白桦反义CCoAOMT基因调控木质素生物合成
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摘要
白桦是我国北方重要的造林树种,但其中的高木质素含量严重制约了它作为造纸资源植物的开发利用。本文利用RACE技术获得了白桦咖啡酰辅酶A-3-O-甲基转移酶(CCoAOMT)基因全长ORF序列,并构建了白桦CCoAOMT基因的反义表达载体,通过农杆菌介导法将其导入到白桦中。PCR检测表明反义CCoAOMT基因已整合到白桦的基因组中。对转化植株的半定量PCR检测显示转基因株系的CCoAOMT基因表达量下降;Wiesner染色发现,与野生型相比,转基因植株木质素含量有所下降。对七年生的转基因白桦和野生型对照进行了化学成分分析,结果表明转基因白桦的苯醇抽提物和Klason木质素显著减少,聚戊糖含量升高。上述结果暗示BpCCoAOMT基因参与白桦木质素的合成,反义表达该基因后木质素含量减少,更易于去除。白桦CCoAOMT基因对木质素的合成起重要作用,这为培育低木质素含量的制浆新品种白桦奠定了基础。
Betula platyphylla is an important forestation tree species in northern China,but its high lignin content severely restricts its development and utilization as a papermaking resource plant.In this paper,the full-length ORF sequence of CCoAOMT(Caffeoyl-coenzyme A-3-O-methyltransferase) gene was obtained using RACE technology.The antisense CCoAOMT expression vector was constructed and transformed into B.platyphylla by Agrobacterium infection method.The molecular detection indicated that the CCoAOMT antisense gene had been successfully integrated into B.platyphylla.By semi-quantitative PCR analysis of the transgenic plants,the CCoAOMT gene expression of the transgenic lines was decreased.Compared to the wild type,by the Wiesner staining,the lignin content of the transgenic plants was decreased.Chemical compositions of seven-year-old transgenic and wild-type birch were analyzed.The phenyl alcohol extracts and Klason lignin of transgenic birch were significantly reduced,and the pentosan content was increased.The BpCCoAOMT gene plays an important role in the synthesis of lignin,which lays a foundation for the cultivation of a new species of birch with low lignin content.
Betula platyphylla is an important forestation tree species in northern China,but its high lignin content severely restricts its development and utilization as a papermaking resource plant.In this paper,the full-length ORF sequence of CCoAOMT(Caffeoyl-coenzyme A-3-O-methyltransferase) gene was obtained using RACE technology.The antisense CCoAOMT expression vector was constructed and transformed into B.platyphylla by Agrobacterium infection method.The molecular detection indicated that the CCoAOMT antisense gene had been successfully integrated into B.platyphylla.By semi-quantitative PCR analysis of the transgenic plants,the CCoAOMT gene expression of the transgenic lines was decreased.Compared to the wild type,by the Wiesner staining,the lignin content of the transgenic plants was decreased.Chemical compositions of seven-year-old transgenic and wild-type birch were analyzed.The phenyl alcohol extracts and Klason lignin of transgenic birch were significantly reduced,and the pentosan content was increased.The BpCCoAOMT gene plays an important role in the synthesis of lignin,which lays a foundation for the cultivation of a new species of birch with low lignin content.
引文
1.宋福南.白桦木质素合成苯丙氨酸途径相关酶基因的表达和功能分析[D].哈尔滨:东北林业大学,2009.Song F N.Expression and functional analysis of correlative enzyme gene of lignin Phenylpropanoid Biosynthetic Pathway in Betula Platphylla[D].Harbin:Northeast Forestry University,2009.
2.Ye Z H.Association of caffeoyl coenzyme A3-O-methyltransferase expression with lignifying tissues in several dicot plants[J].Plant Physiology,1997,115(4):1341-1350.
3.黄春琼,刘国道,郭安平.反义CCo AOMT基因调控烟草木质素的生物合成[J].安徽农业科学,2008,36(19):8026-8027,8043.Huang C Q,Liu G D,Guo A P.Lignin biosynthesis regulated by antisense CCo AOMT gene in tobacco[J].Journal of Anhui Agricultural Sciences,2008,36(19):8026-8027,8043.
4.王玲,田敏,段红平,等.反义CCo AOMT基因转化烟草调控木质素生物合成[J].江苏农业科学,2011,39(2):62-64.Wang L,Tian M,Duan H P,et al.Study on content and ratio of lignin in transformed tobacco plants changed by antisense CCo AOMT[J].Jiangsu Agricultural Science,2011,39(2):62-64.
5.倪志勇,吕萌,范玲.棉花咖啡酰辅酶A-O-甲基转移酶基因的克隆及特征分析[J].西北植物学报,2010,30(6):1083-1091.Ni Z Y,LüM,Fan L.Cloning and characterization of CCo AOMT gene from Gossypium hirsutum L.[J].Acta Botanica Boreali-Occidentalia Sinica,2010,30(6):1083-1091.
6.Guo D J,Chen F,Inoue K,et al.Downregulation of caffeic acid 3-O-methyltransferase and caffeoyl Co A 3-O-methyltransferase in transgenic alfalfa.Impacts on lignin structure and implications for the biosynthesis of G and S lignin[J].The Plant Cell,2001,13(1):73-88.
7.刘红梅,胡尚连,卢学琴,等.青杨CCo AOMT基因的克隆及其生物信息学分析[J].湖北农业科学,2014,53(11):2670-2674.Liu H M,Hu S L,Lu X Q,et al.Cloning and bioinformatics analysis of CCo AOMT gene of Populus cathayana Rehd[J].Hubei Agricultural Science,2014,53(11):2670-2674.
8.赵华燕,魏建华,路静,等.利用反义CCo AOMT基因培育低木质素含量毛白杨的研究[J].自然科学进展,2004,14(9):1067-1071.Zhao H Y,Wei J H,Lu J,et al.Studies on the cultivation of Populus tomentosa with low lignin content by antisense CCo AOMT gene[J].Progress in Natural Sciences,2004,14(9):1067-1071.
9.魏建华,王彦珍,王宏芝,等.抑制CCo AOMT表达的转基因杨树的制浆性能[J].科学通报,2008,53(21):2612-2616.Wei J H,Wang Y Z,Wang H Z,et al.Pulping performance of transgenic poplar with depressed Caffeoyl-CoA O-methyltransferase[J].Chinese Science Bulletin,2008,53(22):3553-3558.
10.张学科,刘文俊,朱明华,等.木质素酚醛树脂的研究进展[J].植物研究,2012,32(6):760-764,769.Zhang X K,Liu W J,Zhu M H,et al.Research progress of phenol-formaldehyde resin[J].Bulletin of Botanical Research,2012,32(6):760-764,769.
11.赵华燕.利用反义RNA技术进行木质素生物合成调控的研究[D].北京:中国科学院研究生院,2004.Zhao H Y.Studies on the regulation of lignin biosynthesis by antisense RNA technique[D].Beijing:Graduate School of Chinese Academy of Sciences:Institute of Botany,2004.
12.Shi R,Sun Y H,Li Q Z,et al.Towards a systems approach for lignin biosynthesis in Populus trichocarpa:transcript abundance and specificity of the monolignol biosynthetic genes[J].Plant and Cell Physiology,2009,51(1):144-163.
13.陈肃.白桦4CL与CCo AOMT基因表达分析及蛋白预测[D].哈尔滨:东北林业大学,2009.Chen S.Expression and forecast of 4CL and CCo AOMT of Birch[D].Harbin:Northeast Forestry University,2009.
14.姜爽.CCo AOMT和4CLc DNA的克隆及转基因紫穗槐的研究[D].长春:吉林大学,2007.Jiang S.Cloning of two c DNA encoding CCo AOMT and 4CLand their transformation in Amorpha fruitcosa[D].Changchun:Jilin University,2007.
15.赵华燕,魏建华,宋艳茹.木质素生物合成及其基因工程研究进展[J].植物生理与分子生物学学报,2004,30(4):361-370.Zhao H Y,Wei J H,Song Y R.Advances in research on lignin biosynthesis and its genetic engineering[J].Journal of Plant Physiology and Molecular Biology,2004,30(4):361-370.
16.Wagner A,Tobimatsu Y,Phillips L,et al.CCo AOMT suppression modifies lignin composition in Pinus radiata[J].The Plant Journal,2011,67(1):119-129.
17.贾彩红.低木质素含量的转基因毛白杨(Populus tomentosa)的获得与毛白杨4CL基因启动子的克隆[D].保定:河北农业大学,2004.Jia C H.Obtaining the transgenic poplars with low lignin content and cloning of 4CL promoter from Chinese white poplar(Populus tomentosa)[D].Baoding:Hebei Agricultural University,2004.
18.徐洋,蔺占兵,马庆虎.木质素与人类生活[J].植物杂志,2002(4):40-41.Xu Y,Lin Z B,Ma Q H.Lignin and human life[J].Plants,2002(4):40-41.
19.Umezawa T.The cinnamate/monolignol pathway[J].Phytochemistry Reviews,2010,9(1):1-17.
20.涂红艳,刘元风.反义RNA的作用机理及其在植物基因工程领域的应用[J].生物磁学,2005(02):32-34.Tu H Y,Liu Y F.The mechanism of antisense RNA and its application in plant gene engineering[J].Biomagnetism.2005(02):32-34.
21.马兴亮,刘耀光.植物CRISPR/Cas9基因组编辑系统与突变分析[J].遗传,2016,38(2):118-125.Ma X L,Liu Y G.CRISPR/Cas9-based genome editing systems and the analysis of targeted genome mutations in plants[J].Hereditas,2016,38(2):118-125.
2.Ye Z H.Association of caffeoyl coenzyme A3-O-methyltransferase expression with lignifying tissues in several dicot plants[J].Plant Physiology,1997,115(4):1341-1350.
3.黄春琼,刘国道,郭安平.反义CCo AOMT基因调控烟草木质素的生物合成[J].安徽农业科学,2008,36(19):8026-8027,8043.Huang C Q,Liu G D,Guo A P.Lignin biosynthesis regulated by antisense CCo AOMT gene in tobacco[J].Journal of Anhui Agricultural Sciences,2008,36(19):8026-8027,8043.
4.王玲,田敏,段红平,等.反义CCo AOMT基因转化烟草调控木质素生物合成[J].江苏农业科学,2011,39(2):62-64.Wang L,Tian M,Duan H P,et al.Study on content and ratio of lignin in transformed tobacco plants changed by antisense CCo AOMT[J].Jiangsu Agricultural Science,2011,39(2):62-64.
5.倪志勇,吕萌,范玲.棉花咖啡酰辅酶A-O-甲基转移酶基因的克隆及特征分析[J].西北植物学报,2010,30(6):1083-1091.Ni Z Y,LüM,Fan L.Cloning and characterization of CCo AOMT gene from Gossypium hirsutum L.[J].Acta Botanica Boreali-Occidentalia Sinica,2010,30(6):1083-1091.
6.Guo D J,Chen F,Inoue K,et al.Downregulation of caffeic acid 3-O-methyltransferase and caffeoyl Co A 3-O-methyltransferase in transgenic alfalfa.Impacts on lignin structure and implications for the biosynthesis of G and S lignin[J].The Plant Cell,2001,13(1):73-88.
7.刘红梅,胡尚连,卢学琴,等.青杨CCo AOMT基因的克隆及其生物信息学分析[J].湖北农业科学,2014,53(11):2670-2674.Liu H M,Hu S L,Lu X Q,et al.Cloning and bioinformatics analysis of CCo AOMT gene of Populus cathayana Rehd[J].Hubei Agricultural Science,2014,53(11):2670-2674.
8.赵华燕,魏建华,路静,等.利用反义CCo AOMT基因培育低木质素含量毛白杨的研究[J].自然科学进展,2004,14(9):1067-1071.Zhao H Y,Wei J H,Lu J,et al.Studies on the cultivation of Populus tomentosa with low lignin content by antisense CCo AOMT gene[J].Progress in Natural Sciences,2004,14(9):1067-1071.
9.魏建华,王彦珍,王宏芝,等.抑制CCo AOMT表达的转基因杨树的制浆性能[J].科学通报,2008,53(21):2612-2616.Wei J H,Wang Y Z,Wang H Z,et al.Pulping performance of transgenic poplar with depressed Caffeoyl-CoA O-methyltransferase[J].Chinese Science Bulletin,2008,53(22):3553-3558.
10.张学科,刘文俊,朱明华,等.木质素酚醛树脂的研究进展[J].植物研究,2012,32(6):760-764,769.Zhang X K,Liu W J,Zhu M H,et al.Research progress of phenol-formaldehyde resin[J].Bulletin of Botanical Research,2012,32(6):760-764,769.
11.赵华燕.利用反义RNA技术进行木质素生物合成调控的研究[D].北京:中国科学院研究生院,2004.Zhao H Y.Studies on the regulation of lignin biosynthesis by antisense RNA technique[D].Beijing:Graduate School of Chinese Academy of Sciences:Institute of Botany,2004.
12.Shi R,Sun Y H,Li Q Z,et al.Towards a systems approach for lignin biosynthesis in Populus trichocarpa:transcript abundance and specificity of the monolignol biosynthetic genes[J].Plant and Cell Physiology,2009,51(1):144-163.
13.陈肃.白桦4CL与CCo AOMT基因表达分析及蛋白预测[D].哈尔滨:东北林业大学,2009.Chen S.Expression and forecast of 4CL and CCo AOMT of Birch[D].Harbin:Northeast Forestry University,2009.
14.姜爽.CCo AOMT和4CLc DNA的克隆及转基因紫穗槐的研究[D].长春:吉林大学,2007.Jiang S.Cloning of two c DNA encoding CCo AOMT and 4CLand their transformation in Amorpha fruitcosa[D].Changchun:Jilin University,2007.
15.赵华燕,魏建华,宋艳茹.木质素生物合成及其基因工程研究进展[J].植物生理与分子生物学学报,2004,30(4):361-370.Zhao H Y,Wei J H,Song Y R.Advances in research on lignin biosynthesis and its genetic engineering[J].Journal of Plant Physiology and Molecular Biology,2004,30(4):361-370.
16.Wagner A,Tobimatsu Y,Phillips L,et al.CCo AOMT suppression modifies lignin composition in Pinus radiata[J].The Plant Journal,2011,67(1):119-129.
17.贾彩红.低木质素含量的转基因毛白杨(Populus tomentosa)的获得与毛白杨4CL基因启动子的克隆[D].保定:河北农业大学,2004.Jia C H.Obtaining the transgenic poplars with low lignin content and cloning of 4CL promoter from Chinese white poplar(Populus tomentosa)[D].Baoding:Hebei Agricultural University,2004.
18.徐洋,蔺占兵,马庆虎.木质素与人类生活[J].植物杂志,2002(4):40-41.Xu Y,Lin Z B,Ma Q H.Lignin and human life[J].Plants,2002(4):40-41.
19.Umezawa T.The cinnamate/monolignol pathway[J].Phytochemistry Reviews,2010,9(1):1-17.
20.涂红艳,刘元风.反义RNA的作用机理及其在植物基因工程领域的应用[J].生物磁学,2005(02):32-34.Tu H Y,Liu Y F.The mechanism of antisense RNA and its application in plant gene engineering[J].Biomagnetism.2005(02):32-34.
21.马兴亮,刘耀光.植物CRISPR/Cas9基因组编辑系统与突变分析[J].遗传,2016,38(2):118-125.Ma X L,Liu Y G.CRISPR/Cas9-based genome editing systems and the analysis of targeted genome mutations in plants[J].Hereditas,2016,38(2):118-125.