COMT、CCOAOMT基因功能分析及三倍体毛白杨组培转化体系的建立
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摘要
木质素是植物体内含量仅次于纤维素的一类次生代谢产物，具有重要的生物学功能。然而，在制浆造纸过程中，通过化学方法去除木质素不但造成严重的环境污染，而且大大增加了造纸的成本。本研究旨在通过反义RNA技术，对木质素的生物合成进行分子水平上的调控，以降低造纸资源植物的木质素含量或改变组分，培育更适于造纸工业用的原料树种。本论文取得如下结果：(1) 通过农杆菌介导法，将反义表达载体APCOA、APCOMT、APCC转化烟草。PCR、PCR-Southern检测证实，外源基因已整合到烟草基因组中。Northern点杂交结果表明外源基因在转录水平表达。将转基因植株移栽入温室培养，三个月后测定Klason木质素含量并进行Wiesner、Maülar组织化学染色。其结果，下调CCoAOMT可有效降低烟草木质素的含量，同时抑制CCoAOMT、COMT对木质素含量及组分具有协同效应。转基因植株在生长过程中未见明显的形态变化。本研究结果进一步证明了两个甲基化酶在木质素生物合成途径中的功能，为通过基因工程调节毛白杨木质素生物合成提供了理论依据；(2) 首次建立了三倍体毛白杨的遗传转化体系，为通过基因工程调控毛白杨木质素生物合成奠定基础。
Lignin is an abundant secondary product after cellulose in plants with important biofuntions. But in the paper pulping process, removing lignin by chemical methods not only lead to very seriously environmental pollution ,but also increase the cost of paper making. The purpose of our study is to regulate the lignin biosynthesis of Chinese white poplar(Populus tomentosa) on molecular level by antisense RNA technology to breed the resource tree species more suitable for paper making in China. Here are the main results of the study: (1) Antisense CCoAOMT cDNA、antisense COMT cDNA and both of them were respectively transformed into tobacco mediated By Agrobacterium tumficense. The results of PCR and PCR-Southern confirmed that the transgenes had integrated into the tobacco genome. Northern dot analysis showed that the antisense cDNAs had expressed at the transcriptional level. After growing about 3 months in the green house, the transgenic plants were conducted for Klason lignin and histochemical anaylesis. The results showed that down-regulating CCoAOMT resulted in the reduction of the lignin content of tobacco efficiently. Simultaneous repression of CCoAOMT and COMT had combinative effects on lignin content. Down-regulating COMT led to the reduction of S-lignin content of the transgenic plants, which demonstrated that COMT was a critical enzyme in the S-lignin biosynthesis. Moreover, the transgenic lines were foundamentally consistent with the control plants during the development and growth. Our study further proved the function of the two OMTs in the lignin biosynthesis and provided theoretical basis for regulating the lignin biosynthesis of Chinese white poplar by the genetic engineering; (2).The system of tissue culture and transformation of the triploid Chinese whitepoplar was established for the first time,which lay a foundation for regulationof the lignin biosynthesis by gene engineering and biotechnology in Chinese white poplar.
Lignin is an abundant secondary product after cellulose in plants with important biofuntions. But in the paper pulping process, removing lignin by chemical methods not only lead to very seriously environmental pollution ,but also increase the cost of paper making. The purpose of our study is to regulate the lignin biosynthesis of Chinese white poplar(Populus tomentosa) on molecular level by antisense RNA technology to breed the resource tree species more suitable for paper making in China. Here are the main results of the study: (1) Antisense CCoAOMT cDNA、antisense COMT cDNA and both of them were respectively transformed into tobacco mediated By Agrobacterium tumficense. The results of PCR and PCR-Southern confirmed that the transgenes had integrated into the tobacco genome. Northern dot analysis showed that the antisense cDNAs had expressed at the transcriptional level. After growing about 3 months in the green house, the transgenic plants were conducted for Klason lignin and histochemical anaylesis. The results showed that down-regulating CCoAOMT resulted in the reduction of the lignin content of tobacco efficiently. Simultaneous repression of CCoAOMT and COMT had combinative effects on lignin content. Down-regulating COMT led to the reduction of S-lignin content of the transgenic plants, which demonstrated that COMT was a critical enzyme in the S-lignin biosynthesis. Moreover, the transgenic lines were foundamentally consistent with the control plants during the development and growth. Our study further proved the function of the two OMTs in the lignin biosynthesis and provided theoretical basis for regulating the lignin biosynthesis of Chinese white poplar by the genetic engineering; (2).The system of tissue culture and transformation of the triploid Chinese white
引文
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2 Vance C P, Kirk T K, and Sherwood R T. Lignification as a mechanism of disease resistance. Annu Rev Phytopath, 1980, 18:259-288
3 Lewis N G, Yamamoto E. Lignin: occurrence, biogenesis and bio-degrada- tion. Annu Rev Plant Physiol and Plant Mol Biol, 1990, 41:455-496
4 Lukas S. Chemical composition of casparian strops isolated from Clivia miniata Reg, root evidence for lignin. Planta, 1996, 199:596-601
5 中野准三（日）编，木质素的化学—基础与应用。高洁，鲍禾，李中正，译。轻工业出版社，1988。
6 Chen C L. Lignin：occurrence in woody tissues，isolation，reactions，and structure. In wood structure and composition. Lewin M, Goldstein IS, eds. New York: Dekker, 1991
7 Sederoff R R, Mackay J J, Ralph J. Unexpected variation in lignin. Current Opin Plant Biol, 1999, 2:145-152
8 Fukuda H. Tracheary element differentiation. Plant Cell, 1997, 9: 1147 -1156
9 Bugos R C, Vincent L C, Campell W H. CDNA cloning, sequence analysis and seasonal expression of lignin-bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase of aspen. Plant Mol Biol, 1991, 17:1203-1215
10 Baucher M, Monties B, Montagu M V, and Boerjan W. Biosynthesis and genetic engineering of lignin. Critical Reviews in Plant Sciences, 1998, 17(2):125-197
11 Sederoff R. Building better trees with antisense RNA technology. Nature Biotechnology, 1998, 17:750-751
12 Rosler J, Krekel F, Amrhein N. Maize phenylalanine ammonia-lyase has tyrosine ammonia-lyase activity. Plant Physiol, 1997, 113:175-179
13 Barriere Y, Argiller O, Chabbert B, Tollier M T, and Monties B. Breeding silage maize with brown-mid-rib genes: Feeding value and biochemical characteristics. Agronomie, 1994, 14:15-25
14 Sewalt V J H, Ni W, Blount J W. Reduced lignin content and altered lignin composition in transgenic tobacco down-regulated in expression of L-phenylalanine ammonia lyase or cinnamate 4-hydrelase. Plant Physiology, 1997,
15 Rugger M, Myer K, Joanne C.C, Chapple C. Regulation of ferulate- 5-hydroxylase expression in Arabidopsis in the context of sinapate esterbisynthesis. Plant Physiology, 1999, 119:101-110
16 Franke R, McMichael C M, Meyer K. Modified lignin in tobacco and poplar plants over-expressing the Arabidopsis gene encoding ferulate 5-hydroxylase. Plant J, 2000, 22:223-234
17 Pakusch A E, Jbeysek R E,Naterb U. S-Adenosyl-L-methionine: trans-caffeoyl- coenzyme A 3-O-methyltransferase from elicitor-treated parsley cell suspension cultures. Arch. Biochem. Biophys, 1989 , 271: 488-494
18 Kuhnl T, Koch U, Heller W. Elicitor induced S-adenosyl-L- methionine: caffeoyl CoA 3-O-methyltransferase from carrot cell suspension cultures. Plant Sci. 1989, 60:21-25
19 Pakusch A E, Matern U and Schiltz E, Elicitor-inducible caffeoyl- coenzyme A 3-O-methyltransferase from Petroselinum cripum cell suspensions. Plant Physiology, 1991a, 95:137-143
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