【作者】 成妮妮；

【导师】 张元湖；

【作者基本信息】 山东农业大学 ， 植物学， 2013， 博士

【摘要】 近年来，在分离和鉴定挥发性萜类合成酶基因及其表达调控和功能特性方面取得了很大的进展，萜类代谢工程已经引起了人们的广泛重视。但是直到现在只对少数几种酶进行了重点研究。α-法尼烯合酶是倍半萜α-法尼烯合成途径中的最终酶。到目前为止，对α-法尼烯及α-法尼烯合酶的研究主要集中于其表达调节与α-法尼烯和虎皮病发生的关系及机制探讨上，关于在植物中过表达α-法尼烯合酶是否影响其它萜类物质的产生及植物生物学特性的研究还未见报道。本研究以已经获得的过表达α-法尼烯合酶的转基因烟草（NC89）T3代纯合体为实验材料，对转基因与野生型植株不同的生长特性及其分子机理进行了研究。利用TAIL-PCR法克隆了该基因的启动子序列，并对其功能进行了初步分析。为研究倍半萜代谢工程对植物生长特性的影响及α-法尼烯合酶基因的表达调控因素提供实验依据。研究结果和主要结论如下：（1）α-AFS过表达影响了转基因烟草的生长特性和激素含量将转基因烟草T3代纯合体与野生型烟草同时播种，同样条件培养后发现，在生长期的前30天，二者无明显差异，但到45天以后，转基因植株生长速率逐渐快于野生型，到60天差异明显，主要表现为株高明显增高，茎节间距离加大，茎变细，叶片数增加，最后出现花蕾和开花时间早于野生型植株50多天。对相同温室条件下相同发育时期的烟草，选取4叶（无差异），8叶、12叶、14~15叶（刚出现花蕾）、16~17叶（开花）五个时期，分别利用ELISA法测其GAs、ABA和IAA含量，发现在发育中前期，转基因和野生型植株激素差异不显著，但到花蕾期和开花期，二者差异显著，前者GAs、ABA和IAA含量分别比后者高102.28%，109.50%，257.37%（花蕾期）和115.75%，223.53%和289.64%（开花期），说明α-AFS基因过表达改变了激素水平，进而影响了转基因植株的株高、开花时间等生长发育特性。（2）α-AFS过表达影响了转基因烟草MVA和MEP途径中关键酶及开花分子标记基因的表达对MVA途径的NtHMGR2和NtFPS及MEP途径的NtDXS和NtDXR主要关键酶基因进行qPCR定量分析表明，过表达α-AFS使NtHMGR2表达量从幼苗到开花期逐渐增高，而NtFPS的表达量是先升后降的趋势，在发育中期达到最高，直到最后又低于野生型。NtDXS表达量在4叶和8叶期高于野生型，后来低于野生型并一直呈下降趋势，NtDXR表达量在4叶期略高于野生型，其它时期都比野生型低。GAs是MEP途径的下游产物，对GAs合成途径相关酶基因表达进行qPCR分析表明，NtGA20ox1在叶片中表达随着生长发育逐渐上升，12叶期时，在三个转基因株系中的表达量已超过野生型植株，相对表达量是野生型的4.87倍，2.40倍和2.46倍，花蕾期和开花期虽有下降但也高于野生型植株。NtGA20ox2表达量在4叶、8叶和12叶期表达量比野生型总体略高，但是到花蕾期和开花期，总体表达量上升较大，尤其是T3-3和T3-5两个株系远高于野生型。NtGA2ox1、NtGA2ox2、NtGA2ox3和NtGA2ox5在幼苗期表达最高，此后呈下降趋势。利用半定量PCR对烟草中开花分子标记基因检测表明，转基因烟草中NtMADS4的表达时期明显早于野生型，NtMADS11表达在转基因植株T3-1、T3-5中表达早于野生型，而T3-3与野生型差异不大。（3）α-AFS过表达影响转基因烟草植株其它萜类、生物碱、类固醇等物质的合成利用GC-MS对固相微萃取顶空收集的烟草挥发物和正己烷提取的物质进行分析表明，转基因和野生型烟草均未检出α-法尼烯，但是转基因植株中检测到另一种倍半萜β-石竹烯的含量是野生型的6倍，转基因植株中还有少量的β-石竹烯氧化产物和穿心莲内酯，而野生型烟草植株未检出。在转基因植株中还发现一种新的二萜物质香紫苏醇。HPLC分析表明，转基因烟草植株中保留时间约10min处物质含量高于野生型，但具体是何种物质还有待于进一步分析。干叶中生物碱含量检测发现，转基因植株的生物碱含量高于野生型植株，而总类固醇含量低于野生型。（4）α-AFS过表达改变了转基因烟草的抗胁迫能力对转基因烟草进行离体自然衰老、MV、NaCl、45C胁迫处理发现，转基因植株叶圆片抗衰老能力高于野生型叶片，但对MV胁迫比野生型敏感。NaCl处理中，种子萌发差异不大，但在100mM NaCl处理中转基因烟草幼苗抗胁迫能力高于野生型幼苗，但到了浓度NaCl达150mM后，二者差异不明显。高温处理转基因植株的净光合速率受影响程度比野生型大，但二者Fv/Fm变化趋势比较相近。转基因植株的相对电导率和伤害度明显低于野生型植株。（5）克隆了α-AFS基因启动子并进行功能分析利用TAIL-PCR方法从青香蕉苹果中克隆了α-AFS基因启动子序列，在线分析表明，该序列具有许多明显的TATA框和CAAT框，同时还具有与激素相关的元件，在ATG下游分别有一个参与热胁迫反应的HSE元件和厌氧诱导的ARE元件等重要顺式作用元件。将启动子分段克隆与GUS报告基因构建融合表达载体转化烟草，转基因烟草GUS活性分析表明，该启动子能够使GUS基因表达，且表达活性因片段长度不同而有差异。利用GA、ABA、SA、MeJA、乙烯利（ethephon）和45C热激处理后对GUS酶活性进行检测表明GA、MeJA、乙烯利处理增强了启动子活性，而SA和ABA处理反之。热激处理对含有HSE的片段2-1较明显，但对其它片段不明显。更多还原

【Abstract】 In recent years, the isolation and identification of volatile terpenoids synthase genes andtheir expression regulation and function characteristics have made great progress, also,terpenoids metabolic engineering has attracted widespread attention. But until now, onlyseveral kinds of enzymes have been studied in detail.α-Farnesene synthase is the final enzyme in the sesquiterpere α-farnesene synthesispathway. Until now, studies on α-farnesene synthase mainly focus on the mechanism ofsuperficial scald production and its relationship with α-farnesene synthase gene expression.Overexpression of α-farnesene synthase in plants, which can influence the production of otherterpenes and plant biological properties, has not been reported.In this study, we studied the difference of growth characters between transgenic plantwhich overexpressed α-farnesene synthase gene from apple tree (Malus×domestica) with35Spromter and wild-type tobaccos, and their molecular mechanisms using homozygous T3generation of transgenic tobacco (NC89). We also isolated the promoter sequence ofα-farnesene synthase gene on genomic DNA from apple by TAIL-PCR method andpreliminarily analyzed its functions. The results will contribute to studies on the influence ofsesquiterpene metabolism engineering on the plant growth and the factors of α-farnesenesynthase gene regulation. The main results and conclusions in this thesis are presented asfollows:(1) α-AFS overexpression has affected the growth characteristics of transgenic tobaccoand hormone levelsT3generation homozygotes of transgenic and wild type tobacco were cultivated in sameconditions. There was no significant difference in morphology after the early30days betweentransgenic and wild-type plants. However, the transgenic plants growth fast after45days, andplant morphology such as plant height, stem internodes, leaf number between transgenic andwild type was significant in difference until60days later, and the flower buds emerging andflowering time of transgenic plant was50days earlier than that of wild type plants.We measured the GAs, ABA and IAA contents at the4leaves,8leaves,12leaves,14leaves and16leaves growth stages by ELISA, and found that in the early development, the hormone levels did not show significant difference in the transgenic and wild-type plants, butin the bud and flowering period, the GAs, ABA and IAA contents in transgenic plants were102.28%,109.50%,257.37%(buds), and115.75%,223.53%and289.64%(blooming) higherthan those in wild-type, which indicated that overexpression of α-AFS gene changed thehormone levels, and then influenced the plant height, flowering time characteristics oftransgenic plants.(2) α-AFS overexpression affected the expression levels of key enzymes in MVA and MEPpathways and the flowering-related genes in transgenic tobaccoWe estimated the transcription levels of the key enzymes NtHMGR2, NtFPS, NtDXS andNtDXR of MVA and MEP pathway by qPCR. The results showed that overexpression ofα-AFS improved the expression level of NtHMGR2gradually from seedling to floweringperiod, while the expression of NtFPS was lower at4and8leaves stages and higher at12leaves stage than wild-type, and then decreased until the flowering time. Transgenic plantsexhibited higher NtDXS expression level from4to8leaf stage, but lower level than the wildtype after8leaf stage, showing a downward trend. NtDXR expression level of transgenic plantwas slightly higher than that of wild-type only at4leaves stage, and was lower at other fourstages.GAs is the downstream product of MEP pathway. The expression levels of the enzymegenes related to the GAs synthesis were analysized by qPCR, the results showed that theNtGA20ox1expression level improved gradually with the development, and at the12leavesstage, the expression levels in all three transgenic plants were4.87,2.40and2.46times towild-type, respectively. Although the NtGA20ox1expression level decreased in budding andflowering period, it was still higher than that in wild-type plants. The expression levels ofNtGA20ox2increased markedly in transgenic plants at the bud and flowering period,especially the T3-3and T3-5strains. NtGA2ox1, NtGA2ox2, NtGA2ox3and NtGA2ox5showedhigher expression level in seedling stage, and then began to decline until flowering.The expression pattern of flowering-related genes in tobacco were detected bysemi-quantitative PCR, the results showed that the expression of NtMADS4in T3-1, T3-3andT3-5and NtMADS11in T3-1, T3-5were all significantly earlier than those in wild-type plants,but T3-3line didn’t show significant difference in expression level comparws to wild-typeplants.(3) α-AFS overexpression affected the synthesis of other terpenoids, alkaloid and sterols intransgenic tobacco.The extractives of transgenic and wild-type lines were analysed by GC-MS, and the results showed that no α-farnesene was detected in all lines, but in transgenic plants, the level ofβ-caryophyllene was increased6fold, trace caryophyllene oxide, andrographolide, and a newditerpene sclareol were also detected. HPLC analysis showed compounds levels were variousbetween transgenic and wild-type tobacco, and what kinds of those compounds are stillunknown.We also found that the alkaloid content of dry leaves was higher but the content of totalsterols was lower in the transgenic plant than the wild-type.(4) α-AFS overexpression improved the stress resistance of transgenic tobaccoUnder treatment with natural senescence, MV, NaCl,45C, compared to wild-type, thenatural senescence resistance ability of leaf discs from transgenic tobacco was higher, butmore sensitive to20μM MV stress, the seed germination rate was no visible difference underNaCl stress, but the seedlings of transgenic plants grew better on the MS mediumsupplemented with100mM NaCl, but there was no obvious difference between them with150mM NaCl stress. Under45C stress, the Pn decreased markedly in the transgenic plantscompared with25C, but the value was similar to the wild type, and had lower relativeconductivity and injury degree than wild-type.(5) The isolation and function analysis of the α-AFS gene promoterWe isolated the α-AFS gene promoter sequence from the ‘white winter pearmain’ apple（Malus x domestica）by TAIL-PCR method, and the online analysis showed that thesequence has many obvious TATA box and CAAT box, the element associated with hormone,and HSE element involved in thermal stress reaction, anaerobic induced element and otherimportant cis-acting elements.The promoter sequence fragment was inserted into the upstream of GUS reporter gene andintroduced into tobacco. GUS activity analysis showed that the promoter can induce GUSgene expression, and the expression activity was different in various length of promotersequence. We treated the transgenic tobacco, which was transformed with the whole promotersequence, with GA, ABA and SA, MeJA, ethephon (ethephon) and45C heat shock, and theresults showed that GA, MeJA, ethephon treatment enhanced the GUS activity, but the SAand ABA treatment exhibited reverse results. Heat shock treatment improved GUS activitywhen promoter sequence contained the HSE element (sequence2-1), but didn’t for otherseuquences.更多还原