杜仲MVA和MEP途径相关基因的亚细胞定位与表达分析

doi:10.7525/j.issn.1673-5102.2017.01.008

摘要/Abstract

摘要： 通过对杜仲基因组分析，筛选并克隆出MVA途径和MEP途径的相关基因全长（EuDXR，EuMCT，EuCMK，EuMDS，EuACOT，EuHMGS和EuHMGR），并通过生物信息学方法分析其结构特征，结果表明上述基因与其他已知物种相应基因的相似度达73%~85%。通过构建亚细胞定位表达载体，并瞬时转化烟草下表皮细胞后激光共聚焦显微镜下观察显示，EuDXR，EuMCT，EuCMK，EuMDS基因编码蛋白定位于叶绿体，EuACOT和EuHMGR基因编码蛋白定位于内质网，EuHMGS基因编码蛋白定位于细胞质膜。利用转录组测序技术分析上述基因的时空表达特性表明，MEP途径相关基因在杜仲叶片中大量表达，而MVA途径相关基因在杜仲幼果中大量表达，且杜仲幼果比叶片中的橡胶含量高，因此，推断MVA途径在杜仲橡胶合成中占主导作用。

关键词: 杜仲, MVA途径, MEP途径, 亚细胞定位, 表达分析

Abstract: The full-long of EuDXR, EuMCT, EuCMK, EuMDS, EuACOT, EuHMGS and EuHMGR genes were isolated from Eucommia ulmoides. The structure characteristics were analyzed by using bioinformatics, which revealed that these deduced proteins had 73%-85% identities with the corresponding protein from other plants. Recombinant plasmid was introduced into the lower epidermal leaf explants of tobacco, and the transient expression of these genes with GFP fusion protein were observed with a Zeiss LSM. The subcellular localization analysis showed that the EuDXR, EuMCT, EuCMK, EuMDS protein were all located in the chlorophyll, EuACOT and EuHMGR protein were both located in the endoplasmic reticulum, EuHMGS protein was located in the plasma membrane. By RNA sequencing, the spatial-temporal expression patterns of these genes were detected. The expression of genes in MEP pathway is higher in leaves, but the expression of genes in MVA pathway is higher in young fruit. The rubber content in young fruit was higher than that in leaves. Therefore, the MVA pathway played a dominant role in the biosynthesis of Eu-rubber.

Key words: Eucommia ulmoides, MVA pathway, MEP pathway, expression analysis, subcellular localization

中图分类号:

Q949.751.5

王淋, 杜红岩, 乌云塔娜. 杜仲MVA和MEP途径相关基因的亚细胞定位与表达分析[J]. 植物研究, 2017, 37(1): 52-62.

WANG Lin, DU Hong-Yan, WUYUN Ta-Na. Subcellular Localization and Expression Analysis of Genes from Eucommia ulmoides Involved in MVA and MEP Pathway[J]. Bulletin of Botanical Research, 2017, 37(1): 52-62.

参考文献

1. 杜红岩,谢碧霞,邵松梅. 杜仲胶的研究进展与发展前景[J]. 中南林学院学报,2003,23(4):95-99.Du H Y,Xie B X,Shao S M. Prospects and research progress of gutta-percha[J]. Journal of Central South Forestry University,2003,23(4):95-99.
2. 杜红岩,胡文臻,俞锐. 杜仲产业绿皮书:中国杜仲橡胶资源与产业发展报告(2014-2015)[M]. 北京:社会科学文献出版社,2015:1-5.Du H Y,Hu W Z,Yu R. Green book of Eucommia industry:Report on development of China's Eucommia rubber resources and industry[M]. Beijing:Social Sciences Academic Press,2015:1-5.
3. 杜红岩,李芳东,杜兰英,等. 果用杜仲良种‘华仲6号’[J]. 林业科学,2010,46(8):182.Du H Y,Li F D,Du L Y,et al. An elite variety for samara use:Eucommia ulmoides Huazhong No. 6[J]. Scientia Silvae Sinicae,2010,46(8):182.
4. Chen R,Harada Y,Bamba T,et al. Overexpression of an isopentenyl diphosphate isomerase gene to enhance trans-polyisoprene production in Eucommia ulmoides Oliver[J]. BMC Biotechnology,2012,12:78.
5. Chappell J. Biochemistry and molecular biology of the isoprenoid biosynthetic pathway in plants[J]. Annual Review of Plant Physiology and Plant Molecular Biology,1995,46(1):521-547.
6. 陈大华,叶和春,李国凤,等. 植物类异戊二烯代谢途径的分子生物学研究进展[J]. 植物学报,2000,42(6):551-558.Chen D H,Ye H C,Li G F,et al. Advances in molecular biology of plant isoprenoid metabolic pathway[J]. Acta Botanica Sinica,2000,42(6):551-558.
7. 马靓,丁鹏,杨广笑,等. 植物类萜生物合成途径及关键酶的研究进展[J]. 生物技术通报,2006,(S1):22-30.Ma L,Ding P,Yang G X,et al. Advances on the plant terpenoid isoprenoid biosynthetic pathway and its key enzymes[J]. Biotechnology Bulletin,2006,(S1):22-30.
8. 祝传书,陈欣,郭嘉,等. 雷公藤1-脱氧-D-木酮糖-5-磷酸还原酶基因(TwDXR)的克隆与表达分析[J]. 农业生物技术学报,2014,22(3):298-308.Zhu C S,Chen X,Guo J,et al. Cloning and expression analysis of 1-deoxy-D-xylulose 5-phosphate reductoisomerase gene(DXR) in Tripterygiun wilfordii[J]. Journal of Agricultural Biotechnology,2014,22(3):298-308.
9. Seetang-Nun Y,Sharkey T D,Suvachittanont W. Molecular cloning and characterization of two cDNAs encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Hevea brasiliensis[J]. Journal of Plant Physiology,2008,165(9):991-1002.
10. Lichtenthaler H K. The 1-deoxy-d-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants[J]. Annual Review of Plant Physiology and Plant Molecular Biology,1999,50(1):47-65.
11. 张琳,谭晓风,胡姣,等. 油茶乙酰CoA酰基转移酶基因cDNA克隆及序列特征分析[J]. 中南林业科技大学学报,2011,31(8):108-112.Zhang L,Tan X F,Hu J,et al. Clonging and sequence characterization of cDNA encoding acetyl-CoA C-acetyltransferase in Camellia oleifera[J]. Journal of Central South University of Forestry & Technology,2011,31(8):108-112.
12. Ahumada I,Cairó A,Hemmerlin A,et al. Characterisation of the gene family encoding acetoacetyl-CoA thiolase in Arabidopsis[J]. Functional Plant Biology,2008,35(11):1100-1111.
13. Suwanmanee P,Sirinupong N,Suvachittanont W. Regulation of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase and rubber biosynthesis of Hevea brasiliensis(B. H. K.) Arg[M].//Bach T J,Rohmer M. Isoprenoid synthesis in plants and microorganisms. New York:Springer,2012:315-327.
14. Wititsuwannakul R,Wititsuwannakul D,Suwanmanee P. 3-Hydroxy-3-methylglutaryl coenzyme a reductase from the latex of Hevea brasiliensis[J]. Phytochemistry,1990,29(5):1401-1403.
15. 杜红岩. 我国的杜仲胶资源及其开发潜力与产业发展思路[J]. 经济林研究,2010,28(3):1-6.Du H Y. Discussion on industry development and exploration potential of gutta-percha resource in China[J]. Nonwood Forest Research,2010,28(3):1-6.
16. 刘万宏. 紫杉醇前体合成途径两个关键酶基因克隆和分析[D]. 重庆:西南大学,2008.Liu W H. Cloning and characterizations of two key enzyme genes involved in the biosynthesis pathway of Taxol Precursors[D]. Chongqing:Southwest University,2008.
17. Laule O,Fürholz A,Chang H S,et al. Crosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis in Arabidopsis thaliana[J]. Proceedings of the National Academy of Sciences of the United States of America,2003,100(11):6866-6871.
18. 邢浩然,刘丽娟,刘国振. 植物蛋白质的亚细胞定位研究进展[J]. 华北农学报,2006,21(S):1-6.Xing H R,Liu L J,Liu G Z. Advancement of protein subcellular localization in plants[J]. Acta Agriculturae Boreali-Sinica,2006,21(S2):1-6.
19. 张付云,陈士云,赵小明,等. NtSKP1-GFP植物表达载体的构建及亚细胞定位[J]. 西北农业学报,2009,18(4):144-148.Zhang F Y,Chen S Y,Zhao X M,et al. Construction of NtSKP1-GFP plant expression vector and subcellular location[J]. Acta Agriculturae Boreali-occidentalis Sinica,2009,18(4):144-148.
20. 杨光,曹雪,房经贵,等. ‘藤稔’葡萄VvGAI基因的克隆、亚细胞定位及时空表达分析[J]. 园艺学报,2011,38(10):1883-1892.Yang G,Cao X,Fang J G,et al. Cloning,subcellular localization and spatiotemporal expression of a VvGAI Gene from Grapevine ‘Fujiminori’[J]. Acta Horticulturae Sinica,2011,38(10):1883-1892.
21. 杨光,曹雪,房经贵,等. 葡萄花发育基因的亚细胞定位和表达分析[J]. 中国农业科学,2011,44(3):641-650.Yang G,Cao X,Fang J G,et al. Sub-cellular localization and expression analysis of genes involved in grapevine floral development[J]. Scientia Agricultura Sinica,2011,44(3):641-650.
22. 李广旭,吴茂森,吴静,等. 受病原细菌诱导表达增强的水稻转录因子基因OsBTF3的分子鉴定[J]. 中国农业科学,2009,42(7):2608-2614.Li G X,Wu M S,Wu J,et al. Molecular identification and characterization of a Rice gene of OsBTF3 encoding a transcriptional factor up-regulated by Xanthomonas oryzae pv. oryzae[J]. Scientia Agricultura Sinica,2009,42(7):2608-2614.
23. 林茂. 蜡梅Cpcor413pm1基因的抗逆性分析与亚细胞定位[D]. 重庆:西南大学,2008.Lin M. Stress tolerance analysis and subcellular localization of Cpcor413pm1 gene[D]. Chongqing:Southwest University,2008.
24. Schuhr C A,Radykewicz T,Sagner S,et al. Quantitative assessment of crosstalk between the two isoprenoid biosynthesis pathways in plants by NMR Spectroscopy[J]. Phytochemistry Reviews,2003,2(1-2):3-16.
25. Bamba T,Murayoshi M,Gyoksen K,et al. Contribution of mevalonate and methylerythritol phosphate pathways to polyisoprenoid biosynthesis in the rubber-producing plant Eucommia ulmoides Oliver[J]. Zeitschriftfür Naturforschung. C,Journal of Biosciences,2010,65(5-6):363-372.
26. 刘卫平,王敏杰,韩玉珍,等. 天然橡胶的生物合成机制[J]. 植物生理学通讯,2002,38(4):382-388.Liu W P,Wang M J,Han Y Z,et al. Biosynthetic mechanism of natural rubber[J]. Plant Physiology Communications,2002,38(4):382-388.
27. Suzuki N,Uefuji H,Nishikawa T,et al. Construction and analysis of EST libraries of the trans-polyisoprene producing plant,Eucommia ulmoides Oliver[J]. Planta,2012,236(5):1405-1417.
28. 杜红岩,杜兰英,李芳东. 杜仲果实内杜仲胶形成积累规律的研究[J]. 林业科学研究,2004,17(2):185-191.Du H Y,Du L Y,Li F D. Dynamic of gutta-percha formation and accumulation in samara of Eucommia ulmoides[J]. Forest Research,2004,17(2):185-191.
29. 杜红岩,杜兰英,陆志科,等. 杜仲无性系叶片含胶特性的差异及其相关性分析[J]. 中南林学院学报,2004,24(4):17-19,23.Du H Y,Du L Y,Lu Z K,et al. Analysis of the difference and co-relation of the gutta-percha content characteristics in Leaves Associated with Eucommia ulmoides Oliv. [J]. Journal of Central South Forestry University,2004,24(4):17-19,23.
30. 杜红岩,杜兰英,陆志科,等. 不同产地杜仲树皮含胶特性的变异规律[J]. 林业科学,2004,40(5):186-190.Du H Y,Du L Y,Lu Z K,et al. Variation dynamic of gutta-percha content characters in barks from different producing areas associated with Eucommia ulmoides[J]. Scientia Silvae Sinicae,2004,40(5):186-190.
31. 杜红岩,杜兰英,傅建敏,等. 杜仲不同器官含胶率的差异及其相关性分析[J]. 中南林学院学报,2006,26(4):1-4.Du H Y,Du L Y,Fu J M,et al. The difference and its pertinence analysis of the gutta-percha content in organs of Eucommia ulmoides Oliv. [J]. Journal of Central South Forestry University,2006,26(4):1-4.
32. 谢碧霞,杜红岩,杜兰英,等. 不同变异类型杜仲果实含胶量变异研究[J]. 林业科学,2005,41(6):144-146.Xie B X,Du H Y,Du L Y,et al. Variations of gutta-percha content in samara from different Eucommia ulmoides forms[J]. Scientia Silvae Sinicae,2005,41(6):144-146.
33. 王淋. 杜仲橡胶合成相关酶基因的克隆及功能研究[D]. 长沙:中南林业科技大学,2014.Wang L. Cloning and function analysis of related genes involved in rubber biosynthesis of Eucommia ulmoides[D]. Changsha:Central South University of Forestry and Technology,2014.
34. 何文广. 杜仲不同器官杜仲胶含量、相对分子质量及其分布的动态研究[D]. 杨凌:西北农林科技大学,2009.He W G. Study on the dynamic change of the content and molecular weight and molecular weight distribution of the gutta-percha in organs of Eucommia ulmoides Oliv. [D]. Yangling:North West Agriculture and Forestry University,2009.

[1]	黄安瀛, 夏德安, 张洋, 那冬晨, 燕青, 魏志刚. PtrWRKY51基因的克隆及抗旱表达特性分析[J]. 植物研究, 2022, 42(6): 1005-1013.
[2]	陈华峰, 代龙军, 刘明洋, 郭冰冰, 杨洪, 王立丰. 橡胶树胶乳高表达热激蛋白HbHSP90.4基因抗逆功能分析[J]. 植物研究, 2022, 42(6): 1023-1032.
[3]	刘明洋, 肖化兴, 王立丰, 梁晓宇, 张宇, 王萌. 橡胶树热激蛋白HbHSP90.8-1基因的克隆与功能分析[J]. 植物研究, 2022, 42(5): 811-820.
[4]	王宏鹏, 李一丹, 汪耀, 谭晓宇, 陈成彬, 张力鹏. 菊叶薯蓣DcPMK基因克隆及互作蛋白筛选[J]. 植物研究, 2022, 42(5): 855-865.
[5]	杨宇宁, 董昊, 董实伟, 王乃锐, 宋跃, 张含国, 李淑娟. 长白落叶松转录因子LobHLH34克隆及表达分析[J]. 植物研究, 2022, 42(1): 112-120.
[6]	马霜, 王博雅, 曹颖, 胡尚连, 高志民. 毛竹扩展蛋白基因的鉴定及其表达分析[J]. 植物研究, 2022, 42(1): 29-38.
[7]	韩连斌, 国庆, 赵凯, 姜廷波, 周博如, 李莉. 小黑杨HD-Zip转录因子PsnHB63基因克隆及表达分析[J]. 植物研究, 2021, 41(6): 1006-1014.
[8]	何凤, 杜红岩, 刘攀峰, 王璐, 庆军, 杜兰英. 干旱胁迫对杜仲叶片结构特征的影响[J]. 植物研究, 2021, 41(6): 947-956.
[9]	彭淑萍, 董诚明, 朱畇昊. 响应内生菌侵染的两个地黄茉莉酸合成关键基因的克隆与表达分析[J]. 植物研究, 2021, 41(2): 294-301.
[10]	王肖肖, 覃碧, 杨玉双, 聂秋海, 张继川, 刘实忠. 橡胶草E2泛素结合酶基因TkUBC2基因的克隆及其表达分析[J]. 植物研究, 2021, 41(1): 98-106.
[11]	吉仁花, 张文波, 林晓飞, 包颖亮, 特日格勒, 包会嘎, 白淑兰. 杂交构树UDP-葡萄糖脱氢酶基因编码蛋白的亚细胞定位及其启动子5'端缺失片段的功能分析[J]. 植物研究, 2020, 40(6): 932-942.
[12]	王万奇, 齐婉竹, 赵秋爽, 曾栋, 刘轶, 付鹏跃, 曲冠证, 赵曦阳. 白桦BpJMJ18基因启动子克隆及表达分析[J]. 植物研究, 2020, 40(5): 751-759.
[13]	王宁, 董莹莹, 袁美丽, 王亚楠, 曾胜蕴. 温水浸种对杜仲种子萌发过程中保护酶活性及内源激素含量变化的影响[J]. 植物研究, 2020, 40(4): 523-529.
[14]	刘晓, 杨佳, 张馨, 马苗苗, 杨静莉. 大青杨PubZIP1基因的克隆及亚细胞定位与抗旱表达特性分析[J]. 植物研究, 2020, 40(2): 233-242.
[15]	朱利利, 杜庆鑫, 何凤, 庆军, 杜红岩. 杜仲雄花芽2个发育时期转录组分析[J]. 植物研究, 2020, 40(2): 284-292.