辽宁省稻瘟病菌无毒基因型鉴定及分析

【目的】鉴定稻瘟病菌（Magnaporthe oryzae）的无毒基因型，了解无毒基因在不同地区流行菌株中的分布情况，为品种布局提供参考。【方法】根据已经克隆且与稻瘟病菌致病性相关的6个无毒基因序列设计引物，选取辽宁省稻瘟病常发区的26株稻瘟病菌单孢菌株，提取各菌株DNA样本作为模板，进行PCR扩增。通过琼脂糖凝胶电泳及PCR产物测序，对6个无毒基因PCR产物进行碱基和氨基酸序列的分析比较。对琼脂糖凝胶电泳未出条带的，设计不同引物进行验证性试验。【结果】在PCR产物电泳检测中，Avr1-CO39、Avr-pia和Avr-pii没有产物条带，对这3个无毒基因设计验证引物，其PCR产物电泳检测仍没有条带出现，其结果证明辽宁省各水稻主产区流行稻瘟病菌中多不携带Avr1-CO39、Avr-pia和Avr-pii；对于其他3个无毒基因 AvrPiz-t、Avr-pik和Avr-pita则有特异性扩增产物，说明这3个无毒基因在各稻区稻瘟病菌中以不同突变类型及不同频率出现。其中，与Pi2、Pi9和Piz-t对应的AvrPiz-t，分别在22个菌株的中被检测到，且有21个菌株的序列与其序列一致，说明该基因遗传相对稳定，也间接证明携带Pi2、Pi9和Piz-t 的水稻品种在辽宁地区的广谱抗性；与基因组序列不同的16号菌株，在DNA序列192 bp处发生一个单碱基C的缺失，从而导致移码突变，且碱基突变导致氨基酸序列至72位氨基酸时提前终止，而使该基因编码的蛋白质失去无毒基因的功能。与Pik、Pik-p、Pik-m和pik-s对应的Avr-pik，电泳检测结果表明各菌株均有特异性条带出现，经测序验证等位基因序列分为4种类型（B、D、F、G），其中12个菌株携带可为Pik或其等位基因Pik-m和pik-p所识别的D类型；9个菌株携带B类型，该等位基因曾被报道，但是否具有无毒基因的功能仍未验证；另有2个菌株携带F类型等位基因，该基因为首次发现，并分别出现在丹东和盘锦地区。其特点在于与D类型基因间存在143（A/G）的碱基差异，氨基酸序列翻译结果显示其为错义突变，即48（G/D）；而其余3个菌株携带G类型等位基因，该基因亦属首次发现，且仅出现在抚顺新宾地区，碱基序列与D类型存在168（G/A）的差异，导致翻译提前终止，基因功能丧失。对于Avr-pita，26个菌株特异性扩增产物一致，但测序检测到5种等位基因类型，且均与Avr-pita有差异，碱基序列的变化多导致错义突变。这5种等位基因的氨基酸序列之间差异由3个氨基酸位点的差异所致，分别为83（D/N）、192（Y/C）和207（K/R），3处突变均在基因结构域范围内，几种等位基因均已见报道。【结论】辽宁稻区流行稻瘟病菌中Avr-pik、AvrPiz-t和Avr-pita分布较为广泛，选育及推广携带相应抗病基因的水稻品种可减轻稻瘟病的危害。

Identification and Analysis of Magnaporthe oryzae Avirulence Genes in Liaoning Province

【Objective】The objective of this study is to identify avirulence genes (Avr-genes), which existed in the prevalent fungus of Magnaporthe oryzae, understand the distribution of Avr-genes in the epidemic strains of different regions, and to provide references for rice cultivars distribution.【Method】According to the sequences of 6 Avr-genes which had been cloned and were associated with the rice blast pathogenicity, the primers were designed to amplify the target sequences of the epidemic rice blast fungus in Liaoning Province. The DNA, extracted from the 26 strains of rice blast fungi, was used as a template for PCR amplification. By analyzing the PCR products by AGE (agarose gel electrophoresis) and sequencing, the sequences of base and amino acid for each Avr-gene were examined and compared. Some candidate primers were designed to check out the Avr-gene which had no PCR product.【Result】There was no PCR product when amplified Avr1-CO39, Avr-pia and Avr-pii by the primers, and there was also no PCR product by using the candidate primers, which showed that Avr1-CO39, Avr-pia and Avr-pii didn’t exist in most of the M. oryzae isolated from the main rice growing regions in Liaoning Province. On the contrary, the PCR products of AvrPiz-t, Avr-pik and Avr-pita were detected by AGE, and different genome types showed the 3 genes below were carried by the fungus in different mutated types and rates. AvrPiz-t related to the resistant genes including Pi2, Pi9 and Piz-t was detected in 22 M. oryzae strains, and the sequences of 21 strains were same to which exited in AvrPiz-t. The results of this experiment illustrated that the rice varieties carrying Pi2, Pi9 or Piz-t could have broad-spectrum disease resistance to rice blast fungi. The sequence of No. 16 strain was not the same as others, because it lost a single base C in the position of 192 bp, which caused the frame-shift mutation. The mutation led the AA (amino acid) sequence to terminate in position of 72nd AA, and made the secretory protein lose the function of avirulence. Avr-pik associated to the resistant genes like Pik, Pik-p, Pik-m and Pik-s were detected by AGE. The results showed that all the strains had the target bands. Their sequence showed that there were 4 genotypes of Avr-pik allele in the strains (B, D, F and G), and genotype D which carried by 12 strains could be recognized by rice blast disease resistance genes of Pik and its alleles as Pik-m and Pik-p. The 9 strains which carried genotype B of Avr-pik had been reported before, but its function of virulence had not been tested. The genotype F which exited in two strains was firstly found to be the new allele of Avr-pik, and they appeared in Dandong and Panjin, respectively. The specific point mutation, the 143 (A/G) of the base sequence, was the difference between genotype F and D. Genotype G which was also firstly detected was carried by the remaining 3 strains which only appeared in the area of Xinbin County in Fushun. The difference between genotype G and D was the point mutation of 168 (G/A) in the base sequence, which caused the protein translation early termination, therefore, genotype G lost its avirulent function. According to the AGE results, the same bands of Avr-pita were detected in all the 26 strains, and the sequences were tested and analyzed. The results showed that the genes in 26 strains had 5 genotypes of alleles, and there were several point mutations between the 5 alleles and the Avr-pita sequence, and the changes of the sequences caused the missense mutation. The difference of AA among the 5 genotypes were the 3 point mutation of 83 (D/N), 192 (Y/C) and 207 (K/R), which had been found in a structural domain of Avr-pita. The genotypes of allele all had been reported before. 【Conclusion】 The M. oryzae who carried Avr-pik, Avr-pita and AvrPiz-t distributed widely in rice growing regions of Liaoning Province. Breeding and cultivating the rice varieties carrying the corresponding resistance genes could alleviate the damage of the rice blast.