大豆疫霉菌(Phytophthora sojae)无毒基因Avr1a、Avr1k及Avr3a的分子鉴定

Molecular identification of avirulent genes of Avr1a, Avr1k and Avr3a in Phytophthora sojae

摘要:

【目的】为大豆疫霉菌(Phytophthora sojae)无毒基因Avr1a、Avr1k和Avr3a快速分子检测提供方法, 也为P. sojae其它无毒基因的快速分子检测研究提供依据。【方法】依据GenBank中公布的P. sojae无毒基因Avr1a、Avr1k和Avr3a的序列设计引物, 分别筛选出特异性引物, 在 PCR反应体系和扩增条件优化基础上, 对已经接种鉴定过无毒基因Avr1a、Avr1k和Avr3a的86株P. sojae进行PCR检测, 建立一套P. sojae无毒基因Avr1a、Avr1k和Avr3a的特异性检测体系。将分子鉴定和接种鉴定结果进行比对, 将扩增出的真阳性条带和假阳性条带分别进行胶回收和克隆测序, 测序结果分别与3个无毒基因的原序列比对, 判定分子标记方法是否适于Avr1a、Avr1k和Avr3a的快速检测。【结果】筛选出的特异性引物均能从含有对应无毒基因的菌株中扩增出约550 bp的条带。Avr1a、Avr1k和Avr3a的分子鉴定及接种鉴定结果符合率依次为45.3%、84.9%和97.7%。3个无毒基因的真阳性条带序列与原序列一致性均达97%以上, Avr1a的假阳性条带与原序列一致性在80%左右, 其余2个基因的都在30%以下。【结论】利用Avr1a、Avr1k和Avr3a基因序列分别设计引物建立的检测体系可以用于Avr3a的快速检测, 不适于Avr1a的快速检测, 是否适合Avr1k的快速检测尚不清楚。

Abstract:

[Objective] To provide method for rapid molecular detection of Avr1a, Avr1k and Avr3a, avirulent genes of Phytophthora sojae, and also to provide some references for rapid molecular detection of other avirulent genes of P. sojae. [Methods] According to Genbank sequence table of Avr1a, Avr1k and Avr3a, avirulent genes of P. sojae, specific primers were filtered out respectively for the three genes by using primer design method. 86 strains of P. sojae which have been identified virulence on the three differential soybean varieties were detected by PCR on the basis of optimized reaction system and amplification conditions. A specific detection system was established for detecting Avr1a, Avr1k and Avr3a, avirulent genes of P. sojae. The results of molecular identification and inoculation identification were compared and analized. And then amplified true and false positive bands were recovered respectively and cloning sequenced and compared with the original sequences of the three avirulent genes, to verify if Avr1a, Avr1k and Avr3a were suitable to be detected by the method of molecular markers. [Results] All the specific primers screened out could amplify a band with length of about 550 bp. The coincidence rate of molecular and inoculation identification for the three avirulent genes were Avr1a-45.3%, Avr1k-84.9% and Avr3a-97.7%. The true positive bands of three avirulent genes had over 97 percent of consistency in sequence with the original one. The false positive bands of Avr1a were about 80 percent, and Avr1k and Avr3a were less than 30 percent of consistency with their original sequences. [Conclusion] The detection system established by using the primers filtered out by utilizing the gene sequence of Avr1a, Avr1k and Avr3a can be used to detect Avr3a rapidly, but not be suitable for detection of Avr1a, whether fit for detection of Avr1k needed further study.