利用反义RNA技术进行木质素生物合成调控的研究
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摘要
木质素是一类酚类次生代谢产物，在植物体内行使重要的生理功能，但它却是
形成造纸污染的主要来源。利用基因工程手段，在分子水平调节木质素的生物合成，
降低木质素的含量或改变组分以培育适合造纸的植物原料树种具有较大的应用价值
和环保效益。本研究利用反义RNA技术，主要围绕木质素合成三种相关酶咖啡酸-O-甲基转移酶(COMT)、咖啡酰辅酶A-O-甲基转移酶(CCoAOMT)、4-香豆酸：辅酶
A连接酶(4CL)的基因对植物木质素生物合成途径调节的研究，取得如下进展：
1． 农杆菌介导法将COMT和CCoAOMT基因的单价和双价的反义表达载体导入烟
草，比较了两个甲基化酶的功能。PCR-Southern和Northern点杂交结果表明反义
基因已整合到烟草基因组DNA上，并在转录水平表达。两种反义基因对木质素生物
合成调节的效果显示，CCoAOMT能更有效地调节木质素生物总量的合成，COMT
仅特异调节S木质素的合成。表达反义CCoAOMT基因的转基因毛白杨，内源
CCoAOMT基因的表达在转录和蛋白水平均受到抑制，最终引起转基因植株木质素
含量普遍降低，最多降低达26． 20％，筛选出木质素含量下降10％以上的转基因毛白
杨株系8个，为源头治理造纸废水污染奠定了基础。
2． 对克隆的．4CL基因进行了表达特性分析， RT-PCR分析表明，分离的毛
白杨4CL基因主要在木质部丰富表达，叶中表达量较少，树皮中不表达。在毛白杨
的一个生长季，该基因表达显示明显的双锋特征，该表达模式与木材早材和晚材的
发育时期相吻合，表明分离的毛白杨4CL基因与木质素的生物合成密切相关。农杆
菌介导法将反义4CL基因导入烟草和毛白杨，利用分子生物学检测手段对转化植株
进行筛选，获得批量转基因植株。Klason木质素含量测定分析表明，抑制内源4CL
基因表达，能有效降低转基因植物中的木质素含量，且不影响植株正常生长和发育
以及碳水化合物的合成。转基因毛白杨的茎杆上一些区域呈红棕色，颜色的深度与
转基因毛白杨木质素含量的下降幅度呈一定的正相关性，颜色变化可作为转基因植
株筛选的一个辅助指标。现己获得木质素含量下降10％以上的转基因株系3个，最
多下降达41． 73％，可供中试与制浆实验，为培育低木质素环保型毛白杨提供理论与
实践依据。
3.为了优化现有的表达框架，使目的基因更有效地调节木质素的生物合成，应
用PCR技术从毛白杨基因组中分离得到‘袱厅(肉桂酸4一轻基化酶)基因启动子片段
(GenBank注册号:AY351673)。GUS荧光活性分析和组织化学染色显示，该启动子在
一些木质化的组织和器官中特异表达，随着组织成熟度和木质化程度的增加，表达
活性逐渐增强，并且该启动子受伤诱导。反义扰b月哪7基因在‘毋偏动子的调控下，
会引起转基因烟草木质素均有不同程度的减少，但不影响碳向碳水化合物的转换合
成，对植物的生长发育也无明显负效应。这些结果证明了从毛白杨中分离的乙丫泞启
动子可以应用于造纸原料树种材性改良的遗传工程操作。
4.首次从水稻中华10号(口娜a sativa L.ssP.joponica)分离了CCO月。彻?
基因家族的三个成员，对其基因结构及表达特性的分析表明，该基因家族的三个成
员与水稻的木质化进程关系密切，研究结果有助于了解单子叶植物中的甲基化途径
发生机制，为高产水稻抗倒伏和茎杆饲料作物的遗传改良奠定了基础。
关键词:木质素生物合成反义RNA制浆造纸
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Lignin is a phenolic polymer and plays important roles in plants, but is the main resource of papermaking pollution. Thus there is of economical and environmental interests in cultivating materials with genetically modified lignin for pulping industry. The effects of downregulation of three lignin related genes COMT, CCoAOMT and 4CL expression on lignin biosyntheisis were studied in this paper. The following presents the main progress.1. To identify the function of two methyltransferases (COMT and CCoAOMT), two single antisense expression vectors containing the cDNA of COMT or CCoAOMT gene and one dual antisense expression vector containing cDNAs of both OMTs genes were transferred into tobacco mediated by Agrobacterium tumefacience. PCR-Southern analysis indicated that antisense cDNAs integrated into the genome of the transgenic tobacco. The antisense genes expressed at transcriptional level displayed by Northern dot analysis. Klason lignin assay showed that CCoAOMT could control lignin content more effectively than COMT. The latter was only specifically responsible for syringyl (S) lignin pathway. The antisense CCoAOMT was also transformed into Chinese white aspen (populus tomentosa). It inhibited the expression of the endogenous target gene at transcriptional and translational levels, and led to the lignin reduction in transgenic poplars finally. Till now eight transgenic lines with more than 10% lignin reduction were obtained, among which the most reduction was 26.20%. These data laid a solid foundation of eliminating pollution resources produced by pulping industry.2. The cDNA encoding 4-coumarate : CoA ligase (4CL) from Populus tomentosa was analyzed. RT-PCR and Northern dot analysis indicated that 4CL mRNA was highly accumulated in xylem and its expression revealed a biphasic pattern in one growing season, almost in phase with the formation of early wood and lately wood. This demonstrated that 4CL gene was involved in lignin biosynthesis. Transgenic researches displayed that the expression of antisense 4CL resulted in the decreasing of lignin content in transgenic tobaccos and Chinese white poplars with little effects on plant growth and
carbohydrate synthesis. But the stems of transgenic poplars with marked lignin reduction turned brown-red. The color darkness was positively related with lignin reduction degree, which could be used as an index for selecting poplars with lignin reduction. Till now three transgenic poplars with more than 10% lignin reduction were obtained, among which the most reduction could be up to 41. 73%.3. To effectively regulate lignin biosynthesis in plants, a C4H promoter fragment (the accession number: AY351673) was isolated from genomic DNA of Populustomentosa by PCR. Fluorometric and histochemical GUS analysis showed that the expression of a C4H-GUS fusion product was temporally and spatially specific. It was mainly expressed in the lignified tissues and its activity gradually increased from the first to nine internodes in stems of tobacco, preceding the lignin deposition. C4H-GUS expression was also inductive by wounding. C4H promoter fused to antisense CCoAOMT cDNA was used for manipulating lignin biosynthesis in transgenic tobacco. Experiments using transgenic plants demonstrated that the expression of the fusion gene could effectively reduce lignin content in plants without any pronounced effect on plant growth and the carbon allocation to carbohydrate pool. These results suggested that the C4H promoter from Populus tomentosa could be employed in genetically improving wood quality.4. To investigate the methylated pathway occurring in monocots, three CCoAOMT genes were firstly isolated from Zhonghua 10 (Oryza sativa L. ssp. japonica) and their gene structure and expression pattern were studied. Results suggested that three rice CCoAOMT genes were closely related with lignin biosynthesis. This opened up the possibility of improving the resistance of rice against lodging and forage digestibility by genetically modifying lignin biosynthesis.
形成造纸污染的主要来源。利用基因工程手段，在分子水平调节木质素的生物合成，
降低木质素的含量或改变组分以培育适合造纸的植物原料树种具有较大的应用价值
和环保效益。本研究利用反义RNA技术，主要围绕木质素合成三种相关酶咖啡酸-O-甲基转移酶(COMT)、咖啡酰辅酶A-O-甲基转移酶(CCoAOMT)、4-香豆酸：辅酶
A连接酶(4CL)的基因对植物木质素生物合成途径调节的研究，取得如下进展：
1． 农杆菌介导法将COMT和CCoAOMT基因的单价和双价的反义表达载体导入烟
草，比较了两个甲基化酶的功能。PCR-Southern和Northern点杂交结果表明反义
基因已整合到烟草基因组DNA上，并在转录水平表达。两种反义基因对木质素生物
合成调节的效果显示，CCoAOMT能更有效地调节木质素生物总量的合成，COMT
仅特异调节S木质素的合成。表达反义CCoAOMT基因的转基因毛白杨，内源
CCoAOMT基因的表达在转录和蛋白水平均受到抑制，最终引起转基因植株木质素
含量普遍降低，最多降低达26． 20％，筛选出木质素含量下降10％以上的转基因毛白
杨株系8个，为源头治理造纸废水污染奠定了基础。
2． 对克隆的．4CL基因进行了表达特性分析， RT-PCR分析表明，分离的毛
白杨4CL基因主要在木质部丰富表达，叶中表达量较少，树皮中不表达。在毛白杨
的一个生长季，该基因表达显示明显的双锋特征，该表达模式与木材早材和晚材的
发育时期相吻合，表明分离的毛白杨4CL基因与木质素的生物合成密切相关。农杆
菌介导法将反义4CL基因导入烟草和毛白杨，利用分子生物学检测手段对转化植株
进行筛选，获得批量转基因植株。Klason木质素含量测定分析表明，抑制内源4CL
基因表达，能有效降低转基因植物中的木质素含量，且不影响植株正常生长和发育
以及碳水化合物的合成。转基因毛白杨的茎杆上一些区域呈红棕色，颜色的深度与
转基因毛白杨木质素含量的下降幅度呈一定的正相关性，颜色变化可作为转基因植
株筛选的一个辅助指标。现己获得木质素含量下降10％以上的转基因株系3个，最
多下降达41． 73％，可供中试与制浆实验，为培育低木质素环保型毛白杨提供理论与
实践依据。
3.为了优化现有的表达框架，使目的基因更有效地调节木质素的生物合成，应
用PCR技术从毛白杨基因组中分离得到‘袱厅(肉桂酸4一轻基化酶)基因启动子片段
(GenBank注册号:AY351673)。GUS荧光活性分析和组织化学染色显示，该启动子在
一些木质化的组织和器官中特异表达，随着组织成熟度和木质化程度的增加，表达
活性逐渐增强，并且该启动子受伤诱导。反义扰b月哪7基因在‘毋偏动子的调控下，
会引起转基因烟草木质素均有不同程度的减少，但不影响碳向碳水化合物的转换合
成，对植物的生长发育也无明显负效应。这些结果证明了从毛白杨中分离的乙丫泞启
动子可以应用于造纸原料树种材性改良的遗传工程操作。
4.首次从水稻中华10号(口娜a sativa L.ssP.joponica)分离了CCO月。彻?
基因家族的三个成员，对其基因结构及表达特性的分析表明，该基因家族的三个成
员与水稻的木质化进程关系密切，研究结果有助于了解单子叶植物中的甲基化途径
发生机制，为高产水稻抗倒伏和茎杆饲料作物的遗传改良奠定了基础。
关键词:木质素生物合成反义RNA制浆造纸
/
Lignin is a phenolic polymer and plays important roles in plants, but is the main resource of papermaking pollution. Thus there is of economical and environmental interests in cultivating materials with genetically modified lignin for pulping industry. The effects of downregulation of three lignin related genes COMT, CCoAOMT and 4CL expression on lignin biosyntheisis were studied in this paper. The following presents the main progress.1. To identify the function of two methyltransferases (COMT and CCoAOMT), two single antisense expression vectors containing the cDNA of COMT or CCoAOMT gene and one dual antisense expression vector containing cDNAs of both OMTs genes were transferred into tobacco mediated by Agrobacterium tumefacience. PCR-Southern analysis indicated that antisense cDNAs integrated into the genome of the transgenic tobacco. The antisense genes expressed at transcriptional level displayed by Northern dot analysis. Klason lignin assay showed that CCoAOMT could control lignin content more effectively than COMT. The latter was only specifically responsible for syringyl (S) lignin pathway. The antisense CCoAOMT was also transformed into Chinese white aspen (populus tomentosa). It inhibited the expression of the endogenous target gene at transcriptional and translational levels, and led to the lignin reduction in transgenic poplars finally. Till now eight transgenic lines with more than 10% lignin reduction were obtained, among which the most reduction was 26.20%. These data laid a solid foundation of eliminating pollution resources produced by pulping industry.2. The cDNA encoding 4-coumarate : CoA ligase (4CL) from Populus tomentosa was analyzed. RT-PCR and Northern dot analysis indicated that 4CL mRNA was highly accumulated in xylem and its expression revealed a biphasic pattern in one growing season, almost in phase with the formation of early wood and lately wood. This demonstrated that 4CL gene was involved in lignin biosynthesis. Transgenic researches displayed that the expression of antisense 4CL resulted in the decreasing of lignin content in transgenic tobaccos and Chinese white poplars with little effects on plant growth and
carbohydrate synthesis. But the stems of transgenic poplars with marked lignin reduction turned brown-red. The color darkness was positively related with lignin reduction degree, which could be used as an index for selecting poplars with lignin reduction. Till now three transgenic poplars with more than 10% lignin reduction were obtained, among which the most reduction could be up to 41. 73%.3. To effectively regulate lignin biosynthesis in plants, a C4H promoter fragment (the accession number: AY351673) was isolated from genomic DNA of Populustomentosa by PCR. Fluorometric and histochemical GUS analysis showed that the expression of a C4H-GUS fusion product was temporally and spatially specific. It was mainly expressed in the lignified tissues and its activity gradually increased from the first to nine internodes in stems of tobacco, preceding the lignin deposition. C4H-GUS expression was also inductive by wounding. C4H promoter fused to antisense CCoAOMT cDNA was used for manipulating lignin biosynthesis in transgenic tobacco. Experiments using transgenic plants demonstrated that the expression of the fusion gene could effectively reduce lignin content in plants without any pronounced effect on plant growth and the carbon allocation to carbohydrate pool. These results suggested that the C4H promoter from Populus tomentosa could be employed in genetically improving wood quality.4. To investigate the methylated pathway occurring in monocots, three CCoAOMT genes were firstly isolated from Zhonghua 10 (Oryza sativa L. ssp. japonica) and their gene structure and expression pattern were studied. Results suggested that three rice CCoAOMT genes were closely related with lignin biosynthesis. This opened up the possibility of improving the resistance of rice against lodging and forage digestibility by genetically modifying lignin biosynthesis.
引文
1． 魏建华，宋艳茹。木质素生物合成途径及调控的研究进展。植物学报，2001，43：771-779
2． Baucher M, Monties B, Van Montagu M and Boerjan W. Biosynthesis and genetic engineering of lignin. Crit Rev Plant Sci, 1998, 17: 125-197
3． Monties B. Lignins. In: Harborne JB, ed. Methods in plant biochemistry, Vol. 1． London: Academic Press, 1989, 113-157
4． Lu F, Ralph J. Detection and determination of p-coumaroylated units in lignins. J Agric Food Chem, 1999,47: 1988-1992
5． Lu F, Ralph J. Preliminary evidence for sinapyl acetate as a lignin monomer in kenaf. Chem Commun, 2002, 1:90-91
6． Grabber JH, Ralph J, Hatfield RD. Model studies of ferulate-coniferyl alcohol cross-product formation in primary maize walls: implications for lignification in grasses. J Agric Food Chem, 2002,50:6008-6016
7． Ralph J, Marita J, Kim H, Lu F, Hatfield RD, Ralph SA, Sederoff RR, Scott JT, MacKay JJ, Yahiaoui N. Elucidation of new structures in lignins of CAD-and COMT-deficient plants by NMR. Phytochemistry, 2001, 57: 993-1003
8． Whiting P, Goring D A I. Chemical characterization of tissue fractions from the middle lamella and secondary wall of black spruce tracheids. Wood Sci Technol, 1982, 16: 261-267
9． Donaldson LA. Lignification and lignin topochemistry-an ultrastructural view. Phytochemistry, 2001,57:859-873
10． Terashima N, Fukushima K. Heterogeneity in formation of lignin. Ⅺ. An autoradiographic study of the heterogeneous formation and structure of pine lignin. Wood Sci Technol, 1988, 22: 259-270
11． Fukushina K, Terashima N. Heterogeneity in formation of lignin. Part x Ⅴ: Formation and structure of lignin in compression wood of Pinus thunbergii studied by microautoradiography. Wood Sci Technol ,1991, 25: 371-381
12
12． Baucher M, Halpin C, Petit-Conil M, Boerjan W. Lignin: genetic engineering and impact on pulping. Crit Rev Biochem Mol Biol, 2003, 38: 305-50
13． Camm E L, Towers G H N.. Phenylalanine ammonia lyase. Phytochemistry ,1973, 12,961-973．
14． Anterola A M, Jeon J H, Davin L B, Lewis NG,. Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism. J Bio Chem, 2002, 277: 18272-18280
15． Sewalt VJH, Ni WT, Blount JW, Jung HG, Masoud SA, Howies PA, Lamb C, Dixon RA. Reduced lignin content and altered lignin composition in transgenic tobacco downregulated in expression of L-phenylalanine ammonia-lyase or cinnamate 4-hydroxylase. Plant Physiol, 1997, 115:41-50
16． Potter S, Moreland DE, Kreuz K, Ward E. Induction of cytochrome P450 genes by ethanol in maize. Drug Metabol Drug Interact, 1995, 12: 317-327 17． Nedelkina S, Jupe SC, Blee KA, Schalk M, Werck-Reichhart D, Bolwell GP. Novel characteristics and regulation of a divergent cinnamate 4-hydroxylase (CYP73A15) from French bean: engineering expression in yeast. Plant Mol Biol, 1999, 39: 1079-1090
18． Logemann E, Parniske M, Hahlbrock K. Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley. Proc Natl Acad Sci USA, 1995,92:5905-5909
19． Bell-Lelong DA, Cusumano JC, Meyer K, Chappie C. Cinnamate-4-hydroxylase expression in Arabidopsis: regulation in response to development and the environment. Plant Physiol, 1997, 113:729-738
20． Mizutani M, Ohta D, Sato R. Isolation of a cDNA and a genomic clone encoding cinnamate 4-hydroxylase from Arabidopsis and its expression manner in planta. Plant Physiol, 1997, 113: 755-763
21． Ro D K, Mah N, Ellis B E, Douglas C J. Functional Characterization and Subcellular Localization of Poplar (Populus trichocarpa 3 Populus deltoides) Cinnamate 4-Hydroxylase. Plant Physio, 126: 317-329
22
22． Ehlting J, Buttner D, Wang Q, Douglas CJ, Somssich I, and Kombrink E. Three 4-coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionary classes in angiosperms. Plant J, 1999, 19: 9-20
23． Hu W J, Kawaoka A, Tsai C J, Lung J, Osakabe K, Ebinuma H, Chiang VL. Compartmentalized expression of two structurally and functionally distinct 4-coumarate: CoA ligase genes in aspen (Populus tremuloides). Proc NatlAcad Sci USA, 1998, 95: 5407-5412
24． Lindermayr C, Mollers B, Fliegmann J, Uhlmann A, Lottspeich F, Meimberg H, Ebel J. Divergent members of a soybean (Glycine max L.) 4-coumarate:coenzyme A ligase gene family. Eur J Biochem, 2002, 269: 1304-1315
25． Allina SM, Pri-Hadash A, Theilmann DA, Ellis BE, and Douglas CJ.. 4-Coumarate:coenzyme A ligase in hybrid poplar. Properties of native enzymes, cDNA cloning, and analysis of recombinant enzymes. Plant Physiol, 1998, 116: 743-754
26． Osakabe K, Tsao CC, Li L, Popko JL, Umezawa T, Carraway DT, Smeltzer RH, Joshi CP, and Chiang VL. Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms. Proc Natl Acad Sci USA, 1999, 96: 8955-8960
27． Humphreys JM, Hemm MR, Chappie C. New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase. Proc NatlAcadSci USA, 1999, 96: 10045-10050
28． Yamauchi K, Yasuda S, Hamada K, Tsutsumi Y, Fukushima K. Multiform biosynthetic pathway of syringyl lignin in angiosperms. Planta, 2003,216:496-501
29． Schoch G, Goepfert S, Morant M, Hehn A, Meyer D, Ullmann P, and Werck-Reichhart D. CYP98A3 from Arabidopsis thaliana is a 3'-hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway. J Biol Chem, 2001, 276: 36566-36574
30． Franke R, Humphreys JM, Hemm MR, Denault JW, Ruegger MO, Cusumano JC, Chappie C. The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism. Plant J, 2002, 30: 33-45
31
31． Hoffmann L, Maury S, Martz F, Geoffrey L P, and Legrand M. Purification, cloning and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism. J Biol Chem, 2002, 278: 95-103
32． Matsui N, Fukushima K, Yasuda S, Terashima N. On the behavior of monolignol glucosides in lignin biosynthesis: Ⅱ. Synthesis of monolignol glucosides labeled with 3H at the hydroxymethyl group of side chain, and incorporation of the label into magnolia and ginkgo lignin. Holzforschung, 1994, 48: 375-380
33． Chen F, Yasuda S, Fukushima K. Evidence for a novel biosynthetic pathway that regulates the ratio of syringyl to guaiacyl residues in lignin in the differentiating xylem of Magnolia kobus DC. Plants, 1999,207:597-603
34． Li L, Popko JL, Umezawa T, Chiang VL. 5-hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms. J Biol Chem, 2000, 275: 6537-6545
35． Schmitt D, Pakusch A-E, Matern U. Molecular cloning, induction, and taxonomic distribution of caffeoyl-CoA 3-O-methyltransferase, an enzyme involved in disease resistance. J Biol Chem, 1991,266: 17416-17423
36． Pakusch A-E, Matern U, Schiltz E. Elicitor-inducible caffeoyl-coenzyme A 3-O-methyltransferase from Petroselinum crispum cell suspensions. Plant Physiol, 1991, 95: 137-143
37． Ye, Z H, Kneusel R E, Matern U. Varner J E. An alternative methylation pathway in lignin biosynthesis in Zinnia. Plant Cell, 1994,6: 1427-1439
38． Ye Z-H. Association of caffeoyl CoA 3-O-methyltransferase expression with lignifying tissues in several dicot plants. Plant Physiol, 1997,115: 1341-1350
39． Inoue K, Sewalt VJH, Murray BG, Ni W, Stu rzer C, Dixon RA. Developmental expression and substrate specificities of alfalfa caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase in relation to lignification. Plant Physiol, 1998, 117: 761-770
40． Martz F, Maury S, Pincon G, Legrand M. cDNA cloning, substrate specificity and expression study of tobacco caffeoyl-CoA 3-O-methyltransferase, a lignin biosynthetic enzyme. Plant Mol Biol, 1998,36:427-437
41． Kersey R, Inoue K, Schubert KR, Dixon RA. Immunolocalization of two lignin O-methyltransferases in stems of alfalfa (Medicago sativa L.). Protoplasma, 1999, 209: 46-57
42． Li L, Osakabe K, Joshi CP, Chiang VL. Secondary xylem-specific expression of caffeoyl-coenzyme A 3-Omethyltransferase plays an important role in the methylation pathway associated with lignin biosynthesis in loblolly pine. Plant Mol Biol, 1999, 40: 555-565
43． Atanassova R, Favet N, Martz F, Chabbert B, Tollier M T, Monties B, Fritig B, Legrand M. Altered lignin composition in transgenic tobacco expressing O-methyltransferase sequences in sense and antisense orientation. Plant J, 1995, 8, 465-477
44． Van Doorsselaere J, Baucher M, Chognot E, Chabbert B, Tollier MT, Petit-Conil M, Inze D, Boerjan W, Jouanin L. A novel lignin in poplar trees with a reduced caffeic acid/5-hydroxyferulic acid-methyltransferase activity. Plant J, 1995, 8: 855-864
45． Goffner D, Campbell MM, Campargue C, Clastre M, Borderies G, Boudet A. Purification and characterization of cinnamoyl-coenzyme A: NADP oxidoreductase in Eucalyptus gunnii. Plant Physiol, 1994, 106: 625-632
46． Lamcombe E, Hawkins S, Van D J, Piquemal J, Goffner D, Poeydomenge O, Boudet AM, Grima-Pettenati J. Cinnamoyl CoA reductase, the first commited enzyme of the lignin branch biosynthesis pathway: cloning, expression and phylogenetic relationships. Plant J, 1997,11: 429-441．
47． Lauvergeat V, Lacomme C, Lacombe E, Lasserre E, Roby D, Grima-Pettenati J Two cinnamoyl-CoA reductase (CCR) genes from Arabidopsis thaliana are di.erentially expressed during development and in response to infection with pathogenic bacteria. Phytochemistry, 2001, 57:1187-1195
48． Piquemal J, Lapierre C, Myton K, O' Connell A, Schuch W, Grima-Pettenati J, Boudet A-M. Down-regulation in cinnamoyl-CoA reductase induces significant changes of lignins profiles in transgenic tobacco plants. Plant,J,1998, 13:71-83
49
49． Anterola AM, Lewis NG. Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity. Phytochemistry, 2002, 61:221-294
50． Li L, Cheng XF, Leshkevich J, Umezawa T, Harding SA , and Chiang VL. The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase. Plant Cell, 2001, 13: 1567-1586
51． Lapierrea C, Pilateb G, Polleta B, Milaa I, Leple' J C, Jouaninc L, Kim H, Ralph J. Signatures of cinnamyl alcohol dehydrogenase deficiency in poplar lignins. Phytochemistry, 2004,65 ,313-321
52． Ranocha P, McDougall G, Hawkins S, Sterjiades R, Borderies G, Stewart D, Cabanes-Macheteau M, Boudet A, Goffner D. Biochemical characterization, molecular cloning and expression of laccases-a divergent gene family-in poplar. Eur J Biochem 1999, 259: 485-495
53． Sarkanen KV, Ludwig CH eds. Lignins: Occurrence, Formation, Structure, and Reactions. New York: Wiley-Intersci., 1971,916
54． Terashima N, Atalla RH, Ralph SA, Landucci L L, Litpierre2 C and Monties B. New peparation of lignin polymer models under conditions that approximate cell wall lignification. Part 1． Holzforschung, 1995, 49: 521-527
55． Terashima N, Seguchi Y. Heterogeneity in formation of lignin. IX. Factors influencing the formation of condensed structures in lignins. Cellulose Chem Technol, 1988, 22:147-54
56． Syrjanen K, Brunow G. Regioselectivity in lignin biosynthesis. The influence of dimerization and crosscoupling. J Chem Soc Perkin Trans, 2000, 1: 183-187
57． Davin LB, Lewis NG. Dirigent proteins and dirigent sites explain the mystery of specificity of radial precursor coupling in lignan and lignin biosynthesis. Plant Physiol, 2000, 123: 453-461
58． Gang DR, Costa MA, Fujita M, Dinkova-Kostova AT, Wang HB, Burlat V, Martin W, Sarkanen S, Davin LB, Lewis NG. Regiochemical control of monolignol radical coupling: a new paradigm for lignin and lignan biosynthesis. J Chem Biol, 1999, 6: 143-51
59
59． Bate N J, Orr J, Ni W, Meromi A, Nadler-Hassar T, Doerner PW, Dixon RA, Lamb CJ and Elkind Y. Quantitative relationship between phenylalanine ammonia-lyase levels and phenylpropanoid accumulation in transgenic tobacco identies a rate determining step in natural product synthesis. Proc Natl Acad Sci USA, 1994, 91: 7608-7612
60． Blount JW, Korth KL, Masoud SA, Rasmussen S, Lamb C, Dixon RA. Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway. Plant Physiol, 2000, 122: 107-116
61． Blee K, Choi JW, O'Connell AP, Jupe SC, Schuch W, Lewis NG, Bolwell GP. Antisense and sense expression of cDNA coding for CYP73A15, a class Ⅱ cinnamate 4-hydroxylase, leads to a delayed and reduced production of lignin in tobacco. Phytochemistry, 2001, 57: 1159-1166
62． Dixon RA, Chen F, Guo D, Parvathi K. The biosynthesis of monolignols: a "metabolic grid," or independent pathways to guaiacyl and syringyl units? Phytochemistry, 2001, 57: 1069-1084
63． Lee D, Meyer K, Chappie C, Douglas CJ. Antisense suppression of 4-coumarate:coenzyme A ligase activity in Arabidopsis leads to altered lignin subunit composition. Plant Cell, 1997, 9: 1985-1998
64． Kajita S, Hishiyama S, Tomimura Y, Katayama Y, Omori S. Structural characterization of modified lignin in transgenic tobacco plants in which the activity of 4-coumarate:coenzyme A ligase is depressed. Plant Physiol, 1997, 114: 871-879
65． Zhao HY, Wei JH, Lu J, Song YR. cDNA clonging and functional analysis of 4-courmarate-CoA :ligase (4CL) gene in Chinese white aspen. Prog Nat Sci, 2003, 13: 895-900
66． Hu W J, Harding S A, Lung J, Popko JL, Ralph J, Stokke DD, Tsai CJ, Chiang VL. Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees. Nat Biotechnol, 1999, 17:808-812
67． Li L, Zhou Y, Cheng X, Sun JY, Marita JM, Ralph J and Chiang VL. Combinatorial modification of multiple lignin traits in trees through multigene cotransformation. Proc Natl Acad Sci USA, 2003, 100:4939-4944
68． 贾彩虹，赵华燕，王宏芝，刑智峰，杜克久，宋艳茹，魏建华。抑制4C1-基因表达获得低 木质素含量的转基因毛白杨。科学通报，2004，49：662-666
69． Kajita S, Ishifuji M, Ougiya H, Hara S,Kawabata H, Morohoshi N, Katayama Y. Improvement in pulping and bleaching properties of xylem from transgenic tobacco plants. J Sci Food Agric, 2002,82: 1216-1223
70． Ralph J, Hatfield RD, Piquemal J, Yahiaoui N, Pean M, Lapierre C, Boudet AM. NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase Proc Natl Acad Sci USA, 1998,95: 12803-12808
71． Chabannes M, Barakate A, Lapierre C, Marita JM, Ralph J, Danoun S, Halpin C, Grima-Pettenati J, Boudet AM. Strong decrease in lignin content without significant alteration of plant development is induced by simultaneous down-regulation of cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) in tobacco plants. Plant J, 2001, 28: 257-270
72． Goujon T, Ferret V, Mila I, Pollet B, Ruel K, Burlat V, Joseleau JP, Barriere Y, Lapierre C, Jouanin L. Down-regulation of the AtCCR1 gene in Arabidopsis thaliana: effects on phenotype, lignins and cell wall degradability. Planta, 2003, 217: 218-228
73． O'Connell A, Holt K, Piquemal J, Grima-Pettenati J, Boudet A, Pollet B, Lapierre C, Petit-Conil M, Schuch W, Halpin C. Improved paper pulp from plants with suppressed cinnamoyl-CoA reductase or cinnamyl alcohol dehydrogenase. Trans Res, 2002, 11: 495-503
74． Ruegger M, Meyer K, Cusumano JC, and Chappie C. Regulation of ferulate-5-hydroxylase expression in Arabidopsis in the context of sinapate ester biosynthesis. Plant Physiol, 1999, 119: 101-110
75． Franke R, McMichael CM, Meyer K, Shirley AM, Cusumano JC, Chappie C. Modified lignin in tobacco and poplar plants over-expressing the Arabidopsis gene encoding ferulate 5-hydroxylase. Plant J, 2000, 22: 223-234
76． Ni W, Paiva N L, Dixon R A. Reduced lignin in transgenic plants containing a caffeic acid O-methyltransferase antisense gene. Trans Res, 1994, 3: 120-126
77
77． Zhong R, Morrison III W H, Negrel J, Ye ZH. Dual methylation pathways in lignin biosynthesis. Plant Cell, 1998, 10: 2033-2045
78． Zhao H Y, Wei J H, Zhang J Y, Liu HR, Song YR (2002) . Lignin biosynthesis by suppression of two O-methyltransferases. Chin Sci Bul, 47: 1092-1095
79． Lapierre C, Pollet B, Petit-Conil M, Toval G, Romero J, Pilate G, Leple JC, Boerjan W, Ferret V, De Nadai V, Jouanin L. Structural alterations of lignins in transgenic poplars with depressed cinnamyl alcohol dehydrogenase or ca.eic acid O-methyltransferase activity have an opposite impact on the efficiency of industrial kraft pulping. Plant Physiol, 1999, 119: 153-164
80． Jouanin L, Goujon T, De Nadai V, Martin MT, Mila I, Vallet C, Pollet B, Yoshinaga A, Chabbert B, Petit-Conil M. Lignification in transgenic poplars with extremely reduced caffeic acid O-methyltransferase activity. Plant Physiol, 2000, 123: 1363-1373
81． Tsai C J, Popko J L, Mielke M R, Hu WJ, Podila GK, Chiang VL. Suppression of O-methyltransferase gene by homologous sense transgene in quaking aspen causes red-brown wood phenotypes. Plant Physiol, 1998, 117: 101-112
82． Guo D, Chen F, Inoue K, Blount JW, Dixon RA. Downregulation of caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa: impacts on lignin structure and implications for the biosynthesis of G and S lignin. Plant Cell, 2001, 13: 73-88
83． Goujon T, Sibout R, Pollet B, Maba B, Nussaume L, Bechtold N, Lu F, Ralph J, Mila I, Barriere Y, Lapierre C, Jouanin L. A new Arabidopsis thaliana mutant deficient in the expression of O-methyltransferase impacts lignins and sinapoyl esters. Plant Mol Biol, 2003, 973-89
84． Zhong R, Morrison III W H, Himmelsbach D S, Poole FL, and Ye ZH. Essential role of caffeoyl coenzyme A O-methyltransferase in lignin biosynthesis in woody poplar plants. Plant Physiol, 2000, 124:563-577
85． Meyermans H, Morreel K, Lapierre C, Pollet B, De Bruyn A, Busson R, Herdewijn P, Devreese B, Van Beeumen J, Marita JM. Modifications in lignin and accumulation of phenolic glucosides in poplar xylem upon down-regulation of caffeoyl-coenzyme A O-methyltransferase, an enzyme involved in lignin biosynthesis. J Bio Chem, 2000, 275: 36899-36909
86
86． Pincon G, Maury S, Hoffmann L, Gefforoy P, Lapierre C, Pollet B, Legrand M. Repression of O-methyltransferase genes in transgenic tobacco affects lignin synthesis and plant growth. Phytochemistry, 2001, 57: 1167-1176
87． Marita JM, Ralph J, Hatfield RD, Chappie C. NMR characterization of lignins in Arabidopsis altered in the activity of ferulate 5-hydroxylase. Proc Natl Acad Sci USA, 1999, 96: 12328-12332
88． Huntley SK., Ellis D, Gilbert M, Chappie C, Mansfield SD. Significant increases in pulping efficiency in C4H-F5H-transformed poplars: improved chemical savings and reduced environmental toxins. J Agric Food Chem, 2003, 51: 6178-6183
89． Tamagnone L, Merida A, Parr A, Mackay S, Culianez-Macia FA, Roberts K, Martin C. The AmMYB308 and AmMYB330 transcription factors from antirrhinum regulate phenylpropanoid and lignin biosynthesis in transgenic tobacco. Plant Cell, 1998, 10: 135-154
90． Kawaoka A, Ebinuma H. Transcriptional control of lignin biosynthesis by tobacco L1M protein. Phytochemistry, 2001, 57: 1149-1157
91． Wei Jian-hua, Zhao Hua-yan, Liu Hui-rong, Song Yan-ru. CCoAOMT cDNA isolation and identification from Populus tomentosa. Acta Bot Sin, 2001, 43: 1179-1183
92． Wei JianHua, Zhao HuaYan, Lu Shan Fa, Wang Tai, Ma QingHu, Song Yan Ru. Cloning of cDNA encoding COMT from Chinese white poplar (Populus tomentosa Carr.), sequence analysis and specific expression. Acta Bot Sin, 2001,43: 326-328
93． Lu Sh F, Zhao H Y, Wei J H, Song Y R. Establishment of Tissue Culture System of Triploid Chinese White Poplar. Acta Bot Sin, 2001,43: 435-437
94． 王关林，方宏筠。植物基因工程(第二版)，科学出版社，2002
95． Nikolaeva T N. On the Relationship between the Activity of O-Methyltransferase and the Content of Lignin in Various Organs of Kidney Bean. Russian Journal of plant physiology, 2001, 48: 464-466
96
96． Whetten RW, Mackay JJ, Sederoff RR. Recent advances in understanding lignin biosynthesis. Annu Rev Plant Mol Biol. 1998, 49: 585-609
97． Douglas C, Hoffmann H, Schulz W, Hahlbrock K. Structure and elicitor or UV-light-simulated expression of two 4-coumarate: CoA ligase genes in parsley. EMBO J, 1987, 1189-1195
98． Becker-AndreM, Schulze-Lefert P, Hahlbrock K. Structural comparison, modes of expression, and putative cis-acting elements of the two 4-coumarate: CoA ligase genes in potato. J Bio Chem. 1991,266,8551-8559
99． Lee D, Douglas C J. Two divergent members of a tobacco 4-coumarate: CoA ligase (4CL) gene family. cDNA structure, gene inheritance and expression, and properties of the recombinant proteins. Plant Physiol, 1996,112,193-205
100． Zhang X H, Chiang V L. Molecular cloning of 4-coumarate: CoA ligase in loblolly pine and the roles of this enzyme in the biosynthesis of lignin in compression wood. Plant Physiol, 1997, 113,65-74
101． Meng H, Campbell W H. Substrate profiles and expression of caffeoyl coenzyme A and caffeic acid O-methyltransferases in secondary xylem of aspen during seasonal development. Plant Mol Bio, 1998, 38, 513-520
102． Kajita S,Katayama Y, Omori S. Alterations in the biosynthesis of lignin in transgenic plants with chimeric genes for 4-coumarate: coenzyme A ligase. Plant cell physiol, 1996, 37: 957-65
103． Harding SA, Leshkevich J, Chiang VL, Tsai CJ. Differential substrate inhibition couples kinetically distinct 4-coumarate: Coenzyme A ligases with spatially distinct metabolic roles in quaking aspen. Plant Physiol, 2002, 128: 428-438
104． Linderman C, Mollers B, Fliegmann J, Uhlmann A, Lottspeich F, Meimberg H, Ebel J. Divergent members of a soybean (Glycine max L.) 4-coumarate: coenzyme A ligase gene family. Eur J Biochem, 2002, 269 : 1304-1315
105． 朱之悌．我国造纸国情的若干特点及其对策．北京林业大学学报，2002，24：284-287
106． Barriere Y, Argiller O, Chabbert B. Breeding silage maize with brown-mid-rib genes: Feeding value and biochemical characteristics. Agronomic, 1994, 14: 15-25
107． Mackay J J, O'Malley D M, Presnell T. Inheritance, gene expression and lignin characterization in a mutant pine deficient in cinnamoyl alcohol dehydrogenase. Pro Natl Acad Sci USA, 1997, 94: 8255-8260
108． Pravan G J, Scobbie L, Chesson A. Characterization of lignin from CAD and OMT deficient Bm mutants of maize. J Sci food Agric, 1997, 73: 133-142
109． Wei J H, Zhao H Y, Zhang J Y, Song Y R. Cloning of cDNA encoding CCoAOMT from Populus tomentosa and down-regulation of lignin content in transgenic plant expressing antisense gene. Acta Bot Sin, 2001, 43:1179-1183
110． Higuchi T, Ito T, Umezawa T, Hibino T, Shibata D. Red-brown color of lignified tissues of transgenic plants with anti-sense CAD gene: Wine-red lignin from coniferyl aldehyde. J Biotechnol, 1994,37: 151-158
111． Williamson JD, Hirsch-Wyncott ME, Larkins BA, Gelvin SB. Differential accumulation of a transcript driven by the CaMV-35S promoter in transgenic tobacco. Plant Physiol, 1989, 90:1570-1576
112． Chappie C. Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenases. Annu Rev Plant Physiol Plant Mol Biol, 1998,49: 311-343
113． Koopmann E, Logemann E, Hahlbrock K. Regulation and functional expression of cinnamate 4-hydroxylase from parsley. Plant Physiol, 1999, 119: 49-56
114． Clark M S．主编，顾红雅瞿礼嘉主译，陈章良主校。植物分子生物学手册--实验手册。 Plant molecular biology--A laboratory mannal．第一版，北京，高等教育出版社，1998，66-80
115． Wei Jian-hua, Zhao Hua-yan, Liu Hui-rong, Song Yan-ru. CCoAOMT cDNA isolation and identification from Populus tomentosa. Acta Bot Sin, 2001, 43:1179-1183
116． Inoue K, Sewalt VJ, Murray GB, Ni W, Sturzer C, Dixon RA. Developmental Expression and Substrate Specificities of Alfalfa Caffeic Acid 3-O-Methyltransferase and Caffeoyl Coenzyme A 3-O-Methyltransferase in Relation to Lignification. Plant Physiol, 1998, 117: 761-770
11
117． Saltveit M E. Wound induced changes in phenolic metabolism and tissue browning are altered by heat shock. Postharvest Biology and Technology, 2000, 21: 61-69
118． Hoffmann L, Maury S, Bergdoll M, Thion L, Erard M, Legrand M. Identification of the enzymatic active site of tobacco caffeoyl-coenzyme A O-methyltransferase by site-directed mutagenesis. J Biol Chem, 2001, 276: 36831-36838
119． Joshi C, Chiang V L. Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases. Plant Mol Biol, 1998, 37: 663-674
120． Maury S, Geoffroy P, Legrand M. Tobacco O-methyltransferases involved in phenylpropanoid metabolism. The different caffeoyl-coenzyme A/5-hydroxyferuloyl-coenzyme A 3/5-O-methyltransferase and caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase classes have distinct substrate specificities and expression patterns. Plant Physiol, 1999, 121: 215-224．
121． Marita JM, Ralph J, Hatfield RD, Guo D, Chen F, Dixon RA. Structural and compositional modifications in lignin of transgenic alfalfa down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase. Phytochemistry, 2003, 62: 53-65．
122． Sambrook J, Fritsch E F, Maniatis T. Molecular Clone: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 1995
123． Xu YY, Chong K, Xu ZH, Tan KH. Practical techniques of in situ hybridization with RNA probe. Chin Bull Bot, 2002, 19: 234-238．
124． Campbell WH, Gowri G. Codon usage in higher plants, green algae and cyanobacteria. Plant Physiol, 1990,92: 1-11
125． Bai Y, Gong W, Liu TY & Zhu YX. Cloning and expressional analyses of a cinnamoyl CoA reductase cDNA from rice seedlings. Chin Sci Bui, 2003, 48 : 2221-2225
126． Ito H, Hiraga S, Tsugawa H, Matsui H, Honma M, Otsuki Y, Murakami T and Ohashi Y. Xylem-specific expression of wound-inducible rice peroxidase genes in transgenic plants. Plant Sci, 2000, 13:210-216
2． Baucher M, Monties B, Van Montagu M and Boerjan W. Biosynthesis and genetic engineering of lignin. Crit Rev Plant Sci, 1998, 17: 125-197
3． Monties B. Lignins. In: Harborne JB, ed. Methods in plant biochemistry, Vol. 1． London: Academic Press, 1989, 113-157
4． Lu F, Ralph J. Detection and determination of p-coumaroylated units in lignins. J Agric Food Chem, 1999,47: 1988-1992
5． Lu F, Ralph J. Preliminary evidence for sinapyl acetate as a lignin monomer in kenaf. Chem Commun, 2002, 1:90-91
6． Grabber JH, Ralph J, Hatfield RD. Model studies of ferulate-coniferyl alcohol cross-product formation in primary maize walls: implications for lignification in grasses. J Agric Food Chem, 2002,50:6008-6016
7． Ralph J, Marita J, Kim H, Lu F, Hatfield RD, Ralph SA, Sederoff RR, Scott JT, MacKay JJ, Yahiaoui N. Elucidation of new structures in lignins of CAD-and COMT-deficient plants by NMR. Phytochemistry, 2001, 57: 993-1003
8． Whiting P, Goring D A I. Chemical characterization of tissue fractions from the middle lamella and secondary wall of black spruce tracheids. Wood Sci Technol, 1982, 16: 261-267
9． Donaldson LA. Lignification and lignin topochemistry-an ultrastructural view. Phytochemistry, 2001,57:859-873
10． Terashima N, Fukushima K. Heterogeneity in formation of lignin. Ⅺ. An autoradiographic study of the heterogeneous formation and structure of pine lignin. Wood Sci Technol, 1988, 22: 259-270
11． Fukushina K, Terashima N. Heterogeneity in formation of lignin. Part x Ⅴ: Formation and structure of lignin in compression wood of Pinus thunbergii studied by microautoradiography. Wood Sci Technol ,1991, 25: 371-381
12
12． Baucher M, Halpin C, Petit-Conil M, Boerjan W. Lignin: genetic engineering and impact on pulping. Crit Rev Biochem Mol Biol, 2003, 38: 305-50
13． Camm E L, Towers G H N.. Phenylalanine ammonia lyase. Phytochemistry ,1973, 12,961-973．
14． Anterola A M, Jeon J H, Davin L B, Lewis NG,. Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism. J Bio Chem, 2002, 277: 18272-18280
15． Sewalt VJH, Ni WT, Blount JW, Jung HG, Masoud SA, Howies PA, Lamb C, Dixon RA. Reduced lignin content and altered lignin composition in transgenic tobacco downregulated in expression of L-phenylalanine ammonia-lyase or cinnamate 4-hydroxylase. Plant Physiol, 1997, 115:41-50
16． Potter S, Moreland DE, Kreuz K, Ward E. Induction of cytochrome P450 genes by ethanol in maize. Drug Metabol Drug Interact, 1995, 12: 317-327 17． Nedelkina S, Jupe SC, Blee KA, Schalk M, Werck-Reichhart D, Bolwell GP. Novel characteristics and regulation of a divergent cinnamate 4-hydroxylase (CYP73A15) from French bean: engineering expression in yeast. Plant Mol Biol, 1999, 39: 1079-1090
18． Logemann E, Parniske M, Hahlbrock K. Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley. Proc Natl Acad Sci USA, 1995,92:5905-5909
19． Bell-Lelong DA, Cusumano JC, Meyer K, Chappie C. Cinnamate-4-hydroxylase expression in Arabidopsis: regulation in response to development and the environment. Plant Physiol, 1997, 113:729-738
20． Mizutani M, Ohta D, Sato R. Isolation of a cDNA and a genomic clone encoding cinnamate 4-hydroxylase from Arabidopsis and its expression manner in planta. Plant Physiol, 1997, 113: 755-763
21． Ro D K, Mah N, Ellis B E, Douglas C J. Functional Characterization and Subcellular Localization of Poplar (Populus trichocarpa 3 Populus deltoides) Cinnamate 4-Hydroxylase. Plant Physio, 126: 317-329
22
22． Ehlting J, Buttner D, Wang Q, Douglas CJ, Somssich I, and Kombrink E. Three 4-coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionary classes in angiosperms. Plant J, 1999, 19: 9-20
23． Hu W J, Kawaoka A, Tsai C J, Lung J, Osakabe K, Ebinuma H, Chiang VL. Compartmentalized expression of two structurally and functionally distinct 4-coumarate: CoA ligase genes in aspen (Populus tremuloides). Proc NatlAcad Sci USA, 1998, 95: 5407-5412
24． Lindermayr C, Mollers B, Fliegmann J, Uhlmann A, Lottspeich F, Meimberg H, Ebel J. Divergent members of a soybean (Glycine max L.) 4-coumarate:coenzyme A ligase gene family. Eur J Biochem, 2002, 269: 1304-1315
25． Allina SM, Pri-Hadash A, Theilmann DA, Ellis BE, and Douglas CJ.. 4-Coumarate:coenzyme A ligase in hybrid poplar. Properties of native enzymes, cDNA cloning, and analysis of recombinant enzymes. Plant Physiol, 1998, 116: 743-754
26． Osakabe K, Tsao CC, Li L, Popko JL, Umezawa T, Carraway DT, Smeltzer RH, Joshi CP, and Chiang VL. Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms. Proc Natl Acad Sci USA, 1999, 96: 8955-8960
27． Humphreys JM, Hemm MR, Chappie C. New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase. Proc NatlAcadSci USA, 1999, 96: 10045-10050
28． Yamauchi K, Yasuda S, Hamada K, Tsutsumi Y, Fukushima K. Multiform biosynthetic pathway of syringyl lignin in angiosperms. Planta, 2003,216:496-501
29． Schoch G, Goepfert S, Morant M, Hehn A, Meyer D, Ullmann P, and Werck-Reichhart D. CYP98A3 from Arabidopsis thaliana is a 3'-hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway. J Biol Chem, 2001, 276: 36566-36574
30． Franke R, Humphreys JM, Hemm MR, Denault JW, Ruegger MO, Cusumano JC, Chappie C. The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism. Plant J, 2002, 30: 33-45
31
31． Hoffmann L, Maury S, Martz F, Geoffrey L P, and Legrand M. Purification, cloning and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism. J Biol Chem, 2002, 278: 95-103
32． Matsui N, Fukushima K, Yasuda S, Terashima N. On the behavior of monolignol glucosides in lignin biosynthesis: Ⅱ. Synthesis of monolignol glucosides labeled with 3H at the hydroxymethyl group of side chain, and incorporation of the label into magnolia and ginkgo lignin. Holzforschung, 1994, 48: 375-380
33． Chen F, Yasuda S, Fukushima K. Evidence for a novel biosynthetic pathway that regulates the ratio of syringyl to guaiacyl residues in lignin in the differentiating xylem of Magnolia kobus DC. Plants, 1999,207:597-603
34． Li L, Popko JL, Umezawa T, Chiang VL. 5-hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms. J Biol Chem, 2000, 275: 6537-6545
35． Schmitt D, Pakusch A-E, Matern U. Molecular cloning, induction, and taxonomic distribution of caffeoyl-CoA 3-O-methyltransferase, an enzyme involved in disease resistance. J Biol Chem, 1991,266: 17416-17423
36． Pakusch A-E, Matern U, Schiltz E. Elicitor-inducible caffeoyl-coenzyme A 3-O-methyltransferase from Petroselinum crispum cell suspensions. Plant Physiol, 1991, 95: 137-143
37． Ye, Z H, Kneusel R E, Matern U. Varner J E. An alternative methylation pathway in lignin biosynthesis in Zinnia. Plant Cell, 1994,6: 1427-1439
38． Ye Z-H. Association of caffeoyl CoA 3-O-methyltransferase expression with lignifying tissues in several dicot plants. Plant Physiol, 1997,115: 1341-1350
39． Inoue K, Sewalt VJH, Murray BG, Ni W, Stu rzer C, Dixon RA. Developmental expression and substrate specificities of alfalfa caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase in relation to lignification. Plant Physiol, 1998, 117: 761-770
40． Martz F, Maury S, Pincon G, Legrand M. cDNA cloning, substrate specificity and expression study of tobacco caffeoyl-CoA 3-O-methyltransferase, a lignin biosynthetic enzyme. Plant Mol Biol, 1998,36:427-437
41． Kersey R, Inoue K, Schubert KR, Dixon RA. Immunolocalization of two lignin O-methyltransferases in stems of alfalfa (Medicago sativa L.). Protoplasma, 1999, 209: 46-57
42． Li L, Osakabe K, Joshi CP, Chiang VL. Secondary xylem-specific expression of caffeoyl-coenzyme A 3-Omethyltransferase plays an important role in the methylation pathway associated with lignin biosynthesis in loblolly pine. Plant Mol Biol, 1999, 40: 555-565
43． Atanassova R, Favet N, Martz F, Chabbert B, Tollier M T, Monties B, Fritig B, Legrand M. Altered lignin composition in transgenic tobacco expressing O-methyltransferase sequences in sense and antisense orientation. Plant J, 1995, 8, 465-477
44． Van Doorsselaere J, Baucher M, Chognot E, Chabbert B, Tollier MT, Petit-Conil M, Inze D, Boerjan W, Jouanin L. A novel lignin in poplar trees with a reduced caffeic acid/5-hydroxyferulic acid-methyltransferase activity. Plant J, 1995, 8: 855-864
45． Goffner D, Campbell MM, Campargue C, Clastre M, Borderies G, Boudet A. Purification and characterization of cinnamoyl-coenzyme A: NADP oxidoreductase in Eucalyptus gunnii. Plant Physiol, 1994, 106: 625-632
46． Lamcombe E, Hawkins S, Van D J, Piquemal J, Goffner D, Poeydomenge O, Boudet AM, Grima-Pettenati J. Cinnamoyl CoA reductase, the first commited enzyme of the lignin branch biosynthesis pathway: cloning, expression and phylogenetic relationships. Plant J, 1997,11: 429-441．
47． Lauvergeat V, Lacomme C, Lacombe E, Lasserre E, Roby D, Grima-Pettenati J Two cinnamoyl-CoA reductase (CCR) genes from Arabidopsis thaliana are di.erentially expressed during development and in response to infection with pathogenic bacteria. Phytochemistry, 2001, 57:1187-1195
48． Piquemal J, Lapierre C, Myton K, O' Connell A, Schuch W, Grima-Pettenati J, Boudet A-M. Down-regulation in cinnamoyl-CoA reductase induces significant changes of lignins profiles in transgenic tobacco plants. Plant,J,1998, 13:71-83
49
49． Anterola AM, Lewis NG. Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity. Phytochemistry, 2002, 61:221-294
50． Li L, Cheng XF, Leshkevich J, Umezawa T, Harding SA , and Chiang VL. The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase. Plant Cell, 2001, 13: 1567-1586
51． Lapierrea C, Pilateb G, Polleta B, Milaa I, Leple' J C, Jouaninc L, Kim H, Ralph J. Signatures of cinnamyl alcohol dehydrogenase deficiency in poplar lignins. Phytochemistry, 2004,65 ,313-321
52． Ranocha P, McDougall G, Hawkins S, Sterjiades R, Borderies G, Stewart D, Cabanes-Macheteau M, Boudet A, Goffner D. Biochemical characterization, molecular cloning and expression of laccases-a divergent gene family-in poplar. Eur J Biochem 1999, 259: 485-495
53． Sarkanen KV, Ludwig CH eds. Lignins: Occurrence, Formation, Structure, and Reactions. New York: Wiley-Intersci., 1971,916
54． Terashima N, Atalla RH, Ralph SA, Landucci L L, Litpierre2 C and Monties B. New peparation of lignin polymer models under conditions that approximate cell wall lignification. Part 1． Holzforschung, 1995, 49: 521-527
55． Terashima N, Seguchi Y. Heterogeneity in formation of lignin. IX. Factors influencing the formation of condensed structures in lignins. Cellulose Chem Technol, 1988, 22:147-54
56． Syrjanen K, Brunow G. Regioselectivity in lignin biosynthesis. The influence of dimerization and crosscoupling. J Chem Soc Perkin Trans, 2000, 1: 183-187
57． Davin LB, Lewis NG. Dirigent proteins and dirigent sites explain the mystery of specificity of radial precursor coupling in lignan and lignin biosynthesis. Plant Physiol, 2000, 123: 453-461
58． Gang DR, Costa MA, Fujita M, Dinkova-Kostova AT, Wang HB, Burlat V, Martin W, Sarkanen S, Davin LB, Lewis NG. Regiochemical control of monolignol radical coupling: a new paradigm for lignin and lignan biosynthesis. J Chem Biol, 1999, 6: 143-51
59
59． Bate N J, Orr J, Ni W, Meromi A, Nadler-Hassar T, Doerner PW, Dixon RA, Lamb CJ and Elkind Y. Quantitative relationship between phenylalanine ammonia-lyase levels and phenylpropanoid accumulation in transgenic tobacco identies a rate determining step in natural product synthesis. Proc Natl Acad Sci USA, 1994, 91: 7608-7612
60． Blount JW, Korth KL, Masoud SA, Rasmussen S, Lamb C, Dixon RA. Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway. Plant Physiol, 2000, 122: 107-116
61． Blee K, Choi JW, O'Connell AP, Jupe SC, Schuch W, Lewis NG, Bolwell GP. Antisense and sense expression of cDNA coding for CYP73A15, a class Ⅱ cinnamate 4-hydroxylase, leads to a delayed and reduced production of lignin in tobacco. Phytochemistry, 2001, 57: 1159-1166
62． Dixon RA, Chen F, Guo D, Parvathi K. The biosynthesis of monolignols: a "metabolic grid," or independent pathways to guaiacyl and syringyl units? Phytochemistry, 2001, 57: 1069-1084
63． Lee D, Meyer K, Chappie C, Douglas CJ. Antisense suppression of 4-coumarate:coenzyme A ligase activity in Arabidopsis leads to altered lignin subunit composition. Plant Cell, 1997, 9: 1985-1998
64． Kajita S, Hishiyama S, Tomimura Y, Katayama Y, Omori S. Structural characterization of modified lignin in transgenic tobacco plants in which the activity of 4-coumarate:coenzyme A ligase is depressed. Plant Physiol, 1997, 114: 871-879
65． Zhao HY, Wei JH, Lu J, Song YR. cDNA clonging and functional analysis of 4-courmarate-CoA :ligase (4CL) gene in Chinese white aspen. Prog Nat Sci, 2003, 13: 895-900
66． Hu W J, Harding S A, Lung J, Popko JL, Ralph J, Stokke DD, Tsai CJ, Chiang VL. Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees. Nat Biotechnol, 1999, 17:808-812
67． Li L, Zhou Y, Cheng X, Sun JY, Marita JM, Ralph J and Chiang VL. Combinatorial modification of multiple lignin traits in trees through multigene cotransformation. Proc Natl Acad Sci USA, 2003, 100:4939-4944
68． 贾彩虹，赵华燕，王宏芝，刑智峰，杜克久，宋艳茹，魏建华。抑制4C1-基因表达获得低 木质素含量的转基因毛白杨。科学通报，2004，49：662-666
69． Kajita S, Ishifuji M, Ougiya H, Hara S,Kawabata H, Morohoshi N, Katayama Y. Improvement in pulping and bleaching properties of xylem from transgenic tobacco plants. J Sci Food Agric, 2002,82: 1216-1223
70． Ralph J, Hatfield RD, Piquemal J, Yahiaoui N, Pean M, Lapierre C, Boudet AM. NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase Proc Natl Acad Sci USA, 1998,95: 12803-12808
71． Chabannes M, Barakate A, Lapierre C, Marita JM, Ralph J, Danoun S, Halpin C, Grima-Pettenati J, Boudet AM. Strong decrease in lignin content without significant alteration of plant development is induced by simultaneous down-regulation of cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) in tobacco plants. Plant J, 2001, 28: 257-270
72． Goujon T, Ferret V, Mila I, Pollet B, Ruel K, Burlat V, Joseleau JP, Barriere Y, Lapierre C, Jouanin L. Down-regulation of the AtCCR1 gene in Arabidopsis thaliana: effects on phenotype, lignins and cell wall degradability. Planta, 2003, 217: 218-228
73． O'Connell A, Holt K, Piquemal J, Grima-Pettenati J, Boudet A, Pollet B, Lapierre C, Petit-Conil M, Schuch W, Halpin C. Improved paper pulp from plants with suppressed cinnamoyl-CoA reductase or cinnamyl alcohol dehydrogenase. Trans Res, 2002, 11: 495-503
74． Ruegger M, Meyer K, Cusumano JC, and Chappie C. Regulation of ferulate-5-hydroxylase expression in Arabidopsis in the context of sinapate ester biosynthesis. Plant Physiol, 1999, 119: 101-110
75． Franke R, McMichael CM, Meyer K, Shirley AM, Cusumano JC, Chappie C. Modified lignin in tobacco and poplar plants over-expressing the Arabidopsis gene encoding ferulate 5-hydroxylase. Plant J, 2000, 22: 223-234
76． Ni W, Paiva N L, Dixon R A. Reduced lignin in transgenic plants containing a caffeic acid O-methyltransferase antisense gene. Trans Res, 1994, 3: 120-126
77
77． Zhong R, Morrison III W H, Negrel J, Ye ZH. Dual methylation pathways in lignin biosynthesis. Plant Cell, 1998, 10: 2033-2045
78． Zhao H Y, Wei J H, Zhang J Y, Liu HR, Song YR (2002) . Lignin biosynthesis by suppression of two O-methyltransferases. Chin Sci Bul, 47: 1092-1095
79． Lapierre C, Pollet B, Petit-Conil M, Toval G, Romero J, Pilate G, Leple JC, Boerjan W, Ferret V, De Nadai V, Jouanin L. Structural alterations of lignins in transgenic poplars with depressed cinnamyl alcohol dehydrogenase or ca.eic acid O-methyltransferase activity have an opposite impact on the efficiency of industrial kraft pulping. Plant Physiol, 1999, 119: 153-164
80． Jouanin L, Goujon T, De Nadai V, Martin MT, Mila I, Vallet C, Pollet B, Yoshinaga A, Chabbert B, Petit-Conil M. Lignification in transgenic poplars with extremely reduced caffeic acid O-methyltransferase activity. Plant Physiol, 2000, 123: 1363-1373
81． Tsai C J, Popko J L, Mielke M R, Hu WJ, Podila GK, Chiang VL. Suppression of O-methyltransferase gene by homologous sense transgene in quaking aspen causes red-brown wood phenotypes. Plant Physiol, 1998, 117: 101-112
82． Guo D, Chen F, Inoue K, Blount JW, Dixon RA. Downregulation of caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa: impacts on lignin structure and implications for the biosynthesis of G and S lignin. Plant Cell, 2001, 13: 73-88
83． Goujon T, Sibout R, Pollet B, Maba B, Nussaume L, Bechtold N, Lu F, Ralph J, Mila I, Barriere Y, Lapierre C, Jouanin L. A new Arabidopsis thaliana mutant deficient in the expression of O-methyltransferase impacts lignins and sinapoyl esters. Plant Mol Biol, 2003, 973-89
84． Zhong R, Morrison III W H, Himmelsbach D S, Poole FL, and Ye ZH. Essential role of caffeoyl coenzyme A O-methyltransferase in lignin biosynthesis in woody poplar plants. Plant Physiol, 2000, 124:563-577
85． Meyermans H, Morreel K, Lapierre C, Pollet B, De Bruyn A, Busson R, Herdewijn P, Devreese B, Van Beeumen J, Marita JM. Modifications in lignin and accumulation of phenolic glucosides in poplar xylem upon down-regulation of caffeoyl-coenzyme A O-methyltransferase, an enzyme involved in lignin biosynthesis. J Bio Chem, 2000, 275: 36899-36909
86
86． Pincon G, Maury S, Hoffmann L, Gefforoy P, Lapierre C, Pollet B, Legrand M. Repression of O-methyltransferase genes in transgenic tobacco affects lignin synthesis and plant growth. Phytochemistry, 2001, 57: 1167-1176
87． Marita JM, Ralph J, Hatfield RD, Chappie C. NMR characterization of lignins in Arabidopsis altered in the activity of ferulate 5-hydroxylase. Proc Natl Acad Sci USA, 1999, 96: 12328-12332
88． Huntley SK., Ellis D, Gilbert M, Chappie C, Mansfield SD. Significant increases in pulping efficiency in C4H-F5H-transformed poplars: improved chemical savings and reduced environmental toxins. J Agric Food Chem, 2003, 51: 6178-6183
89． Tamagnone L, Merida A, Parr A, Mackay S, Culianez-Macia FA, Roberts K, Martin C. The AmMYB308 and AmMYB330 transcription factors from antirrhinum regulate phenylpropanoid and lignin biosynthesis in transgenic tobacco. Plant Cell, 1998, 10: 135-154
90． Kawaoka A, Ebinuma H. Transcriptional control of lignin biosynthesis by tobacco L1M protein. Phytochemistry, 2001, 57: 1149-1157
91． Wei Jian-hua, Zhao Hua-yan, Liu Hui-rong, Song Yan-ru. CCoAOMT cDNA isolation and identification from Populus tomentosa. Acta Bot Sin, 2001, 43: 1179-1183
92． Wei JianHua, Zhao HuaYan, Lu Shan Fa, Wang Tai, Ma QingHu, Song Yan Ru. Cloning of cDNA encoding COMT from Chinese white poplar (Populus tomentosa Carr.), sequence analysis and specific expression. Acta Bot Sin, 2001,43: 326-328
93． Lu Sh F, Zhao H Y, Wei J H, Song Y R. Establishment of Tissue Culture System of Triploid Chinese White Poplar. Acta Bot Sin, 2001,43: 435-437
94． 王关林，方宏筠。植物基因工程(第二版)，科学出版社，2002
95． Nikolaeva T N. On the Relationship between the Activity of O-Methyltransferase and the Content of Lignin in Various Organs of Kidney Bean. Russian Journal of plant physiology, 2001, 48: 464-466
96
96． Whetten RW, Mackay JJ, Sederoff RR. Recent advances in understanding lignin biosynthesis. Annu Rev Plant Mol Biol. 1998, 49: 585-609
97． Douglas C, Hoffmann H, Schulz W, Hahlbrock K. Structure and elicitor or UV-light-simulated expression of two 4-coumarate: CoA ligase genes in parsley. EMBO J, 1987, 1189-1195
98． Becker-AndreM, Schulze-Lefert P, Hahlbrock K. Structural comparison, modes of expression, and putative cis-acting elements of the two 4-coumarate: CoA ligase genes in potato. J Bio Chem. 1991,266,8551-8559
99． Lee D, Douglas C J. Two divergent members of a tobacco 4-coumarate: CoA ligase (4CL) gene family. cDNA structure, gene inheritance and expression, and properties of the recombinant proteins. Plant Physiol, 1996,112,193-205
100． Zhang X H, Chiang V L. Molecular cloning of 4-coumarate: CoA ligase in loblolly pine and the roles of this enzyme in the biosynthesis of lignin in compression wood. Plant Physiol, 1997, 113,65-74
101． Meng H, Campbell W H. Substrate profiles and expression of caffeoyl coenzyme A and caffeic acid O-methyltransferases in secondary xylem of aspen during seasonal development. Plant Mol Bio, 1998, 38, 513-520
102． Kajita S,Katayama Y, Omori S. Alterations in the biosynthesis of lignin in transgenic plants with chimeric genes for 4-coumarate: coenzyme A ligase. Plant cell physiol, 1996, 37: 957-65
103． Harding SA, Leshkevich J, Chiang VL, Tsai CJ. Differential substrate inhibition couples kinetically distinct 4-coumarate: Coenzyme A ligases with spatially distinct metabolic roles in quaking aspen. Plant Physiol, 2002, 128: 428-438
104． Linderman C, Mollers B, Fliegmann J, Uhlmann A, Lottspeich F, Meimberg H, Ebel J. Divergent members of a soybean (Glycine max L.) 4-coumarate: coenzyme A ligase gene family. Eur J Biochem, 2002, 269 : 1304-1315
105． 朱之悌．我国造纸国情的若干特点及其对策．北京林业大学学报，2002，24：284-287
106． Barriere Y, Argiller O, Chabbert B. Breeding silage maize with brown-mid-rib genes: Feeding value and biochemical characteristics. Agronomic, 1994, 14: 15-25
107． Mackay J J, O'Malley D M, Presnell T. Inheritance, gene expression and lignin characterization in a mutant pine deficient in cinnamoyl alcohol dehydrogenase. Pro Natl Acad Sci USA, 1997, 94: 8255-8260
108． Pravan G J, Scobbie L, Chesson A. Characterization of lignin from CAD and OMT deficient Bm mutants of maize. J Sci food Agric, 1997, 73: 133-142
109． Wei J H, Zhao H Y, Zhang J Y, Song Y R. Cloning of cDNA encoding CCoAOMT from Populus tomentosa and down-regulation of lignin content in transgenic plant expressing antisense gene. Acta Bot Sin, 2001, 43:1179-1183
110． Higuchi T, Ito T, Umezawa T, Hibino T, Shibata D. Red-brown color of lignified tissues of transgenic plants with anti-sense CAD gene: Wine-red lignin from coniferyl aldehyde. J Biotechnol, 1994,37: 151-158
111． Williamson JD, Hirsch-Wyncott ME, Larkins BA, Gelvin SB. Differential accumulation of a transcript driven by the CaMV-35S promoter in transgenic tobacco. Plant Physiol, 1989, 90:1570-1576
112． Chappie C. Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenases. Annu Rev Plant Physiol Plant Mol Biol, 1998,49: 311-343
113． Koopmann E, Logemann E, Hahlbrock K. Regulation and functional expression of cinnamate 4-hydroxylase from parsley. Plant Physiol, 1999, 119: 49-56
114． Clark M S．主编，顾红雅瞿礼嘉主译，陈章良主校。植物分子生物学手册--实验手册。 Plant molecular biology--A laboratory mannal．第一版，北京，高等教育出版社，1998，66-80
115． Wei Jian-hua, Zhao Hua-yan, Liu Hui-rong, Song Yan-ru. CCoAOMT cDNA isolation and identification from Populus tomentosa. Acta Bot Sin, 2001, 43:1179-1183
116． Inoue K, Sewalt VJ, Murray GB, Ni W, Sturzer C, Dixon RA. Developmental Expression and Substrate Specificities of Alfalfa Caffeic Acid 3-O-Methyltransferase and Caffeoyl Coenzyme A 3-O-Methyltransferase in Relation to Lignification. Plant Physiol, 1998, 117: 761-770
11
117． Saltveit M E. Wound induced changes in phenolic metabolism and tissue browning are altered by heat shock. Postharvest Biology and Technology, 2000, 21: 61-69
118． Hoffmann L, Maury S, Bergdoll M, Thion L, Erard M, Legrand M. Identification of the enzymatic active site of tobacco caffeoyl-coenzyme A O-methyltransferase by site-directed mutagenesis. J Biol Chem, 2001, 276: 36831-36838
119． Joshi C, Chiang V L. Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases. Plant Mol Biol, 1998, 37: 663-674
120． Maury S, Geoffroy P, Legrand M. Tobacco O-methyltransferases involved in phenylpropanoid metabolism. The different caffeoyl-coenzyme A/5-hydroxyferuloyl-coenzyme A 3/5-O-methyltransferase and caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase classes have distinct substrate specificities and expression patterns. Plant Physiol, 1999, 121: 215-224．
121． Marita JM, Ralph J, Hatfield RD, Guo D, Chen F, Dixon RA. Structural and compositional modifications in lignin of transgenic alfalfa down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase. Phytochemistry, 2003, 62: 53-65．
122． Sambrook J, Fritsch E F, Maniatis T. Molecular Clone: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 1995
123． Xu YY, Chong K, Xu ZH, Tan KH. Practical techniques of in situ hybridization with RNA probe. Chin Bull Bot, 2002, 19: 234-238．
124． Campbell WH, Gowri G. Codon usage in higher plants, green algae and cyanobacteria. Plant Physiol, 1990,92: 1-11
125． Bai Y, Gong W, Liu TY & Zhu YX. Cloning and expressional analyses of a cinnamoyl CoA reductase cDNA from rice seedlings. Chin Sci Bui, 2003, 48 : 2221-2225
126． Ito H, Hiraga S, Tsugawa H, Matsui H, Honma M, Otsuki Y, Murakami T and Ohashi Y. Xylem-specific expression of wound-inducible rice peroxidase genes in transgenic plants. Plant Sci, 2000, 13:210-216