| Cucumber(Cucumis sativus L.)is one of the most cultivated vegetable crops in the world for the scent of its flavor and crispy taste.In recent years,the cultivation of protected cucumber has regional and professional characteristics.With continuous cultivating of sole cucumber plants,the diseases caused by soil-borne fungi and nematodes occur seriously.Grafting could alleviate soil-borne disease effectively in cucumber cultivation.It has been studied that the disease resistance,growth and yield of cucumber are probable to be improved after grafted.Sweetness and aroma are two important flavor qualities in cucumber fruit.Moreover,whether or not grafting could affect the two qualities items,we are ought to study its mechanisms in depth.Thus,figleaf gourd(Cucurbita ficifolia)is a traditional rootstock and specified for cucumber.Weisheng NO.1 pumpkin(Cucurbita moschata × Cucurbita moschata hybrids)is a rootstock processed yellow seed.In this study,a cucumber scion was grafted onto the two rootstocks respectively and self-grafted was used as the control to explore the differences on plant development and fruit flavor quality.In addition,screen for the different expressed genes and pathways which play important roles in regulating the formation of fruit flavor quality by the transcriptome sequencing technology.Based on all the results from fruit flavor and transcriptome sequence,a gene,CsADH7-like,related to aroma synthesis in cucumber fruit was screened and cloned.Bioinformatics analysis and the gene function study were conducted subsequently.The main results are as follows:1.Figleaf gourd and Weisheng No.1 pumpkin were used as rootstocks,Jindi No.1(Cucumis sativus var.)was used as the scion,and self-grafted cucumber was used as the control,comparing between the two grafted combinations of the physiological and biochemical characteristics of grafted plants and fruit at different growth stages.The result was showed that after grafting onto the two rootstocks,the plant heights were enhanced and number of leaves were increased in the seedling stage,the initial flowering stage,the initial stage of fruit setting and the fruit setting period,as well as,the photosynthetic characteristics,the dry matter content of different organs of the plants,the root bleeding sap and the absorption of N,P and Ca elements are elevated during the fruiting period.The improvements of fig leaf gourd were more dramatic than those of Weisheng No.1 grafted combination,but the content of K element was lower.2.The types and contents of volatile substances and sugars in cucumber fruits at different growth periods were analyzed by gas chromatography-mass spectrometry(GC-MS)binded with headspace solid phase micro-extraction(SPME)and and high performance liquid chromatography(HPLC).The contents of total esters,alcohols,aldehydes and alkenes were increased in cucumber fruit after grafting onto Weisheng No.1 rootstock,and were decreased in figleaf gourd.By the principal component analysis,we found that these volatile compounds,including(E)-2-Nonenal,(E,Z)-2,6-Nonadienal,Cyclooctane-l-ol,(2E,6E)-2,6-Nonadien-l-ol,Caryophyllene are the main volatile substances.(E)-2-Nonenal and(E,Z)-2,6-Nonadienal are two most typical aromas in cucumber fruit and their contents are detected higher in the grafting combination of Weisheng No.1,moreover,theses contents of glucose,fructose and soluble solids in the fruit of 9 d after flowering were also improved.All of these substances in figleaf gourd were lower than the control.No significant difference in sucrose content among the three grafted combinations.3.The cucumber fruits obtained from the three grafting combinations were sampled at 9 d after anthesis to use as test material,moreover,the transcriptome high-throughput sequencing technology was conducted to screen the metabolic pathways and differentially expressed genes related to the synthesis of aroma and sugar in cucumber fruit from the three grafted combinations.GO enrichment and KEGG functional analysis were indicated that fructose,aldehydes and alcohols in cucumber fruit were synthesized via the three pathways,fructose,glycolysis/gluconeogenesis and a-linolenic acid metabolic pathways.In order to vertify the sequencing accuracy,20 differentially expressed genes were selected at random from the three pathways and conducted by qRT-PCR.The result was indicated that the reliability of sequence was up to 90%,so we can carry out the follow-up test.Grafted cucumber fruits from 3th day to 15th day after anthesis,the enzymes activities of Alcohol dehydrogenases(ADH),Aldehyde dehydrogenase(ALDH),Fructose 1,6-diphosphate aldolase(FDA)and 6-phosphofructokinase 1(PFK1)in G2 were higher than G1 and SG.Pyruvate kinase(PK)activity was in G1 was the highest.Moreover,in ?-linolenic acid and fructose metabolisms,the four genes,Csa5G147470?Csa7G075600?Csa4G023020 and Csa1G701210,were possessed highly expressed,while the relative expressions of Csa5G580610,Csa3G827250,Csa5G517170 and Csa6G000100 in glycolysis/gluconeogenesis pathway rose gradually.Accordingly,the changes of fruit qualities in cucumber grafted onto different rootstocks were likely to correlate with the regulation of differentially expressed genes and activities of different enzymes.4.According to previous studies,alcohols and aldehydes in cucumber fruit were affected largely owing to different rootstocks.It was obtained that CsADH7-like gene(Csa6G361260,EC:1.1.1.1)screened from sequencing result in ?-linolenic acid pathway was up-expressed in G2 and significantly-between G1 and G2.ADH is a key enzyme in the transition of alcohols and aldehydes in the lipoxygenase(LOX)pathway of aroma synthesis.Members of the alcohol aldehydrogenases(ADHs)gene family members in melon fruit have been identified and their functions have been initially studied,while ADH genes study in cucumber fruit has not been reported yet.The CsADH7-like gene was differentially expressed in the sequencing result.In order to analyze its function,the gene was most homologous to the CmADH10 gene by aligning to melon ADHs gene family.RNA was extracted from cucumber fruit in self-grafted combination,along with reverse transcription,cloned and bioinformatically analyze the full-length sequence of CsADH7-like gene,the gene fragment of 1150 bp was detected.It was also indicated that CsADH7-like belongs to the medium chain alcohol dehydrogenase gene family with 384 Amino acids.It was a hydrophobic protein and predicted to be localized in the cytoplasm with a predicted protein size of 45 kDa.5.CsADH7-like gene was ligated into a prokaryotic expression vector,induced and obtained a purified recombinant protein with a size of 45 kDa,which was consistent with our expected result.The optimal substrate for this protein is acetaldehyde and hexanal.The affinity for branched aldehydes and aromatic aldehydes is poor.The relative enzyme activity is higher at pH=6.0 and temperature 30?.By transient overexpression method,CsADH7-like gene was transfected into the plant overexpression vector.The Agrobacterium carrying the recombinant plasmid was transferred into cucumber fruit by injection infestation,and the luciferase was used to test the infected effect in the plant living imaging system.The result was showed that the infection began to appear 0.5 d after infected,the effect was most significant at 2.5 d and weakest in 3.5 d.In addition,the infection effects of the pedicle and the middle part of the fruit were better than that of near the stalk.After infected,the relative ADH enzyme activity appeared peaked 0.5 d later than the gene relative expression level,but the trends were consistent and exhibited higher than the control with the level of low-high-low.These results were indicated that the CsADH7-like gene has a regulatory role LOX in aromatic synthesis pathway in cucumber,but the associations with its upstream and downstream genes remain to be further studied.6.By GenBank database and PlantCare software,cis-acting elements that relate to hormone response in CsADH7-like gene promoter were found,including abscisic acid(ABA)responsive(ABRE),Methyl jasmonate(MeJA)responsive(TGACG-motif and CGTCA-motif).According to the two cis-acting elements,9d mesocarp of cucumber fruit for sarcocarp incubation was sampled and the expression mode to different hormones treatments was studied.The base buffer was added into 50?mol/L ABA and MeJA solution,mixed uniformly and tested in 12 h,24 h,36 h and 48 h.The result was showed that the relative expression of CsADH7-like gene were up-regulated by the two hormones,LOX and ADH activities were up to the maxmum at 24 h,increased up-regulated the aldehydes content was increased significantly in 24 h and up to the maximum in 36 h,declined to the lowest value in 48 h.The alcohols content was higher than SG slightly but appeared a state of gradual decline.It was inferred that CsADH7-like gene was likely to involve in the regulation of hormones by responsing ABA and MeJA.In summary,with Jindi No.1 as scion and figleaf gourd as rootstock,the grafted combination was significantly promoted plant growth,plant uptake of nutrient elements,improved fruit quality and fruit type index,while Weisheng No.1 pumpkin as rootstock,it had a little facilitation.Morever,the contents of volatile aroma compounds and sugar in Cucumber fruits were increased in the grafted combination of Weisheng No.1 pumpkin and decreased in that of figleaf gourd.Combining the results of these physiological indicators with transcriptome sequencing,CsADH7-like gene was screened from the pathway of aroma synthesis in Cucumber fruits,a-linolenic acid metabolism.The gene function was preliminarily verified by real-time fluorescence quantitative analysis,prokaryotic expression,transient overexpression and hormone treatment.It was speculated that CsADH7-like gene was possible to play a prominant role in regulating the characteristic aroma compounds in grafted cucumber,including alcohols and aldehydes,which might be related to the differences in flavor quality of cucumber fruit caused by different rootstocks. |
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