Identification and Functions of Caffeic Acid O-methyltransferase Genes in Maize
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摘要:
目的 咖啡酸O-甲基转移酶（Caffeic acid O-methyltransferase，COMT）是褪黑素生物合成中的关键酶，在植物的生长、发育和抵御胁迫中发挥着重要的作用。本研究旨在探究COMT在玉米全基因组中的分布与盐胁迫响应情况，鉴定玉米咖啡酸O-甲基转移酶（COMT）基因。 方法 通过分析玉米COMT基因家族成员的结构特征、系统发育关系、基因结构、表达模式等，对ZmCOMTs基因进行系统分析，并通过过表达ZmCOMT12拟南芥对ZmCOMT的功能进行初步验证。 结果 在玉米全基因组中共鉴定出了28个COMT基因，根据系统发育分析分为2个分支（分支Ⅰ～II），大部分的COMT成员具有相似的motif组成和基因结构特征。利用玉米数据库分析ZmCOMTs的表达模式发现部分基因存在组织特异性，实时荧光定量PCR发现ZmCOMTs被盐胁迫诱导表达，说明ZmCOMTs成员参与了盐胁迫的调控，过表达ZmCOMT12拟南芥提高了褪黑素的含量和耐盐性。 结论 通过序列比对、亲缘关系、蛋白结构分析得到了假定的褪黑素合成基因ZmCOMT12，过表达拟南芥发现该基因与褪黑素的合成和耐盐性相关。 -
关键词:
- 玉米 /
- 咖啡酸O-甲基转移酶 /
- 褪黑素 /
- 非生物胁迫 /
- 成员鉴定
Abstract:Objective Localization in genome and response to salt stress of the genes of caffeic acid O-methyltransferase (COMT), a key melatonin biosynthesis enzyme relating to plant growth, development, and stress resistance, in maize were studied. Method Structure, phylogenetics, and expressions of the COMT family in maize (ZmCOMT) were analyzed. Functions of the genes were verified by overexpressing ZmCOMT12 in Arabidopsis thaliana. Result Twenty-eight COMTs were identified in the maize genome and phylogenetically divided into Branch Ⅰ and Branch II. Most of them were structurally similar with a like motif composition, and some had tissue specific expressions. As revealed by RT-PCR, ZmCOMTs could be induced by salt stress, indicating its association with the stress regulation. The overexpressed ZmCOMT12 significantly elevated the melatonin content and salt tolerance of Arabidopsis. Conclusion ZmCOMT12 was hypothesized as the melatonin synthesizing gene by the sequence alignment, genetic relationship, and protein structure analyses. -
Key words:
- Maize /
- caffeic acid O-methyltransferase /
- melatonin /
- abiotic stress /
- member identification
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图 1 28个玉米ZmCOMTs的系统发育分析
A：28个玉米ZmCOMTs的系统发育分析；B：拟南芥AtCOMT与玉米ZmCOMTs的系统发育分析。
Figure 1. Phylogenetic analysis on 28 ZmCOMTs
A: Phylogenetic analysis on 28 ZmCOMTs; B: phylogenetic analyses on AtCOMT and ZmCOMTs.
图 2 玉米ZmCOMTs的进化关系及motif组成与基因结构的关联分析
A：ZmCOMTs的系统发育分析；B：ZmCOMTs的蛋白motif组成；C：ZmCOMTs的基因结构。
Figure 2. Evolutionary relationships and association between motif composition and gene structure of ZmCOMTs in maize
A: Phylogenetic analysis on ZmCOMTs; B: protein motif composition of ZmCOMTs; C: gene structure of ZmCOMTs.
图 3 ZmCOMTs的染色体定位与基因复制
Figure 3. Chromosomal localization and gene duplication of ZmCOMTs
图 4 ZmCOMTs在不同组织中的表达
a：节间6~7；b：节间7~8；c：叶片区1；d：叶片区2；e：叶片区3；f：成熟叶片8；g：果皮糊粉27 d；h：初生根5 d；i：根皮层5 d；j：根伸长区5 d；k：根分生区5 d；l：次生根7~8 d。
Figure 4. Expressions of ZmCOMTs in different tissues
a: internode 6–7; b: internode 7–8; c: Leaf zone 1; d: Leaf zone 2; e: Leaf zone 3; f: Mature leaf 8; g: Pericarp aleurone 27 d; h: Primary root 5 d; i: Root cortex 5 d; j: Root elongation zone 5 d; k: Root meristem zone 5 d; l: Secondary root 7–8 d.
图 5 ZmCOMTs在盐胁迫下的表达
*、**分别表示与对照相比差异显著（P<0.05）和极显著（P<0.01）。
Figure 5. Expression of ZmCOMTs under salt stress
* and ** mean significant difference (P<0.05) and extremely signigifant difference (P<0.01) with control.
图 6 ZmCOMT12的功能研究
A：ZmCOMT12过表达植株的表型；B：ZmCOMT12的相对表达水平；C：褪黑素含量检测；D：POD酶活检测；E：SOD酶活检测。**P ＜ 0.01。
Figure 6. Functions of ZmCOMT12
A: Phenotypes of ZmCOMT12 overexpressed plants; B: relative expression of ZmCOMT12; C: melatonin content determination; D: POD enzyme activity determination; E: SOD enzyme activity assay. **: P＜0.01.
表 1 引物信息
Table 1. Information on primer
基因
Gene上游引物 5′-3′
Forward primer 5′-3′下游引物 5′-3′
Reverse primer 5′-3′ZmCOMT4 TACCGTCTCCAGCATCTT CGTCCTTGAACCACTCTT ZmCOMT6 TCTGTTCCACGAGAGCAT TGTCATCACCGAAGTTACC ZmCOMT13 ATGTTGGCGGAGATATGTT GATAGATGGCTCGGAAGG ZmCOMT22 GCTGATGCCGTTCTTCTAA GCCGATACCATTCTTCCTC ZmCOMT23 GGACAGCCACTTCATCAT GCAGTTCCTCAGTATCTTCA ZmCOMT24 CACGAACGCCATACTGAA GACTCATCATATCCAACTACCA ZmCOMT25 GGCTGATGAACCTGAACA CGCTCGGAATGTACTCAA Actin1 GCAGGTATTGTGATGGATTC CATTAGGTGGTCGGTGAG 
下载: 导出CSV
表 2 ZmCOMT家族成员的基本信息
Table 2. Basic information on ZmCOMTs
基因
Gene编号
ID氨基酸数量
Amino
acid amount等电点
Isoelectric
point分子质量
Molecular
weight/DaZmCOMT1 Zm00001eb018630 365 6.04 39538.69 ZmCOMT2 Zm00001eb018640 375 5.72 40568.66 ZmCOMT3 Zm00001eb018660 372 7.27 40187.3 ZmCOMT4 Zm00001eb040570 372 4.97 40596.44 ZmCOMT5 Zm00001eb090230 365 6.37 40018.77 ZmCOMT6 Zm00001eb090830 363 5.65 40252.51 ZmCOMT7 Zm00001eb092770 366 5.53 39785.78 ZmCOMT8 Zm00001eb164750 354 5.52 39183.45 ZmCOMT9 Zm00001eb168290 364 5.54 39700.6 ZmCOMT10 Zm00001eb169520 391 5.41 42526.25 ZmCOMT11 Zm00001eb170590 371 5.66 40477.9 ZmCOMT12 Zm00001eb172420 364 5.48 39553.44 ZmCOMT13 Zm00001eb190920 374 5.15 39590.18 ZmCOMT14 Zm00001eb191590 417 5.07 45060.54 ZmCOMT15 Zm00001eb198140 364 5.57 39200.14 ZmCOMT16 Zm00001eb198150 379 5.77 41100.43 ZmCOMT17 Zm00001eb202200 365 5.23 39345.35 ZmCOMT18 Zm00001eb292840 359 5.83 38600.51 ZmCOMT19 Zm00001eb292850 355 5.37 38002.74 ZmCOMT20 Zm00001eb292880 356 5.42 38880.9 ZmCOMT21 Zm00001eb292890 358 5.46 38816.89 ZmCOMT22 Zm00001eb306700 365 4.87 39122.97 ZmCOMT23 Zm00001eb353110 364 5.61 39605.53 ZmCOMT24 Zm00001eb392340 362 5.54 39456.21 ZmCOMT25 Zm00001eb400190 365 5.66 39687.64 ZmCOMT26 Zm00001eb405160 405 5.76 43159.01 ZmCOMT27 Zm00001eb405180 397 5.76 42617.41 ZmCOMT28 Zm00001eb412260 364 5.44 39950.98
下载: 导出CSV
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[1] ARNAO M B, HERNÁNDEZ-RUIZ J. Melatonin: A new plant hormone and/or a plant master regulator? [J]. Trends in Plant Science, 2019, 24(1): 38−48. doi: 10.1016/j.tplants.2018.10.010 [2] GALANO A, TAN D X, REITER R J. Melatonin as a natural ally against oxidative stress: A physicochemical examination [J]. Journal of Pineal Research, 2011, 51(1): 1−16. doi: 10.1111/j.1600-079X.2011.00916.x [3] ZHANG N, SUN Q Q, ZHANG H J, et al. Roles of melatonin in abiotic stress resistance in plants [J]. Journal of Experimental Botany, 2015, 66(3): 647−656. doi: 10.1093/jxb/eru336 [4] CHEN Y E, MAO J J, SUN L Q, et al. Exogenous melatonin enhances salt stress tolerance in maize seedlings by improving antioxidant and photosynthetic capacity [J]. Physiologia Plantarum, 2018, 164(3): 349−363. doi: 10.1111/ppl.12737 [5] BYEON Y, LEE H Y, LEE K, et al. Caffeic acid O-methyltransferase is involved in the synthesis of melatonin by methylating N-acetylserotonin in Arabidopsis [J]. Journal of Pineal Research, 2014, 57(2): 219−227. doi: 10.1111/jpi.12160 [6] SCHUBERT H L, BLUMENTHAL R M, CHENG X D. Many paths to methyltransfer: A chronicle of convergence [J]. Trends in Biochemical Sciences, 2003, 28(6): 329−335. doi: 10.1016/S0968-0004(03)00090-2 [7] YANG W J, DU Y T, ZHOU Y B, et al. Overexpression of TaCOMT improves melatonin production and enhances drought tolerance in transgenic Arabidopsis [J]. International Journal of Molecular Sciences, 2019, 20(3): 652. doi: 10.3390/ijms20030652 [8] LI W, LU J X, LU K, et al. Cloning and phylogenetic analysis of Brassica napus L. caffeic acid O-methyltransferase 1 gene family and its expression pattern under drought stress [J]. PLoS One, 2016, 11(11): e0165975. doi: 10.1371/journal.pone.0165975 [9] YAN Y Y, SUN S S, ZHAO N, et al. COMT1 overexpression resulting in increased melatonin biosynthesis contributes to the alleviation of carbendazim phytotoxicity and residues in tomato plants[J]. Environmental Pollution, 2019, 252(Pt A): 51-61. [10] ZHANG Y X, FAN Y P, RUI C, et al. Melatonin improves cotton salt tolerance by regulating ROS scavenging system and Ca2 + signal transduction [J]. Frontiers in Plant Science, 2021, 12: 693690. doi: 10.3389/fpls.2021.693690 [11] GÓMEZ-MERINO F C, BREARLEY C A, ORNATOWSKA M, et al. AtDGK2, a novel diacylglycerol kinase from Arabidopsis thaliana, phosphorylates 1-stearoyl-2-arachidonoyl-sn-glycerol and 1, 2-dioleoyl-sn-glycerol and exhibits cold-inducible gene expression [J]. The Journal of Biological Chemistry, 2004, 279(9): 8230−8241. doi: 10.1074/jbc.M312187200 [12] CHEN C J, CHEN H, ZHANG Y, et al. TBtools: An integrative toolkit developed for interactive analyses of big biological data [J]. Molecular Plant, 2020, 13(8): 1194−1202. doi: 10.1016/j.molp.2020.06.009 [13] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method [J]. Methods, 2001, 25(4): 402−408. doi: 10.1006/meth.2001.1262 [14] ZHANG X R, HENRIQUES R, LIN S S, et al. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method [J]. Nature Protocols, 2006, 1(2): 641−646. doi: 10.1038/nprot.2006.97 [15] LIU Y S, WANG Y Z, PEI J B, et al. Genome-wide identification and characterization of COMT gene family during the development of blueberry fruit [J]. BMC Plant Biology, 2021, 21(1): 5. doi: 10.1186/s12870-020-02767-9 [16] GUO C L, GUO R R, XU X Z, et al. Evolution and expression analysis of the grape (Vitis vinifera L. ) WRKY gene family [J]. Journal of Experimental Botany, 2014, 65(6): 1513−1528. doi: 10.1093/jxb/eru007 [17] LIU Y, JIANG H Y, CHEN W J, et al. Genome-wide analysis of the auxin response factor (ARF) gene family in maize (Zea mays) [J]. Plant Growth Regulation, 2011, 63(3): 225−234. doi: 10.1007/s10725-010-9519-0 [18] ZHENG X D, TAN D X, ALLAN A C, et al. Chloroplastic biosynthesis of melatonin and its involvement in protection of plants from salt stress [J]. Scientific Reports, 2017, 7: 41236. doi: 10.1038/srep41236 [19] LI C, WANG P, WEI Z W, et al. The mitigation effects of exogenous melatonin on salinity-induced stress in Malus hupehensis [J]. Journal of Pineal Research, 2012, 53(3): 298−306. doi: 10.1111/j.1600-079X.2012.00999.x -
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