84K杨PagC3H3基因表达模式分析
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摘要
木质素作为木材的主要组成成分,通常是由3种单体聚合而成,在其生物合成过程中,共有10个酶家族参与负责将苯丙胺酸转化为单体木质素,其中C3H是在对-香豆酰辅酶A(p-coumaroyl CoA)到咖啡酰辅酶A(caffeoyl CoA)的羟基化过程和G/S单体形成中的关键控制酶类,探究PagC3H3基因表达模式,对于进一步了解该基因功能具有重要意义。该研究通过定量PCR对PagC3H3基因的组织特异性表达进行分析;克隆得到了长度为2 035 bp的PagC3H3的启动子序列,预测含有多个顺式作用元件;同时,将获得的PagC3H3的启动子序列构建植物表达载体pBI121-PagC3H3pro::GUS,进行拟南芥瞬时转化,结果显示PagC3H3基因在84K杨的根、中部茎节和基部茎节中的表达量较高;瞬时转化拟南芥,GUS染色表明:在下胚轴和根中GUS活性较强,由此推测PagC3H3基因在木质素合成过程中发挥作用。
As the main component of wood,lignin is usually polymerized from three monomers. During its biosynthesis,a total of ten enzyme families are involved in the conversion of phenylalanine to monomeric lignin,of which C3H is the key control enzymes in the hydroxylation process of p-coumaroyl CoA to caffeoyl CoA and in the formation of G/S monomer. Exploring the expression pattern of PagC3H3 gene is of great significance for further understanding of the function of this gene. In this study,the tissue-specific expression of PagC3H3 gene was analyzed by quantitative PCR; the promoter sequence of PagC3H3 with a length of 2 035 bp was cloned,and it was predicted to contain multiple cis-acting elements. At the same time,the promoter sequence of PagC3H3 was constructed to construct a plant expression vector pBI121-PagC3H3 pro: : GUS for transient transformation of Arabidopsis thaliana. The expression of PagC3H3 gene was higher in the roots,middle stem segments and basal stem segments of Populus alba × P. grandulos; transient transformation of A. thaliana showed that GUS activity was stronger in hypocotyls and roots,suggesting that PagC3H3 gene plays a role in lignin synthesis.
As the main component of wood,lignin is usually polymerized from three monomers. During its biosynthesis,a total of ten enzyme families are involved in the conversion of phenylalanine to monomeric lignin,of which C3H is the key control enzymes in the hydroxylation process of p-coumaroyl CoA to caffeoyl CoA and in the formation of G/S monomer. Exploring the expression pattern of PagC3H3 gene is of great significance for further understanding of the function of this gene. In this study,the tissue-specific expression of PagC3H3 gene was analyzed by quantitative PCR; the promoter sequence of PagC3H3 with a length of 2 035 bp was cloned,and it was predicted to contain multiple cis-acting elements. At the same time,the promoter sequence of PagC3H3 was constructed to construct a plant expression vector pBI121-PagC3H3 pro: : GUS for transient transformation of Arabidopsis thaliana. The expression of PagC3H3 gene was higher in the roots,middle stem segments and basal stem segments of Populus alba × P. grandulos; transient transformation of A. thaliana showed that GUS activity was stronger in hypocotyls and roots,suggesting that PagC3H3 gene plays a role in lignin synthesis.
引文
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15 . Brown G R,Gill G P,Kuntz R J,et al. Nucleotide diversity and linkage disequilibrium in loblolly pine[J]. Proceedings of the National Academy of Sciences of the United States of America,2004,101(42):15255-15260.
16 . Gang D R,Beuerle T,Ullmann P,et al. Differential production of meta hydroxylated phenylpropanoids in sweet basil peltate glandular trichomes and leaves is controlled by the activities of specific acyltransferases and hydroxylases[J].Plant Physiology,2002,130(3):1536-1544.
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19 .丁浩,郑唐春,梁德洋,等.一种简易高效提取多种植物纯净DNA的方法[J].植物研究,2015,35(3):457-461.Ding H,Zheng T C,Liang D Y,et al. A simple and efficient method of DNA extraction from different plant samples[J]. Bulletin of Botanical Research,2015,35(3):457-461.
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21 . Li L G,Cheng X F,Leshkevich J,et al. The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase[J]. The Plant Cell,2001,13(7):1567-1585.
22 . Coleman H D,Park J Y,Nair R,et al. RNAi-mediated suppression of p-coumaroyl-CoA 3'-hydroxylase in hybrid poplar impacts lignin deposition and soluble secondary metabolism[J]. Proceedings of the National Academy of Sciences of the United States of America,2008,105(11):4501-4506.
23 .李杰,张福城,王文泉,等.高等植物启动子的研究进展[J].生物技术通讯,2006,17(4):658-661.Li J,Zhang F C,Wang W Q,et al. Advance in the study of higher plant promoter[J]. Letters in Biotechnology,2006,17(4):658-661.
24 .聂丽娜,夏兰琴,徐兆师,等.植物基因启动子的克隆及其功能研究进展[J].植物遗传资源学报,2008,9(3):385-391.Nie L N,Xia L Q,Xu Z S,et al. Progress on cloning and functional study of plant gene promoters[J]. Journal of Plant Genetic Resources,2008,9(3):385-391.
25 .李一琨,王金发.高等植物启动子研究进展[J].植物学通报,1998,15(S1):1-6.Li Y K,Wang J F. Advances of the studies on plant promoter[J]. Chinese Bulletin of Botany,1998,15(S1):1-6.
2 . Chiang V L. From rags to riches[J]. Nature Biotechnology,2002,20(6):557-558.
3 . Lee B R,Kim K Y,Jung W J,et al. Peroxidases and lignification in relation to the intensity of water-deficit stress in white clover(Trifolium repens L.)[J]. Journal of Experimental Botany,2007,58(6):1271-1279.
4 . Jubany-MaríT,Munné-Bosch S,López-Carbonell M,et al.Hydrogen peroxide is involved in the acclimation of the Mediterranean shrub,Cistus albidus L. to summer drought[J]. Journal of Experimental Botany,2009,60(1):107-120.
5 . Xu Q,Yin X R,Zeng J K,et al. Activator-and repressortype MYB transcription factors are involved in chilling injury induced flesh lignification in loquat via their interactions with the phenylpropanoid pathway[J]. Journal of Experimental Botany,2014,65(15):4349-4359.
6 . Scurfield G. Reaction Wood:its structure and function:Lignification may generate the force active in restoring the trunks of leaning trees to the vertical[J]. Science,1973,179(4074):647-655.
7 . Timell T E. Compression wood in gymnosperms. Volume 1:bibliography,historical background,determination,structure,chemistry,topochemistry,physical properties,origin,and formation of compression wood[J]. The Quarterly Review of Biology,1987,62(4):449.
8 . Dixon R A. Natural products and plant disease resistance[J]. Nature,2001,411(6839):843-847.
9 .高原,陈信波,张志扬.木质素生物合成途径及其基因调控的研究进展[J].生物技术通报,2007,(2):47-51.Gao Y,Chen X B,Zhang Z Y. Advances in research on lignin biosynthesis and its molecular regulation[J]. Biotechnology Bulletin,2007,(2):47-51.
10 . Hu W J,Harding S A,Lung J,et al. Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees[J]. Nature Biotechnology,1999,17(8):808-812.
11 .王雪霞,薛永常,赵文超.木质素生物合成中C3H/HCT的研究进展[J].生命的化学,2008,28(5):650-653.Wang X X,Xue Y C,Zhao W C. The research progress of C3H/HCT in lignin biosynthesis[J]. Chemistry of Life,2008,28(5):650-653.
12 . Schoch G,Goepfert S,Morant M,et al. CYP98A3 from Arabidopsis thaliana is a 3'-hydroxylase of phenolic esters,a missing link in the phenylpropanoid pathway[J]. Journal of Biological Chemistry,2001,276(39):36566-36574.
13 . Franke R,Humphreys J M,Hemm M R,et al. The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism[J]. The Plant Journal,2002,30(1):13-45.
14 . Anterola A M,Jeon J H,Davin L B,et al. Transcriptional control of monolignol biosynthesis in Pinus taeda:factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism[J]. The Journal of Biological Chemistry,2002,277(21):18272-18280.
15 . Brown G R,Gill G P,Kuntz R J,et al. Nucleotide diversity and linkage disequilibrium in loblolly pine[J]. Proceedings of the National Academy of Sciences of the United States of America,2004,101(42):15255-15260.
16 . Gang D R,Beuerle T,Ullmann P,et al. Differential production of meta hydroxylated phenylpropanoids in sweet basil peltate glandular trichomes and leaves is controlled by the activities of specific acyltransferases and hydroxylases[J].Plant Physiology,2002,130(3):1536-1544.
17 . Fao(Food and Agriculture Organization of the United Nations). State of the World’s Forests 2012[R]. Rome,Italy:FAO,2012.
18 . Chen F,Dixon R A. Lignin modification improves fermentable sugar yields for biofuel production[J]. Nature Biotechnology,2007,25(7):759-761.
19 .丁浩,郑唐春,梁德洋,等.一种简易高效提取多种植物纯净DNA的方法[J].植物研究,2015,35(3):457-461.Ding H,Zheng T C,Liang D Y,et al. A simple and efficient method of DNA extraction from different plant samples[J]. Bulletin of Botanical Research,2015,35(3):457-461.
20 . Ji X Y,Zheng L,Liu Y J,et al. A transient transformation system for the functional characterization of genes involved in stress response[J]. Plant Molecular Biology Reporter,2014,32(3):732-739.
21 . Li L G,Cheng X F,Leshkevich J,et al. The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase[J]. The Plant Cell,2001,13(7):1567-1585.
22 . Coleman H D,Park J Y,Nair R,et al. RNAi-mediated suppression of p-coumaroyl-CoA 3'-hydroxylase in hybrid poplar impacts lignin deposition and soluble secondary metabolism[J]. Proceedings of the National Academy of Sciences of the United States of America,2008,105(11):4501-4506.
23 .李杰,张福城,王文泉,等.高等植物启动子的研究进展[J].生物技术通讯,2006,17(4):658-661.Li J,Zhang F C,Wang W Q,et al. Advance in the study of higher plant promoter[J]. Letters in Biotechnology,2006,17(4):658-661.
24 .聂丽娜,夏兰琴,徐兆师,等.植物基因启动子的克隆及其功能研究进展[J].植物遗传资源学报,2008,9(3):385-391.Nie L N,Xia L Q,Xu Z S,et al. Progress on cloning and functional study of plant gene promoters[J]. Journal of Plant Genetic Resources,2008,9(3):385-391.
25 .李一琨,王金发.高等植物启动子研究进展[J].植物学通报,1998,15(S1):1-6.Li Y K,Wang J F. Advances of the studies on plant promoter[J]. Chinese Bulletin of Botany,1998,15(S1):1-6.
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