【作者】 赵丽丽；

【导师】 齐红岩；

【作者基本信息】 沈阳农业大学 ， 蔬菜学， 2019， 博士

【摘要】 黄瓜(Cucumis sativus L.)果实风味清香,口感爽脆,是世界上栽培面积较大的蔬菜作物之一。但是,由于其根系浅,木栓化早,不耐连作的特点,在设施内连年种植易产生连作障碍,造成土传病害大量发生,严重影响黄瓜的产量和品质。嫁接是一项能够缓解土传病害的重要措施。研究发现,嫁接换根后,黄瓜植株抗病性增强,长势和产量提高,但是风味品质却受到影响。目前,嫁接影响黄瓜果实风味品质的机制尚不明确。因此,本研究以黑籽南瓜(传统的黄瓜嫁接专用砧木)和’威盛1号’南瓜(黄籽南瓜砧木)为砧木,分别嫁接于同一黄瓜接穗,比较嫁接后对黄瓜植株生长和果实风味品质的影响,并且利用转录组学测序技术筛选调控嫁接黄瓜果实风味品质的代谢路径和差异基因。综合果实风味测定结果和测序分析,筛选出一个与黄瓜果实香气合成相关的基因CsADH7-like,克隆该基因并进行生物信息学分析,以及对基因功能进行了初步研究。主要研究结果如下:1.分别以黑籽南瓜和’威盛1号’南瓜为砧木,’金地1号’黄瓜为接穗,以自嫁接黄瓜为对照,比较3个嫁接组合在不同生长发育时期的植株和果实生理生化特性。结果表明,两个砧木嫁接均提高了不同发育时期植株的株高和叶片数,以及盛果期植株的光合特性、各器官干物质的积累量、伤流量和对N、P、Ca元素的吸收量,黑籽南瓜砧木嫁接提高的幅度大,但是对K元素的吸收量却低于’威盛1号’南瓜嫁接组合。两个砧木嫁接均提高了黄瓜果实的纵径和单瓜质量,黑籽南瓜嫁接组合提高的幅度较大。2.利用气质联用仪(GC-MS)结合顶空固相微萃取技术和液相色谱技术(HPLC)分析两个不同砧木嫁接对不同生长时期黄瓜果实中的挥发性物质及糖的种类、含量的影响。’威盛1号’砧木嫁接提高了黄瓜果实中总酯类、醇类、醛类、烯类物质含量,而黑籽南瓜嫁接的黄瓜果实中这些物质含量均低于’威盛1号’砧木和对照。主成分分析得出,反-2-壬醛、反-2,顺6-壬二辛醛、环辛醇、反-2,顺6-壬二烯醇、丁香烯是两个嫁接组合中主要的挥发性物质,黄瓜果实中具有典型香气特征的反-2-壬醛和反-2,顺6-壬二辛醛的含量在’威盛1号’嫁接组合中含量均较高。’威盛1号’砧木嫁接提高了花后9 d黄瓜果实中可溶性固形物的含量,其中葡萄糖和果糖含量较高,蔗糖含量无差异,而黑籽南瓜砧木降低了果实中可溶性固形物的含量。3.以3个嫁接组合花后9 d黄瓜果实为试材,运用转录组高通量测序技术筛选调控果实香气物质和糖合成的代谢路径和基因。通过GO富集和KEGG功能分析,发现果糖代谢、糖酵解代谢和α-亚麻酸代谢三条代谢途径能够分别调控果糖、挥发性物质中的醛类物质、醇类物质合成。从这三条途径上随机选择20个差异表达基因,运用qRT-PCR的方法验证测序的可信度达到90%。对糖和香气合成相关的酶活性和基因表达分析发现,在黄瓜花后3d至15 d期间,’威盛1号’嫁接的黄瓜果实中乙醇脱氢酶(Alcohol dehydrogenases,ADH)、乙醛脱氢酶(Aldehyde dehydrogenase,ALDH)、果糖 1,6-二磷酸醛缩酶(Fructose 1,6-diphosphate aldolase,FDA)和磷酸果糖激酶(6-phosphofructokinase 1,PFK1)活性高于黑籽南瓜和对照,而黑籽南瓜嫁接提高了果实中丙酮酸酸激酶(Pyruvate kinase,PK)活性。并且,在α-亚麻酸代谢和果糖代谢途径中,Csa5G147470、Csa7G075600、Csa4G023020 和 CsalG701210基因的表达量显著高于其他基因,糖酵解代谢通路上的Csa5G580610、Csa3G827250、Csa5G517170和Csa6G000100基因均在花后6 d开始表达量逐渐升高。由此表明,不同砧木嫁接引起黄瓜果实风味品质的改变可能与这些代谢途径上基因的差异调控和酶活性有关。4.不同砧木嫁接对黄瓜果实中醇类和醛类物质影响较大,通过对测序数据分析发现在α-亚麻酸代谢通路上有一个基因,CsADH7-like(Csa6G361260,EC:1.1.1.1)在’威盛1号’嫁接组合中上调表达,并且黑籽南瓜和’威盛1号’中差异表达达到显著。将该基因与甜瓜ADHs基因家族成员进行序列比对后发现与CmADH10基因同源性最高。从自嫁接黄瓜中提取RNA、反转录,克隆基因全长,检测基因片段长度为1150 bp,利用多种生物信息学软件分析基因的生物学特性表明,CsDH7-like属于中链醇脱氢酶基因家族,具有384个氨基酸,疏水性蛋白,预测蛋白大小为45 KDa,预测其定位在细胞质中。5.将CsADH7-like基因连接至原核表达载体,诱导获得纯化的重组蛋白,大小为45 KDa,与预期结果一致。该蛋白的最适反应底物为乙醛和己醛,对支链醛和芳香醛亲和力较差,在pH=6.0和温度30℃条件下相对酶活性较高。利用瞬时超表达的方法将黄瓜CsADH7-like基因转入超表达载体中,通过注射侵染的方法将携带重组质粒的农杆菌转入黄瓜果实中,利用荧光素酶在植物活体成像系统中的表达检测侵染效果。结果表明,侵染后0.5 d开始出现表达,2.5 d侵染效果显著,3.5 d效果降低,而且在果实瓜蒂区域和果实中部区域的侵染效果优于近瓜柄区域。对侵染后的基因表达和酶活性分析发现,相对酶活性出现峰值时间比基因表达延迟0.5 d,但是两者的趋势是一致的,均高于对照且呈现的趋势为低-高-低。对样品进行测定分析发现,瞬时超表达CsADH7-like基因使醛类物质含量增加、醇类物质含量减少,两者均呈现低-高-低的状态,3d时达到峰值,这与CsADH7-like基因超表达后基因的相对表达和酶活性的结果吻合。亚细胞定位发现该蛋白定位在细胞膜和细胞核中。表明CsADH7-like蛋白具有将醇转化为醛的作用。6.利用GenBank数据库和PlantCare软件分析CsADH7-like基因启动子顺式作用元件,结果发现响应激素的元件有3个,包括脱落酸(Abscisic acid,ABA)应答(ABRE)和茉莉酸甲酯(Methyl jasmonate,MeJA)应答(TGACG-motif 和 CGTCA-motif)。根据启动子上存在的这两个激素应答元件,进行黄瓜果实圆片孵育试验。结果表明,在处理期间,两种激素处理均能够诱导CsADH7-like基因上调表达、LOX和ADH活性提高且在24 h出现峰值。对挥发性物质测定发现,醛类物质含量在24 h时显著增加并在36 h时达到峰值,在48 h时降到最低值达到与对照接近水平,醇类物质含量稍高于对照,但是一直呈现逐渐降低的状态。由此表明,CsADH7-like基因可能通过参与激素调控通路来响应脱落酸和茉莉酸甲酯的诱导。综上所述,以’金地1号’黄瓜作为接穗,黑籽南瓜砧木嫁接后显著促进植株生长、植株对营养元素的吸收、提高单果质量和果型指数,’威盛1号’南瓜砧木嫁接的促进作用较小。’威盛1号’南瓜砧木嫁接提高了黄瓜果实中挥发性香气物质和糖的含量,黑籽南瓜砧木嫁接则降低了这些风味物质的含量。将这些生理指标测定结果与转录组测序结果相结合进行分析,从黄瓜果实香气物质合成途径——α-亚麻酸代谢途径上筛选获得CsADH7-like基因,运用实时荧光定量、原核表达、瞬时超表达、激素处理的方法对基因功能进行初步验证,推测该基因在调控嫁接黄瓜果实特征性香气物质——醇类与醛类物质转化中可能起着重要作用,与不同砧木嫁接引起黄瓜果实风味品质上的差异存在关联。更多还原

【Abstract】 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 α-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 α-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 50μmol/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.更多还原