Cloning and Expression Analysis of RsPhmGene inRhizoctoniasolani AG-1ⅠA of Rice Sheath Blight Pathogen

doi:10.16819/j.1001-7216.2018.7094

Abstract

Abstract:

【Objective】In order to elucidate the functions of phenol 2-monooxygenase(RsPhm)gene in melanization of RhizoctoniasolaniKühnAG-1ⅠA, the causal agent of rice sheath blight,【Method】the gene was cloned by routine PCR andRT-PCR techniques, and the bioinformatics analysis of this gene was conducted; furthermore, therelative expression levelunder catecholstresswas determinedby using fluorescence quantitative real-time PCR (qRT-PCR) technique.【Result】Bioinformatics analysis showed that the full-lengthDNA and cDNA sequences of RsPhmgene were 2628 bp and 1983 bp, respectively, which encode660 amino acids. The phylogenetic tree analysis showed thatRsPhm gene hada close relationship in different anastomosis groups (AGs) ofR.solani, and a certainevolutionary conservation amongdifferent fungal species. Results of qRT-PCR indicated that the exposure to exogenous catecholcould improvethe expression level ofRsPhm gene, and which peaked at 12.5 µg/mL of catechol,with a significant increase of 35.7 times, 19.1times and 28.4 timesup-regulated at 25 µg/mL and 50 g/mL,respectively, but only 2.1 times up-regulated at 100 g/mL.【Conclusion】The full-length sequence ofRsPhm gene was obtained, its basic biological information was understood, and its expression pattern under catechol stress was clarified.Thesefindings will lay abasis for the scientific and systematic elucidation of regulatorymechanism of melanin formation byRsPhmgene ofR. solaniAG-1ⅠA.

Key words: RhizoctoniasolaniKühnAG-1ⅠA;, RsPhm gene, gene clone, expression analysis

摘要：

【目的】为了阐明苯酚2-单加氧酶基因(RsPhm)在水稻纹枯病菌(Rhizoctoniasolani AG-1ⅠA)黑化中的功能,【方法】采用常规PCR和RT-PCR技术对该基因进行克隆和生物信息学分析,通过荧光定量PCR(qRT-PCR)技术检测在儿茶酚胁迫下该基因的相对表达量。【结果】生物信息学分析结果表明,RsPhm基因的DNA和cDNA全长序列分别为2628 bp和1983 bp,编码660个氨基酸。系统进化树分析显示,RsPhm基因在立枯丝核菌(R. solani)不同融合群中具有较近的亲缘关系,在不同真菌之间在进化上具有一定保守性。通过qRT-PCR技术分析了在不同浓度儿茶酚胁迫下水稻纹枯病菌RsPhm基因的转录表达情况。外源儿茶酚能提高RsPhm基因的表达量,在12.5 µg/mL浓度下表达量最高,极显著上调35.7倍,在25 µg/mL和50 µg/mL浓度下表达量分别上调19.1倍和28.4倍,但在100 µg/mL浓度下表达量仅上调2.1倍。【结论】获得了RsPhm基因全长序列,了解了其基本生物学信息,明确了其在儿茶酚胁迫下的表达模式。研究结果为科学、系统地阐明水稻纹枯病菌RsPhm基因调控黑色素形成机制奠定了理论基础。

关键词: 水稻纹枯病菌, 苯酚2-单加氧酶基因, 基因克隆, 表达分析

CLC Number:

Shaofeng JIANG, Chenjiaozi WANG, Canwei SHU, Erxun ZHOU. Cloning and Expression Analysis of RsPhmGene inRhizoctoniasolani AG-1ⅠA of Rice Sheath Blight Pathogen[J]. Chinese Journal OF Rice Science, 2018, 1(1): 111-118.

江绍锋, 王陈骄子, 舒灿伟, 周而勋. 水稻纹枯病菌RsPhm基因的克隆及其表达分析[J]. 中国水稻科学, 2018, 1(1): 111-118.

Figures/Tables 6

Table 1 Sequences of oligonucleotide primers used in this study.

引物 Primer	序列 Sequence(5′-3′)
DF01905	ATGCCCGCTGTTGAATCTAA
DR01905	TCACGCGGAAGCCATAAACC
F01905	CCTGATATCCGAAACCGTACC
R01905	TCATCTTGCTCCTGTCCATTC
GAPDHF	TACTCCGCAATGCTATCG
GAPDHR	TACTCGGTCCCAGTGGT

Fig.1. PCR products of RsPhm gene in Rhizoctoniasolani. M, 250 bp DNA marker; 1, The PCR product of RsPhmusing DNA as template; 2, The PCR product of RsPhmusing cDNA as template.

Fig. 2. Comparison of amino acid sequences ofRsPhm proteins in different anastomosis groups ofRhizoctoniasolani. RsPhm is the result of this study, the NCBI GenBank accession numbers of R. solani AG-1ⅠA, R. solani AG-1ⅠB, R. solani AG22ⅢB, R. solani AG-3, R. solani AG-1ⅠB and R. solani 123EareELU44059.1, CEL56393.1, CUA74266.1, EUC54794.1, CCO31161.1 and KEP48781.1, respectively.

Fig. 3. Three-dimensional structure prediction of RsPhm protein in RhizoctoniasolaniAG-1 IA.

Fig. 4. Phylogenetic tree of amino acid sequences of RsPhm proteins from Rhizoctoniasolaniand other related fungi.

Fig. 5. Expression levelof RsPhm geneinRhizoctoniasolani AG-1 IA at different concentrations of catechol. Values areMean±SE of three replicates. *and**, Significantly different at 5% or 1% levelscompared to the control, respectively.

References 29

[1]	Webb K M, Freeman C, Broeckling C D.Metabolome profiling to understand the defense response of sugar beet (Beta vulgaris) to Rhizoctoniasolani AG 2-2 IIIB. PhysiolMol Plant Pathol, 2016, 94: 108-117.
[2]	HannukkalaA O, Rastas M, Laitinen P, Latvala S.Rhizoctoniasolani injuries in oilseed crops in Finland and impacts of different crop management practices on disease incidence and severity. Ann ApplBiol, 2016, 169(2): 257-273.
[3]	邹成佳, 唐芳, 杨媚, 贺晓霞, 李献军, 周而勋. 华南3省(区)水稻纹枯病菌的生物学性状与致病力分化研究. 中国水稻科学, 2011, 25(2): 206-212.
	Zou C J, Tang F, Yang M, He X X, Li X J, Zhou E X.Studies on biological characteristics and pathogenicity differentiation of rice sheath blight pathogen from three provinces in South China.Chin J Rice Sci,2011, 25(2): 206-212. (in Chinese with English abstract)
[4]	Feng S J, Shu C W, Wang C J Z, Jiang S H, Zhou E X. Survival of Rhizoctoniasolani AG-1 IA, the causal agent of rice sheath blight, under different environmental conditions. J Phytopathol, 2017, 165(1): 44-52.
[5]	刘卫东. Pseudomonas putid DLL-E4 1,2,4 -苯三酚1,2-双加氧酶的晶体结构及两种农药降解相关酶的蛋白质晶体学研究. 南京: 南京农业大学, 2010.
	Liu W D.Structural analysis of hydroxyquinol 1, 2-dioxygenase from Pseudomonas putida DLL-E4 and crystallographic study on two pesticide degradation related enzymes. Nanjing: Nanjing Agricultural University,2010. (in Chinese with English abstract)
[6]	Neujahr H Y, Gaal A.Phenol hydroxylase from yeast. Sulfhydryl groups in phenol hydroxylase from Trichosporoncutaneum. Eur J Biochem,1975, 58(2): 351-357.
[7]	Yotinov I, Todorova Y, Schneider I, Daskalova E, Topalova Y.The effect of nanodiamonds on phenol biodegradation by Pseudomonassp. strain isolated from polluted sediments. J NanosciNanotechnol, 2016, 16(7): 7696-7706.
[8]	Nakagawa H, Takeda Y.Phenol hydroxylase.BiochimBiophysActa, 1962, 62(2): 423-426.
[9]	Yang R D, Humphrey A E.Dynamic and steady state studies of phenol degradation in pure and mixed cultures.BiotechnolBioeng, 1975, 17(8): 1211-1235.
[10]	Hamada T, Asanagi M, Satozawa T, Araki N, Banba S, Higashimura N, Akase T, Hirase K.Action mechanism of the novel rice blast fungicide tolprocarb distinct from that of conventional melanin biosynthesis inhibitors.J PesticSci, 2014, 39(3): 152-158.
[11]	Chai L Y A, Netea M G, Sugui J, Vonk A G, van de Sande W W J, Warris A, Kwon-Chung K J, Jan Kullberg B.Aspergillus fumigatus conidial melanin modulates host cytokine response. Immunobiology, 2010, 215(11): 915-920.
[12]	Soares A R, de Lourdes Lucio Ferrarese M, de CássiaSiqueira-Soares R, Marchiosi R, Finger-Teixeira A, Ferrarese-Filho O. The allelochemical1-dopa increases melanin production and reduces reactive oxygen species in soybean roots.J Chem Ecol, 2011, 37(8): 891-898.
[13]	Piattelli M, Fattorusso E, Magno S, Nicolaus RA.Ustilago melanin, a naturally occurring catechol melanin. Tetrahedron Lett, 1963, 4(15): 997-998.
[14]	Chen J Y, Wang C J Z, Shu C W, Zhu M H, Zhou E X. Isolation and characterization of a melanin from Rhizoctoniasolani, the causal agent of rice sheath blight. Eur J Plant Pathol, 2015, 142(2): 281-290.
[15]	Yang Y Q, Yang M, Li M H, Zhou E X.Cloning and functional analysis of an endo-PG-encoding gene Rrspg1 of Rhizoctoniasolani, the causal agent of rice sheath blight. Eur J Plant Pathol, 2012, 34(3): 1-12.
[16]	Zheng A P, Lin R M, Zhang D H, Qin P G, Xu L Z, Ai P, Ding L, Wang Y R, Chen Y, Liu Y, Sun Z G, Feng H T, Liang X X, Fu R G, Tang C Q, Li Q, Zhang J, Xie Z L, Deng Q M, Li S C, Wang S Q, Zhu J, Wang L X, Liu H N, Li P.The evolution and pathogenic mechanisms of the rice sheath blight pathogen.Nat Commun, 2013, 4: 1424.
[17]	Wojcieszynska D, Gren I, Labuzek S, Respondek M.Substrate specificity and sensitiveness of phenol monooxygenase from Stenotrophomonasmaltophilia strain KB2 versus their potential application to bioremediation of the environment. Biotechnologia, 2007, 2: 181-191.
[18]	李朦. 嗜酸硫化芽孢杆菌苯酚羟化酶还原酶组分的重组表达与性质研究. 厦门: 国家海洋局第三海洋研究所, 2016.
	Li M.Expression and characterization of the phenol hydroxylase reductase from Sulfobacillus acidophilus TPY. Xiamen: The Third Institute of Oceanography,StateOceanicAdministration, 2016. (in Chinese with English abstract)
[19]	沈文静, 张静, 曹慧, 崔中利. 恶臭假单胞菌DLL-E4对硝基苯酚降解途径关键基因pnpC的突变分析. 生态与农村环境学报, 2008, 24(4): 77-82.
	Shen W J, Zhang J, Cao H,Cui Z L.p-nitrophenol degradation characteristics of hydroxyquinol1, 2-D ioxygenase gene (pnpC) knock-out mutant of Pseudomonas putida DLL-E4. J Ecol Rural Environ, 2008, 24(4): 77-82. (in Chinese with English abstract)
[20]	Kasak L, Hôrak R, Nurk A, Talvik K, Kivisaar M.Regulation of the catechol 1,2-dioxygenase- and phenol monooxygenase-encodingpheBA operon in Pseudomonas putida PaW85. J Bacteriol, 1993, 175(24): 8038-8042.
[21]	Tuan N N, Hsieh H C, Lin Y W, Huang S L.Analysis of bacterial degradation pathways for long-chain alkylphenols involving phenol hydroxylase, alkylphenol monooxygenase and catechol dioxygenase genes.BioresourceTechnol, 2011, 102(5): 4232-4240.
[22]	Lu L, Shu C W, Liu C J Z, Zhou E X. The impacts of natural antioxidants on sclerotial differentiation and development in RhizoctoniasolaniAG-1 IA. Eur J Plant Pathol, 2016, 146(4): 729-740.
[23]	Weijn A, Bastiaan-Net S, Wichers H J, Mes J J.Melanin biosynthesis pathway in Agaricusbisporus mushrooms. Fungal Genet Biol, 2013, 55(6): 42-53.
[24]	Butler M J, Gardiner R B, Day A W.Melanin synthesis by Sclerotiniasclerotiorum.Mycologia, 2009, 101(3): 296.
[25]	Butler M J, Day A W.Destruction of fungal melanins by ligninases of Phanerochaetechrysosporiumand other white rot fungi.Int J Plant Sci, 1998, 159(6): 989-995.
[26]	Casadevall A, Rosas A L, Nosanchuk J D.Melanin and virulence in Cryptococcus neoformans. CurrOpinMicrobiol, 2000, 3(4): 354-358.
[27]	Eisenman H C, Chow S, Tsé K K, McClelland E, Casadevall A. The effect of L-DOPA on Cryptococcus neoformans growth and gene expression. Virulence, 2014, 2(4): 329-336.
[28]	Hyakumachi M, Yokoyama K, Ui T.Role of melanin in susceptibility and resistance of Rhizoctoniasolani to microbial lysis. T BritMycolSoc, 1987, 89(1): 27-33.
[29]	Kim H T, Chung Y R, Cho K Y.Mycelial melanization of RhizoctoniasolaniAG1 affecting pathogenicity in rice. Plant Pathol J, 2001, 17: 210-215.