五种园林植物与花香及胁迫防御相关的挥发性萜类物质的调控与合成
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摘要
萜类物质是植物中存在的种类最多、分布最广的一类次生代谢物质。其中，半萜、单萜、倍半萜、萜烯同系物及部分二萜物质具有挥发性，是植物与其它生物交流的媒介，对植物自身的繁殖和防御具有重要的生物学和生态学意义。本论文旨在系统全面的研究萜类物质在不同进化层次园林植物中的成分、调控因素、生物学效应以及生物合成，进而探究萜类物质在植物中的起源与进化。文中以代表性园林植物千日红、紫藤、莎草、毛果杨和江南卷柏为材料，主要对以下内容进行了研究：草本植物千日红和藤本植物紫藤的花香萜类挥发性物质成分、释放影响因素；生物与非生物胁迫对单子叶草本植物莎草及双子叶木本植物毛果杨中萜类物质合成的影响；以杨树为模式植物，通过功能与比较基因组学对与间接防御有关的倍半萜类合成酶的功能与进化的研究；生物与非生物胁迫对非种子植物江南卷柏中萜类物质合成的影响及三萜鲨烯合成酶的功能与进化的研究。主要研究结果如下：
（1）从千日红4个品种‘Fireworks’、‘Las Vegas White’、‘Las Vegas Pink’、‘Las Vegas Purple’的花香中分别鉴定出5、2、1和2种萜类挥发物质，占各自总挥发性物质释放量的63.3%、25.8%、19.3%和22.4%。‘Fireworks’花香中萜类物质种类最多，且释放量最大，其中单萜物质罗勒烯的释放量占总释放量的52.2%，为花中最主要的挥发性物质成分。此外，千日红花中萜类物质的释放表现出昼夜节律，最高量出现在下午13:00pm—17:00pm，最低量出现在凌晨21:00pm—1:00am。进一步研究表明，这种昼夜节律是由植物自身生物钟所控制的，不受光照影响。外施硫代硫酸银作为乙烯抑制剂在短期(4h)和长期(24h)都能够促进萜类物质的释放；茉莉酸和水杨酸两个防御信号分子短期处理(4h)能提高花香萜类物质的释放，长期处理(24h)则抑制萜类物质的释放。
（2）从中国紫藤中鉴定出6种萜类物质，包括3种单萜、1种倍半萜和2种萜烯同系物，总释放量为16.9μg·h~(-1)·g~(-1)。从日本紫藤中鉴定出10种萜类物质，包括3种单萜、6种倍半萜和1种萜烯同系物，总释放量为16.1μg·h~(-1)·g~(-1)。其中，单萜物质芳樟醇和罗勒烯在两个品种中，都为花中挥发性物质最主要成分。分别从中国紫藤的花瓣、萼片、雌雄蕊和花梗中鉴定出5、3、4和4种萜类物质，多数萜类物质的释放都存在组织特异性，其中花瓣是萜类物质释放的主要器官。中国紫藤花中萜类物质的释放表现出昼夜节律，最高量出现在白天9:00am—10:00am，最低量出现在凌晨1:00am—2:00am。进一步研究表明，萜类物质释放的昼夜节律是由光照控制的。外施硫代硫酸银对中国紫藤花香萜类物质的释放在短期(2h)和长期(24h)都没有明显的作用；茉莉酸长期处理(24h)能显著提高中国紫藤花香萜类物质的释放量。
（3）在未成熟期，碎米莎草叶、根中倍半萜总浓度在播种后70天达到最高，然后持续降低直到花期。而且倍半萜主要在新生和生长活力高的组织中积累，表现出发育阶段和组织的特异性。单萜物质只在播种90天后的叶和花中积累，其中花是单萜物质积累的主要组织。茉莉酸甲酯处理能显著提高碎米莎草叶中倍半萜的总量，并且能够特异性的诱导单萜芳樟醇和倍半萜橙花叔醇的合成。利用PEG8000模拟干旱胁迫，能够显著提高叶中萜类物质总浓度，而水杨酸处理和机械损伤对萜类物质的积累没有明显影响。甜菜夜蛾咬食后，能够显著提高碎米莎草叶中萜类物质总浓度，而且能够诱导合成单萜物质柠檬烯、β-罗勒烯、芳樟醇，和倍半萜物质大牻牛儿烯D、古芸烯。通过对处理后碎米莎草叶中倍半萜物质的浓度进行聚类分析，推测4个倍半萜合成酶参与了其倍半萜物质的合成。播种70天后的碎米莎草叶片中的挥发性物质能够有效抑制真菌禾谷镰刀菌的生长。
（4）茉莉酸甲酯、水杨酸、丙甲菌素的短期处理（2h）对毛果杨叶片中萜类物质的释放都没有影响，而其长期处理（24h）却均能显著提高叶中萜类物质的释放量。其中丙甲菌素处理24h能够诱导单萜罗勒烯，倍半萜α-香柠檬烯、橙花叔醇，萜烯同系物DMNT((E)-4,8-dimethyl-1,3,7-nonatriene)和TMTT的释放。柳圆叶甲侵食24h后能够显著提高萜类物质的释放，并能诱导单萜罗勒烯，倍半萜β-石竹烯、大牻牛儿烯D、α-葎草萜、橙花叔醇，萜烯同系物DMNT和TMTT的合成。通过与已知倍半萜合成酶序列的比对及系统发育树分析，筛选出4个毛果杨倍半萜合成酶基因，PtTPS1—PtTPS4。半定量PCR基因表达显示PtTPS1和PtTPS2在所有组织中都有表达，且在茎中表达量最大；PtTPS3只在幼叶中表达；PtTPS4在所有组织中都表达，且在幼叶中表达量最大。此外，PtTPS1的表达能够被水杨酸处理所诱导；柳圆叶甲侵食、丙甲菌素及水杨酸短期处理能够显著提高PtTPS2的表达量；PtTPS3的表达只能被柳圆叶甲侵食所诱导。对4个基因的全长序列进行克隆并测序后发现，其中PtTPS1为假基因，其它3个基因为功能基因。对PtTPS2、PtTPS3和PtTPS4进行原核表达及生化活性分析，发现3个基因表达蛋白均能催化底物FPP，产生倍半萜物质。其中PtTPS2催化的主要产物为β-法尼烯和榄香醇；PtTPS3的主要产物为β-石竹烯和α-葎草萜；PtTPS4的主要产物为榄香醇、β-桉叶醇和α-桉叶醇。此3个合成酶的产物包含了柳圆叶甲侵害后叶中释放的几乎所有主要的倍半萜物质，从而证明PtTPS2、PtTPS3和PtTPS4为毛果杨倍半萜合成酶，并且参与柳圆叶甲侵食后的间接防御。
（5）丙甲菌素、水杨酸、茉莉酸甲酯、脱落酸溶液、机械损伤和热激处理均能诱导江南卷柏释放唯一的萜类物质—鲨烯。其中，丙甲菌素处理后的鲨烯释放量最大。用拟南芥鲨烯合成酶为模版搜索江南卷柏基因组数据库，得到一个与鲨烯合成酶相似的基因，命名为江南卷柏鲨烯合成酶(SmSQS)。克隆SmSQS cDNA全长，在大肠杆菌中进行原核表达，发现SmSQS融合蛋白在NADPH和Mg~(2+)的存在下能够通过两步环化将底物FPP转化成鲨烯。半定量PCR基因表达显示，SmSQS在根中表达量高，在叶中表达量较低。丙甲菌素处理24h和热激处理4h后，叶中SmSQS的表达量显著提高；而水杨酸、茉莉酸甲酯、脱落酸溶液、机械损伤处理24h后的表达量也有所提高。SmSQS蛋白序列缺失了其他物种鲨烯合成酶在C段保守的30个氨基酸跨膜转移信号肽。对SmSQS基因进行亚细胞定位，发现其定位在细胞质中。对SmSQS基因上游-2000bp的启动子序列元件进行分析，发现其包含了多处重要的响应胁迫信号的顺式作用元件。将启动子-SmSQS在拟南芥中进行表达，通过抗生素筛选和PCR验证，筛选出12株阳性植株。转基因T1代植株鲨烯的释放量显著高于对照植株，且T1代幼苗对重金属镉胁迫具有明显的抗性。
（6）通过对五种园林植物中与花香合成及胁迫防御有关的萜类挥发性物质成分的对比发现：蕨类植物卷柏中合成和释放的萜类物质最少，只在胁迫条件下释放鲨烯。在种子植物中，莎草叶、杨树叶和紫藤花中释放的萜类物质种类相近，包含有6个相同的物质（1种单萜和5种倍半萜）。此外，萜类物质的合成和积累方式在不同物种间表现出多样性：一部分萜类物质（单萜罗勒烯、1,8-桉叶素，倍半萜石竹烯α-葎草萜、β-法尼烯、榄香醇）的合成在不同器官中是系统发生的，即在不同物种的叶和花中普遍存在；一部分萜类物质（花中的香叶基丙酮、丙酸叶醇酯、异戊酸香叶酯，叶中的γ-桉叶醇、β-桉叶醇、α-桉叶醇）的合成具有组织特异性，在不同组织中发挥特有的生物学与生态学作用；另一部分萜类物质（马兜铃烯、大牻牛儿烯D、橙花叔醇）的合成只能被生物胁迫（植食昆虫侵害和真菌侵染）所诱导。
Terpenoids constitute the largest class of secondary metabolites made by plants. Part ofthe terpenoids exhibit the volatile property, including semiterpenes, monoterpenes,sesquiterpenes and part of the diterpenes. Terpenoids play as the role of the interaction withother organisms in the environment, which is significant to the reproduction and defence forthe plants. Taking Gomphrena globosa, Wisteria, Cyperus Iria, Populus trichocarpa andSelaginella moellendorffii as the model, the purpose of this study is to investigate thecomposition, factors of regulation, biological function and biosynthesis of the terpenoids inthe landscape plants with different evolution level in taxonomy to elucidate the origin andevolution of the terpenoids in plants. The main contents in this dessertation contain thecomposition and regulation of the volatile terpenoids in the floral scent of the herbalG.globosa and the woody Wisteria, the effects of the biotic and abiotic stress to the emissionand biosynthesis of the terpenoids in monocot C.Iria and dicot P.trichocarpa, the effects ofthe biotic and abiotic stress to the emission and biosynthesis of the terpenoids in lycopodS.moellendorffii.The main results are as follows:
(1) Volatile chemicals emitted from the flowers of four G.globosa cultivars werecollected using a dynamic headspace technique and analyzed using gas chromatography–massspectrometry.5,2,1,2voaltile terpenoids were identified from ‘Fireworks’,‘Las VegasWhite’,‘Las Vegas Pink’ and ‘Las Vegas Purple’ respectively. The emssion rates ofterpenoid account for63.3%,25.8%,19.3%and22.4%in the total floral volatiles in thesefour cultivars respectively.‘FW’ showed the highest level of emission of terpenoids amongthe four cultivars, with the monoterpene β-ocimene as the dominant compound in the floralscent which accounts for52.2%of the total floral volatile compounds. The emission rate ofthe terpenoids in ‘FW’ showed a diurnal pattern with the maximum emission occurring at lateafternoon13:00pm-17:00pm and the lowest level of emission occurring at the night21:00pm-1:00am. Further analysis showed that this diurnal emission of G.globosa floral terpenoidsvolatiles is independent of light and regulated by circadian clock. The emission of floralvolatiles from ‘FW’ flowers that were treated with several chemicals was also analyzed. Thetreatment with silver thiosulphate (STS), an ethylene inhibitor, led to enhanced emission of total volatiles. In contrast, the treatments with salicylic acid (SA) and jasmonic acid (JA) ledto enhanced emission of total floral volatiles at4h but reduced emission at24h after thetreatment.
(2) Volatile chemicals emitted from the flowers of chinese wisteria (W.sinenesis) andjapanese wisteria (W.floribunda) were collected using a dynamic headspace technique andidentified using gas chromatography–mass spectrometry.6terpenoids compounds weredetected from chinese wisteria flowers, including3monoterpenes,1sesquiterpene and2homoterpenes with the total emission rate as16.9μg·h~(-1)·g~(-1), while10terpenoids compoundswere detected from japanese wisteria flowers, including3monoterpnes,6sesquiterpenes and1homoterpene with the total emission rate as16.1μg·h~(-1)·g~(-1). Two monoterpenes, β-ocimeneand linalool were the most abundant compounds emitted from both species. Chinese wisteriawas selected as a model for further study of volatile emission from different parts of flowers,emission dynamics, and regulation of terpenoid production.5,3,4,4terpenoids wereidentified from the petals, sepals, stamens coupled with pistils, and pedicels. Although somevolatiles were detected from all parts of chinese wisteria flowers including monoterpeneβ-ocimene, linalool, and homoterpene TMTT, other terpenoids showed tissue specificity.Petals showed the highest level of total emission rate of terpenoid. The emission of floralterpenoids displayed a diurnal pattern with the maximal emissions occurring at9:00am-10:00am and the lowest level of emission occurring at1:00am-2:00am. This rhythmic pattern wasdetermined to be light-dependent. Regulation of floral terpenoids emission by exogenouschemicals, including STS, SA and JA, also was analyzed. Generally, jasmonic acid promotedthe emission of floral volatiles. In contrast, neither silver thiosulphate nor salicylic acidshowed a significant effect on floral terpenoids emission.
(3) Composition of these terpenoids and their concentrations in leaves, flowers and rootsof C.Iria at different developmental stages through the whole lifetime, were determined.During the immature stage, the total concentration of sesquiterpenoids increased and reachedthe maximum level at70d and then decreased until the emergence of inflorescence.Monoterpenes started to accumulate from90d only in leaves. During the mature stage, thetotal concentration of sesquiterpenoids increased dramatically. It indicates that thesesquiterpenoids were mainly produced in the new-born and more dynamic tissues. Somesesquiterpenoids showed temporal and saptial specificity. The total amount of monoterpenes,mainly accumulated in flower, kept increaseing until130d, displaying temporal and saptialspecificity. The effect of several biotic or abiotic stress to the produce of terpenoids wasstudied. The concentration of sesquiterpenoids can be promoted significantly by methyljasmonate (MeJA) in leaves. In contrast, SA had no significant effect to accumulation of the total sesquiterpenoids in leaves. Polyethylene Glycol8000(PEG8000) used to imitate thestress of drought could promote the concentration of total sesquiterpenoids significantly inleave. Infestation of beet armyworm in leaves promoted the concentration of totalsesquiterpenoids significantly and could induce the producing of more variety ofmonoterpenes and sesquiterpenoids. However, no effect of neither the fall armyworminfestation nor physical wounding to the concentration of terpenoid in C. Iria leaves can bedectected. The volatile of C.iria leaves was found to possess the anti-fungal effect toFusarium graminearum.
(4) The threatment of MeJA, SA and alamethicin (Ala) showed signifiacant effect ofpromotion on the emission of terpenoids volatile from the leaves of P.trichocarpa in the longterm (24h). Moreover, the production of monoterpene (β-Ocimene), sesquiterpenes(α-Bergamotene, Nerolidol Z and E), homoterpenes (DMNT and TMTT) can be inducedspecificly by the treatment of Ala after24h. The infestation of the willow leaf beetle (WLB)larve could promote the emission rate of terpenoids from the leaves of poplar and couldinduce the producing of monoterpne (β-Ocimene), sesquiterpenes (β-Caryophyllene,Germacrene D, α-Humulene, Nerolidol Z and E) and homoterpenes (DMNT and TMTT)specificly. Four putative sesquiterpene synthase genes, PtTPS1-PtTPS4, were identified andcharacterized by the alignment and phylogenetic analysis of terpene synthase (TPS) candidategenes in P.trichocarpa. The semi-quantitatively expression analysis showed that PtTPS1andPtTPS2could express in all the tisssues with the highest expression in the stem; PtTPS3could only express in the young leaves; PtTPS4could express in all the tisssues with thehighest expression in the young leaves. PtTPS1only expressed in the leaves with SAtreatment after both2h and24h; PtTPS2expressed in the leaves of all the treatment exceptthe control after24h; PtTPS3only expressed in the leaves infested by WLB; PtTPS4couldexpress in the leaves after all the treatments.The full length of PtTPS1-4were cloned. Theresults of sequencing found that PtTPS1is a pseudogene, while other3genes are functionalbased on their protein sequence. PtTPS2-PtTPS4were expressed in E.coli and theirbiochemical activity assays were conducted. It displayed that all these three expressedrecombinant protein could catalyze to produce the sesquiterpene with FPP as the substrate.The main products of PtTPS2were β-Farnesene and Elemol; The main products of PtTPS3were β-Caryophyllene and α-Humulene; The main products of PtTPS4were Elemol,β-Eudesmol and α-Eudesmol.The products of sesquiterpene of PtTPS2, PtTPS3and PtTPS4included almost all the sesquiterpene products emitted from the leaves infested by WLB,suggesting they are the sesquiterpene synthase in P.trichocarpa involved in the indirectdefence after the infestation of WLB.
(5) After being treated with Ala, a fungal peptide antibiotic, S.moellendorffii plants areinduced to emit a triterpene—squalene as the only volatile terpenoid. The emission ofsqualene from S.moellendorffii also can be induced by a number of stress factors, includingheat, physical wounding, abscisic acid (ABA), SA and MeJA. To understand the biosynthesisof stress-induced squalene, a single squalene synthase (SQS) gene was identified from theS.moellendorffii genome. The encoded protein, SmSQS, shows high level of sequencesimilarity to SQSs from higher plants. Gene expression analysis verified that SmSQStranscript accumulation induced by various stress factors correlates well with squaleneemission under the same treatments. The stress induced SmSQS gene expression and a basallevel expression of SmSQS in untreated control plants suggests that SmSQS functions in bothprimary metabolism and specialized metabolism. The full-length cDNA of SmSQS wascloned and expressed in E. coli. Recombinant SmSQS was shown to convert FPP to squalene.Expression of green fluorescent protein (GFP)-SmSQS in onion epidermal cells demonstratedthat SmSQS is targeted to the cytoplasm rather than endoplasmic reticulum (ER) membranedue to the absence of the C-terminal hydrophobic transmembrane domain.The analysis ofSmSQS promoter regions revealed the presence of elements that are related to plant responseto stresses. The transgenic Arabidopsis expressed with the fusion of promoter-SmSQSshowed higher emission of squalene and improved resistance to the stress of Cd.
(6) The composition of the volatile terpenoids from five species studied in thisdessertation were compared systematically. The non-seed, S.moellendorffii, produces the leastvariety of volatile terpenoids, with the triterpene squalene as the unique isoprene compoundsafter stresses. The profile of the volatile terpenoids from the leaves of C.iria, P.trichocarpaand flowers of Wisteria showed similarity to some extent, sharing6terpnoids (1monoterpeneand5sesquiterpenes). There are diverse types of biosynthesis and accumulation of theterpenoid compounds in these five species. Some terpenoids were present in both the leavesand flowers, including1,8-Cineole,(E)-β-ocimene, β-Caryophyllene, α-Humulene,β-Farnesene and Elemol. Part of the volatile terpenoids showed tissue specifity, includingGeranyl acetone, Geranyl propionate and Geranyl isovalerate only in flowers, γ-eudesmol,β-Eudesmol and α-Eudesmol only in leaves. Others can only be emitted by the induction ofbiotic and abiotic stresses, including Germacrene D, Nerolidol,(-)-Aristolene and squalene.
（1）从千日红4个品种‘Fireworks’、‘Las Vegas White’、‘Las Vegas Pink’、‘Las Vegas Purple’的花香中分别鉴定出5、2、1和2种萜类挥发物质，占各自总挥发性物质释放量的63.3%、25.8%、19.3%和22.4%。‘Fireworks’花香中萜类物质种类最多，且释放量最大，其中单萜物质罗勒烯的释放量占总释放量的52.2%，为花中最主要的挥发性物质成分。此外，千日红花中萜类物质的释放表现出昼夜节律，最高量出现在下午13:00pm—17:00pm，最低量出现在凌晨21:00pm—1:00am。进一步研究表明，这种昼夜节律是由植物自身生物钟所控制的，不受光照影响。外施硫代硫酸银作为乙烯抑制剂在短期(4h)和长期(24h)都能够促进萜类物质的释放；茉莉酸和水杨酸两个防御信号分子短期处理(4h)能提高花香萜类物质的释放，长期处理(24h)则抑制萜类物质的释放。
（2）从中国紫藤中鉴定出6种萜类物质，包括3种单萜、1种倍半萜和2种萜烯同系物，总释放量为16.9μg·h~(-1)·g~(-1)。从日本紫藤中鉴定出10种萜类物质，包括3种单萜、6种倍半萜和1种萜烯同系物，总释放量为16.1μg·h~(-1)·g~(-1)。其中，单萜物质芳樟醇和罗勒烯在两个品种中，都为花中挥发性物质最主要成分。分别从中国紫藤的花瓣、萼片、雌雄蕊和花梗中鉴定出5、3、4和4种萜类物质，多数萜类物质的释放都存在组织特异性，其中花瓣是萜类物质释放的主要器官。中国紫藤花中萜类物质的释放表现出昼夜节律，最高量出现在白天9:00am—10:00am，最低量出现在凌晨1:00am—2:00am。进一步研究表明，萜类物质释放的昼夜节律是由光照控制的。外施硫代硫酸银对中国紫藤花香萜类物质的释放在短期(2h)和长期(24h)都没有明显的作用；茉莉酸长期处理(24h)能显著提高中国紫藤花香萜类物质的释放量。
（3）在未成熟期，碎米莎草叶、根中倍半萜总浓度在播种后70天达到最高，然后持续降低直到花期。而且倍半萜主要在新生和生长活力高的组织中积累，表现出发育阶段和组织的特异性。单萜物质只在播种90天后的叶和花中积累，其中花是单萜物质积累的主要组织。茉莉酸甲酯处理能显著提高碎米莎草叶中倍半萜的总量，并且能够特异性的诱导单萜芳樟醇和倍半萜橙花叔醇的合成。利用PEG8000模拟干旱胁迫，能够显著提高叶中萜类物质总浓度，而水杨酸处理和机械损伤对萜类物质的积累没有明显影响。甜菜夜蛾咬食后，能够显著提高碎米莎草叶中萜类物质总浓度，而且能够诱导合成单萜物质柠檬烯、β-罗勒烯、芳樟醇，和倍半萜物质大牻牛儿烯D、古芸烯。通过对处理后碎米莎草叶中倍半萜物质的浓度进行聚类分析，推测4个倍半萜合成酶参与了其倍半萜物质的合成。播种70天后的碎米莎草叶片中的挥发性物质能够有效抑制真菌禾谷镰刀菌的生长。
（4）茉莉酸甲酯、水杨酸、丙甲菌素的短期处理（2h）对毛果杨叶片中萜类物质的释放都没有影响，而其长期处理（24h）却均能显著提高叶中萜类物质的释放量。其中丙甲菌素处理24h能够诱导单萜罗勒烯，倍半萜α-香柠檬烯、橙花叔醇，萜烯同系物DMNT((E)-4,8-dimethyl-1,3,7-nonatriene)和TMTT的释放。柳圆叶甲侵食24h后能够显著提高萜类物质的释放，并能诱导单萜罗勒烯，倍半萜β-石竹烯、大牻牛儿烯D、α-葎草萜、橙花叔醇，萜烯同系物DMNT和TMTT的合成。通过与已知倍半萜合成酶序列的比对及系统发育树分析，筛选出4个毛果杨倍半萜合成酶基因，PtTPS1—PtTPS4。半定量PCR基因表达显示PtTPS1和PtTPS2在所有组织中都有表达，且在茎中表达量最大；PtTPS3只在幼叶中表达；PtTPS4在所有组织中都表达，且在幼叶中表达量最大。此外，PtTPS1的表达能够被水杨酸处理所诱导；柳圆叶甲侵食、丙甲菌素及水杨酸短期处理能够显著提高PtTPS2的表达量；PtTPS3的表达只能被柳圆叶甲侵食所诱导。对4个基因的全长序列进行克隆并测序后发现，其中PtTPS1为假基因，其它3个基因为功能基因。对PtTPS2、PtTPS3和PtTPS4进行原核表达及生化活性分析，发现3个基因表达蛋白均能催化底物FPP，产生倍半萜物质。其中PtTPS2催化的主要产物为β-法尼烯和榄香醇；PtTPS3的主要产物为β-石竹烯和α-葎草萜；PtTPS4的主要产物为榄香醇、β-桉叶醇和α-桉叶醇。此3个合成酶的产物包含了柳圆叶甲侵害后叶中释放的几乎所有主要的倍半萜物质，从而证明PtTPS2、PtTPS3和PtTPS4为毛果杨倍半萜合成酶，并且参与柳圆叶甲侵食后的间接防御。
（5）丙甲菌素、水杨酸、茉莉酸甲酯、脱落酸溶液、机械损伤和热激处理均能诱导江南卷柏释放唯一的萜类物质—鲨烯。其中，丙甲菌素处理后的鲨烯释放量最大。用拟南芥鲨烯合成酶为模版搜索江南卷柏基因组数据库，得到一个与鲨烯合成酶相似的基因，命名为江南卷柏鲨烯合成酶(SmSQS)。克隆SmSQS cDNA全长，在大肠杆菌中进行原核表达，发现SmSQS融合蛋白在NADPH和Mg~(2+)的存在下能够通过两步环化将底物FPP转化成鲨烯。半定量PCR基因表达显示，SmSQS在根中表达量高，在叶中表达量较低。丙甲菌素处理24h和热激处理4h后，叶中SmSQS的表达量显著提高；而水杨酸、茉莉酸甲酯、脱落酸溶液、机械损伤处理24h后的表达量也有所提高。SmSQS蛋白序列缺失了其他物种鲨烯合成酶在C段保守的30个氨基酸跨膜转移信号肽。对SmSQS基因进行亚细胞定位，发现其定位在细胞质中。对SmSQS基因上游-2000bp的启动子序列元件进行分析，发现其包含了多处重要的响应胁迫信号的顺式作用元件。将启动子-SmSQS在拟南芥中进行表达，通过抗生素筛选和PCR验证，筛选出12株阳性植株。转基因T1代植株鲨烯的释放量显著高于对照植株，且T1代幼苗对重金属镉胁迫具有明显的抗性。
（6）通过对五种园林植物中与花香合成及胁迫防御有关的萜类挥发性物质成分的对比发现：蕨类植物卷柏中合成和释放的萜类物质最少，只在胁迫条件下释放鲨烯。在种子植物中，莎草叶、杨树叶和紫藤花中释放的萜类物质种类相近，包含有6个相同的物质（1种单萜和5种倍半萜）。此外，萜类物质的合成和积累方式在不同物种间表现出多样性：一部分萜类物质（单萜罗勒烯、1,8-桉叶素，倍半萜石竹烯α-葎草萜、β-法尼烯、榄香醇）的合成在不同器官中是系统发生的，即在不同物种的叶和花中普遍存在；一部分萜类物质（花中的香叶基丙酮、丙酸叶醇酯、异戊酸香叶酯，叶中的γ-桉叶醇、β-桉叶醇、α-桉叶醇）的合成具有组织特异性，在不同组织中发挥特有的生物学与生态学作用；另一部分萜类物质（马兜铃烯、大牻牛儿烯D、橙花叔醇）的合成只能被生物胁迫（植食昆虫侵害和真菌侵染）所诱导。
Terpenoids constitute the largest class of secondary metabolites made by plants. Part ofthe terpenoids exhibit the volatile property, including semiterpenes, monoterpenes,sesquiterpenes and part of the diterpenes. Terpenoids play as the role of the interaction withother organisms in the environment, which is significant to the reproduction and defence forthe plants. Taking Gomphrena globosa, Wisteria, Cyperus Iria, Populus trichocarpa andSelaginella moellendorffii as the model, the purpose of this study is to investigate thecomposition, factors of regulation, biological function and biosynthesis of the terpenoids inthe landscape plants with different evolution level in taxonomy to elucidate the origin andevolution of the terpenoids in plants. The main contents in this dessertation contain thecomposition and regulation of the volatile terpenoids in the floral scent of the herbalG.globosa and the woody Wisteria, the effects of the biotic and abiotic stress to the emissionand biosynthesis of the terpenoids in monocot C.Iria and dicot P.trichocarpa, the effects ofthe biotic and abiotic stress to the emission and biosynthesis of the terpenoids in lycopodS.moellendorffii.The main results are as follows:
(1) Volatile chemicals emitted from the flowers of four G.globosa cultivars werecollected using a dynamic headspace technique and analyzed using gas chromatography–massspectrometry.5,2,1,2voaltile terpenoids were identified from ‘Fireworks’,‘Las VegasWhite’,‘Las Vegas Pink’ and ‘Las Vegas Purple’ respectively. The emssion rates ofterpenoid account for63.3%,25.8%,19.3%and22.4%in the total floral volatiles in thesefour cultivars respectively.‘FW’ showed the highest level of emission of terpenoids amongthe four cultivars, with the monoterpene β-ocimene as the dominant compound in the floralscent which accounts for52.2%of the total floral volatile compounds. The emission rate ofthe terpenoids in ‘FW’ showed a diurnal pattern with the maximum emission occurring at lateafternoon13:00pm-17:00pm and the lowest level of emission occurring at the night21:00pm-1:00am. Further analysis showed that this diurnal emission of G.globosa floral terpenoidsvolatiles is independent of light and regulated by circadian clock. The emission of floralvolatiles from ‘FW’ flowers that were treated with several chemicals was also analyzed. Thetreatment with silver thiosulphate (STS), an ethylene inhibitor, led to enhanced emission of total volatiles. In contrast, the treatments with salicylic acid (SA) and jasmonic acid (JA) ledto enhanced emission of total floral volatiles at4h but reduced emission at24h after thetreatment.
(2) Volatile chemicals emitted from the flowers of chinese wisteria (W.sinenesis) andjapanese wisteria (W.floribunda) were collected using a dynamic headspace technique andidentified using gas chromatography–mass spectrometry.6terpenoids compounds weredetected from chinese wisteria flowers, including3monoterpenes,1sesquiterpene and2homoterpenes with the total emission rate as16.9μg·h~(-1)·g~(-1), while10terpenoids compoundswere detected from japanese wisteria flowers, including3monoterpnes,6sesquiterpenes and1homoterpene with the total emission rate as16.1μg·h~(-1)·g~(-1). Two monoterpenes, β-ocimeneand linalool were the most abundant compounds emitted from both species. Chinese wisteriawas selected as a model for further study of volatile emission from different parts of flowers,emission dynamics, and regulation of terpenoid production.5,3,4,4terpenoids wereidentified from the petals, sepals, stamens coupled with pistils, and pedicels. Although somevolatiles were detected from all parts of chinese wisteria flowers including monoterpeneβ-ocimene, linalool, and homoterpene TMTT, other terpenoids showed tissue specificity.Petals showed the highest level of total emission rate of terpenoid. The emission of floralterpenoids displayed a diurnal pattern with the maximal emissions occurring at9:00am-10:00am and the lowest level of emission occurring at1:00am-2:00am. This rhythmic pattern wasdetermined to be light-dependent. Regulation of floral terpenoids emission by exogenouschemicals, including STS, SA and JA, also was analyzed. Generally, jasmonic acid promotedthe emission of floral volatiles. In contrast, neither silver thiosulphate nor salicylic acidshowed a significant effect on floral terpenoids emission.
(3) Composition of these terpenoids and their concentrations in leaves, flowers and rootsof C.Iria at different developmental stages through the whole lifetime, were determined.During the immature stage, the total concentration of sesquiterpenoids increased and reachedthe maximum level at70d and then decreased until the emergence of inflorescence.Monoterpenes started to accumulate from90d only in leaves. During the mature stage, thetotal concentration of sesquiterpenoids increased dramatically. It indicates that thesesquiterpenoids were mainly produced in the new-born and more dynamic tissues. Somesesquiterpenoids showed temporal and saptial specificity. The total amount of monoterpenes,mainly accumulated in flower, kept increaseing until130d, displaying temporal and saptialspecificity. The effect of several biotic or abiotic stress to the produce of terpenoids wasstudied. The concentration of sesquiterpenoids can be promoted significantly by methyljasmonate (MeJA) in leaves. In contrast, SA had no significant effect to accumulation of the total sesquiterpenoids in leaves. Polyethylene Glycol8000(PEG8000) used to imitate thestress of drought could promote the concentration of total sesquiterpenoids significantly inleave. Infestation of beet armyworm in leaves promoted the concentration of totalsesquiterpenoids significantly and could induce the producing of more variety ofmonoterpenes and sesquiterpenoids. However, no effect of neither the fall armyworminfestation nor physical wounding to the concentration of terpenoid in C. Iria leaves can bedectected. The volatile of C.iria leaves was found to possess the anti-fungal effect toFusarium graminearum.
(4) The threatment of MeJA, SA and alamethicin (Ala) showed signifiacant effect ofpromotion on the emission of terpenoids volatile from the leaves of P.trichocarpa in the longterm (24h). Moreover, the production of monoterpene (β-Ocimene), sesquiterpenes(α-Bergamotene, Nerolidol Z and E), homoterpenes (DMNT and TMTT) can be inducedspecificly by the treatment of Ala after24h. The infestation of the willow leaf beetle (WLB)larve could promote the emission rate of terpenoids from the leaves of poplar and couldinduce the producing of monoterpne (β-Ocimene), sesquiterpenes (β-Caryophyllene,Germacrene D, α-Humulene, Nerolidol Z and E) and homoterpenes (DMNT and TMTT)specificly. Four putative sesquiterpene synthase genes, PtTPS1-PtTPS4, were identified andcharacterized by the alignment and phylogenetic analysis of terpene synthase (TPS) candidategenes in P.trichocarpa. The semi-quantitatively expression analysis showed that PtTPS1andPtTPS2could express in all the tisssues with the highest expression in the stem; PtTPS3could only express in the young leaves; PtTPS4could express in all the tisssues with thehighest expression in the young leaves. PtTPS1only expressed in the leaves with SAtreatment after both2h and24h; PtTPS2expressed in the leaves of all the treatment exceptthe control after24h; PtTPS3only expressed in the leaves infested by WLB; PtTPS4couldexpress in the leaves after all the treatments.The full length of PtTPS1-4were cloned. Theresults of sequencing found that PtTPS1is a pseudogene, while other3genes are functionalbased on their protein sequence. PtTPS2-PtTPS4were expressed in E.coli and theirbiochemical activity assays were conducted. It displayed that all these three expressedrecombinant protein could catalyze to produce the sesquiterpene with FPP as the substrate.The main products of PtTPS2were β-Farnesene and Elemol; The main products of PtTPS3were β-Caryophyllene and α-Humulene; The main products of PtTPS4were Elemol,β-Eudesmol and α-Eudesmol.The products of sesquiterpene of PtTPS2, PtTPS3and PtTPS4included almost all the sesquiterpene products emitted from the leaves infested by WLB,suggesting they are the sesquiterpene synthase in P.trichocarpa involved in the indirectdefence after the infestation of WLB.
(5) After being treated with Ala, a fungal peptide antibiotic, S.moellendorffii plants areinduced to emit a triterpene—squalene as the only volatile terpenoid. The emission ofsqualene from S.moellendorffii also can be induced by a number of stress factors, includingheat, physical wounding, abscisic acid (ABA), SA and MeJA. To understand the biosynthesisof stress-induced squalene, a single squalene synthase (SQS) gene was identified from theS.moellendorffii genome. The encoded protein, SmSQS, shows high level of sequencesimilarity to SQSs from higher plants. Gene expression analysis verified that SmSQStranscript accumulation induced by various stress factors correlates well with squaleneemission under the same treatments. The stress induced SmSQS gene expression and a basallevel expression of SmSQS in untreated control plants suggests that SmSQS functions in bothprimary metabolism and specialized metabolism. The full-length cDNA of SmSQS wascloned and expressed in E. coli. Recombinant SmSQS was shown to convert FPP to squalene.Expression of green fluorescent protein (GFP)-SmSQS in onion epidermal cells demonstratedthat SmSQS is targeted to the cytoplasm rather than endoplasmic reticulum (ER) membranedue to the absence of the C-terminal hydrophobic transmembrane domain.The analysis ofSmSQS promoter regions revealed the presence of elements that are related to plant responseto stresses. The transgenic Arabidopsis expressed with the fusion of promoter-SmSQSshowed higher emission of squalene and improved resistance to the stress of Cd.
(6) The composition of the volatile terpenoids from five species studied in thisdessertation were compared systematically. The non-seed, S.moellendorffii, produces the leastvariety of volatile terpenoids, with the triterpene squalene as the unique isoprene compoundsafter stresses. The profile of the volatile terpenoids from the leaves of C.iria, P.trichocarpaand flowers of Wisteria showed similarity to some extent, sharing6terpnoids (1monoterpeneand5sesquiterpenes). There are diverse types of biosynthesis and accumulation of theterpenoid compounds in these five species. Some terpenoids were present in both the leavesand flowers, including1,8-Cineole,(E)-β-ocimene, β-Caryophyllene, α-Humulene,β-Farnesene and Elemol. Part of the volatile terpenoids showed tissue specifity, includingGeranyl acetone, Geranyl propionate and Geranyl isovalerate only in flowers, γ-eudesmol,β-Eudesmol and α-Eudesmol only in leaves. Others can only be emitted by the induction ofbiotic and abiotic stresses, including Germacrene D, Nerolidol,(-)-Aristolene and squalene.
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