棉花学报 ›› 2019, Vol. 31 ›› Issue (2): 101-113.doi: 10.11963/1002-7807.swhjf.20190322
收稿日期:
2019-02-01
出版日期:
2019-03-15
发布日期:
2019-03-15
通讯作者:
高峰,黄家风
E-mail:335298645@qq.com;gaofenggra@sohu.com;jiafeng_huang@163.com
作者简介:
宋雯(1994-),女,硕士研究生, 基金资助:
Song Wen(),Wang Chunqiao,Yu Yan,Gao Feng*(
),Huang Jiafeng*(
)
Received:
2019-02-01
Online:
2019-03-15
Published:
2019-03-15
Contact:
Gao Feng,Huang Jiafeng
E-mail:335298645@qq.com;gaofenggra@sohu.com;jiafeng_huang@163.com
摘要:
【目的】明确棉花黄萎病菌中鸟氨酸脱羧酶抗酶蛋白(OAZ)基因(VdOAZ)的功能。【方法】以大丽轮枝菌野生型菌株V592的基因组DNA和cDNA为模板,对VdOAZ基因全长进行克隆并测序。构建针对VdOAZ基因的敲除载体和互补载体,通过农杆菌介导的遗传转化筛选VdOAZ基因敲除菌株和和互补菌株。以野生型菌株V592为对照,对VdOAZ基因敲除突变体和互补菌株的菌落生长速率、产孢量、微菌核产量及对棉花的致病力进行测定;通过实时荧光定量逆转录聚合酶链式反应测定致病相关的其他基因在VdOAZ基因敲除突变体中的表达量,及亚精胺诱导条件下,V592菌株中VdOAZ基因及致病相关的其他基因的相对表达量。【结果】从棉花黄萎病菌中克隆到VdOAZ基因的全长为1 006 bp,具有2个开放阅读框(Open reading frame,ORF),ORF2编码的蛋白具有OAZ所特有的ODC-AZ保守结构域。与野生型菌株V592和互补菌株相比,VdOAZ基因敲除突变体的菌落生长速率降低、微菌核产量及产孢量明显减少,对棉花的致病力下降,表明VdOAZ基因与大丽轮枝菌分生孢子和微菌核的产生有关,并参与大丽轮枝菌致病。在VdOAZ基因敲除突变体中,VdPKAC1、VMK1、VdNLP1、VdNLP2和VdSge1基因表达量显著上调;V592菌株经亚精胺诱导培养后,VdOAZ基因的表达量显著上调,而上述5个致病相关基因的表达量均明显下调,表明VdOAZ基因对其表达具有负调控作用。【结论】VdOAZ基因响应多胺水平改变,通过调控VdPKAC1、VMK1、VdNLP1、VdNLP2、VdSge1的表达影响大丽轮枝菌孢子产生、微菌核形成和致病过程。
宋雯,王春巧,俞燕,高峰,黄家风. 棉花黄萎病菌鸟氨酸脱羧酶抗酶蛋白基因VdOAZ的功能分析[J]. 棉花学报, 2019, 31(2): 101-113.
Song Wen,Wang Chunqiao,Yu Yan,Gao Feng,Huang Jiafeng. Functional Analysis of an Ornithine Decarboxylase Antizyme Gene VdOAZ in Verticillium dahliae Isolated from Cotton[J]. Cotton Science, 2019, 31(2): 101-113.
表1
供试引物"
引物名称 Primer name | 引物序列 ( 5′→3′ ) Primer sequence (5′→3′) |
VdPKAC1-F | CCCTCACCGATTTCGACCTG |
VdPKAC1-R | CTCGGCGGCATAAAACTTGG |
VMK1-F | CGCAGCAACGCCCCTAATC |
VMK1-R | GGCAGTGGTCATCGGAGAGGT |
VdNLP1-F | TCGGTCTTTGCCCTCGTC |
VdNLP1-R | GCCTGGTTTGCGTTGTTC |
VdNLP2-F | AAGCCGTACCTCAAGGTGTTCA |
VdNLP2-R | CCGACCCAAAGTCCGTGTTCT |
VdCYC8-F | GGATGCCCTCGATGCTTACT |
VdCYC8-R | CGTCGCTGATCTGGTTGTTG |
VGB-F | CGGTGCCTGCGATGCTTT |
VGB-R | GGAGGTGATGCCACAGAGGA |
VdSge1-F | CATGGATCCTTCCGAGGCATCTAG |
VdSge1-R | GATGATGCGGGACGCTTCTGAAC |
VdHog1-F | AGGACCACGTCAACCAGTTC |
VdHog1-R | AGCATCGTTGAAGCTCCAGT |
VDH1-F | CTATTGCGATTGCTCTG |
VDH1-R | GAGCTCAAGGTTTTCGTG |
β-tubulin-F | TCACCAGCCGTGGCAAGGTTG |
β-tubulin-R | AGCAAAGGGCGGTCTGGACGTTG |
表2
VdOAZ敲除突变体在PDA培养基上的菌落生长速率"
菌株 Strains | 第5天菌落直径 Colony diameter on the fifth day/mm | 第9天菌落直径 Colony diameter on the ninth day/mm | 菌落平均生长速率 Colony growth rate/(mm·d-1) |
V592 | 18.17±0.27 | 30.17±1.02 | 3.00 |
vdoaz-1 | 17.59±0.29 | 28.53±0.38 | 2.73* |
vdoaz-2 | 17.67±0.26 | 28.72±0.09 | 2.76* |
vdoaz-3 | 18.24±0.08 | 28.86±0.31 | 2.66* |
EC-oaz-1 | 17.58±0.65 | 29.08±0.15 | 2.88 |
EC-oaz-2 | 17.75±0.19 | 29.67±0.27 | 2.98 |
[1] |
刘琳琳, 张文文, 周易, 等. 棉花与番茄抗棉花黄萎病不依赖于Ve1[J]. 中国科学: 生命科学, 2014, 44(8): 803-814. https://doi.org/10.1360/052014-90
doi: https://doi.org/10.1360/052014-90 |
Liu L L, Zhang W W, Zhou Y, et al. Resistance of cotton and tomato to Verticillium dahliae from cotton is independent on Ve1[J]. Scientia Sinica Vitae, 2014, 44(8): 803-814.
doi: https://doi.org/10.1360/052014-90 |
|
[2] | Schnathorst W C. Life cycle and epidemiology of verticillium. Fungal wilt diseases of plants[M]. San Fransisco: Academic Press, 1981: 81-111. |
[3] |
Hoppenau C E, Tran V, Kusch H, et al. Verticillium dahliae VdTHI4, involved in thiazole biosynthesis, stress response and DNA repair functions, is required for vascular disease induction in tomato[J]. Environmental and Experimental Botany, 2014, 108: 14-22. http://dx.doi.org/10.1016/j.envexpbot.2013.12.015
doi: http://dx.doi.org/10.1016/j.envexpbot.2013.12.015 |
[4] |
Qi X L, Su X F, Guo H M, et al. VdThit, a thiamine transport protein, is required for pathogenicity of the vascular pathogen Verticillium dahliae[J]. Molecular Plant Microbe Interactions, 2016, 29(7): 545-559. http://dx.doi.org/10.1094/MPMI-03-16-0057-R
doi: http://dx.doi.org/10.1094/MPMI-03-16-0057-R |
[5] |
Maruthachalam K, Klosterman S J, Kang S, et al. Identification of pathogenicity-related genes in the vascular wilt fungus Verticillium dahliae by Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis[J]. Molecular Biotechnology, 2011, 49(3): 209-221. https://doi.org/10.1007/s12033-011-9392-8
doi: 10.1007/s12033-011-9392-8 pmid: 21424547 |
[6] |
Zhao Y L, Zhou T T, Guo H S. Hyphopodium-specific VdNoxB/VdPls1-dependent ROS-Ca2+ signaling is required for plant infection by Verticillium dahliae[J/OL]. PLoS Pathogens, 2016, 12(7): e1005793. https://doi.org/10.1371/journal.ppat.1005793
doi: https://doi.org/10.1371/journal.ppat.1005793 |
[7] |
Li Z F, Liu Y J, Feng Z L, et al. VdCYC8, encoding CYC8 glucose repression mediator protein, is required for microsclerotia formation and full virulence in Verticillium dahliae[J/OL]. PLoS ONE, 2015, 10(12): e0144020. https://doi.org/10.1371/journal.pone.0144020
doi: https://doi.org/10.1371/journal.pone.0144020 |
[8] |
Gao F, Zhou B J, Li G Y, et al. A glutamic acid-rich protein identified in Verticillium dahliae from an insertional mutagenesis affects microsclerotial formation and pathogenicity[J/OL]. PLoS ONE, 2010, 5(12): e15319. https://doi.org/10.1371/journal.pone.0015319
doi: https://doi.org/10.1371/journal.pone.0015319 |
[9] |
Tzima A K, Paplomatas E J, Rauyaree P, et al. VdSNF1, the sucrose nonfermenting protein kinase gene of Verticillium dahliae, is required for virulence and expression of genes involved in cell-wall degradation[J]. Molecular Plant Microbe Interactions, 2011, 24(1): 129-142. https://doi.org/10.1094/MPMI-09-09-0217
doi: https://doi.org/10.1094/MPMI-09-09-0217 |
[10] |
Zhang Y L, Mao J C, Huang J F, et al. A uracil-DNA glycosylase functions in spore development and pathogenicity of Verticillium dahliae[J]. Physiological and Molecular Plant Pathology, 2015, 92: 148-153. http://dx.doi.org/10.1016/j.pmpp.2015.05.001
doi: http://dx.doi.org/10.1016/j.pmpp.2015.05.001 |
[11] |
Jonge R D, Esse H P V, Maruthachalam K, et al. Tomato immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing[J]. Proceedings of the National Academy of Sciences, 2012, 109(13): 5110-5115. https://doi.org/10.1073/pnas.1119623109
doi: https://doi.org/10.1073/pnas.1119623109 |
[12] |
Liu T L, Song T Q, Zhang X, et al. Unconventionally secreted effectors of two filamentous pathogens target plant salicylate biosynthesis[J]. Nature Communications, 2014, 5(1): 4686. https://doi.org/10.1038/ncomms5686
doi: https://doi.org/10.1038/ncomms5686 |
[13] |
Santhanam P, Esse H P V, Albert I, et al. Evidence for functional diversification within a fungal NEP1-like protein family[J]. Molecular Plant Microbe Interactions, 2013, 26(3): 278-286. http://dx.doi.org/10.1094/MPMI-09-12-0222-R
doi: http://dx.doi.org/10.1094/MPMI-09-12-0222-R |
[14] |
Zhou B J, Jia P S, Gao F, et al. Molecular characterization and functional analysis of a necrosis- and ethylene-inducing, protein-encoding gene family from Verticillium dahliae[J]. Molecular Plant Microbe Interactions, 2012, 25(7): 964-975.
doi: 10.1094/MPMI-12-11-0319 |
[15] |
Gui Y J, Zhang W Q, Zhang D D, et al. A Verticillium dahliae extracellular cutinase modulates plant immune responses[J]. Molecular Plant Microbe Interactions, 2018, 31(2): 260-273. https://doi.org/10.1094/MPMI-06-17-0136-R
doi: https://doi.org/10.1094/MPMI-06-17-0136-R |
[16] |
Rauyaree P, Ospina-Giraldo M D, Kang S, et al. Mutations in VMK1, a mitogen-activated protein kinase gene, affect microsclerotia formation and pathogenicity in Verticillium dahliae[J]. Current Genetics, 2005, 48(2): 109-116. http://dx.doi.org/10.1007/s00294-005-0586-0.
doi: http://dx.doi.org/10.1007/s00294-005-0586-0 |
[17] |
Tian L L, Xu J, Zhou L, et al. VdMsb regulates virulence and microsclerotia production in the fungal plant pathogen Verticillium dahliae[J]. Gene, 2014, 550(2): 238-244. http://dx.doi.org/10.1016/j.gene.2014.08.035
doi: http://dx.doi.org/10.1016/j.gene.2014.08.035 |
[18] |
Wang Y L, Tian L Y, Xiong D G, et al. The mitogen-activated protein kinase gene, VdHog1, regulates osmotic stress response, microsclerotia formation and virulence in Verticillium dahliae[J]. Fungal Genetics and Biology, 2016, 88: 13-23. http://dx.doi.org/10.1016/j.fgb.2016.01.011
doi: http://dx.doi.org/10.1016/j.fgb.2016.01.011 |
[19] |
Qi X Y, Zhou S, Shang X G, et al. VdSho 1 regulates growth, oxidant adaptation and virulence in Verticillium dahliae[J]. Journal of Phytopathology, 2016, 164(11-12): 1064-1074. https://doi.org/10.1111/jph.12527
doi: https://doi.org/10.1111/jph.12527 |
[20] |
Tzima A, Paplomatas E J, Rauyaree P, et al. Roles of the catalytic subunit of cAMP-dependent protein kinase A in virulence and development of the soilborne plant pathogen Verticillium dahliae[J]. Fungal Genetics and Biology, 2010, 47(5): 406-415. https://doi.org/10.1016/j.fgb.2010.01.007
doi: https://doi.org/10.1016/j.fgb.2010.01.007 |
[21] |
Tzima A K, Paplomatas E J, Tsitsigiannis D I, et al. The G protein β subunit controls virulence and multiple growth- and development- related traits in Verticillium dahliae[J]. Fungal Genetics and Biology, 2012, 49(4): 271-283. https://doi.org/10.1016/j.fgb.2012.02.005
doi: https://doi.org/10.1016/j.fgb.2012.02.005 |
[22] |
Zhang T, Zhang B S, Hua C L, et al. VdPKS 1 is required for melanin formation and virulence in a cotton wilt pathogen Verticillium dahliae[J]. Science China Life Sciences, 2017, 60(8): 868-879. https://doi.org/10.1007/s11427-017-9075-3
doi: 10.1007/s11427-017-9075-3 pmid: 28755294 |
[23] |
Santhanam P, Thomma B P H J. Verticillium dahliae Sge 1 differentially regulates expression of candidate effector genes[J]. Molecular Plant Microbe Interactions, 2013, 26(2): 249-256. http://dx.doi.org/10.1094/MPMI-08-12-0198-R
doi: http://dx.doi.org/10.1094/MPMI-08-12-0198-R |
[24] | 高云, 孟佩, 张婷, 等. 棉花黄萎病菌VdSge1基因的敲除与功能分析[J]. 棉花学报, 2015, 27(4): 346-353. |
Gao Y, Meng P, Zhang T, et al. Knockout and functional analysis of VdSge1 in Verticillium dahliae[J]. Cotton Science, 2015, 27(4): 346-353. | |
[25] |
Luo X M, Mao H Q, Wei Y M, et al. The fungal-specific transcription factor Vdpf influences conidia production, melanized microsclerotia formation and pathogenicity in Verticillium dahliae[J]. Molecular Plant Pathology, 2016, 17(9): 1364-1381. https://doi.org/10.1111/mpp.12367
doi: https://doi.org/10.1111/mpp.12367 |
[26] |
Xiong D G, Wang Y L, Tian L Y, et al. MADS-Box transcription factor VdMcm1 regulates conidiation, microsclerotia formation, pathogenicity, and secondary metabolism of Verticillium dahliae[J]. Frontiers in Microbiology, 2016, 7: 1192. https://doi.org/10.3389/fmicb.2016.01192
doi: https://doi.org/10.3389/fmicb.2016.01192 |
[27] |
Xiong D G, Wang Y L, Tang C, et al. VdCrz 1 is involved in microsclerotia formation and required for full virulence in Verticillium dahliae[J]. Fungal Genetics and Biology, 2015, 82: 201-212. http://dx.doi.org/10.1016/j.fgb.2015.07.011
doi: http://dx.doi.org/10.1016/j.fgb.2015.07.011 |
[28] |
Zhang W Q, Gui Y J, Short D P G, et al. Verticillium dahliae transcription factor VdFTF 1 regulates the expression of multiple secreted virulence factors and is required for full virulence in cotton[J]. Molecular Plant Pathology, 2018, 19(4): 841-857. https://doi.org/10.1111/mpp.12569
doi: https://doi.org/10.1111/mpp.12569 |
[29] |
Wallace H M, Fraser A V, Hughes A. A perspective of polyamine metabolism[J]. Biochemical Journal, 2003, 376(1): 1-14. https://doi.org/10.1042/BJ20031327
doi: https://doi.org/10.1042/BJ20031327 |
[30] |
Casero J R A, Pegg A E. Polyamine catabolism and disease[J]. Biochemical Journal, 2009, 421(3): 323-338. https://doi.org/10.1042/BJ20090598
doi: https://doi.org/10.1042/BJ20090598 |
[31] |
Thomas T, Thomas T J. Polyamine metabolism and cancer[J]. Journal of Cellular and Molecular Medicine, 2003, 2(7): 113-126. https://doi.org/10.1111/j.1582-4934.2003.tb00210.x
doi: https://doi.org/10.1111/j.1582-4934.2003.tb00210.x |
[32] |
Schipper R G, Cuijpers V M J I, De Groot L H J M, et al. Intracellular localization of ornithine decarboxylase and its regulatory protein, antizyme-1[J]. Journal of Histochemistry and Cytochemistry, 2004, 52(10): 1259-1266. https://doi.org/10.1369/jhc.4C6389.2004
doi: https://doi.org/10.1369/jhc.4C6389.2004 pmid: 15385572 |
[33] |
蔡富强, 王艳林. 鸟氨酸脱羧酶抗酶Ⅰ与多胺代谢[J]. 生命科学, 2011(10) : 1002-1008. https://doi.org/10.13376/j.cbls/2011.10.016
doi: https://doi.org/10.13376/j.cbls/2011.10.016 |
Cai F Q, Wang Y L. Ornithine decarboxylase antizyme Ⅰ and polyamine metabolism[J]. Chinese Bulletin of Life Sciences, 2011(10): 1002-1008.
doi: https://doi.org/10.13376/j.cbls/2011.10.016 |
|
[34] |
Wang S, Xing H Y, Hua C L, et al. An improved single-step cloning strategy simplifies the Agrobacterium tumefaciens-mediated transformation (ATMT)-based gene-disruption method for Verticillium dahliae[J]. Phytopathology, 2016, 106(6): 645-652. https://doi.org/10.1094/PHYTO-10-15-0280-R
doi: https://doi.org/10.1094/PHYTO-10-15-0280-R |
[35] |
Klimes A, Dobinson K F. A hydrophobin gene, VDH1, is involved in microsclerotial development and spore viability in the plant pathogen Verticillium dahliae[J]. Fungal Genetics and Biology, 2006, 43(4): 283-294. https://doi.org/10.1016/j.fgb.2005.12.006
doi: https://doi.org/10.1016/j.fgb.2005.12.006 pmid: 16488633 |
[1] | 杨可心,陈秀叶,刘畅,鹿秀云,郭庆港,马平. 棉花枯萎病菌新生理型菌株毒素鉴定及其活性测定[J]. 棉花学报, 2021, 33(3): 258-268. |
[2] | 李社增,牛露欣,李博超,陈秀叶,刘畅,鹿秀云,郭庆港,马平,马峙英. 大丽轮枝菌与陆地棉互作过程中棉花次生代谢产物分析[J]. 棉花学报, 2020, 32(6): 501-521. |
[3] | 李敏,李彩红,赵瑞元,刘冰蕾,张志刚. 湖南省棉花黄萎病菌致病力分化及致病型分布研究[J]. 棉花学报, 2019, 31(2): 129-137. |
[4] | 陈丽华, 何鹏飞, 袁德超, 欧晓慧, 吴毅歆, 何月秋. 一种防治棉花黄萎病的生物复合种衣剂的研制[J]. 棉花学报, 2018, 30(3): 282-290,封三. |
[5] | 张希鹤, 李岩, 郁凯, 霍钰阳, 王友华, 陈兵林. 黄萎病胁迫影响棉花幼苗光合及叶绿素荧光特性的机理[J]. 棉花学报, 2018, 30(2): 136-144. |
[6] | 张帆, 李晓林, 张敬泽, 祝水金. 病原物协同致病性对棉花黄萎病严重度的影响[J]. 棉花学报, 2018, 30(2): 188-196. |
[7] | 崔伟业, 周雷, 单柳颖, 张君, 戴小枫, 郭维. 大丽轮枝菌致病缺陷型T-DNA插入突变体的筛选与鉴定[J]. 棉花学报, 2018, 30(1): 29-37. |
[8] | 许琦, 武林琳, 王咪, 李晓萍, 郭文治, 裴蕾. 抗黄萎病海岛棉叶片在大丽轮枝菌胁迫下的蛋白组学分析[J]. 棉花学报, 2017, 29(6): 533-540. |
[9] | 刘廷利, 惠慧, 阚家亮, 卢轩宇, 花晟, 范春晨, 凌溪铁, 王金彦, 陈天子, 杨郁文, 艾尼江, 赵建军, 张保龙. 新疆北部棉花黄萎病菌培养特性、致病型、致病性分化及ISSR遗传变异研究[J]. 棉花学报, 2017, 29(6): 541-549. |
[10] | 陈丽华, 袁德超, 吴毅歆, 何鹏飞, 何月秋. 棉花黄萎病生防内生芽孢杆菌LH-L3的分离鉴定[J]. 棉花学报, 2017, 29(6): 550-559. |
[11] | 王奕丁, 姜婷婷, 王全. 棉花黄萎病拮抗细菌Bacillus subtilis ZL2-70抗菌蛋白的理化性质和抑菌机理[J]. 棉花学报, 2017, 29(6): 560-569. |
[12] | 张志东,段兴鹏,周玉梅,李建福,韩松,高正银,赵佳薇,左开井. 大丽轮枝菌侵染陆地棉早期的转录组分析[J]. 棉花学报, 2017, 29(3): 253-260. |
[13] | 王全, 王占利, 高同国, 李术娜. 响应面法对解淀粉芽孢杆菌(Bacillus amyloliquefaciens)12-7产抗菌蛋白条件的优化[J]. 棉花学报, 2016, 28(3): 283-290. |
[14] | 高云, 孟佩, 张婷, 黄家风, 高峰. 棉花黄萎病菌VdSge1基因的敲除与功能分析[J]. 棉花学报, 2015, 27(4): 346-353. |
[15] | 徐明, 陈捷胤, 马雪峰, 王新艳, 郭维, 戴小枫. 高毒力大丽轮枝菌VDG1特异分泌蛋白HSSP致病相关功能分析[J]. 棉花学报, 2015, 27(4): 337-345. |
|