黑龙江省稻瘟病菌无毒基因AVR-Pita及其同源基因的检测与分析

【目的】为了检测黑龙江省稻瘟病菌无毒基因AVR-Pita及其同源基因分布情况与变异机制,了解其变异类型的致病表型。【方法】采用3个无毒基因AVR-Pita1、AVR-Pita2和AVR-Pita3的特异性引物,对202个采自黑龙江省各稻区的稻瘟病菌单孢分离菌株的DNA进行PCR扩增,通过琼脂糖凝胶电泳检测分析,挑选不同带型和不同地区代表菌株的PCR产物进行测序。测序结果与相应无毒基因序列进行碱基与氨基酸序列的比较分析,并利用水稻抗性单基因系,对不同变异类型的稻瘟病菌株进行功能验证。【结果】AVR-Pita1的出现频率为36.14％,AVR-Pita3出现频率为59.41％。AVR-Pita2在黑龙江省202个菌株DNA中未扩增出目的条带。对AVR-Pita1和AVR-Pita3的部分PCR产物进行序列分析,检测出AVR-Pita1有5种变异类型,它们是AVR-Pita1-1、AVR-Pita1-A、AVR-Pita1-B、AVR-Pita1-C和AVR-Pita1-D。经功能验证,AVR-Pita1-1、AVR-Pita1-A、AVR-Pita1-B和AVR-Pita1-D无毒功能丧失。而无毒基因AVR-Pita3未检测出变异菌株。【结论】AVR-Pita1变异能力较强,导致大多数菌株无毒功能丧失,需与其他抗性基因搭配使用。在黑龙江省稻瘟病菌生理小种中未发现AVR-Pita2基因。AVR-Pita3基因序列在菌株中比较稳定。

Detection and Analysis of Magnaporthe oryzae Avirulent Gene AVR-Pita and Its Homologous Genes in Heilongjiang Province

【Objective】We aim to investigate the distribution and variation of avirulent AVR-Pita family of Magnaporthe oryzae from Heilongjiang Province, and to understand the pathogenic phenotypes of avirulence alleles.【Methods】Specific primers of the three avirulent genes AVR-Pita1, AVR-Pita2 and AVR-Pita3 were used. 202 M. oryzae single-spore isolates were obtained from different regions of Heilongjiang Province, their DNA were PCR-amplified by using avirulent genes primers in 2017. PCR products of representative strains falling into different types and from different aresa were selected and sequenced by using agarose gel electrophoresis detection. The base and amino acid sequences were compared with those of the corresponding avirulent genes, and the rice resistant single gene line was used to verify the function of different strains of Magnaporthe oryzae.【Results】The frequency of AVR-Pita1 is 36.14%, and that of AVR-Pita3 is 59.41%. The target band of AVR-Pita2 could not be amplified from the DNA of 202 strains from Heilongjiang Province. Sequence analysis of some PCR products of AVR-Pita1 and AVR-Pita3 were carried out. Five variation types of AVR-Pita1 were detected; they were AVR-Pita1-1, AVR-Pita1-A, AVR-Pita1-B, AVR-Pita1-C and AVR-Pita1-D. Functional verification showed that AVR-Pita1-1, AVR-Pita1-A, AVR-Pita1-B and AVR-Pita1-D lost their function. No mutant strain was detected in the virulent gene AVR-Pita3. 【Conclusion】 AVR-Pita1 had strong mutation ability, which led to the loss of avirulent function of most strains, this should be used in conjunction with other resistant genes. AVR-Pita2 was not found in Heilongjiang Province. The sequence of AVR-Pita3 gene was stable in the strain.