叶桉COMT和CCoAOMT基因定向调控木质素单体合成的烟草转化研究*

doi:10.13523/j.cb.20180304

中国生物工程杂志

2018, Vol. 38

Issue (3): 24-32 DOI: 10.13523/j.cb.20180304

研究报告

叶桉COMT和CCoAOMT基因定向调控木质素单体合成的烟草转化研究^*

陈博雯(

),刘海龙,肖玉菲,覃子海,张烨,张晓宁

广西壮族自治区林业科学研究院国家林业局中南速生材繁育实验室广西优良用材林资源培育重点实验室南宁 530002

Directional Regulation of Lignin Monomer Synthesis in Tobacco by Using COMT Gene and CCoAOMT Gene of Eucalyptus urophylla

Bo-wen CHEN(

),Hai-long LIU,Yu-fei XIAO,Zi-hai QIN,Ye ZHANG,Xiao-ning ZHANG

Guangxi Forestry Research Institute, Key Laboratory of Central South Fast-growing Timber Cultivation of Forestry Ministry of China, Guangxi Key Laboratory of Superior Timber Trees Resource Cultivation, Nanning 530002, China

全文: PDF(1258 KB) HTML

摘要： 目的

利用烟草遗传转化体系,研究叶桉(Eucalyptus urophylla)咖啡酸氧甲基转移酶基因(EuCOMT)和咖啡酰CoA氧甲基转移酶基因(EuCCoAOMT)对木质素单体合成的定向调控效果。

方法

分别利用EuCOMT的正义片段、EuCCoAOMT的全长RNAi片段进行单基因和二价基因的烟草转化研究,并对转基因烟草植株中目标基因表达水平、木质素和纤维素的含量、茎部解剖结构及木质素单体含量进行检测。

结果

分别获得了转基因植株C-S(转EuCOMT正义片段)、CR(转EuCCoAOMT全长RNAi片段)、C-CR(EuCOM和EuCCoAOMT二价基因转化)。烟草中转入的正义EuCOMT的片段能够正常表达,而EuCCoAOMT的全长RNAi片段对烟草CCoAOMT基因引发了强烈的抑制。转基因烟草的生长形态、木质素、纤维含量及解剖结构与野生型无显著差异。转基因植株C-S中G木质素含量升高17.72%,S/G值降低17.99%;CR中S/G值升高61.62%,C-CR中G木质素降幅达到57.38%,S/G比值升幅达到114.94%。

结论

抑制CCoAOMT对G木质素合成具有显著的抑制效果,EuCOMT和EuCCoAOMT二价基因转化对S/G比值的定向调控效果最为理想。

关键词： 二价基因转化; 木质素单体合成; 定向调控; 烟草转化

Abstract:

Directional regulation effect of Eucalyptus urophylla COMT gene and CCoAOMT gene on lignin monomer synthesis were studied by tobacco transformation system. Transgenic plants C-S(contain sense EuCOMT fragment), CR(contain full-length RNAi fragment of EuCCoAOMT), C-CR(contain EuCOM and EuCCoAOMT dual-gene) were obtained, and the expression of sense EuCOMT fragment was detected in transgenic tobacco, while the RNAi fragment of EuCCoAOMT caused strong inhibition on tobacco CCoAOMT gene. The growth morphology, lignin content, fiber content and anatomical structure of transgenic tobacco were not significantly different from those of wild type. The results of lignin monomer detection showed the G lignin content increased 17.72% and S/G ratio decreased 17.99% in transgenic plant C-S, meanwhile the S/G ratio increased 61.62% in CR. In C-CR, G lignin content decreased 57.38%, and S/G ratio increased 114.94%. The result showed that the inhibition of CCoAOMT had a significant inhibitory effect on lignin synthesis in G, and the dual-gene transformation of EuCOMT and EuCCoAOMT showing the best directional regulation effect.

Key words: Dual-gene transformation Lignin monomer synthesis Directional regulation Tobacco transformation

收稿日期: 2017-11-28 出版日期: 2018-04-04

ZTFLH:

Q78

基金资助: 国家自然科学基金(31400522);广西林业科技项目(桂林科字[2014]第33号);广西林业科技项目(桂林科字[2016]第11号)

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	陈博雯
	刘海龙
	肖玉菲
	覃子海
	张烨
	张晓宁

引用本文:

陈博雯,刘海龙,肖玉菲,覃子海,张烨,张晓宁. 叶桉COMT和CCoAOMT基因定向调控木质素单体合成的烟草转化研究^*[J]. 中国生物工程杂志, 2018, 38(3): 24-32.

Bo-wen CHEN,Hai-long LIU,Yu-fei XIAO,Zi-hai QIN,Ye ZHANG,Xiao-ning ZHANG. Directional Regulation of Lignin Monomer Synthesis in Tobacco by Using COMT Gene and CCoAOMT Gene of Eucalyptus urophylla. China Biotechnology, 2018, 38(3): 24-32.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180304 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I3/24

图1 表达载体结构示意图

表1 本研究所使用的引物

图2 烟草转化苗PCR验证

图3 烟草遗传转化

图4 转基因烟草中基因表达水平分析

表2 转基因烟草中木质素、纤维素含量

表3 转化植株茎部切片测量结果

图5 木质部M?ule染色

表4 转基因烟草茎部木质素单体含量分析

[1]	何柏华, 付强, 曾嵘. 广西南部桉树工业原料林立地质量评价. 广西林业科学, 2017, 46(3):325-328.
	He B H, Fu Q, Zeng R. Site quality evaluation of eucalyptus industrial raw material forest in Southern Guangxi. Guangxi Forestry Science, 2017, 46(3):325-328.
[2]	黎怀玲, 付朝阳, 廖仁雅,等. 6年生杂交桉无性系对比试验. 广西林业科学, 2016, 45(2):195-198.
	Li H L,Fu C Y, Liao R Y, et al.A Comparison Trial of 6-Year-Old Hybrid Eucalyptus Clones. Guangxi Forestry Science, 2016, 45(2):195-198.
[3]	赵艳玲. 正义、反义和RNA干扰技术调控杨树木质素生物合成途径. 北京: 北京林业大学,2006.
	Zhao Y L.Modulation of Lignin Biosynthesis of Poplar by Sense, Antisense and RNA Interference Technology. Beijing: Beijing Forestry University,2006.
[4]	Trabucco G M, Matos D A, Lee S J, et al.Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon. Bmc Biotechnology, 2013, 13(1): 1-18. doi: 10.1186/1472-6750-13-1
[5]	Tu Y, Rochfort S, Liu Z, et al.Functional analyses of caffeic acid O-methyltransferase and cinnamoyl-coa-reductase genes from perennial ryegrass (Lolium perenne). Plant Cell, 2010, 22(10): 3357-3373. doi: 10.1105/tpc.109.072827 pmid: 20952635
[6]	Guo D, Chen F, Inoue K, et al.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(1): 73-88. doi: 10.1105/tpc.13.1.73
[7]	Piquemal J, Chamayou S, Nadaud I, et al.Down-regulation of caffeic acid O-methyltransferase in maize revisited using a transgenic approach. Plant Physiology, 2003, 130(4): 1675-1685.
[8]	Zhong R, Himmelsbach D S, Ye Z H.Essential role of caffeoyl coenzyme a O-methyltransferase in lignin biosynthesis in woody poplar plants. Plant Physiology, 2000, 124(2): 563-577. doi: 10.1104/pp.124.2.563
[9]	魏建华, 王彦珍, 王宏芝, 等. 抑制CCoAOMT表达的转基因杨树的制浆性能. 科学通报, 2008,53(21): 2612-2616.
	Wei J H, Wang Y Z, Wang H Z, et al.Pulping properties of transgenic poplar with inhibition of CCoAOMT expression. Chinese Science Bulletin, 2008,53(21): 2612-2616.
[10]	Lee Y, Chen F, Gallegogiraldo L, et al.Integrative analysis of transgenic alfalfa (Medicago sativa L.) suggests new metabolic control mechanisms for monolignol biosynthesis. Plos Computational Biology, 2011, 7(5): e1002047. doi: 10.1371/journal.pcbi.1002047 pmid: 21625579
[11]	Zhou R.Distinct cinnamoyl coa reductases involved in parallel routes to lignin in Medicago truncatula. Proceedings of the National Academy of Sciences, 2010, 107(41): 17803-17808.
[12]	赵华燕, 张景昱, 刘惠荣, 等. 抑制COMT与CCoAOMT调控植物木质素的生物合成. 科学通报, 2002, 47(8): 604-607. doi: 10.3321/j.issn:0023-074X.2002.08.011
	Zhao H Y, Zhang J Y, Liu H R, et al.Regulation of lignin synthesis by inhibition of COMT and CCoAOMT. Chinese Science Bulletin, 2002, 47(8): 604-607. doi: 10.3321/j.issn:0023-074X.2002.08.011
[13]	陈洪章. 纤维素生物技术.北京: 化学工业出版社, 2005.
	Chen H Z. Cellulosic Biotechnology.Beijing: Chemical Industry Press, 2005.
[14]	王林风, 程远超. 硝酸乙醇法测定纤维素含量. 化学研究, 2011, 22(4):52-55.
	Wang L F, Cheng C Y.Determination the content of cellulose by nitric acid-ethanol method. Chemical Research, 2011, 22(4):52-55.
[15]	李正理. 植物制片技术. 北京: 科学出版社, 1987.
	Li Z L. Plant Sectioning Technique.Beijing: Science Press, 1987.
[16]	Tian X, Xie J, Zhao Y, et al.Sense-, antisense- and RNAi-4CL1 regulate soluble phenolic acids, cell wall components and growth in transgenic Populus tomentosa Carr. Plant Physiology & Biochemistry, 2013, 65(6):111. doi: 10.1016/j.plaphy.2013.01.010 pmid: 23434928
[17]	邓建华, 管梦灵, 刘美芹. 高粱茎秆中木质素分布的显微技术研究. 植物学研究, 2014, 3: 188-194. doi: 10.12677/br.2014.35024
	Deng J H, Guan M L, Liu M Q.Study on microscopy techniques for identifying lignin distribution in the stem of Sorghum. Botanical Research, 2014, 3:188-194. doi: 10.12677/br.2014.35024
[18]	Barros-Rios J, Malvar R A, Jung H J G, et al. Cell wall composition as a maize defense Mechanism against corn borers. Phytochemistry, 2011, 72(4-5): 365-371. doi: 10.1016/j.phytochem.2011.01.004 pmid: 21281952
[19]	Lothar K, Frankw T.Biomechanics and transgenic wood. American Journal of Botany, 2006, 93(10): 1433-1438. doi: 10.3732/ajb.93.10.1433
[20]	Buanafina M M, Fescemyer H W.Modification of esterified cell wall phenolics increases vulnerability of tall fescue to herbivory by the fall armyworm. Planta, 2012, 236(2): 513-523. doi: 10.1007/s00425-012-1625-y pmid: 22434315
[21]	Meyermans H.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. Journal of Biological Chemistry, 2000, 275(47): 36899. doi: 10.1074/jbc.M006915200
[22]	Chapple C.Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenases. Annual Review of Plant Biology, 1998, 49(1): 311. doi: 10.1146/annurev.arplant.49.1.311
[23]	Jackson L A, Shadle G L, Zhou R, et al.Improving saccharification efficiency of Alfalfa stems through modification of the terminal stages of monolignol biosynthesis. BioEnergy Research, 2008, 1(3): 180. doi: 10.1007/s12155-008-9020-z

[1]	陈鑫洁,钱芷兰,刘启,赵清,张元兴,蔡孟浩. 毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸^*[J]. 中国生物工程杂志, 2021, 41(10): 52-61.
[2]	王伟东,杜加茹,张运尚,樊剑鸣. CRISPR/Cas9在人病毒感染相关疾病治疗研究中的应用^*[J]. 中国生物工程杂志, 2020, 40(12): 18-24.
[3]	徐应永. 基因治疗产品的开发现状与挑战[J]. 中国生物工程杂志, 2020, 40(12): 95-103.
[4]	王玥,牟彦双,刘忠华. 基于CRISPR/Cas系统的单碱基编辑技术研究进展^*[J]. 中国生物工程杂志, 2020, 40(12): 58-66.
[5]	雷海英,赵青松,白凤麟,宋慧芳,王志军. 利用CRISPR/Cas9鉴定玉米发育相关基因ZmCen^*[J]. 中国生物工程杂志, 2020, 40(12): 49-57.
[6]	薛瑞,姚林,王瑞,罗正山,徐虹,李莎. 重组贻贝足蛋白的研究进展与应用^*[J]. 中国生物工程杂志, 2020, 40(11): 82-89.
[7]	陈庆宇,王鲜忠,张姣姣. 基因技术在治疗2型糖尿病中的应用^*[J]. 中国生物工程杂志, 2020, 40(11): 73-81.
[8]	何秀娟,胡凤枝,刘秋丽,刘玉萍,祝玲,郑文云. 乳腺癌细胞QSOX1的CRISPR/Cas9基因编辑及其对增殖侵袭的影响研究^*[J]. 中国生物工程杂志, 2020, 40(11): 1-9.
[9]	吕一凡,李更东,薛楠,吕国梁,时邵辉,王春生. **LbCpf1基因的原核表达、纯化与体外切割检测 ^***[J]. 中国生物工程杂志, 2020, 40(8): 41-48.
[10]	张保惠,熊华龙,张天英,袁权. 基于水疱性口炎病毒(VSV)的溶瘤病毒研究进展 ^*[J]. 中国生物工程杂志, 2020, 40(6): 53-62.
[11]	武瑞君,李治非,张鑫,濮润,敖翼,孙燕荣. 新冠病毒抗体药物研发进展及展望分析[J]. 中国生物工程杂志, 2020, 40(5): 1-6.
[12]	刘迪,张洪春. 慢性阻塞性肺疾病基因工程动物模型研究进展 ^*[J]. 中国生物工程杂志, 2020, 40(4): 59-68.
[13]	程平,张洋子,马翾,陈旭,朱保庆,许文涛. 刺激响应型DNA水凝胶的性质及其应用 ^*[J]. 中国生物工程杂志, 2020, 40(3): 132-143.
[14]	郭胜楠, 李信晓, 王峰, 刘昆梅, 丁娜, 扈启宽, 孙涛. 海马与新皮质组织特异性GABRG2基因敲除小鼠模型的构建及其在遗传性癫痫伴热性惊厥附加症中的初步研究 ^*[J]. 中国生物工程杂志, 2020, 40(3): 9-20.
[15]	郭晶,侯占铭. **Folpcs1基因对尖孢镰刀菌亚麻专化型的无性繁殖和营养生长的调控 ^***[J]. 中国生物工程杂志, 2020, 40(3): 48-64.

Viewed

Full text

Abstract

Cited

Shared

Discussed