of the bonded aroma,thus improved the aroma of'Naguoli' pear fruit.4.DGE analysis of 'Nanguoli' fruit was performed at three important stages,including calcium treatments and without calcium treatment.The total number of mapped reads were in the range of 16,661,134-18,800,372 with an average of 69.81%of all clean data for the six libraries could be mapped to the pear genome.A total of 10,776 differentially expressed genes between the poor aroma and intense aroma were categorized into 36 functional groups and 37 pathways according to GO and KEGG database,respectively.Genes involved in fatty acid pathway encoding FAD,ADH,PDC,LOX and lipase had higher expression in the intense aroma fhuit.Some transcription factors were identified as having high correlation with aroma biosynthesis in pear.We speeulate that ABA may play an important role in the process of calcium improving 'Nanguoli' aroma.In addition,candidate genes were verified by quantitative real-time PCR.We screened a number of candidate genes,and provide an important reference for further study of molecular mechanisms of pear fruit aroma regulation.On this basis,we also used aroma components,metabolic and expression of associated gene data spectrum analysis,screened 'Nanguoli'aroma synthesis and release eandidate genes,which provided important reference data for further study of molecular mechanisms of pear fruit aroma regulation.5.ADH is a key enzyme in the pear fruit aroma formation.This study used homologous cloning technology,cloned gene encoding Aldehyde dehydrogenase family 3 member I1(Pbr027959.1),named PuADH1.Using bioinformatics methods,I performed nucleotide.and amino acid sequences encoding the physicochemical properties,domain analysis and phylogenetic tree construction.PuADH1 has a complete open reading frame(ORF),with the size of 1683bp,encoding 561 amino acids.Pear PuADH1 gene and apple evolutionary relationship is close.The fusion expression vector of PuADH1 gene and green fluorescent protein was constructed and transformed into Arabidopsis thaliana by agrobacterip?m mediated transformation.PuADHl gene was localized in the cytoplasm.Four transgenic lines were obtained by construction of plant expression vector PRl101-PuDH1 for verification by transgenic tomato.I compared morphological phenotype of transgenic plants and wild-type tomato plants,but over expression of PuADH1 tomato plants had not obvious changing.Signiflcant change took place in the transgenic tomato leaves and fruits volatile aroma compounds,total contents of aroma compounds,alcohols and aldehydes content increased significantly. |
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