薄荷茉莉酸受体McCOI1a基因的克隆与表达模式分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0789

摘要/Abstract

摘要：

【目的】茉莉酸（jasmonic acid, JA）受体COI1在调节植物生长发育、逆境响应方面具有重要作用。克隆薄荷McCOI1a基因，并分析其蛋白特征与表达模式，为薄荷分子育种提供基因资源。【方法】基于转录组数据从薄荷叶片中克隆McCOI1a，并通过生物信息学分析、烟草叶片瞬时表达、实时荧光定量PCR技术对McCOI1a的蛋白特性、亚细胞定位、基因表达模式进行分析。【结果】McCOI1a基因全长1 842 bp，编码613个氨基酸；McCOI1a蛋白具有保守的F-box和LRR结构域，与丹参SmCOI1蛋白同源性最高；亚细胞定位结果显示McCOI1a蛋白定位于细胞核；McCOI1a在不同组织中均有表达，在根中表达量最高，在叶序中表达呈逐渐上升的趋势；在叶片中，McCOI1a在MeJA、干旱、NaCl、AlCl₃、CdCl₂、CuCl₂处理下的表达呈不同程度的上调，并且AlCl₃处理下其上调最明显，表达量最高可达1 206倍；在根中，McCOI1a的表达在CuCl₂处理下呈先下调而后上调的模式，在其余处理下，McCOI1a的表达都呈现出不同程度的下调。【结论】McCOI1a响应MeJA和非生物逆境胁迫，可能在调控薄荷生长发育、逆境响应方面发挥重要作用。

关键词: 薄荷, McCOI1a基因, 蛋白特征, 胁迫响应, 表达分析

Abstract:

【Objective】The jasmonic acid（JA）receptor COI1 plays a vital role in regulating plant growth and development, and stress response in plants. Cloning and analysis of the protein characterization, and analyzing expressions of McCOI1a from Mentha canadensis L. may provide the gene resources for its molecular breeding. 【Method】The McCOI1a gene was cloned from the leaves of M. canadensis based on transcriptome data, and then the characteristics, subcellular localization, and expression profiles of McCOI1a were analyzed through bioinformatics analysis, transient expression in the leaves of tobacco, and real-time quantitative PCR. 【Result】The full-length of McCOI1a gene was 1 842 bp and it encoded 613 amino acids. McCOI1a protein possessed the conserved F-box and LRR domains and that was highly homologous with Salvia miltiorrhiza SmCOI1. Subcellular localization results showed that McCOI1a protein was located in the nucleus. The qRT-PCR results showed that McCOI1a was broadly expressed in different tissues, with the highest expression in the root; McCOI1a expression gradually increased during leaf development. In addition, the expressions of McCOI1a in the leaves were up-regulated to varying degrees under MeJA, drought, NaCl, AlCl₃, CdCl₂, and CuCl₂ treatments, and the up-regulation was the most obvious under AlCl₃treatment, with the highest expression reaching 1 206-fold. And McCOI1a expression in the root was firstly down-regulated and then up-regulated under CuCl₂ treatment, while the expression of McCOI1a showed varying degrees of down-regulation under other treatments. 【Conclusion】McCOI1a responds to MeJA and abiotic stress, and may play an important role in regulating the growth and development, and stress response in M. canadensis.

Key words: Mentha canadensis L., McCOI1a gene, protein characteristics, stress response, expression analysis

唐伟林, 康琴, 汪霞, 谌明洋, 孙欣江, 王棵, 侯凯, 吴卫, 徐东北. 薄荷茉莉酸受体McCOI1a基因的克隆与表达模式分析[J]. 生物技术通报, 2024, 40(1): 270-280.

TANG Wei-lin, KANG Qin, WANG Xia, SHEN Ming-yang, SUN Xin-jiang, WANG Ke, HOU Kai, WU Wei, XU Dong-bei. Cloning and Expression Pattern Analysis of Jasmonic Acid Receptor Gene McCOI1a in Mentha canadensis L.[J]. Biotechnology Bulletin, 2024, 40(1): 270-280.

图/表 8

图1 薄荷McCOI1a编码基因及其蛋白结构分析 A：McCOI1a基因及蛋白保守结构域示意图，a：McCOI1a基因全长；b：蛋白全长及保守结构域F-box，TIR1和AMN1位置；c：氨基酸长度比例尺，bp：碱基对。B：McCOI1a蛋白二级结构，蓝色：α-螺旋；绿色：β-转角；红色：延伸链；紫色：随机卷曲。C：McCOI1a蛋白三级结构，淡蓝色：α-螺旋；紫色：β-折叠；绿色：配体周围的残基；红色：配体；黄色虚线：氢键

Fig. 1 McCOI1a gene and its protein structure analysis in M. canadensis A: The diagram of McCOI1a gene and the conserved domain of McCOI1a protein. a: The full-length of McCOI1a; b: the full-length sequence of protein and the positions of conserved domain F-box, TIR1, and AMN1; c: the scale bar of the length of amino acid; bp: base pair. B: Secondary structure of McCOI1a protein. Blue: α-helix; Green: beta-turn; Red: extended-strand; Purple: random coil. C: Tertiary structure of McCOI1a protein. Baby blue: α-helix. Purple: beta-turn; Green: the residue around the ligand; Red: ligand; The dotted yellow line: hydrogen bond

图2 McCOI1a与其同源蛋白的氨基酸序列对比黑色方框：F-box结构域；红线下划线：亮氨酸富集重复序列（LRR）

Fig. 2 Amino acid sequence comparison of McCOI1a and its homologous proteins Black box: F-box domain; red underline: leucine-rich repeat sequence（LRR）

图3 McCOI1a与其同源蛋白保守基序分析

Fig. 3 Analysis of conserved motifs of McCOI1a and its homologous proteins

图4 薄荷与其它物种中COI1蛋白进化树分析

Fig. 4 Phylogenetic analysis of COI1 proteins from M. canadensis and other species

图5 McCOI1a亚细胞定位结果

Fig. 5 Subcellular localization results of McCOI1a

图6 McCOI1a在薄荷不同组织及叶序中的表达分析 A：McCOI1a在薄荷根、茎、幼叶、老叶、花、蕾、茎尖中的表达。B：McCOI1a在薄荷不同部位叶片中的表达，叶1-叶8：形态学从上到下的第1至第8片叶子。数据为3个生物学重复的平均值±SE

Fig. 6 Expression analysis of McCOI1a gene in different tissues and phyllotaxis of M. canadensis A: Expressions of McCOI1a in the roots, stems, young leaf, old leaf, flower, bud, apical shoot of M. canadensis. B: Relative expressions of McCOI1a in the leaves at different positions. Leaf 1-leaf 8: The first to eighth leaf morphologically from top to bottom. The data are of ±SE of three biological replicates

图7 茉莉酸和非生物胁迫处理下薄荷McCOI1a基因在叶片中的表达分析数据为3个生物学重复的平均值±SE（n = 3）。方差分析采用邓肯检验，小写字母代表0.05水平上显著差异。下同

Fig. 7 Expression analysis of McCOI1a in the leaves of M. canadensis under MeJA and abiotic stress treatments The data ±SE（n=3）of three biological replicates. Duncan test was used in ANOVA, the lowercase letters indicate significant differences at the 0.05 level. The same below

图8 茉莉酸和非生物胁迫处理下薄荷McCOI1a基因在根中的表达分析

Fig. 8 Expression analysis of McCOI1a in the roots of M. canadensis under MeJA and abiotic stress treatments

参考文献 39

[1]	陈智坤, 梁呈元, 任冰如, 等. 薄荷属植物挥发性成分及药理作用研究进展[J]. 天然产物研究与开发, 2013, 25(6): 856-861, 865.
	Chen ZK, Liang CY, Ren BR, et al. Advances in the studies of chemical constituents and pharmacological activities of volatile components from Mentha L[J]. Nat Prod Res Dev, 2013, 25(6): 856-861, 865.
[2]	Yu X, Liang CY, Chen J, et al. The effects of salinity stress on morphological characteristics, mineral nutrient accumulation and essential oil yield and composition in Mentha canadensis L[J]. Sci Hortic, 2015, 197: 579-583. doi: 10.1016/j.scienta.2015.10.023 URL
[3]	Azimychetabi Z, Sabokdast Nodehi M, Karami Moghadam T, et al. Cadmium stress alters the essential oil composition and the expression of genes involved in their synthesis in peppermint(Mentha piperita L.)[J]. Ind Crops Prod, 2021, 168: 113602. doi: 10.1016/j.indcrop.2021.113602 URL
[4]	Singh R, Luxmi S, Charak A, et al. Effects of intermittent drought on the essential oil yield, contents, and nutrient status of Mentha longifolia(L.) huds[J]. J Essent Oil Bear Plants, 2022, 25(3): 626-638. doi: 10.1080/0972060X.2022.2091957 URL
[5]	Yu XX, Zhang WJ, Zhang Y, et al. The roles of methyl jasmonate to stress in plants[J]. Funct Plant Biol, 2019, 46(3): 197-212. doi: 10.1071/FP18106 pmid: 32172764
[6]	Yan JB, Zhang C, Gu M, et al. The Arabidopsis CORONATINE INSENSITIVE1 protein is a jasmonate receptor[J]. Plant Cell, 2009, 21(8): 2220-2236. doi: 10.1105/tpc.109.065730 URL
[7]	Wasternack C, Song SS. Jasmonates: biosynthesis, metabolism, and signaling by proteins activating and repressing transcription[J]. J Exp Bot, 2017, 68(6): 1303-1321. doi: 10.1093/jxb/erw443 pmid: 27940470
[8]	Xie DX, Feys BF, James S, et al. COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility[J]. Science, 1998, 280(5366): 1091-1094. doi: 10.1126/science.280.5366.1091 pmid: 9582125
[9]	Ye M, Luo SM, Xie JF, et al. Silencing COI1 in rice increases susceptibility to chewing insects and impairs inducible defense[J]. PLoS One, 2012, 7(4): e36214. doi: 10.1371/journal.pone.0036214 URL
[10]	Lee SH, Sakuraba Y, Lee T, et al. Mutation of Oryza sativa CORONATINE INSENSITIVE 1b(OsCOI1b)delays leaf senescence[J]. J Integr Plant Biol, 2015, 57(6): 562-576. doi: 10.1111/jipb.v57.6 URL
[11]	Bai JF, Wang YK, Wang P, et al. Genome-wide identification and analysis of the COI gene family in wheat(Triticum aestivum L.)[J]. BMC Genomics, 2018, 19(1): 754. doi: 10.1186/s12864-018-5116-9
[12]	Qi XL, Guo SW, Wang D, et al. ZmCOI2a and ZmCOI2b redundantly regulate anther dehiscence and gametophytic male fertility in maize[J]. Plant J, 2022, 110(3): 849-862. doi: 10.1111/tpj.v110.3 URL
[13]	Sun TT, Meng YT, Cen GL, et al. Genome-wide identification and expression analysis of the coronatine-insensitive 1(COI1)gene family in response to biotic and abiotic stresses in Saccharum[J]. BMC Genomics, 2022, 23(1): 38. doi: 10.1186/s12864-021-08255-0
[14]	Chen QF, Dai LY, Xiao S, et al. The COI1 and DFR genes are essential for regulation of jasmonate-induced anthocyanin accumulation in Arabidopsis[J]. J Integr Plant Biol, 2007, 49(9): 1370-1377. doi: 10.1111/jipb.2007.49.issue-9 URL
[15]	Shen Q, Lu X, Yan TX, et al. The jasmonate-responsive AaMYC2 transcription factor positively regulates artemisinin biosynthesis in Artemisia annua[J]. New Phytol, 2016, 210(4): 1269-1281. doi: 10.1111/nph.2016.210.issue-4 URL
[16]	Zhou YY, Sun W, Chen JF, et al. SmMYC2a and SmMYC2b played similar but irreplaceable roles in regulating the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza[J]. Sci Rep, 2016, 6: 22852. doi: 10.1038/srep22852
[17]	Zhang HT, Hedhili S, Montiel G, et al. The basic helix-loop-helix transcription factor CrMYC2 controls the jasmonate-responsive expression of the ORCA genes that regulate alkaloid biosynthesis in Catharanthus roseus[J]. Plant J, 2011, 67(1): 61-71. doi: 10.1111/tpj.2011.67.issue-1 URL
[18]	Zhang HB, Bokowiec MT, Rushton PJ, et al. Tobacco transcription factors NtMYC2a and NtMYC2b form nuclear complexes with the NtJAZ1 repressor and regulate multiple jasmonate-inducible steps in nicotine biosynthesis[J]. Mol Plant, 2012, 5(1): 73-84. doi: 10.1093/mp/ssr056 URL
[19]	Xu DB, Ma YN, Qin TF, et al. Transcriptome-wide identification and characterization of the JAZ gene family in Mentha canadensis L[J]. Int J Mol Sci, 2021, 22(16): 8859. doi: 10.3390/ijms22168859 URL
[20]	Ma YN, Xu DB, Li L, et al. Jasmonate promotes artemisinin biosynthesis by activating the TCP14-ORA complex in Artemisia annua[J]. Sci Adv, 2018, 4(11): eaas9357. doi: 10.1126/sciadv.aas9357 URL
[21]	Ruan JJ, Zhou YX, Zhou ML, et al. Jasmonic acid signaling pathway in plants[J]. Int J Mol Sci, 2019, 20(10): 2479. doi: 10.3390/ijms20102479 URL
[22]	Chini A, Boter M, Solano R. Plant oxylipins: COI1/JAZs/MYC2 as the core jasmonic acid-signalling module[J]. FEBS J, 2009, 276(17): 4682-4692. doi: 10.1111/j.1742-4658.2009.07194.x pmid: 19663905
[23]	Trang Nguyen H, Thi Mai To H, Lebrun M, et al. Jasmonates-the master regulator of rice development, adaptation and defense[J]. Plants, 2019, 8(9): 339. doi: 10.3390/plants8090339 URL
[24]	Chen J, Yang HT, Ma S, et al. HbCOI1 perceives jasmonate to trigger signal transduction in Hevea brasiliensis[J]. Tree Physiol, 2021, 41(3): 460-471. doi: 10.1093/treephys/tpaa124 URL
[25]	Hu S, Yu KM, Yan JB, et al. Jasmonate perception: ligand-receptor interaction, regulation, and evolution[J]. Mol Plant, 2023, 16(1): 23-42. doi: 10.1016/j.molp.2022.08.011 URL
[26]	付鸿博, 向成刚, 陶宏征, 等. 灯盏花COI1基因的克隆及表达分析[J]. 分子植物育种, 2023. http://kns.cnki.net/kcms/detail/46.1068.s.20220707.1441.006.html.
	Fu HB, Xiang CG, Tao HZ, et al. Cloning and expression analysis of COI1 gene in Erigeron breviscapus[J]. Molecular Plant Breeding, 2023. http://kns.cnki.net/kcms/detail/46.1068.s.20220707.1441.006.html.
[27]	Lee HY, Seo JS, Cho JH, et al. Oryza sativa COI homologues restore jasmonate signal transduction in Arabidopsis coi1-1 mutants[J]. PLoS One, 2013, 8(1): e52802. doi: 10.1371/journal.pone.0052802 URL
[28]	吴晨雨, 何俊娜, 钟雄辉, 等. 唐菖蒲茉莉素受体基因GhCOI1的克隆与表达分析[J]. 园艺学报, 2015, 42(2): 289-300.
	Wu CY, He JN, Zhong XH, et al. Cloning and expression analysis of coronatine insensitive gene encoding a jasmonate receptor from Gladiolus hybridus[J]. Acta Hortic Sin, 2015, 42(2): 289-300.
[29]	Shan XY, Wang JX, Chua L, et al. The role of Arabidopsis Rubisco activase in jasmonate-induced leaf senescence[J]. Plant Physiol, 2011, 155(2): 751-764. doi: 10.1104/pp.110.166595 URL
[30]	Chen YN, Feng PP, Tang BY, et al. The AP2/ERF transcription factor SlERF.F5 functions in leaf senescence in tomato[J]. Plant Cell Rep, 2022, 41(5): 1181-1195. doi: 10.1007/s00299-022-02846-1 pmid: 35238951
[31]	Liao YC, Wei JH, Xu YH, et al. Cloning, expression and characterization of COI1 gene(AsCOI1)from Aquilaria sinensis(Lour.) Gilg[J]. Acta Pharm Sin B, 2015, 5(5): 473-481. doi: 10.1016/j.apsb.2015.05.009 URL
[32]	Liu R, Wang JB, Xiao M, et al. AaCOI1, encoding a CORONATINE INSENSITIVE 1-like protein of Artemisia annua L., is involved in development, defense, and anthocyanin synthesis[J]. Genes, 2020, 11(2): 221. doi: 10.3390/genes11020221 URL
[33]	Yastreb TO, Kolupaev YE, Shkliarevskyi MA, et al. Involvement of jasmonate signaling components in salt stress-induced stomatal closure in Arabidopsis thaliana[J]. Cytol Genet, 2020, 54(4): 318-323. doi: 10.3103/S009545272004012X
[34]	Gupta A, Bhardwaj M, Tran LS P. JASMONATE ZIM-DOMAIN family proteins: important nodes in jasmonic acid-abscisic acid crosstalk for regulating plant response to drought[J]. Curr Protein Pept Sci, 2021, 22(11): 759-766. doi: 10.2174/1389203722666211018114443 URL
[35]	Chen YJ, Chen Y, Shi ZJ, et al. Biosynthesis and signal transduction of ABA, JA, and BRs in response to drought stress of Kentucky bluegrass[J]. Int J Mol Sci, 2019, 20(6): 1289. doi: 10.3390/ijms20061289 URL
[36]	Valenzuela CE, Acevedo-Acevedo O, Miranda GS, et al. Salt stress response triggers activation of the jasmonate signaling pathway leading to inhibition of cell elongation in Arabidopsis primary root[J]. J Exp Bot, 2016, 67(14): 4209-4220. doi: 10.1093/jxb/erw202 pmid: 27217545
[37]	Yang ZB, He CM, Ma YQ, et al. Jasmonic acid enhances Al-induced root growth inhibition[J]. Plant Physiol, 2017, 173(2): 1420-1433. doi: 10.1104/pp.16.01756 URL
[38]	Zhang CH, Huang RQ, Zhan NH, et al. Methyl jasmonate and selenium synergistically mitigative cadmium toxicity in hot pepper(Capsicum annuum L.) plants by improving antioxidase activities and reducing Cd accumulation[J]. Environ Sci Pollut Res Int, 2023, 30(34): 82458-82469. doi: 10.1007/s11356-023-28273-7
[39]	Hanaka A, Wójcik M, Dresler S, et al. Does methyl jasmonate modify the oxidative stress response in Phaseolus coccineus treated with Cu?[J]. Ecotoxicol Environ Saf, 2016, 124: 480-488. doi: 10.1016/j.ecoenv.2015.11.024 URL