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【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。
【Objective】 The objective of this study was to provide a method for the rapid molecular detection of avirulence genes Avr1a, Avr1k and Avr3a in Phytophthora sojae and to provide a basis for the rapid molecular detection of other non-toxic genes in P. sojae. 【Method】 According to the sequence of Avr1a, Avr1k and Avr3a of P. sojae published in GenBank, primers were designed and specific primers were screened respectively. Based on the optimization of PCR reaction system and amplification conditions, A total of 86 strains of P.sojae, Avr1a, Avr1k and Avr3a, were detected by PCR and a set of specific detection systems for Avr1a, Avr1k and Avr3a were established. The molecular identification and inoculation identification results were compared, the amplified positive-positive bands and false-positive bands were gel recovery and cloning sequencing, sequencing results were compared with the original sequence of three non-toxic genes to determine the molecular Whether the labeling method is suitable for the rapid detection of Avr1a, Avr1k and Avr3a. 【Result】 The results showed that the selected specific primers could amplify a band of about 550 bp from the strain containing the corresponding non-toxic gene. The coincidence rates of Avr1a, Avr1k and Avr3a in molecular identification and inoculation were 45.3%, 84.9% and 97.7%, respectively. The true positive bands of three non-toxic genes were all above 97% identity with the original sequence, the false positive bands of Avr1a were about 80% identical to the original sequence, and the remaining two genes were below 30%. 【Conclusion】 The detection system based on the sequences of Avr1a, Avr1k and Avr3a can be used for the rapid detection of Avr3a, which is not suitable for the rapid detection of Avr1a. It is unclear whether it is suitable for the rapid detection of Avr1k.