尾巨桉挥发油含量测定及HMGR基因的克隆与表达分析
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摘要
桉树挥发油是世界产量最大的精油之一,广泛运用于制药、香料和工业行业。目前,国际市场的桉树挥发油基本全部由中国提供,一方面人们对桉树挥发油的需求越来越大,另一方面桉树挥发油的产率却低至1%左右,目前通过基因工程提高桉树挥发油产率是一个有效的措施之一。因此,本研究提取尾巨桉不同组织的挥发油并对其中的1,8-桉叶素和β-桉叶醇含量进行测定；同时克隆了尾巨桉树挥发油代谢途径上的相关基因,为尾巨桉遗传转化和挥发油含量检测提供理论依据。
本研究使用Clevenger apparatus分别提取尾巨桉根、茎和叶组织挥发油,结果表明叶的挥发油产量最高,其总油量为252.6 mg·100g-1,出油率为0.25%：茎的总油量为18.2mg·100g-1,出油率为0.018%,根中没有挥发油存在。经高效液相色谱检测表明,1,8-桉叶素在叶和茎挥发油总量中分别占0.06%和0.04%；β-桉叶醇在叶和茎挥发油总量中分别占6.69%和11.5%,说明β-桉叶醇是尾巨桉挥发油的主要成分之一,1,8-桉叶素含量相对较低。
桉树挥发油由单萜和倍半萜类化合物组成,通过甲羟戊酸(MVA)途径和甲基赤藓醇4-磷酸(MEP)途径可形成单萜类化合物的前体物质香叶基二磷酸和倍半萜类化合物的前体物质法呢基二磷酸,香叶基二磷酸和法呢基二磷酸在其它酶的作用下发生去磷酸化、异构、环化和原子重排等反应形成挥发油的各种成分。本实验通过cDNA末端快速扩增技术克隆了甲羟戊酸途径上的第一个关键限速酶3-羟基-3-甲基戊二酰CoA还原酶(HMGR)基因,该基因全长1955bp,包含66bp的5'-UTR、329 bp的3'-UTR、poly A尾巴和1560 bp的开放阅读框,该基因编码519个氨基酸组成的蛋白质,其分子质量为55.1202kD,理论等电点pI为5.37。通过在线NCBI中的Blast P比对分析表明,尾巨桉HMGR基因与青钱柳、橡胶树、喜树、沙梨的HMGR基因同源性均达100%。尾巨桉HMGR生物信息学分析表明：HMGR为疏水性蛋白质；位于A60、A62、A63、A83、A111、A121、A147、A149、A191、A282、A344、A363、A437、A510、A511共15个丝氨酸残基,A144、A197、A237、A350、A362共5个苏氨酸残基和A120、A158、A172共3个酪氨酸残基是蛋白质翻译后磷酸化修饰位点；存在1个信号肽,由位于A1～A25氨基酸残组成,信号肽最有可能的酶剪切位于A25(Ser)残基与第A26(Leu)残基之间；亚细胞定位分析表明,HMGR属于次生代谢途径上的分泌型酶,具有一信号肽；存在1个跨膜结构域位于A5(V)～A27(I)残基之间：位于A167～A176氨基酸残基和A196～A203氨基酸残基分别存在一个HMG-COA结合motif:EMPVGYVQLP和TTEGCLVA,其中TTEGCLVA中的谷氨酸(Glu)在HMGR催化中起着重要作用,位于A292～A298氨基酸残基和A441～A447氨基酸残基分别存在一个NADP(H)结合motif:RCDGHEH和GTVGGGT;分子系统进化树显示,尾巨桉HMGR类聚于植物群,表明所得到的HMGR准确可靠；二级结构由47.40%的α-螺旋、15.41%的延伸链、7.13%的β-转角、30.06%的无规卷曲组成。以人的HMGR为基础进行同源建模得到尾巨桉HMGR三维空间构象呈“V”形,包含了N-结构域、S-结构域和L-结构域。
HMGR基因结构分析表明,HMGR基因在基因组中存在一个大小为319bp的内含子,尚不明确此内含子的作用。实时定量PCR分析表明,HMGR基因在枝中的表达量最高,茎次之,在根中表达量最低。原核表达表明,HMGR基因能够在大肠杆菌M15中通过IPTG诱导表达约55kD的目的蛋白。
Eucalyptus essential oil, which is one of the most volatile oil output in the world, is used for medicinal, perfumery and industrial purposes. Now, the eucalyptus essential oil is mainly supplied by China in international market. On one hand the people's requirement is increasing, on the other hand the eucalyptus essential oil yield is about 1%lowly. It's an effective method to improve the yield by genetic engineering. So, the volatile oil from different tissue were isolated and the composition contents,1,8-cineol andβ-eudesmol, were determined; Simultaneously the relative HMGR gene was cloned in the essention oil metabolic pathway in this study. It can provide some information to transformation of Eucalyptus and determination of essential oil composition.
Eucalyptus essential oil from root, stem and leaf of Eucalyptus urophylla×E. grandis were extracted by Clevenger apparatus. The result indicated the total oil from leaf was more than any other tissue and there wasn't essential oil in root. The oil quantity from leaf and stem was 252.6 mg·100g-1 and 18.2 mg·100g-1 respectively, corresponding the oil yield was 0.25%and 0.018%; The proportion of 1,8-cineol in total oil from leaf and stem was 0.06% and 0.04%, and the proportion ofβ-eudesmol in total oil from leaf and stem was 6.69% and 11.5% respectively by HPLC. The result indicatedβ-eudesmol was the mainly composition in Eucalyptus urophylla×E. grandis volatile oil while 1,8-cineol was low relatively.
Monoterpenes and sesquiterpenes constitute of the eucalyptus essential oil. GPP and FPP, whose precursors of monoterpenes and sesquiterpenes, are synthesized in both mevalonate pathway and methylerythritol 4-phosphate pathway. The precursors generate the different composition by abscising phosphorylation, isomerization, cyclization and atom rearrangement. The gene encoding 3-Hydroxy-3-methylglutaryl-CoA reductase regarded as the first key enzyme in MVA pathway was cloning through Rapid Amplification of cDNA Ends technology from Eucalyptus urophylla×E. grandis. The full-length HMGR cDNA was 1955 bp. containing 66 bp 5'untranslated region,329 bp 3'untranslated region, poly A tail and a 1560 bp open reading frame encoding a peptide of 519 amino acids with a calculated molecular mass of 55.1202 kDa and an isoelectric point of 5.37. HMGR protein predicted from Eucalyptus urophyla×E.grandis showed 100% identities with Cyclocarya paliurus, Hevea brasiliensis, Camptotheca acuminate and Pyrus pyrifolia by Blast P on-line NCBI. By bioinformatics analysis, HMGR belonged to hydrophobic protein; The 23 amino acid residues, including 15 Serines localized in A60, A62, A63, A83, A111, A121, A147, A149, A191, A282, A344, A363, A437, A510, A511,5 Threonines localized in A144, A197, A237, A350, A362 and 3 Tyrosines localized in A120, A158, A172, were the phosphorylation sites after being translated; There were a signal peptide composed of the A1 to A25 amino acid residues at N-terminus which max possible cleavage site was between the A25 (S) and A26(L) and a transmembrane localized in A5(V) to A27(I); The subcellular prediction found that HMGR hadn't transit peptide at N-terminus and localized in secondary metabolism pathway with a signal peptide; There were two HMG-CoA binding motifs: EMPVGYVQLP and TTEGCLVA, localized in A167 to A176 and A196 to A203 amino acid residues respectively, the G(Glu) amino acid residue had an important promotion; Also there were two NADP(H) binding motifs:RCDGHEH and GTVGGGT localized in A292 to A298 and A441 to A447 amino acid residues respectively; HMGR from Eucalyptus urophylla×E.grandis belonged to plants group by the phylogenetic tree of HMGRs from other plants, the result was accurate and reliable; The secondary structure analysis revealed that HMGR was composed of 47.40% alpha helix,15.41% extended strand,7.13% beta turn and 30.06% random coil. HMGR had a three dimensional structure with "V" homology-based on the HMGR model of Homo sapiens, containing N-domain, S-domain and L-domain.
Through the gene construction analysis, there was an intron of 319 bp in genome DNA, the function of the intron was indefinite. Tissue expression profile analysis by Real Time quantitative PCR indicated HMGR expression was the highest in branch, following leaf and the lowest in root. The protein,55.1202 kD of HMGR predicted, was induced expression by IPTG in E. coli M15 successfully.
本研究使用Clevenger apparatus分别提取尾巨桉根、茎和叶组织挥发油,结果表明叶的挥发油产量最高,其总油量为252.6 mg·100g-1,出油率为0.25%：茎的总油量为18.2mg·100g-1,出油率为0.018%,根中没有挥发油存在。经高效液相色谱检测表明,1,8-桉叶素在叶和茎挥发油总量中分别占0.06%和0.04%；β-桉叶醇在叶和茎挥发油总量中分别占6.69%和11.5%,说明β-桉叶醇是尾巨桉挥发油的主要成分之一,1,8-桉叶素含量相对较低。
桉树挥发油由单萜和倍半萜类化合物组成,通过甲羟戊酸(MVA)途径和甲基赤藓醇4-磷酸(MEP)途径可形成单萜类化合物的前体物质香叶基二磷酸和倍半萜类化合物的前体物质法呢基二磷酸,香叶基二磷酸和法呢基二磷酸在其它酶的作用下发生去磷酸化、异构、环化和原子重排等反应形成挥发油的各种成分。本实验通过cDNA末端快速扩增技术克隆了甲羟戊酸途径上的第一个关键限速酶3-羟基-3-甲基戊二酰CoA还原酶(HMGR)基因,该基因全长1955bp,包含66bp的5'-UTR、329 bp的3'-UTR、poly A尾巴和1560 bp的开放阅读框,该基因编码519个氨基酸组成的蛋白质,其分子质量为55.1202kD,理论等电点pI为5.37。通过在线NCBI中的Blast P比对分析表明,尾巨桉HMGR基因与青钱柳、橡胶树、喜树、沙梨的HMGR基因同源性均达100%。尾巨桉HMGR生物信息学分析表明：HMGR为疏水性蛋白质；位于A60、A62、A63、A83、A111、A121、A147、A149、A191、A282、A344、A363、A437、A510、A511共15个丝氨酸残基,A144、A197、A237、A350、A362共5个苏氨酸残基和A120、A158、A172共3个酪氨酸残基是蛋白质翻译后磷酸化修饰位点；存在1个信号肽,由位于A1～A25氨基酸残组成,信号肽最有可能的酶剪切位于A25(Ser)残基与第A26(Leu)残基之间；亚细胞定位分析表明,HMGR属于次生代谢途径上的分泌型酶,具有一信号肽；存在1个跨膜结构域位于A5(V)～A27(I)残基之间：位于A167～A176氨基酸残基和A196～A203氨基酸残基分别存在一个HMG-COA结合motif:EMPVGYVQLP和TTEGCLVA,其中TTEGCLVA中的谷氨酸(Glu)在HMGR催化中起着重要作用,位于A292～A298氨基酸残基和A441～A447氨基酸残基分别存在一个NADP(H)结合motif:RCDGHEH和GTVGGGT;分子系统进化树显示,尾巨桉HMGR类聚于植物群,表明所得到的HMGR准确可靠；二级结构由47.40%的α-螺旋、15.41%的延伸链、7.13%的β-转角、30.06%的无规卷曲组成。以人的HMGR为基础进行同源建模得到尾巨桉HMGR三维空间构象呈“V”形,包含了N-结构域、S-结构域和L-结构域。
HMGR基因结构分析表明,HMGR基因在基因组中存在一个大小为319bp的内含子,尚不明确此内含子的作用。实时定量PCR分析表明,HMGR基因在枝中的表达量最高,茎次之,在根中表达量最低。原核表达表明,HMGR基因能够在大肠杆菌M15中通过IPTG诱导表达约55kD的目的蛋白。
Eucalyptus essential oil, which is one of the most volatile oil output in the world, is used for medicinal, perfumery and industrial purposes. Now, the eucalyptus essential oil is mainly supplied by China in international market. On one hand the people's requirement is increasing, on the other hand the eucalyptus essential oil yield is about 1%lowly. It's an effective method to improve the yield by genetic engineering. So, the volatile oil from different tissue were isolated and the composition contents,1,8-cineol andβ-eudesmol, were determined; Simultaneously the relative HMGR gene was cloned in the essention oil metabolic pathway in this study. It can provide some information to transformation of Eucalyptus and determination of essential oil composition.
Eucalyptus essential oil from root, stem and leaf of Eucalyptus urophylla×E. grandis were extracted by Clevenger apparatus. The result indicated the total oil from leaf was more than any other tissue and there wasn't essential oil in root. The oil quantity from leaf and stem was 252.6 mg·100g-1 and 18.2 mg·100g-1 respectively, corresponding the oil yield was 0.25%and 0.018%; The proportion of 1,8-cineol in total oil from leaf and stem was 0.06% and 0.04%, and the proportion ofβ-eudesmol in total oil from leaf and stem was 6.69% and 11.5% respectively by HPLC. The result indicatedβ-eudesmol was the mainly composition in Eucalyptus urophylla×E. grandis volatile oil while 1,8-cineol was low relatively.
Monoterpenes and sesquiterpenes constitute of the eucalyptus essential oil. GPP and FPP, whose precursors of monoterpenes and sesquiterpenes, are synthesized in both mevalonate pathway and methylerythritol 4-phosphate pathway. The precursors generate the different composition by abscising phosphorylation, isomerization, cyclization and atom rearrangement. The gene encoding 3-Hydroxy-3-methylglutaryl-CoA reductase regarded as the first key enzyme in MVA pathway was cloning through Rapid Amplification of cDNA Ends technology from Eucalyptus urophylla×E. grandis. The full-length HMGR cDNA was 1955 bp. containing 66 bp 5'untranslated region,329 bp 3'untranslated region, poly A tail and a 1560 bp open reading frame encoding a peptide of 519 amino acids with a calculated molecular mass of 55.1202 kDa and an isoelectric point of 5.37. HMGR protein predicted from Eucalyptus urophyla×E.grandis showed 100% identities with Cyclocarya paliurus, Hevea brasiliensis, Camptotheca acuminate and Pyrus pyrifolia by Blast P on-line NCBI. By bioinformatics analysis, HMGR belonged to hydrophobic protein; The 23 amino acid residues, including 15 Serines localized in A60, A62, A63, A83, A111, A121, A147, A149, A191, A282, A344, A363, A437, A510, A511,5 Threonines localized in A144, A197, A237, A350, A362 and 3 Tyrosines localized in A120, A158, A172, were the phosphorylation sites after being translated; There were a signal peptide composed of the A1 to A25 amino acid residues at N-terminus which max possible cleavage site was between the A25 (S) and A26(L) and a transmembrane localized in A5(V) to A27(I); The subcellular prediction found that HMGR hadn't transit peptide at N-terminus and localized in secondary metabolism pathway with a signal peptide; There were two HMG-CoA binding motifs: EMPVGYVQLP and TTEGCLVA, localized in A167 to A176 and A196 to A203 amino acid residues respectively, the G(Glu) amino acid residue had an important promotion; Also there were two NADP(H) binding motifs:RCDGHEH and GTVGGGT localized in A292 to A298 and A441 to A447 amino acid residues respectively; HMGR from Eucalyptus urophylla×E.grandis belonged to plants group by the phylogenetic tree of HMGRs from other plants, the result was accurate and reliable; The secondary structure analysis revealed that HMGR was composed of 47.40% alpha helix,15.41% extended strand,7.13% beta turn and 30.06% random coil. HMGR had a three dimensional structure with "V" homology-based on the HMGR model of Homo sapiens, containing N-domain, S-domain and L-domain.
Through the gene construction analysis, there was an intron of 319 bp in genome DNA, the function of the intron was indefinite. Tissue expression profile analysis by Real Time quantitative PCR indicated HMGR expression was the highest in branch, following leaf and the lowest in root. The protein,55.1202 kD of HMGR predicted, was induced expression by IPTG in E. coli M15 successfully.
引文
[1]廖志华.紫杉醇前体生物合成的分子生物学和抗癌萜类吲哚生物碱的代谢工程.博士研究生论文.2004,上海：复旦大学.
[2]陈剑勇,宋建英,林威,等.尾赤桉组织培养研究[J].西南林学院学报,2006,26(3)：44-46.
[3]莫晓勇.中国桉树引种驯化技术对策分析[J].桉树科技,2000,2：21-25.
[4]李俊,许兴.桉树生物质能源的价值研究[J].企业技术开发,2008,27(9)：96-98.
[5]董文海.全球造纸工业发展趋势分析[J].造纸信息,2010,1：44-49.
[6]陈少雄,刘杰锋,孙正军,等.桉树生物质能源的优势、现状和潜力[J].生物质化学工程,2006.1：119-128.
[7]梁川.中国桉树种植与制浆造纸研讨会在海南海口召开[J].造纸信息,2005.10：35.
[8]吴继林,林方良.桉树木屑袋栽食用菌试验初报[J].桉树科技,2001(1)：40-44.
[9]张明华,黄文,钟祝烂,等.桉树木屑栽培食用菌试验初报[J].食用菌,2001,23(5)：21-22.
[10]中国电力网.www. chinapower. com. cn.
[11]潘少军.生物质发电如何走出困境?[N].人民日报,2009.
[12]活性炭网http://www.active-carbons.com/Info/IndustrialNews/75893.htm.
[13]经贸资讯网.http://www.macauhub.com.mo/gb/news.php?ID=9071.
[14]郑岚岚,符安平.粤电湛江生物质发电项目遂溪开工[N].湛江日报.2010.
[15]邓勇,房俊民,陈方,等.生物燃料最新发展态势分析[J].中国生物工程杂志,2008,28(8)：142-147.
[16]肖正春,张卫民,张广伦,等.生物柴油植物桉树类叶油的开发利用[J].中国野生植物资源,2008,27(2)：19-20.
[17]陈培栋.可替代能源——石油树[N].中国绿色时报.2005.
[18]中国生物技术信息网.http://www.biotech.org.cn/news/news/show.php?id=57876.
[19]国际新能源网.http://www.in-en.com/newenergy/html/newenergy-1031103165576814.html.
[20]宋永芳.桉树叶的利用[M].1990,北京：中国林业出版社.
[21]田玉红,刘雄民,陶明有,等.巨尾桉叶挥发性成分的提取及成分分析[J].广西科学院学报,2006,22(z1)：466-468.
[22]周燕园,韦志英,钟振国,等.GC-MS对广西细叶桉叶及果实挥发油成分研究[J].中药材,2009,32(2)：216-219.
[23]Ogunwande I A, Olawore N O, Adelekel K A, et al. Volatile constituents from the leaves of Eucalyptus cloeziana F. Muell and E.propinqua Deane & Maiden from Nigeria. Flavour and Fragrance Journal,2005,20(6):637-639.
[24]田玉红,张祥民,黄泰松,等.桉叶油的研究进展[J].食品与发酵工业,2007,33(10)：139-143.
[25]吴志民.真空催促精馏提纯1,8-桉叶油素研究[D].硕士论文.2007,昆明：昆明理工大学.
[26]http://en.wikipedia.org/wiki/Steam_distillation.
[27]王健英.1,8-按叶油素的提取和提纯[D].硕士论文.2004,天津：天津大学.
[28]Lassak E V, Brophy J J. Steam volatile leaf oils of some western Australian species of the family Myrtaceae. Flavour and Fragrance Journal,2004,19(1):12-16.
[29]Li H, Madden J L. Analysis of Leaf Oils from a Eucalyptus Species Trial. Biochemical Systemadcs and Ecology,1995,23(2):167-177.
[30]Nathan S S. The use of Eucalyptus tereticornis Sm. (Myrtaceae) oil (leaf extract) as a natural
larvicidal agent against the malaria vector Anopheles stephensi Liston (Diptera:Culicidae). Bioresource Technology,2007,98(9):1856-1860.[31]国家药典委员会,中国药典2005年版一部[S].2005,北京：化学工业出版社.[32]傅梅红,王金华,张颖.气相色谱法测定苍术中有效成分β-桉叶醇含量的方法学研究[J].中国中药杂志,2000,25(11)：135-137.
[33]陈炎明,俞桂新,王峥涛.RP-HPLC测定茅苍术中β-桉叶醇的含量[J].中国中药杂志,2007,32(21)：2265-2267.
[34]Pourmortazavi S M, Hajimirsadeghi S S. Supercritical fluid extraction in plant essential and volatile oil analysis. Journal of Chromatography A,2007,1163(1-2):2-24.
[35]李昶红,李薇,李旭红,等.超临界二氧化碳萃取中试装置的工艺改进[J].化工进展,2004,23(11)：1248-1251.
[36]G Della Porta, S Porcedda, B Marongiu, et al, Isolation of eucalyptus oil by supercritical fluid extraction. Flavour and Fragrance Journal,1999,14:214-218.
[37]编者.桉叶油综述[J].国内外香化信息,2005,1：8-9.
[38]李士雨,’王静康.桉叶素应用与制各[J].精细化工,1995,12(5)：12-15.
[39]孙少文,杨光军,丁建生,等.熔融结晶工艺开发[J].化学工程,2008,36(12)：18-24.
[40]王志刚,吴佳,和国红.等.高纯桉叶素的制备方法及其装置[P],发明专利,2003：中国.
[41]李士雨.枝叶素的纯化[J].精细化工,2006,23(1)：35-37.
[42]符乃光,林昭华,田庆,等.桉叶油蒸馏装置的初步研制[C].第四届十三省区市机械工程学会科技论坛暨2008海南机械科技论坛论文集.2008,出版者不详：海南.
[43]郭文生,郭放,佟健,等.包结物晶析法选择分离辛夷挥发油中1,8-桉叶素[J].高等学校化学学报,2005,26(5)：883-885.
[44]谭新智,李炳强,揭新明,等.桉叶油素回收技术[P].1988：中国.
[45]王莉,史玲玲,张艳霞,等.植物次生代谢途径及其研究进展[J].武汉植物学研究,2007,25(5)：500-508.
[46]田玉红,刘雄民,周永红,等,赤桉和本泌桉叶精油的化学成分研究[J].精细化工,2005.22(12)：920-923.
[47]田玉红,刘雄民,周永红,等.圆角桉叶精油的化学成分[J].食品科学,2007,28(1)：36-38.
[48]殷清华,梁振益,陈祎平,等.桉树叶挥发油化学成分的研究[J].化学分析计量,2008,17(3)：30-31.
[49]Elaissi A, Medini H, Marzouki H, et al. Variation in Volatile Leaf Oils of Twelve Eucalyptus Species Harvested from Hajeb Layoun Arboreta (Tunisia). Chemistry & Biodiversity,2010, 7(3):705-716.
[50]http://www.genome.jp/kegg/pathway.html.
[51]付佳,王洋,阎秀峰.萜类化合物的生理生态功能及经济价值[J].东北林业大学学报,2003,31(6)：59-62.
[52]潘瑞炽.植物生理学[M].1979,北京：高等教育出版社.
[53]彭少麟,南蓬,钟扬,等.植物中的萜类化合物及其在生态系统中的作用[J].生态学杂志,2002,2l(3)：33-38.
[54]Withers S T, Keasling J D. Biosynthesis and engineering of isoprenoid small molecules. Applied Microbiology and Biotechnology,2007,73(5):980-990.
[55]Hsieh M H, Goodman H M. The Arabidopsis IspH homolog is involved in the plastid nonmevalonate pathway of isoprenoid biosynthesis. Plant Physiology,2005,138(2): 641-653.
[56]Douglas J, McGarvey, Rodney Croteau:Terpenoid Metabolism. The Plant Cell,1995,7(7): 1015-1026.
[57]Park H, Denbow C J, Cramer C L. Cramer. Structure and nucleotide sequence of tomato HMG2 encoding 3-hydroxy-3-methyl-glutaryl coenzyme A reductase. Plant Molecular Biology, 1992,20:327-331.
[58]Istvan E S, Deisenhofer J. The structure of the catalytic portion of human HMG-CoA reductase. Achieves of Biochemistry and Biophysics,2000,1529:9-18.
[59]Ruiz-Albert J, Cerda-Olmedo E, Corrochano L M. Corrochano. Genes for mevalonate biosynthesis in Phycomyces. Molecular Genetics and Genomics,2002,266(5):768-777.
[60]Enjuto M, Balcells L, Campos N, et al. Campos N,et al. Arabidopsis thaliana contains two differentially expressed 3-hydroxy-3-methylglutaryl-CoA reductase genes, which encode microsomal forms of the enzyme. Proc Natl Acad Sci USA,1994,91(3):927-31.
[61]Lin J, Jin Y J, Zhou M Q, et al. Molecular cloning, characterization and functional analysis of a 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Jatropha curcas. African Journal of Biotechnology,2009,8(15):3455-3462.
[62]Liao Z H, Tan Q M, Chai Y R, et al. Cloning and characterisation of the gene encoding HMG-CoA reductase from Taxus media and its functional identification in yeast. Functional Plant Biology,2004,31(1):73-81.
[63]Sando T, Takaoka C, Mukai Y, et al. Cloning and characterization of mevalonate pathway genes in a natural rubber producing plant, Hevea brasiliensis. Bioscience Biotechnology and Biochemistry,2008,72(8):2049-2060.
[64]Chye M-L, Kush A, Tan C-T, et al. Characterization of cDNA and genomic clones encoding 3-hydroxy-3-methyiglutaryl-coenzyme A reductase from Hevea brasiliensis. Plant Molecular Biology,1991,16:567-577.
[65]Chye M L, Tan C T, Chua N H. Three genes encode 3-hydroxy-3-methylglutaryl-coenzyme A reductase in Hevea brasiliensis:hmgl and hmg3 are differentially expressed. Plant Molecular Biology,1992,19:473-484.
[66]Eisenreich W, Rohdich F, Bacher A. Deoxyxylulose phosphate pathway to terpenoids. TRENDS in Plant Science,2001,6:78-84.
[67]Kuzuyama T, Shimizu T, Takahashi S, et al. Fosmidomycin, a Specific Inhibitor of 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase in the Nonmevalonate Pathway for Terpenoid Biosynthesis. Tetrahedron Letters,1998,39:7913-7916.
[68]Lange B M, Croteau R. Isoprenoid Biosynthesis via a Mevalonate-Independent Pathway in Plants:Cloning and Heterologous Expression of 1-Deoxy-D-xylulose-5-phosphate Reductoisomerase from Peppermint. Archives of Biochemistry and Biophysics,1999, 365(1):170-174.
[69]Carretero-Paulet L, Ahumada I, Cunillera N, et al. Expression and molecular analysis of the Arabidopsis DXR gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the first committed enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway. Plant Physiology,2002,129(4):1581-1591.
[70]Yoonram K, Takahashi S, Rattanapittayaporn A, et al. cDNA, from Hevea brasiliensis latex, encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase. Plant Science,2008,175(5): 694-700.
[71]Yao H Y, Gong Y F, Zuo K J, et al. Molecular cloning, expression profiling and functional
analysis of a DXR gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Camptotheca acuminata. Journal of Plant Physiology,2008,165(2):203-213. [72] Mahmoud S S, Croteau R B. Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase. The Proceedings of the National Academy of Sciences of the United States of American,2001,98 (15):8915-8920.
[73]Hasunuma T, Takeno S, Hayashi, S, et al. Overexpression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene in chloroplast contributes to increment of isoprenoid production. Journal of Bioscience and Bioengineering,2008,105(5):p.518-526.
[74]Clastre M, Bantignies B, Feron C, et al. Purification and Characterization of Geranyl Diphosphate Synthase from Vitis vinifera L. cv Muscat de Frontignan Cell Cultures. Plant Physiology,1993,102:205-211.
[75]Tholl D, Croteau R, Gershenzon J. Partial Purification and Characterization of the Short-Chain Prenyltransferases, Geranyl Diphosphate Synthase and Farnesyl Diphosphate Synthase,from Abies grandis (Grand Fir). Archives of Biochemistry and Biophysics,2001,386(2): 233-242.
[76]Burke C C, Wildung M R, Croteau R. Geranyl diphosphate synthase:Cloning, expression,and characterization of this prenyltransferase as a heterodimer. PNAS,1999,96(23): 13062-13067.
[77]Bouvier F, Suire C, Harlingue A D, et al. Molecular cloning of geranyl diphosphate synthase and compaftmentation of monoterpene synthesis in plant cells. The plant Journal,2000, 24(2):241-252.
[78]Delourme D, Lacroute F, Karst F. Cloning of an Arabidopsis thaliana cDNA coding for farnesyl diphosphate synthase by functional complementation in yeast. Plant Molecular Biology, 1994,26:1867-1873.
[79]Adiwilaga K, Kush A. Cloning and characterization of cDNA encoding farnesyl diphosphate synthase from rubber tree (Hevea brasiliensis). Plant Molecular Biology,1996,30:935-946.
[80]Kim O T, Ahn J C, Hwang S J, et al. Cloning and expression of a farnesyl diphosphate synthase in Centella asiatica (L.) urban. Molecules and Cells,2005,19(2):294-299.
[81]Matsushita Y, Kang W, Charlwood B V. Cloning and analysis of a cDNA encoding farnesyl diphosphate synthase from Artemisia annua. Gene,1996,172:207-209.
[82]KrisansSO S K, Ericssonnll J, Edwardsn P A, et al. Farnesyl-diphosphate Synthase Is Localized in Peroxisomes. The journal of biological chemistry,1994,269(19):14165-14169.
[83]Marrero P F, Poulter C D, Edwards P A. Effects of Site-directed Mutagenesis of the Highly Conserved Aspartate Residues in Domain II of Farnesyl Diphosphate Synthase Activity. The journal of biological chemistry,1992,267(30):21873-21878.
[84]Masferrer A, Arro M, Manzano D, et al. Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase (FPS1S) in transgenic Arabidopsis induces a cell death/senescence-like response and reduced cytokinin levels. Plant Journal,2002,30(2): 123-132.
[85]Degenhardt J, Kollner T G, and Gershenzon J. Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry,2009,70(15-16):21-1637.
[86]Yang T, Li J, Wang H X, et al. A geraniol-synthase gene from Cinnamomum tenuipilum. Phytochemistry,2005,66:285-293.
[87]Landmann C, Fink B, Festner M,et al. Cloning and functional characterization of three terpene synthases from lavender (Lavandula angustifolia). Archives of Biochemistry and Biophysics,2007,465(2):417-429.
[88]唐丽,唐芳,段经华,等.金桂芳樟醇合成酶基因的克隆与序列分析.林业科学,2009,45(5)：11-19.
[89]Lane A, Boecklemann A, Woronuk G N, et al. A genomics resource for investigating regulation of essential oil production in Lavandula angustifolia. Planta,2010,231(4):835-845.
[90]Colby S M, Alonso W R, Katahira E J, et al.4S-Limonene Synthase from the Oil Glands of Spearmint (Mentha spicata). The journal of biological chemistry,1993,268(31): 23016-23024.
[91]Turner G, Gershenzon J, Nielson E E, et al. Limonene Synthase, the Enzyme Responsible for Monoterpene Biosynthesis in Peppermint, Is Localized to Leucoplasts of Oil Gland Secretory Cells. Plant Physiology,1999,120:879-886.
[92]Krasnyanski S, May R A, Loskutov A, et al. Transformation of the limonene synthase gene into peppermint(Mentha piperita L.) and preliminary studies on the essential oil profiles of single transgenic plants. Theoretical and Applied Genetics,1999,99:676-682.
[93]Munoz-Bertomeu J, Ros R, Arrillaga I, et al. Expression of spearmint limonene synthase in transgenic spike lavender results in an altered monoterpene composition in developing leaves. Metabolic Engineering,2008,10(3-4):166-177.
[94]Fraga B M. Natural sesquiterpenoids. Natural Product Reports,2009,26(9):1125-1155.
[95]《化学化工辞典》编委会.化学化工大辞典(上、下册).2003,北京：化学工业出版社.
[96]刘彦,叶和春,李国凤.一个新高产青蒿倍半萜合酶基因的克隆、表达和分析.植物学报,2002,44(12)：1450-1455.
[97]Crock J, Wildung M, Croteau R. Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha×piperita, L.) that produces the aphid alarm pheromone (E)-β-farnesene. PNAS,1997,94(24):12833-12838.
[98]Picaud S, Brodelius M, Brodelius P E. Expression, purification and characterization of recombinant (E)-β-farnesene synthase from Artemisia annua. Phytochemistry,2005,66: 961-967.
[99]Bennett M H, Mansfield J W, Lewis M J, et al. Cloning and expression of sesquiterpene synthase genes from lettuce (Lactuca sativa L.). Phytochemistry,2002,60(3):255-261.
[100]Bouwmeester H J, Kodde J, Verstappen F W A, et al. Isolation and characterization of two germacrene A synthase cDNA clones from chicory. Plant Physiology,2002,129(1): 134-144.
[101]Kim M Y, Chang Y J, Bang M H, et al. cDNA isolation and characterization of (+)-germacrene A synthase from Ixeris dentata form, albiflora Hara. Journal of Plant Biology,2005,48(2): 178-186.
[102]Bertea C M, Voster A, Verstappen F W A, et al. Isoprenoid biosynthesis in Artemisia annua: Cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library. Archives of Biochemistry and Biophysics,2006,448(1-2):3-12.
[103]Yu F, Harada H, Yamasaki K, et al. Isolation and functional characterization of a beta-eudesmol synthase, a new sesquiterpene synthase from Zingiber zerumbet Smith. Febs Letters,2008,582(5):565-572.
[104]Teulleres C, Feuillet C, Boude A M. Differential characteristics of cell suspension cultures
initiated from Eucalyptus gunnii clones differing by their frost tolerance. Plant Cell Reports, 1989,8(7):407-410. [105] Teulleres C, Boude A M. Isolation of protoplasts from different Eucalyptus species and preliminary studies on regeneration. Plant Cell, Tissue and Organ Culture,1991,25(2): 133-140.
[106]Leborgne N, Teulleres C, Travert S. Introduction of specific carbohydrates into Eucalyptus gunnii cells increases their freezing tolerance. European Journal of Biochemistry,1995,229: 710-717.
[107]Prakash M G, Gurumurthi K. Organogenesis and plant regeneration from seedling explants of Eucalyptus tereticornis. Plant Cell Biotechnology and Molecular Biology,2005,6:121-126.
[108]Azmi A, Noin M, Landre P, et al. High frequency plant regeneration from Eucalyptus globulus Labill hypocotyls:Ontogenesis and ploidy level of the regenerants. Plant Cell Tissue and Organ Culture,1997,51:9-16.
[109]Dibax R, Eisfeld C D, Cuquel F L, et al. Plant regeneration from cotyledonary explants of Eucalyptus camaldulensis. Scientia Agricola,2005,62(4):406-412.
[110]Bandyopadhyay S, Cane K, Rasmussen G, et al. Efficient plant regeneration from seedling explants of two commercially important temperate eucalyptus species-Eucalyptus nitens and E.globulus. Plant Science,1999,140(2):189-198.
[111]Hajari E, Watt M P, Mycock D J, et al. Plant regeneration from induced callus of improved Eucalyptus clones. South African Journal of Botany,2006,72(2):195-201.
[112]肖尊安.植物生物技术[M].2004,北京：化学工业出版社.
[113]Nugent G, Chandler S F, Whiteman P, et al. Somatic embryogenesis in Eucalyptus globulus. Plant Cell,Tissue and Organ Culture,2001,67:85-88.
[114]Prakash M G, Gurumurthi K. Effect of age of explants on somatic embryogenesis and plant regeneration in Eucalyptus tereticornis Sm. Plant Cell Biotechnology and Molecular Biology,2004,5(1-2):39-44.
[115]Prakash M G, Gurumurthi K. Somatic embryogenesis and plant regeneration in Eucalyptus tereticornis Sm. Current Science,2005,88(8):1311-1316.
[116]王关琳,方宏筠.植物基因工程[M].2002,北京：科学出版社.
[117]Macrae S, Staden J V. Agrobacterium rhizogenes-mediated transformation to improve rooting ability of eucalypts. Tree Physiology,1993,12:411-418.
[118]Lucianade O R M, Gisele M A, Luis P B C, et al. Agrobacterium strain specificity and shooty tumour formation in Eucalyptus(Eucalyptus grandis×E.urophylla). Plant Cell Reports, 1997,16(5):299-303.
[119]Azmi A, Drevet C, Vanonckelen H, et al. Bud regeneration from Eucalyptus globules clones and seedlings through hormoneal imbalances induced by Agrobacterium tumefaciens strain 82.139. Plant Science,1997,127(1):81-90.
[120]Mullins K V, Llewellyn D J, Hartney V J, et al. Regeneration and transformation of Eucalyptus camaldulensis. Plant Cell Reports,1997,16(11):787-791.
[121]Ho C-K, Chang S-H, Tsay J-Y, et al. Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants. Plant Cell Reports,1998,17: 675-680.
[122]Tournier V, Grat S, Marque C, et al. An efficient procedure to stably introduce genes into an economically important pulp tree (Eucalyptus grandis×Eucalyptus urophylla). Transgenic Research,2003,12(4):403-411.
[123]Spokevicius A V, Van Beveren K, Leitch M M, et al. Agrobacterium-mediated in vitro transformation of wood-producing stem segments in eucalypts. Plant Cell Reports,2005, 23(9):617-624.
[124]Serranol L, Rochange F, Semblat J P, et al. Genetic transformation of Eucalyptus globulus through biolistics:complementary development of procedures for organogenesis from zygotic embryos and stable transformation of corresponding proliferating tissue. Journal of Experimental Botany,1996,47(295):285-290.
[125]Sartoretto L M, Cid L P B, Brasileiro A C M. Biolistic transformation of Eucalyptus grandis×E.urophylla callus. Functional Plant Biology,2002.29(8):917-924.
[126]Teulieres C, Cid L P B, Boudet A M, et al. Transient foreign gene expression in polyethylenelglycol treated or electropulsated Eucalyptus gunnii protoplast. Plant Cell Tissue and Organ Culture,1991,25(1):125-132.
[127]Manders G, Santos A V P, UtraVaz F B, et al. Transient gene expression in electroporated protoplasts of Eucalyptus citriodora Hook. Plant Cell,Tissue and Organ Culture,1992, (30): 67-75.
[128]Chen Z Z, Tsay J Y, Chung J D, et al. Callus Culture of Eucalyptus grandis×urophylla and Preliminary Studies on Organogenesis and Agrobacterium-mediated Transformation. Taiwan Journal of Forest Science,1996,11(3):43-52.
[129]Esteban R G, Alexander A, Ana L B, et al. Production of transgenic Eucalyptus grandis×E.urophylla using the sonication-assisted Agrobacterium transformation (SAAT) system. Funct Plant Biol,2002,29:97-102.
[130]J.莎姆布鲁克,黄培堂.分子克隆实验指南第三版(上、下册).2002,北京：科学出版社.
[131]TAKARA公司.大连宝生物工程有限公司2007-2008版商品目录.2007,大连：大连宝生物工程有限公司.
[132]Sefidkon F, Assareh M H, Abravesh Z, et al. Chemical composition of the essential oils of four cultivated Eucalyptus species in Iran as medicinal plants (E.microtheca, E.spathulata, E.largiflorens and E.torquata). Iranian Journal of Pharmaceutical Research,2007,6(2): 135-140.
[133]谢培山.中药色谱指纹图谱.2005,北京：人民卫生出版社.
[134]Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol,2002, 3(7):RESEARCH0034.
[135]Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic messenger-RNAs. Nucleic Acids Research,1984,12(2):p.857-872.
[136]王镜岩,朱圣庚,徐长法.生物化学第三版[M].2002,北京：高等教育出版社.
[137]Geourjon C, Deleage G. SOPMA:Significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Computer Applications in the Biosciences,1995,11(6):681-684.
[138]Arnold K, Bordoli L, Kopp J, et al. The SWISS-MODEL workspace:a web-based environment for protein structure homology modelling. Bioinformatics,2006,22(2): 195-201.
[139]Schwede, T, Kopp J, Guex N, et al. SWISS-MODEL:an automated protein homology-modeling server. Nucleic Acids Research,2003,31(13):3381-3385.
[140]Guex N, Peitsch M C. SWISS-MODEL and the Swiss-PdbViewer:An environment for comparative protein modeling. Electrophoresis,1997,18(15):2714-2723.
[141]Pfefferkorn J A, Choi C, Song Y, et al. Design and synthesis of novel, conformationally restricted HMG-CoA reductase inhibitors. Bioorganic & Medicinal Chemistry Letters, 2007,17(16):4531-4537.
[142]Istvan E S, Deisenhofer J, The structure of the catalytic portion of human HMG-CoA reductase. Biochimica Et Biophysica Acta-Molecular and Cell Biology of Lipids,2000, 1529(1-3):9-18.
[143]李嵘,王喆之.植物萜类合成酶3-羟基-3-甲基戊二酰辅酶A还原酶的生物信息学分析[J].广西植物,2006,26(5)：464-473.
[144]Larkin M A, Blackshields G, Brown N P, et al. Clustal W and clustal X version 2.0. Bioinformatics,2007,23:2947-2948.
[145]Cao X Y, Zong Z M, Ju X Y, et al. Molecular cloning, characterization and function analysis of the gene encoding HMG-CoA reductase from Euphorbia Pekinensis Rupr. Molecular Biology Reports,2010,37(3):1559-1567.
[146]朱玉贤,李毅.现代分子生物学[M].1997,北京：高等教育出版社.
[147]Gachon C, Mingam A C, Charrier B. Real-time PCR:what relevance to plant studies? Journal of Experimental Botany,2004,55(402):1445-1554.
[148]Pabinger S, Thallinger G G, Snajder R, et al. QPCR:Application for real-time PCR data management and analysis. Bmc Bioinformatics,2009,10.
[2]陈剑勇,宋建英,林威,等.尾赤桉组织培养研究[J].西南林学院学报,2006,26(3)：44-46.
[3]莫晓勇.中国桉树引种驯化技术对策分析[J].桉树科技,2000,2：21-25.
[4]李俊,许兴.桉树生物质能源的价值研究[J].企业技术开发,2008,27(9)：96-98.
[5]董文海.全球造纸工业发展趋势分析[J].造纸信息,2010,1：44-49.
[6]陈少雄,刘杰锋,孙正军,等.桉树生物质能源的优势、现状和潜力[J].生物质化学工程,2006.1：119-128.
[7]梁川.中国桉树种植与制浆造纸研讨会在海南海口召开[J].造纸信息,2005.10：35.
[8]吴继林,林方良.桉树木屑袋栽食用菌试验初报[J].桉树科技,2001(1)：40-44.
[9]张明华,黄文,钟祝烂,等.桉树木屑栽培食用菌试验初报[J].食用菌,2001,23(5)：21-22.
[10]中国电力网.www. chinapower. com. cn.
[11]潘少军.生物质发电如何走出困境?[N].人民日报,2009.
[12]活性炭网http://www.active-carbons.com/Info/IndustrialNews/75893.htm.
[13]经贸资讯网.http://www.macauhub.com.mo/gb/news.php?ID=9071.
[14]郑岚岚,符安平.粤电湛江生物质发电项目遂溪开工[N].湛江日报.2010.
[15]邓勇,房俊民,陈方,等.生物燃料最新发展态势分析[J].中国生物工程杂志,2008,28(8)：142-147.
[16]肖正春,张卫民,张广伦,等.生物柴油植物桉树类叶油的开发利用[J].中国野生植物资源,2008,27(2)：19-20.
[17]陈培栋.可替代能源——石油树[N].中国绿色时报.2005.
[18]中国生物技术信息网.http://www.biotech.org.cn/news/news/show.php?id=57876.
[19]国际新能源网.http://www.in-en.com/newenergy/html/newenergy-1031103165576814.html.
[20]宋永芳.桉树叶的利用[M].1990,北京：中国林业出版社.
[21]田玉红,刘雄民,陶明有,等.巨尾桉叶挥发性成分的提取及成分分析[J].广西科学院学报,2006,22(z1)：466-468.
[22]周燕园,韦志英,钟振国,等.GC-MS对广西细叶桉叶及果实挥发油成分研究[J].中药材,2009,32(2)：216-219.
[23]Ogunwande I A, Olawore N O, Adelekel K A, et al. Volatile constituents from the leaves of Eucalyptus cloeziana F. Muell and E.propinqua Deane & Maiden from Nigeria. Flavour and Fragrance Journal,2005,20(6):637-639.
[24]田玉红,张祥民,黄泰松,等.桉叶油的研究进展[J].食品与发酵工业,2007,33(10)：139-143.
[25]吴志民.真空催促精馏提纯1,8-桉叶油素研究[D].硕士论文.2007,昆明：昆明理工大学.
[26]http://en.wikipedia.org/wiki/Steam_distillation.
[27]王健英.1,8-按叶油素的提取和提纯[D].硕士论文.2004,天津：天津大学.
[28]Lassak E V, Brophy J J. Steam volatile leaf oils of some western Australian species of the family Myrtaceae. Flavour and Fragrance Journal,2004,19(1):12-16.
[29]Li H, Madden J L. Analysis of Leaf Oils from a Eucalyptus Species Trial. Biochemical Systemadcs and Ecology,1995,23(2):167-177.
[30]Nathan S S. The use of Eucalyptus tereticornis Sm. (Myrtaceae) oil (leaf extract) as a natural
larvicidal agent against the malaria vector Anopheles stephensi Liston (Diptera:Culicidae). Bioresource Technology,2007,98(9):1856-1860.[31]国家药典委员会,中国药典2005年版一部[S].2005,北京：化学工业出版社.[32]傅梅红,王金华,张颖.气相色谱法测定苍术中有效成分β-桉叶醇含量的方法学研究[J].中国中药杂志,2000,25(11)：135-137.
[33]陈炎明,俞桂新,王峥涛.RP-HPLC测定茅苍术中β-桉叶醇的含量[J].中国中药杂志,2007,32(21)：2265-2267.
[34]Pourmortazavi S M, Hajimirsadeghi S S. Supercritical fluid extraction in plant essential and volatile oil analysis. Journal of Chromatography A,2007,1163(1-2):2-24.
[35]李昶红,李薇,李旭红,等.超临界二氧化碳萃取中试装置的工艺改进[J].化工进展,2004,23(11)：1248-1251.
[36]G Della Porta, S Porcedda, B Marongiu, et al, Isolation of eucalyptus oil by supercritical fluid extraction. Flavour and Fragrance Journal,1999,14:214-218.
[37]编者.桉叶油综述[J].国内外香化信息,2005,1：8-9.
[38]李士雨,’王静康.桉叶素应用与制各[J].精细化工,1995,12(5)：12-15.
[39]孙少文,杨光军,丁建生,等.熔融结晶工艺开发[J].化学工程,2008,36(12)：18-24.
[40]王志刚,吴佳,和国红.等.高纯桉叶素的制备方法及其装置[P],发明专利,2003：中国.
[41]李士雨.枝叶素的纯化[J].精细化工,2006,23(1)：35-37.
[42]符乃光,林昭华,田庆,等.桉叶油蒸馏装置的初步研制[C].第四届十三省区市机械工程学会科技论坛暨2008海南机械科技论坛论文集.2008,出版者不详：海南.
[43]郭文生,郭放,佟健,等.包结物晶析法选择分离辛夷挥发油中1,8-桉叶素[J].高等学校化学学报,2005,26(5)：883-885.
[44]谭新智,李炳强,揭新明,等.桉叶油素回收技术[P].1988：中国.
[45]王莉,史玲玲,张艳霞,等.植物次生代谢途径及其研究进展[J].武汉植物学研究,2007,25(5)：500-508.
[46]田玉红,刘雄民,周永红,等,赤桉和本泌桉叶精油的化学成分研究[J].精细化工,2005.22(12)：920-923.
[47]田玉红,刘雄民,周永红,等.圆角桉叶精油的化学成分[J].食品科学,2007,28(1)：36-38.
[48]殷清华,梁振益,陈祎平,等.桉树叶挥发油化学成分的研究[J].化学分析计量,2008,17(3)：30-31.
[49]Elaissi A, Medini H, Marzouki H, et al. Variation in Volatile Leaf Oils of Twelve Eucalyptus Species Harvested from Hajeb Layoun Arboreta (Tunisia). Chemistry & Biodiversity,2010, 7(3):705-716.
[50]http://www.genome.jp/kegg/pathway.html.
[51]付佳,王洋,阎秀峰.萜类化合物的生理生态功能及经济价值[J].东北林业大学学报,2003,31(6)：59-62.
[52]潘瑞炽.植物生理学[M].1979,北京：高等教育出版社.
[53]彭少麟,南蓬,钟扬,等.植物中的萜类化合物及其在生态系统中的作用[J].生态学杂志,2002,2l(3)：33-38.
[54]Withers S T, Keasling J D. Biosynthesis and engineering of isoprenoid small molecules. Applied Microbiology and Biotechnology,2007,73(5):980-990.
[55]Hsieh M H, Goodman H M. The Arabidopsis IspH homolog is involved in the plastid nonmevalonate pathway of isoprenoid biosynthesis. Plant Physiology,2005,138(2): 641-653.
[56]Douglas J, McGarvey, Rodney Croteau:Terpenoid Metabolism. The Plant Cell,1995,7(7): 1015-1026.
[57]Park H, Denbow C J, Cramer C L. Cramer. Structure and nucleotide sequence of tomato HMG2 encoding 3-hydroxy-3-methyl-glutaryl coenzyme A reductase. Plant Molecular Biology, 1992,20:327-331.
[58]Istvan E S, Deisenhofer J. The structure of the catalytic portion of human HMG-CoA reductase. Achieves of Biochemistry and Biophysics,2000,1529:9-18.
[59]Ruiz-Albert J, Cerda-Olmedo E, Corrochano L M. Corrochano. Genes for mevalonate biosynthesis in Phycomyces. Molecular Genetics and Genomics,2002,266(5):768-777.
[60]Enjuto M, Balcells L, Campos N, et al. Campos N,et al. Arabidopsis thaliana contains two differentially expressed 3-hydroxy-3-methylglutaryl-CoA reductase genes, which encode microsomal forms of the enzyme. Proc Natl Acad Sci USA,1994,91(3):927-31.
[61]Lin J, Jin Y J, Zhou M Q, et al. Molecular cloning, characterization and functional analysis of a 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Jatropha curcas. African Journal of Biotechnology,2009,8(15):3455-3462.
[62]Liao Z H, Tan Q M, Chai Y R, et al. Cloning and characterisation of the gene encoding HMG-CoA reductase from Taxus media and its functional identification in yeast. Functional Plant Biology,2004,31(1):73-81.
[63]Sando T, Takaoka C, Mukai Y, et al. Cloning and characterization of mevalonate pathway genes in a natural rubber producing plant, Hevea brasiliensis. Bioscience Biotechnology and Biochemistry,2008,72(8):2049-2060.
[64]Chye M-L, Kush A, Tan C-T, et al. Characterization of cDNA and genomic clones encoding 3-hydroxy-3-methyiglutaryl-coenzyme A reductase from Hevea brasiliensis. Plant Molecular Biology,1991,16:567-577.
[65]Chye M L, Tan C T, Chua N H. Three genes encode 3-hydroxy-3-methylglutaryl-coenzyme A reductase in Hevea brasiliensis:hmgl and hmg3 are differentially expressed. Plant Molecular Biology,1992,19:473-484.
[66]Eisenreich W, Rohdich F, Bacher A. Deoxyxylulose phosphate pathway to terpenoids. TRENDS in Plant Science,2001,6:78-84.
[67]Kuzuyama T, Shimizu T, Takahashi S, et al. Fosmidomycin, a Specific Inhibitor of 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase in the Nonmevalonate Pathway for Terpenoid Biosynthesis. Tetrahedron Letters,1998,39:7913-7916.
[68]Lange B M, Croteau R. Isoprenoid Biosynthesis via a Mevalonate-Independent Pathway in Plants:Cloning and Heterologous Expression of 1-Deoxy-D-xylulose-5-phosphate Reductoisomerase from Peppermint. Archives of Biochemistry and Biophysics,1999, 365(1):170-174.
[69]Carretero-Paulet L, Ahumada I, Cunillera N, et al. Expression and molecular analysis of the Arabidopsis DXR gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the first committed enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway. Plant Physiology,2002,129(4):1581-1591.
[70]Yoonram K, Takahashi S, Rattanapittayaporn A, et al. cDNA, from Hevea brasiliensis latex, encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase. Plant Science,2008,175(5): 694-700.
[71]Yao H Y, Gong Y F, Zuo K J, et al. Molecular cloning, expression profiling and functional
analysis of a DXR gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Camptotheca acuminata. Journal of Plant Physiology,2008,165(2):203-213. [72] Mahmoud S S, Croteau R B. Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase. The Proceedings of the National Academy of Sciences of the United States of American,2001,98 (15):8915-8920.
[73]Hasunuma T, Takeno S, Hayashi, S, et al. Overexpression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene in chloroplast contributes to increment of isoprenoid production. Journal of Bioscience and Bioengineering,2008,105(5):p.518-526.
[74]Clastre M, Bantignies B, Feron C, et al. Purification and Characterization of Geranyl Diphosphate Synthase from Vitis vinifera L. cv Muscat de Frontignan Cell Cultures. Plant Physiology,1993,102:205-211.
[75]Tholl D, Croteau R, Gershenzon J. Partial Purification and Characterization of the Short-Chain Prenyltransferases, Geranyl Diphosphate Synthase and Farnesyl Diphosphate Synthase,from Abies grandis (Grand Fir). Archives of Biochemistry and Biophysics,2001,386(2): 233-242.
[76]Burke C C, Wildung M R, Croteau R. Geranyl diphosphate synthase:Cloning, expression,and characterization of this prenyltransferase as a heterodimer. PNAS,1999,96(23): 13062-13067.
[77]Bouvier F, Suire C, Harlingue A D, et al. Molecular cloning of geranyl diphosphate synthase and compaftmentation of monoterpene synthesis in plant cells. The plant Journal,2000, 24(2):241-252.
[78]Delourme D, Lacroute F, Karst F. Cloning of an Arabidopsis thaliana cDNA coding for farnesyl diphosphate synthase by functional complementation in yeast. Plant Molecular Biology, 1994,26:1867-1873.
[79]Adiwilaga K, Kush A. Cloning and characterization of cDNA encoding farnesyl diphosphate synthase from rubber tree (Hevea brasiliensis). Plant Molecular Biology,1996,30:935-946.
[80]Kim O T, Ahn J C, Hwang S J, et al. Cloning and expression of a farnesyl diphosphate synthase in Centella asiatica (L.) urban. Molecules and Cells,2005,19(2):294-299.
[81]Matsushita Y, Kang W, Charlwood B V. Cloning and analysis of a cDNA encoding farnesyl diphosphate synthase from Artemisia annua. Gene,1996,172:207-209.
[82]KrisansSO S K, Ericssonnll J, Edwardsn P A, et al. Farnesyl-diphosphate Synthase Is Localized in Peroxisomes. The journal of biological chemistry,1994,269(19):14165-14169.
[83]Marrero P F, Poulter C D, Edwards P A. Effects of Site-directed Mutagenesis of the Highly Conserved Aspartate Residues in Domain II of Farnesyl Diphosphate Synthase Activity. The journal of biological chemistry,1992,267(30):21873-21878.
[84]Masferrer A, Arro M, Manzano D, et al. Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase (FPS1S) in transgenic Arabidopsis induces a cell death/senescence-like response and reduced cytokinin levels. Plant Journal,2002,30(2): 123-132.
[85]Degenhardt J, Kollner T G, and Gershenzon J. Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry,2009,70(15-16):21-1637.
[86]Yang T, Li J, Wang H X, et al. A geraniol-synthase gene from Cinnamomum tenuipilum. Phytochemistry,2005,66:285-293.
[87]Landmann C, Fink B, Festner M,et al. Cloning and functional characterization of three terpene synthases from lavender (Lavandula angustifolia). Archives of Biochemistry and Biophysics,2007,465(2):417-429.
[88]唐丽,唐芳,段经华,等.金桂芳樟醇合成酶基因的克隆与序列分析.林业科学,2009,45(5)：11-19.
[89]Lane A, Boecklemann A, Woronuk G N, et al. A genomics resource for investigating regulation of essential oil production in Lavandula angustifolia. Planta,2010,231(4):835-845.
[90]Colby S M, Alonso W R, Katahira E J, et al.4S-Limonene Synthase from the Oil Glands of Spearmint (Mentha spicata). The journal of biological chemistry,1993,268(31): 23016-23024.
[91]Turner G, Gershenzon J, Nielson E E, et al. Limonene Synthase, the Enzyme Responsible for Monoterpene Biosynthesis in Peppermint, Is Localized to Leucoplasts of Oil Gland Secretory Cells. Plant Physiology,1999,120:879-886.
[92]Krasnyanski S, May R A, Loskutov A, et al. Transformation of the limonene synthase gene into peppermint(Mentha piperita L.) and preliminary studies on the essential oil profiles of single transgenic plants. Theoretical and Applied Genetics,1999,99:676-682.
[93]Munoz-Bertomeu J, Ros R, Arrillaga I, et al. Expression of spearmint limonene synthase in transgenic spike lavender results in an altered monoterpene composition in developing leaves. Metabolic Engineering,2008,10(3-4):166-177.
[94]Fraga B M. Natural sesquiterpenoids. Natural Product Reports,2009,26(9):1125-1155.
[95]《化学化工辞典》编委会.化学化工大辞典(上、下册).2003,北京：化学工业出版社.
[96]刘彦,叶和春,李国凤.一个新高产青蒿倍半萜合酶基因的克隆、表达和分析.植物学报,2002,44(12)：1450-1455.
[97]Crock J, Wildung M, Croteau R. Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha×piperita, L.) that produces the aphid alarm pheromone (E)-β-farnesene. PNAS,1997,94(24):12833-12838.
[98]Picaud S, Brodelius M, Brodelius P E. Expression, purification and characterization of recombinant (E)-β-farnesene synthase from Artemisia annua. Phytochemistry,2005,66: 961-967.
[99]Bennett M H, Mansfield J W, Lewis M J, et al. Cloning and expression of sesquiterpene synthase genes from lettuce (Lactuca sativa L.). Phytochemistry,2002,60(3):255-261.
[100]Bouwmeester H J, Kodde J, Verstappen F W A, et al. Isolation and characterization of two germacrene A synthase cDNA clones from chicory. Plant Physiology,2002,129(1): 134-144.
[101]Kim M Y, Chang Y J, Bang M H, et al. cDNA isolation and characterization of (+)-germacrene A synthase from Ixeris dentata form, albiflora Hara. Journal of Plant Biology,2005,48(2): 178-186.
[102]Bertea C M, Voster A, Verstappen F W A, et al. Isoprenoid biosynthesis in Artemisia annua: Cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library. Archives of Biochemistry and Biophysics,2006,448(1-2):3-12.
[103]Yu F, Harada H, Yamasaki K, et al. Isolation and functional characterization of a beta-eudesmol synthase, a new sesquiterpene synthase from Zingiber zerumbet Smith. Febs Letters,2008,582(5):565-572.
[104]Teulleres C, Feuillet C, Boude A M. Differential characteristics of cell suspension cultures
initiated from Eucalyptus gunnii clones differing by their frost tolerance. Plant Cell Reports, 1989,8(7):407-410. [105] Teulleres C, Boude A M. Isolation of protoplasts from different Eucalyptus species and preliminary studies on regeneration. Plant Cell, Tissue and Organ Culture,1991,25(2): 133-140.
[106]Leborgne N, Teulleres C, Travert S. Introduction of specific carbohydrates into Eucalyptus gunnii cells increases their freezing tolerance. European Journal of Biochemistry,1995,229: 710-717.
[107]Prakash M G, Gurumurthi K. Organogenesis and plant regeneration from seedling explants of Eucalyptus tereticornis. Plant Cell Biotechnology and Molecular Biology,2005,6:121-126.
[108]Azmi A, Noin M, Landre P, et al. High frequency plant regeneration from Eucalyptus globulus Labill hypocotyls:Ontogenesis and ploidy level of the regenerants. Plant Cell Tissue and Organ Culture,1997,51:9-16.
[109]Dibax R, Eisfeld C D, Cuquel F L, et al. Plant regeneration from cotyledonary explants of Eucalyptus camaldulensis. Scientia Agricola,2005,62(4):406-412.
[110]Bandyopadhyay S, Cane K, Rasmussen G, et al. Efficient plant regeneration from seedling explants of two commercially important temperate eucalyptus species-Eucalyptus nitens and E.globulus. Plant Science,1999,140(2):189-198.
[111]Hajari E, Watt M P, Mycock D J, et al. Plant regeneration from induced callus of improved Eucalyptus clones. South African Journal of Botany,2006,72(2):195-201.
[112]肖尊安.植物生物技术[M].2004,北京：化学工业出版社.
[113]Nugent G, Chandler S F, Whiteman P, et al. Somatic embryogenesis in Eucalyptus globulus. Plant Cell,Tissue and Organ Culture,2001,67:85-88.
[114]Prakash M G, Gurumurthi K. Effect of age of explants on somatic embryogenesis and plant regeneration in Eucalyptus tereticornis Sm. Plant Cell Biotechnology and Molecular Biology,2004,5(1-2):39-44.
[115]Prakash M G, Gurumurthi K. Somatic embryogenesis and plant regeneration in Eucalyptus tereticornis Sm. Current Science,2005,88(8):1311-1316.
[116]王关琳,方宏筠.植物基因工程[M].2002,北京：科学出版社.
[117]Macrae S, Staden J V. Agrobacterium rhizogenes-mediated transformation to improve rooting ability of eucalypts. Tree Physiology,1993,12:411-418.
[118]Lucianade O R M, Gisele M A, Luis P B C, et al. Agrobacterium strain specificity and shooty tumour formation in Eucalyptus(Eucalyptus grandis×E.urophylla). Plant Cell Reports, 1997,16(5):299-303.
[119]Azmi A, Drevet C, Vanonckelen H, et al. Bud regeneration from Eucalyptus globules clones and seedlings through hormoneal imbalances induced by Agrobacterium tumefaciens strain 82.139. Plant Science,1997,127(1):81-90.
[120]Mullins K V, Llewellyn D J, Hartney V J, et al. Regeneration and transformation of Eucalyptus camaldulensis. Plant Cell Reports,1997,16(11):787-791.
[121]Ho C-K, Chang S-H, Tsay J-Y, et al. Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants. Plant Cell Reports,1998,17: 675-680.
[122]Tournier V, Grat S, Marque C, et al. An efficient procedure to stably introduce genes into an economically important pulp tree (Eucalyptus grandis×Eucalyptus urophylla). Transgenic Research,2003,12(4):403-411.
[123]Spokevicius A V, Van Beveren K, Leitch M M, et al. Agrobacterium-mediated in vitro transformation of wood-producing stem segments in eucalypts. Plant Cell Reports,2005, 23(9):617-624.
[124]Serranol L, Rochange F, Semblat J P, et al. Genetic transformation of Eucalyptus globulus through biolistics:complementary development of procedures for organogenesis from zygotic embryos and stable transformation of corresponding proliferating tissue. Journal of Experimental Botany,1996,47(295):285-290.
[125]Sartoretto L M, Cid L P B, Brasileiro A C M. Biolistic transformation of Eucalyptus grandis×E.urophylla callus. Functional Plant Biology,2002.29(8):917-924.
[126]Teulieres C, Cid L P B, Boudet A M, et al. Transient foreign gene expression in polyethylenelglycol treated or electropulsated Eucalyptus gunnii protoplast. Plant Cell Tissue and Organ Culture,1991,25(1):125-132.
[127]Manders G, Santos A V P, UtraVaz F B, et al. Transient gene expression in electroporated protoplasts of Eucalyptus citriodora Hook. Plant Cell,Tissue and Organ Culture,1992, (30): 67-75.
[128]Chen Z Z, Tsay J Y, Chung J D, et al. Callus Culture of Eucalyptus grandis×urophylla and Preliminary Studies on Organogenesis and Agrobacterium-mediated Transformation. Taiwan Journal of Forest Science,1996,11(3):43-52.
[129]Esteban R G, Alexander A, Ana L B, et al. Production of transgenic Eucalyptus grandis×E.urophylla using the sonication-assisted Agrobacterium transformation (SAAT) system. Funct Plant Biol,2002,29:97-102.
[130]J.莎姆布鲁克,黄培堂.分子克隆实验指南第三版(上、下册).2002,北京：科学出版社.
[131]TAKARA公司.大连宝生物工程有限公司2007-2008版商品目录.2007,大连：大连宝生物工程有限公司.
[132]Sefidkon F, Assareh M H, Abravesh Z, et al. Chemical composition of the essential oils of four cultivated Eucalyptus species in Iran as medicinal plants (E.microtheca, E.spathulata, E.largiflorens and E.torquata). Iranian Journal of Pharmaceutical Research,2007,6(2): 135-140.
[133]谢培山.中药色谱指纹图谱.2005,北京：人民卫生出版社.
[134]Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol,2002, 3(7):RESEARCH0034.
[135]Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic messenger-RNAs. Nucleic Acids Research,1984,12(2):p.857-872.
[136]王镜岩,朱圣庚,徐长法.生物化学第三版[M].2002,北京：高等教育出版社.
[137]Geourjon C, Deleage G. SOPMA:Significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Computer Applications in the Biosciences,1995,11(6):681-684.
[138]Arnold K, Bordoli L, Kopp J, et al. The SWISS-MODEL workspace:a web-based environment for protein structure homology modelling. Bioinformatics,2006,22(2): 195-201.
[139]Schwede, T, Kopp J, Guex N, et al. SWISS-MODEL:an automated protein homology-modeling server. Nucleic Acids Research,2003,31(13):3381-3385.
[140]Guex N, Peitsch M C. SWISS-MODEL and the Swiss-PdbViewer:An environment for comparative protein modeling. Electrophoresis,1997,18(15):2714-2723.
[141]Pfefferkorn J A, Choi C, Song Y, et al. Design and synthesis of novel, conformationally restricted HMG-CoA reductase inhibitors. Bioorganic & Medicinal Chemistry Letters, 2007,17(16):4531-4537.
[142]Istvan E S, Deisenhofer J, The structure of the catalytic portion of human HMG-CoA reductase. Biochimica Et Biophysica Acta-Molecular and Cell Biology of Lipids,2000, 1529(1-3):9-18.
[143]李嵘,王喆之.植物萜类合成酶3-羟基-3-甲基戊二酰辅酶A还原酶的生物信息学分析[J].广西植物,2006,26(5)：464-473.
[144]Larkin M A, Blackshields G, Brown N P, et al. Clustal W and clustal X version 2.0. Bioinformatics,2007,23:2947-2948.
[145]Cao X Y, Zong Z M, Ju X Y, et al. Molecular cloning, characterization and function analysis of the gene encoding HMG-CoA reductase from Euphorbia Pekinensis Rupr. Molecular Biology Reports,2010,37(3):1559-1567.
[146]朱玉贤,李毅.现代分子生物学[M].1997,北京：高等教育出版社.
[147]Gachon C, Mingam A C, Charrier B. Real-time PCR:what relevance to plant studies? Journal of Experimental Botany,2004,55(402):1445-1554.
[148]Pabinger S, Thallinger G G, Snajder R, et al. QPCR:Application for real-time PCR data management and analysis. Bmc Bioinformatics,2009,10.