【作者】 刘佳；

【导师】 邵建柱；

【作者基本信息】 河北农业大学 ， 果树学， 2020， 硕士

【摘要】 植物花香除了能提升植物的观赏价值,还在吸引昆虫传粉、防御天敌,以及香精香料生产与医疗保健上发挥着重要作用。在我国,一直有“海棠无香”的说法,大多数海棠品种香气清淡,关于其花香的研究更鲜有报道,尚未引起人们的重视。本试验以浓香型八棱海棠实生后代(称为香海棠)为试材,以淡香型八棱海棠实生后代(称为B-1海棠)及八棱海棠为对照,对不同开花阶段及香海棠主要释香时期花器官不同部位的挥发性物质进行分析,并对其小气球期、开放初期、开放后期花朵进行转录组测序,旨在探究香海棠的主要释香时期及特征香气物质,揭示香海棠形成浓郁香气的分子机理,为海棠花香改良及新品种培育奠定基础。主要研究结果如下:1.采用了顶空固相微萃取(HS-SPME)与气相色谱质谱联用技术(GC-MS),测定了香海棠、B-1海棠及八棱海棠四个开花阶段(小气球期、大气球期、开放初期、开放后期)花朵中挥发性物质的种类及含量,并计算香气值。结果表明:开放初期为3种海棠主要释香时期,其中香海棠在该时期的挥发性物质含量最高,为5293.68ng/g,分别是同时期B-1海棠香气的2.7倍,八棱海棠的3.0倍;在主要释香时期,香海棠中的苯甲酸甲酯、芳樟醇、乙酸叶醇酯、邻氨基苯甲酸甲酯共4种化合物的香气值大于1,为香海棠的主要香气成分物质。2.采用HS-SPME结合GC-MS技术,对香海棠开放初期花朵的花瓣、雄蕊、雌蕊、花萼+花托的挥发性物质进行测定及分析,共检测出26种化合物,其中苯丙烷/苯环型化合物的相对含量分别为77.07%,80.59%,96.01%和76.58%,为主要的挥发性化合物类型。苯甲醇、邻氨基苯甲酸甲酯、苯甲酸甲酯3种物质在各部位均有检测到,且苯甲醇、邻氨基苯甲酸甲酯相对含量位于各部位含量中的前两位。雌蕊中邻氨基苯甲酸甲酯及苯甲酸甲酯的相对含量最高,分别为26.80%和6.01%;雄蕊中芳樟醇相对含量最高为4.58%;而乙酸叶醇酯在花瓣中含量最高为11.84%。3.利用RNA-Seq技术测定并分析了 3种海棠花朵在开放过程中(小气球期、开放初期、开放后期)转录组变化。27个转录组共得到209.51G的clean bases。其中,香海棠与B-1海棠相比,共有10324个基因表达水平发生了显著变化,在小气球期、开放初期、开放后期分别有2456、2016、1096个基因上调表达,1907、1797、1052个基因下调表达;香海棠与八棱海棠相比,共有36047个基因表达水平发生了显著变化,在小气球期、开放初期、开放后期分别有7367、6135和5653个基因上调表达,5850、5548和5493个基因下调表达;对差异基因进行GO注释,其中在各时期注释的差异基因数量从大到小均为MF>CC>BP,最终显著富集到核糖体、核糖核酸蛋白复合体、启动子的选择因子活性、丝氨酸类羧肽酶活性、DNA整合等24个GO功能注释上;对差异基因进行KEGG注释,差异基因被注释到1 13条代谢通路中,其中显著富集到缬氨酸、亮氨酸和异亮氨酸的降解(Valine,leucine and isoleucine degradation),核糖体(Ribosome)植物昼夜节律(Circadian rhythm-plant),β-丙氨酸代谢(beta-Alanine metabolism),硫代谢(Sulfur metabolism),光合作用(Photosynthesis),抗坏血酸和醛酸代谢(Ascorbate and aldarate metabolism)7 条代谢通路上。4.在所有差异基因富集到的代谢通路中,与乙酸叶醇酯合成相关的通路为α-亚麻酸代谢(alpha-Linolenic acidmetabolism),涉及调控磷脂酶、脂氧合酶、氢过氧化物裂解酶、乙醇脱氢酶等酶合成的14个相关的差异基因上下调表达;与苯甲酸甲酯合成相关的通路为苯丙烷代谢(Phenylalanine metabolism),涉及调控天冬氨酸转氨酶、酪氨酸转氨酶等酶合成的7个相关的差异基因上下调表达;与邻氨基苯甲酸甲酯合成相关的通路为苯丙氨酸、酪氨酸和色氨酸的生物合成(Phenylalanine,tyrosine and tryptophan biosynthesis),氨基酸的生物合成(Biosynthesis of amino acids),涉及调控莽草酸激酶、邻氨基苯甲酸合成酶等酶合成的7个相关的差异基因上下调表达;与芳樟醇合成相关的通路为萜类骨干生物合成(Terpenoid backbone biosynthesis),涉及1-脱氧-D-木酮糖-5-磷酸还原异构酶、2-甲基-D-赤藓糖-4-磷酸胱氨酰转移酶等酶合成的12个相关的差异基因上下调表达。5.将四种挥发性物质的含量及变化趋势与代谢通路中差异基因的上下调情况进行关联分析,选定脂氧合酶相关基因HF16930、HF18003、HF10743,乙醇脱氢酶基因HF31864;双功能天冬氨酸转氨酶与谷氨酸/天冬氨酸-预苯酸转氨酶相关基因HF35423,天冬氨酸转氨酶相关基因HF32183;邻氨基苯甲酸合成酶相关的基因HF43372;1-脱氧-D-木酮糖-5-磷酸还原异构酶基因HF41298为香海棠形成浓郁香气的候选基因。更多还原

【Abstract】 Flower fragrance not only improves the ornamental value of plants,but also plays an important role in attracting insects to pollinating,defending natural enemies,as well as the production of flavors and health care.The original varieties of crabapple in China are light in aroma,just like an old saying that crabapple has no fragrance since ancient times.There are little researches on crabapple yet.The volatiles of different flowering stages(small ballon stage,balloon stage,early flowering stage and late flowering stage)and different parts of flower(calyx and receptacle,petal,stamen and pistil)in the main aroma releasing stage were studied in this experiment.The purpose of this study is to determine the main volatiles releasing period and characteristic aroma substances of the crabapple.Then the transcriptome of the three crabapples were also studied to reveal the molecular mechanism of the formation of the strong aroma and to lay a foundation for the improvement of flower fragrance and the cultivation of new varieties.The results showed as follows:1.Headspace solid phase microextraction(HS-SPME)combined with gas chromatography-mass spectrometry(GC-MS)were used to analyze the types and contents of volatiles in the flowers of Xianghaitang,B-1 and Baleng crabapples in four flowering stages(small balloon stage,balloon stage,early flowering stage and late flowering stage),then the aroma values were calculated.The results showed that:the early flowering stage was the main aroma releasing stage among the three crabapples,in which the volatile content of Xianghaitang was 1.7 times higher than B-1 crabapple and 2.0 times higher than Baleng crabapple,reaching 5293.68ng/g;in the main aroma releasing period,the aroma values of methyl benzoate,linalool,(Z)-3-Hexen-l-ol acetate,and methyl anthranilate were higher than 1,which meaned they were the main aroma components of Xianghaitang.2.Using HS-SPME and GC-MS,the volatiles of petal,stamens pistil,calyx and receptacle of flowers in the early flowering stage of Xianghaitang were analyzed.26 compounds were detected,among which the main volatile compounds were phenylpropanoid/benzenoid,accounting for 77.07%,80.59%,96.01%and 76.58%,respectively.Benzyl alcohol,methyl anthranilate and methyl benzoate were detected in all parts,and the relative contents of benzyl alcohol and methyl anthranilate were in the first two places.Among them,the relative contents of methyl anthranilate and methyl benzoate were the highest in pistil,accounting for 26.80%and 6.01%respectively;the relative content of linalool was the highest in stamen,accounting for 4.58%;while the content of(Z)-3-Hexen-1-ol acetate was the highest in petal,accounting for 11.84%.3.RNA-Seq technique was used to detect and analyze the transcriptome changes of three kinds of crabapple flowers in the opening process(small ballon stage,early flowering stage and late flowering stage).A total of 209.51G clean bases were obtained from 27 transcriptomes.Compared to B-1 crabapple,there were 10324 genes differently expressing in Xianghaitang.Among them,2456,2016 and 1096 genes were up-regulated and 1907,1797 and 1052 genes were down regulated in small ballon stage,early flowering stage and late flowering stage respectively;Compared to Baleng crabapple,there were 36047 genes differently expressing in Xianghaitang.Among them,7367,6135 and 5653 genes were up-regulated and 5850,5548 and 5493 genes were down regulated in three flowering stages respectively;GO annotation was carried out for the differential genes,among which the number of differential genes annotated in each period was MF>CC>BP,and finally significantly enriched to 24 GO terms,such as ribosome,ribonucleoprotein complex,promoter selection factor activity,serine-type carboxypeptidase activity,DNA integration and so on.In KEGG pathways annotation,genes were annotated into 113 pathways,among which Valine,leucine and isoleucine,Ribosome,Circadian rhythm-plant,beta-Alanine metabolism,Sulfur metabolism,Photosynthesis,and Ascorbate and aldarate metabolism 7 pathways were enriched significantly.4.Among all the metabolic pathways enriched by different genes,the alpha-Linolenic acid metabolism was related to the synthesis of 3-hexen-l-ol acetate,involving 14 different genes about phospholipase,lipoxygenase,hydroperoxide lyase,alcohol dehydrogenase and so on;Phenylalanine metabolism was related to the synthesis of methyl benzoate,involving 7 different genes about aspartate aminotransferase,tyrosine aminotransferase and so on;Phenylalanine,tyrosine and tryptophan biosynthesis and biosynthesis of amino acids were related to the synthesis of methylanthranilate,involving 7 different genes about shikimate kinase,anthranilate synthase and so on;Terpenoid backbone biosynthesis was related to the synthesis of linalool,including 14 different genes about 1-deoxy-D-xylulose-5-phosphate reductoisomerase,2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase and so on.5.According to the four volatiles and the related genes in the metabolic pathways.,the genes HF16930,HF18003,HF10743 related to lipoxygenase,HF31864 related to alcohol dehydrogenase;HF35423 related to bifunctional aspartate aminotransferase and glutamate/aspartate-prephenate aminotransferase,HF32183 related to aspartate aminotransferase;HF43372 related to anthranilate synthase;HF41298 related to 1-deoxy-D-xylulose-5-phosphate reductoisomerase were selected as the candidate genes for the reasons why there is special aroma in Xianghaitang.更多还原