谷瘟病菌无毒基因型鉴定及分析

doi:10.3864/j.issn.0578-1752.2018.06.007

摘要/Abstract

摘要： 【目的】鉴定不同地区谷瘟病菌（Magnaporthe oryzae）所含无毒基因的类型，确定无毒基因在菌株中的分布及变异情况，为深入研究谷瘟病菌无毒基因变异机制提供参考。【方法】从中国北方谷子主产区不同区域内采集并分离76个谷瘟病菌的单孢菌株，提取其基因组DNA，根据目前已成功克隆的稻瘟病菌的7个无毒基因的核苷酸序列设计特异性引物，进行PCR扩增及电泳检测，并对部分菌株的无毒基因进行测序分析。【结果】在76个谷瘟病菌中，无毒基因ACE1、Avr-pita、Avr1-CO39和AvrPiz-t的扩增率为100％，无毒基因Avr-pik、Avr-pia和Avr-pii的扩增率分别为63.2%、42.1%和21.1%。在谷瘟病菌菌株P11和P34中，Avr1-CO39的扩增条带较预期片段大490 bp，测序结果发现菌株P11和P34中的Avr1-CO39基因序列完全一致，均在启动子区插入了490 bp核苷酸，该插入序列与non-LTR retrotransposon: Mg-SINE的相似度达99.16％。Avr-pita的测序结果发现，谷瘟病菌菌株中的Avr-pita基因序列变异较为丰富，其变异形式主要为单核苷酸的变异，包括单碱基的插入、缺失及多位点的SNP。Avr-pia的变异类型主要为整个无毒基因的缺失，经测序验证等位基因序列分为4种类型。Avr-pia-A与参考序列（AB498873.1）一致，包含10个菌株；Avr-pia-B包含20个菌株，在-116、-109和-16 bp处分别存在C/T、G/T和C/A变异，但与参考序列的CDS区序列相同；Avr-pia-C仅包含菌株P10，在+150 bp处存在T/G变异，但为同义突变；Avr-pia-D仅包含菌株P18，在+212 bp位点处存在C/T变异，导致该变异位点由编码苏氨酸突变为编码异亮氨酸。谷瘟病菌Avr-pii包含3种等位基因类型。Avr-pii-A型与参考序列（AB498874.1）一致，共包含14个菌株；Avr-pii-B型和Avr-pii-C型分别在+139和+64 bp处存在A/G变异，核苷酸的变异导致该位点由编码苏氨酸改为丙氨酸。Avr-pii-B型和Avr-pii-C型变异均为首次报道。单元型分析表明，AG2包含23个菌株，占供试菌株的30.2%，为优势单元型。【结论】明确了不同地区的谷瘟病菌中无毒基因ACE1、Avr-pita、Avr1-CO39和AvrPiz-t不存在地理来源的差异；而无毒基因Avr-pik、Avr-pia和Avr-pii在各地分布有差异。谷瘟病菌AG2单元型为优势单元型，其次是单元型AG1和AG5。

关键词: 谷子, 谷瘟病菌, 无毒基因, 变异

Abstract: 【Objective】The objective of this study is to identify avirulence genes (Avr-genes) of Magnaporthe oryzae, understand the distribution and variation of Avr-genes in the epidemic strains of different regions, and to provide a reference for further study of the mechanism of Avr-genes variation in M. oryzae. 【Method】 A total of 76 strains of M. oryzae were isolated from different regions of millet growing area in northern China. The genomic DNA of 76 M. oryzae strains was extracted and used as a template for PCR amplification using the specific primers designed according to the 7 Avr-genes cloned from rice blast. AGE (agarose gel electrophoresis) analysis and sequencing of the PCR products were used to analyze the distribution of Avr-genes in M. oryzae strains of different regions from foxtail millet. 【Result】 The amplification rate of ACE1, Avr-pita, Avr1-CO39 and AvrPiz-t genes was 100% in 76 strains of M. oryzae and the amplification rate of avirulent genes Avr-pik, Avr-pia and Avr-pii was 63.2%, 42.1% and 21.1%, respectively. The amplification of Avr1-CO39 in the strains P11 and P34 was about 490 bp larger than the expected fragments. Sequencing of the Avr1-CO39 in the strains P11 and P34 showed that the Avr1-CO39 sequences in strains P11 and P34 were identical and the promoter region contained a 490 bp inserted fragment which shared 99.16% similarity with non-LTR retrotransposon: Mg-SINE. A high level of Avr-pita polymorphisms were found in M. oryzae and the major variation was single nucleotide variation, including single base insertion, deletion, and SNP of multiple loci. The main sequence variation of Avr-pia was the absence of the whole Avr-gene, and their sequencing verification results showed that there were 4 genotypes of Avr-pia allele in the strains. The Avr-pia-A was identical to the reference sequence (AB498873.1) and contained 10 strains. Avr-pia-B contained 20 strains, and there were C/T, G/T and C/A mutations at -116, -109, and -16 bp sites, but the CDS region of Avr-pia-B was the same as the reference sequence. The Avr-pia-C type contained only the strain P10, and there was a T/G mutation at +150 bp, but it is a synonymous mutation. The Avr-pia-D contained only the strain P18, and there was a C/T mutation at +212 bp site, which resulted in the mutation of coding amino acid from threonine to isoleucine. The Avr-pii of M. oryzae contained 3 alleles. The Avr-pii-A was the same as the reference sequence (AB498874.1) and contained 14 strains. An A/G mutation was found in Avr-pii-B and Avr-pii-C at +139 and +64 bp sites, respectively, which caused the amino sequence in the site to mutate from threonine to alanine. Avr-pii-B and Avr-pii-C were both reported for the first time. The haplotype analysis showed that AG2 containing 23 strains was the dominant type, which accounted for 30.2% of the tested strains. 【Conclusion】 Avr-genes ACE1, Avr-pita, Avr1-CO39 and AvrPiz-t had no geographical origin difference and the frequency distribution of Avr-genes Avr-pik, Avr-pia and Avr-pii varied in different regions. The AG2 haplotype was predominant in M. oryzae from foxtail millet, followed by AG1 and AG5 haplotypes.

Key words: foxtail millet, Magnaporthe oryzae, avirulence gene, mutation

任世龙，白辉，王永芳，全建章，董志平，李志勇，邢继红. 谷瘟病菌无毒基因型鉴定及分析[J]. 中国农业科学, 2018, 51(6): 1079-1088.

REN ShiLong, BAI Hui, WANG yongFang, QUAN JianZhang, DONG ZhiPing, LI ZhiYong, XING JiHong. Identification and Analysis of Magnaporthe oryzae of Foxtail Millet Avirulence Genes[J]. Scientia Agricultura Sinica, 2018, 51(6): 1079-1088.

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