|本期目录/Table of Contents|

[1]孙婷婷,王文举,刘峰,等.甘蔗茉莉酸合成关键酶基因ScOPR1的克隆、亚细胞定位及表达[J].应用与环境生物学报,2018,24(06):1365-1374.[doi:10.19675/j.cnki.1006-687x.2018.01005]
　SUN Tingting,et al..Molecular cloning, subcellular localization, and expression analysis of jasmonic acid synthesis gene ScOPR1 from sugarcane[J].Chinese Journal of Applied & Environmental Biology,2018,24(06):1365-1374.[doi:10.19675/j.cnki.1006-687x.2018.01005]
点击复制

甘蔗茉莉酸合成关键酶基因ScOPR1的克隆、亚细胞定位及表达()
分享到：

《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
24卷
期数:
2018年06期
页码:
1365-1374
栏目:
研究论文
出版日期:
2018-12-25

文章信息/Info

Title:
Molecular cloning, subcellular localization, and expression analysis of jasmonic acid synthesis gene ScOPR1 from sugarcane
作者:
孙婷婷;  王文举;  刘峰;  王玲;  李竹;  戴明剑;  阙友雄;  许莉萍;  苏亚春
1福建农林大学农业部福建甘蔗生物学与遗传育种重点实验室 福州 350002 2福建农林大学教育部作物遗传育种与综合利用重点实验室 福州 350002
Author(s):
SUN Tingting;  et al.
1 Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, 350002, China 2 Key Laboratory of Crop Genetics and Breeding and Comprehensive Utilization, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
关键词:
甘蔗; 12-氧-植物二烯酸还原酶; 生物信息学分析; 亚细胞定位; 定量表达
Keywords:
sugarcane;  12-oxo-phytodienoic acid;  bioinformatic analysis;  subcellular localization;  quantitative expression
分类号:
Q786 : S566.1
DOI:
10.19675/j.cnki.1006-687x.2018.01005
摘要:
12-氧-植物二烯酸还原酶（12-Oxo-phytodienoic acid reductase，OPR）是十八碳烯酸途径的关键酶之一，控制着茉莉酸合成的最后一个步骤. 目前，甘蔗（Saccharum spp.）OPR基因的研究尚未见报道. 从课题组构建的甘蔗转录组unigene注释库中筛选到一个OPR基因，利用RT-PCR技术从ROC22接种黑穗病菌48 h的蔗芽中扩增获得全长cDNA序列，命名为ScOPR1（GenBank Accession Number：MG755745），并对该基因的序列特征、亚细胞定位、组织特异性表达及其在不同植物激素胁迫和不同基因型甘蔗与黑穗病菌互作过程中的表达情况进行分析. 生物信息学分析发现，ScOPR1基因全长1 287 bp，包含1个1 116 bp的开放阅读框，编码371个氨基酸，理论等电点为6.01，含有底物结合活性、FMN结合活性和催化活性的氨基酸保守位点以及OYE家族的保守结构域，且与高粱（Sorghum bicolor）OPR蛋白（XP_002447901.2）的氨基酸序列一致性高达96.23%，推测其为酸性稳定亲水性非分泌OPRⅠ蛋白. 本氏烟（Nicotiana benthamiana）瞬时表达显示，ScOPR1::GFP融合蛋白定位于细胞质和细胞膜. qRT-PCR分析结果显示，ScOPR1基因在甘蔗不同组织中均有表达，其在根中的表达量最高，其次为叶和蔗皮，而在蔗芽和蔗肉中的表达量最低. 茉莉酸甲酯和水杨酸处理后，随着胁迫时间的延长（3-24 h），ScOPR1基因显著上调表达；该基因在脱落酸处理0.5 h时被诱导表达，为对照的1.54倍. 此外，ScOPR1基因在抗黑穗病品种崖城05-179受黑穗病菌侵染初期（24 h）显著上调表达，但在感黑穗病品种ROC22中下调表达；48-72 h时ScOPR1基因的表达量在两个甘蔗基因型中较对照有所增加，但在抗病品种中高于感病品种. 本研究表明，ScOPR1基因积极响应茉莉酸甲酯、水杨酸和脱落酸信号分子以及黑穗病菌的胁迫，结果可为进一步分析ScOPR1基因的功能和甘蔗抗病基因工程提供参考. （图8 表1 参42）
Abstract:
12-Oxo-phytodienoic acid reductase (OPR), one of the key enzymes in the octadecanoid pathway, controls the last step of jasmonic acid synthesis. At present, OPR gene in sugarcane (Saccharum spp.) has not yet been reported. An OPR gene was selected from our established sugarcane transcriptome unigene annotation library. Its full-length cDNA sequence was obtained from ROC22 bud infected with Sporisorium scitamineum for 48 h by using RT-PCR and named as ScOPR1 (GenBank Accession Number: MG755745). Characteristics of ScOPR1 gene sequence, subcellular localization, tissue specific expression, ScOPR1 gene expression level under different plant hormone stresses, and in the interactions of different sugarcane genotypes with smut fungus were analyzed. Bioinformatic analysis showed that the full length of ScOPR1 gene was 1 287 bp, which had an open reading frame of 1 116 bp, encoding 371 amino acids. The pI value of ScOPR1 was 6.01. The conservative amino acids sites, including a catalytic activity, a substrate binding activity, an FMN binding activity and an OYE family structure domain, were discovered in ScOPR1. The similarity of amino acid sequences between ScOPR1 and Sorghum bicolor OPR protein (XP_002447901.2) was 96.23%. Therefore, ScOPR1 was predicted as an acid, stable, hydrophilic, and non-secretory OPRI protein. Transient expression in Nicotiana benthamiana indicated that the ScOPR1::GFP fusion protein was located in the cytoplasm and cell membrane. The qRT-PCR analysis revealed that ScOPR1 was constitutively expressed in different sugarcane organizations, with the highest expression level in root, followed by that in leaf and epidermis, and the minimum amount was noted in bud and stem pith. After methyl jasmonate (MeJA) and salicylic acid (SA) treatments, ScOPR1 gene was significantly up-regulated along with an increase of stress time (3-24 h). At 0.5 h after abscisic acid (ABA) treatment, the expression of ScOPR1 was induced, which was 1.54-fold higher than that of the control. In addition, ScOPR1 gene was significantly up-regulated at an early infection stage (24 h) in the smut-resistant variety Yacheng05-179 post-inoculation with S. scitamineum but was down-regulated in the smut-susceptible variety ROC22. At 48-72 h, the expression level of ScOPR1 was increased in both the sugarcane genotypes, but its transcripts in the resistant variety were higher than those in the susceptible one. These results showed that ScOPR1 gene responded positively to the signal molecules MeJA, SA, and ABA, as well as smut pathogen infection, which laid a foundation for further study of the function of ScOPR1 gene and the genetic engineering of sugarcane disease resistance.

参考文献/References:

1 Chen RK, Yuan ZN. Sugarcane production and research in China [J]. Int Sugar J, 2010, 112 (1340): 452-458
2 Que YX, Xu LP, Wu QB, Liu YF, Ling H, Liu YH, Zhang YY, Guo JL, Su YC, Chen JB, Wang SS, Zhang CG. Genome sequencing of Sporisorium scitamineum provides insights into the pathogenic mechanisms of sugarcane smut [J]. BMC Genomics, 2014, 15: 996
3 Sundar AR, Barnabas EL, Malathi P, Viswanathan R. A Mini-review on Smut Disease of Sugarcane Caused by Sporisorium scitamineum [M]. Botany Rijeka, Croatia: InTech, 2012: 109-128
4 许莉萍, 陈如凯. 甘蔗黑穗病及其抗病育种的现状与展望[J]. 福建农业学报, 2000, 15 (2): 26-31 [Xu LP, Chen RK. Current status and prospects of smut and smut resistance breeding in sugarcane [J]. Fujian J Agric, 2000, 15 (2): 26-31]
5 苏亚春. 甘蔗应答黑穗病菌侵染的转录组与蛋白组研究及抗性相关基因挖掘[D]. 福州: 福建农林大学, 2014 [Su YC. Transcriptomics and proteomics of sugarcane response to Sporisorium scitamineum infection and mining of resistance-related genes [D]. Fuzhou: Fujian Agriculture and Forestry University, 2014]
6 Wasternack C, Hause B. Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development [J]. Ann Bot-London, 2013, 111 (6): 1021-1058
7 Conconi A, Smerdon MJ, Howe GA, Ryan CA. The octadecanoid signalling pathway in plants mediates a response to ultraviolet radiation [J]. Nature, 1996, 383 (6603): 826-829
8 Browse J. The power of mutants for investigating jasmonate biosynthesis and signaling [J]. Phytochemistry, 2009, 70 (13-14): 1539-1546
9 蒋科技, 皮妍, 侯嵘, 唐克轩. 植物内源茉莉酸类物质的生物合成途径及其生物学意义[J]. 植物学报, 2010, 45 (2): 137-148 [Jiang KJ, Pi Y, Hou R, Tang KX. Jasmonate biosynthetic pathway: its physiological role and potential application in plant secondary metabolic engineering [J]. Bull Bot, 2010, 45 (2): 137-148]
10 Seo HS, Song JT, Cheong JJ, Lee YH, Lee YW, Hwang I, Lee JS, Choi YD. Jasmonic acid carboxyl methyltransferase: a key enzyme for jasmonate-regulated plant responses [J]. PNAS, 2001, 98 (8): 4788-4793
11 Breithaupt C, Kurzbauer R, Lilie H, Schaller A, Strassner J, Huber R, Macheroux P, Clausen T. Crystal structure of 12-oxophytodienoate reductase 3 from tomato: self-inhibition by dimerization [J]. PNAS, 2006, 103 (39): 14337-14342
12 李文燕. 植物OPR基因家族系统发育分析及水稻OPR家族基因分子生物学功能研究[D]. 广州: 中山大学, 2010 [Li WY. Phylogenetic analysis of OPR gene family in plants and study of molecular biological functions of OPR family genes in rice [D]. Guangzhou: Sun Yat-sen University, 2010]
13 许奕, 徐碧玉, 宋顺, 刘菊华, 张建斌, 贾彩红, 金志强. 香蕉茉莉酸合成关键酶基因MaOPR的克隆和表达分析[J]. 园艺学报, 2013, 40 (2): 237-246 [Xu Y, Xu BY, Song S, Liu JH, Zhang JB, Jia CH, Jin ZQ. Molecular cloning and expression analysis of MaOPR gene from banana [J]. Acta Hortic Sin, 2013, 40 (2): 237-246]
14 Schaller F, Weiler EW. Molecular cloning and characterization of 12-oxophytodienoate reductase, an enzyme of the octadecanoid signaling pathway from Arabidopsis thaliana structural and functional relationship to yeast old yellow enzyme [J]. J Biol Chem, 1997, 272 (44): 28066-28072
15 Schaller F, Hennig P, Weiler EW. 12-oxophytodienoate-10,11-reductase: occurrence of two isoenzymes of different specificity against stereoisomers of 12-oxophytodienoic acid [J]. Plant Physiol, 1998, 118 (4): 1345-1351
16 Schaller F, Biesgen C, Müssig C, Altmann T, Weiler EW. 12-oxophytodienoate reductase 3 (OPR3) is the isoenzyme involved in jasmonate biosynthesis [J]. Planta, 2000, 210 (6): 979-984
17 Strassner J, Fürholz A, Macheroux P, Amrhein N, Schaller A. A homolog of old yellow enzyme in tomato: spectral properties and substrate specificity of the recombinant protein [J]. J Biol Chem, 1999, 274 (49): 35067-35073
18 Biesgen C, Weiler EW. Structure and regulation of OPR1 and OPR2, two closely related genes encoding 12-oxophytodienoic acid-10, 11-reductases from Arabidopsis thaliana [J]. Planta, 1999, 208 (2): 155-165
19 Agrawal GK, Jwa NS, Shibato J, Han O, Iwahashi H, Rakwal R. Diverse environmental cues transiently regulate OsOPR1 of the “octadecanoid pathway” revealing its importance in rice defense/stress and development [J]. Biochem Bioph Res Commun, 2003, 310 (4): 1073-1082
20 Sobajima H, Takeda M, Sugimori M, Kobashi N, Kiribuchi K, Cho EM, Akimoto C, Yamaguchi T, Minami E, Shibuya N, Schaller F, Weiler EW, Yoshihara T, Nishida H, Nojiri H, Omori T, Nishiyama M, Yamane H. Cloning and characterization of a jasmonic acid-responsive gene encoding 12-oxophytodienoic acid reductase in suspension-cultured rice cells [J]. Planta, 2003, 216 (4): 692-698
21 Zhang J, Simmons C, Yalpani N, Crane V, Wilkinson HH, Kolomiets M. Genomic analysis of the 12-oxo-phytodienoic acid reductase gene family of Zea mays [J]. Plant Mol Biol, 2005, 59 (2): 323-343
22 Matsui H, Nakamura G, Ishiga Y, Toshima H, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y. Structure and expression of 12-oxophytodienoate reductase (subgroup I) genes in pea, and characterization of the oxidoreductase activities of their recombinant products [J]. Mol Genet Genomics, 2004, 271 (1): 1-10
23 Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, Uknes S, Ward E, Kessmann H, Ryals J. Requirement of salicylic acid for the induction of systemic acquired resistance [J]. Science, 1993, 261 (5122): 754-756
24 Liu B, Xue XD, Cui SP, Zhang XY, Han QM, Zhu L, Liang XF, Wang XJ, Huang LL, Chen XM, Kang ZS. Cloning and characterization of a wheat β-1,3-glucanase gene induced by the stripe rust pathogen Puccinia striiformis f. sp. Tritici [J]. Mol Biol Rep, 2010, 37 (2): 1045-1052
25 Mauch-Mani B, Mauch F. The role of abscisic acid in plant-pathogen interactions [J]. Curr Opin Plant Biol, 2005, 8 (4): 409-414
26 Cai Q, Yuan Z, Chen M, Luo Z, Zhao X, Liang W, Hu J, Zhang D. Jasmonic acid regulates spikelet development in rice [J]. Nat Commun, 2014, 5 (3): 3476
27 Mcconn M, Creelman RA, Bell E, Mullet JE, Browse J. Jasmonate is essential for insect defense in Arabidopsis [J]. PNAS, 1997, 94 (10): 5473-5477
28 Zhu F, Xi DH, Yuan S, Xu F, Zhang DW, Lin HH. Salicylic acid and jasmonic acid are essential for systemic resistance against tobacco mosaic virus in Nicotiana benthamiana [J]. Mol Plant Microbe Interact, 2014, 27 (6): 567-577
29 Fan J, Hill L, Crooks C, Doerner P, Lamb C. Abscisic acid has a key role in modulating diverse plant-pathogen interactions [J]. Plant Physiol, 2009, 150 (4): 1750-1761
30 王志卫, 贝学军, 朱世平, 马岩岩, 杨润婷. 植物激素在植物抗病过程中的作用研究进展[J]. 安徽农业科学, 2011, 39 (15): 9035-9038 [Wang ZW, Bei XJ, Zhu SP, Ma YY, Yang RT. Recent advances in phytohormone regulated plant resistance to pathogens [J]. J Anhui Agric Sci, 2011, 39 (15): 9035-9038]
31 Su YC, Guo JL, Ling H, Chen SS, Wang SS, Xu LP, Allan AC, Que YX. Isolation of a novel peroxisomal catalase gene from sugarcane, which is responsive to biotic and abiotic stresses [J]. Plos One, 2014, 9 (1): e84426
32 肖新换, 黄宁, 张玉叶, 杨宗锋, 凌辉, 黄珑, 苏炜华, 阙友雄. 甘蔗光合系统Ⅰ亚基O基因的克隆与表达分析[J]. 应用与环境生物学报, 2015, 21 (2): 208-214 [Xiao XH, Huang N, Zhang YY, Yang ZF, Ling H, Huang L, Su WH, Que YX. Cloning and expression of photosystem I subunit O gene from sugarcane [J]. Chin J Appl Environ Biol, 2015, 21 (2): 208-214]
33 苏亚春, 王竹青, 李竹, 刘峰, 许莉萍, 阙友雄, 戴明剑, 陈允浩. 甘蔗过氧化物酶基因ScPOD02的克隆与功能鉴定[J]. 作物学报, 2017, 43 (4): 510-521 [Su YC, Wang ZQ, Li Z, Liu F, Xu LP, Que YX, Dai MJ, Chen YH. Molecular cloning and functional identification of peroxidase gene ScPOD02 in sugarcane [J]. Acta Agron Sin, 2017, 43 (4): 510-521]
34 董蔚. 十八碳烷酸合成途径基因的逆境胁迫应答研究[D]. 济南: 山东大学, 2012 [Dong W. Study on octadecanoid pathway genes of Triticum aestivum responding to abiotic stresss [D]. Jinan: Shandong University, 2012]
35 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method [J]. Methods, 2001, 25 (4): 402-408
36 文锴, 王远, 胡蝶, 袁敬平, 侯喜林, 李英. 不结球白菜BcOPR3基因的克隆与功能分析[J]. 南京农业大学学报, 2017, 40 (5): 804-811 [Wen K, Wang Y, Hu D, Yuan JP, Hou XL, Li Y. Cloning and expression analysis of BcOPR3 gene in non-heading Chinese cabbage [J]. J Nanjing Agric Univ, 2017, 40 (5): 804-811]
37 Stintzi A, Browse J. The Arabidopsis male-sterile mutant, opr3, lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis [J]. PNAS, 2000, 97 (19): 10625-10630
38 Mussig C, Biesgen C, Lisso J, Uwer U, Weiler EW, Altmann T. A novel stress-inducible 12-oxophytodienoate reductase from Arabidopsis thaliana provides a potential link between brassinosteroid-action and jasmonic-acid synthesis [J]. J Plant Physiol, 2000, 157: 143-152
39 Wang YK, Yuan GL, Yuan SH, Duan WJ, W. TaOPR2 encodes a 12-oxo-phytodienoic acid reductase involved in the biosynthesis of jasmonic acid in wheat (Triticum aestivum L.) [J]. Biochem Bioph Res Commun, 2016, 470 (1): 233-238
40 Wasternack C. Jasmonates: an update on biosynthesis, signal transduction and action in plant stress response, growth and development [J]. Ann Bot-London, 2007, 100 (4): 681-697
41 Alexander K, Ramakrishnan K. Infection of the bud, establishment in the host and production of whips in sugarcane smut (Ustilago scitaminea) of sugarcane [J]. Proc Int Soc Sug Technol, 1980, 17: 1453-1455
42 莫凤连, 杨丽涛, 潘如科, 宋修鹏, 李杨瑞. 甘蔗黑穗病菌胁迫对甘蔗内源激素含量的影响[J]. 南方农业学报, 2012, 43 (11): 1676-1681 [Mo FL, Yang LT, Pan RK, Song XP, Li YR. Changes of endogenous hormone content in sugarcane under smut pathogen stress [J]. J S Agric, 2012, 43 (11): 1676-1681]

相似文献/References:

[1]罗俊,袁照年,张华,等.宿根甘蔗产量性状的稳定性分析[J].应用与环境生物学报,2009,15(04):488.[doi:10.3724/SP.J.1145.2009.00488]
　LUO Jun,YUAN Zhaonian,ZHANG Hua,et al.Stability Analysis on Yield Characters of Sugarcane Ratoon[J].Chinese Journal of Applied & Environmental Biology,2009,15(06):488.[doi:10.3724/SP.J.1145.2009.00488]
[2]叶冰莹,邱思,周平,等.甘蔗蔗糖磷酸合成酶SPSⅡ cDNA片段克隆与表达分析[J].应用与环境生物学报,2011,17(05):673.[doi:10.3724/SP.J.1145.2011.00673]
　YE Bingying,QIU Si,ZHOU Ping,et al.Cloning and Expression Analysis of Sucrose-phosphate Synthase II Gene from Sugarcane[J].Chinese Journal of Applied & Environmental Biology,2011,17(06):673.[doi:10.3724/SP.J.1145.2011.00673]
[3]黄祖新,黄镇,叶冰莹,等.宿根甘蔗根际土壤细菌多样性分析中培养法与非培养法比较研究[J].应用与环境生物学报,2011,17(05):742.[doi:10.3724/SP.J.1145.2011.00742]
　HUANG Zuxin,HUANG Zhen,YE Bingying,et al.Comparison of Culture-dependent and -independent Approaches for Diversity Analysis of Soil Bacteria in the Rhizosphere of Sugarcane[J].Chinese Journal of Applied & Environmental Biology,2011,17(06):742.[doi:10.3724/SP.J.1145.2011.00742]
[4]罗俊,邓祖湖,阙友雄,等.国家甘蔗第七轮区域试验品种的丰产性及稳定性[J].应用与环境生物学报,2012,18(05):734.[doi:10.3724/SP.J.1145.2012.00734]
　LUO Jun,DENG Zuhu,QUE Youxiong,et al.Productivity and Stability of Sugarcane Varieties in the 7th Round National Regional Trial of China[J].Chinese Journal of Applied & Environmental Biology,2012,18(06):734.[doi:10.3724/SP.J.1145.2012.00734]
[5]苏亚春,凌辉,王恒波,等.甘蔗SCoT-PCR反应体系优化与多态性引物筛选及应用[J].应用与环境生物学报,2012,18(05):810.[doi:10.3724/SP.J.1145.2012.00810]
　SU Yachun,LIN Hui,WANG Hengbo,et al.Optimization of SCoT-PCR Reaction System, and Screening and Utilization of Polymorphic Primers in Sugarcane[J].Chinese Journal of Applied & Environmental Biology,2012,18(06):810.[doi:10.3724/SP.J.1145.2012.00810]
[6]肖新换,黄宁,张玉叶,等.甘蔗光合系统Ⅰ亚基O基因的克隆与表达分析[J].应用与环境生物学报,2015,21(02):208.[doi:10.3724/SP.J.1145.2014.09033]
　XIAO Xinhuan,HUANG Ning,ZHANG Yuye,et al.Cloning and expression of photosystem I subunit O gene from sugarcane[J].Chinese Journal of Applied & Environmental Biology,2015,21(06):208.[doi:10.3724/SP.J.1145.2014.09033]
[7]肖新换,黄珑,黄宁,等.甘蔗ScBAK1基因及其可变剪接体的克隆与表达分析[J].应用与环境生物学报,2015,21(05):872.[doi:10.3724/SP.J.1145.2015.03005]
　XIAO Xinhuan,HUANG Long,HUANG Ning,et al.Cloning and expression analysis of ScBAK1 gene and its alternative spliceosome in sugarcane[J].Chinese Journal of Applied & Environmental Biology,2015,21(06):872.[doi:10.3724/SP.J.1145.2015.03005]
[8]阙万才,黄宁,刘峰,等.甘蔗真核生物翻译起始因子5A基因的克隆与表达分析[J].应用与环境生物学报,2015,21(06):1120.[doi:10.3724/SP.J.1145.2015.04008]
　QUE Wancai,HUANG Ning,LIU Feng,et al.Isolation and expression of a eukaryotic translation initiation factor 5A gene from sugarcane[J].Chinese Journal of Applied & Environmental Biology,2015,21(06):1120.[doi:10.3724/SP.J.1145.2015.04008]
[9]苏炜华# 黄 珑# 黄 宁 刘 峰 苏亚春 肖新换 凌 辉 阙友雄.甘蔗细胞色素P450还原酶基因的RT-PCR扩增与表达分析[J].应用与环境生物学报,2016,22(02):173.[doi:10.3724/SP.J.1145.2015.07029]
　SU Weihua#,HUANG Long#,HUANG Ning,et al.RT-PCR amplification and expression analysis of a cytochrome P450 reductasegene from sugarcane[J].Chinese Journal of Applied & Environmental Biology,2016,22(06):173.[doi:10.3724/SP.J.1145.2015.07029]
[10]苏炜华,黄宁,凌辉,等.甘蔗乙醇脱氢酶基因的克隆与表达分析[J].应用与环境生物学报,2017,23(03):474.[doi:2016.0704]
　SU Weihua,HUANG Ning,LING Hui,et al.Cloning and expression of ScADH from sugarcane[J].Chinese Journal of Applied & Environmental Biology,2017,23(06):474.[doi:2016.0704]

更新日期/Last Update: 2018-12-25