水稻纹枯病菌RsPhm基因的克隆及其表达分析

doi:10.16819/j.1001-7216.2018.7094

摘要/Abstract

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

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

中图分类号:

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.

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.

图/表 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

图1 水稻纹枯病菌RsPhm基因的PCR产物 M―250 bp DNA标记; 1―以DNA为模板的RsPhm基因PCR产物; 2―以cDNA为模板的RsPhm基因PCR产物。

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.

图2 立枯丝核菌不同融合群RsPhm蛋白氨基酸序列的比较 RsPhm为本研究结果,R. solani AG-1ⅠA、R. solani AG-1ⅠB、R. solani AG22ⅢB、R. solani AG-3、R. solani AG-1ⅠB和R. solani 123E的NCBI GenBank注册号分别为ELU44059.1、CEL56393.1、CUA74266.1、EUC54794.1、CCO31161.1和KEP48781.1。

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.

图3 水稻纹枯病菌RsPhm蛋白三维结构预测

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

图4 立枯丝核菌与其他近缘真菌的RsPhm蛋白氨基酸序列的系统进化树

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

图5 不同儿茶酚浓度处理下水稻纹枯病菌RsPhm基因的相对表达量表中数据为3次重复的平均值±标准误。*和**分别表示相对于对照在5%和1%上差异显著。

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.

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