摘要
为建立鉴定双孢蘑菇(Agaricus bisporus)核型的方法,根据GeneBank公布的其基因组信息,针对交配型基因序列设计特异性引物,以W192两个不同核型的同核体菌株A1和A2的菌丝体基因组DNA为模板进行PCR扩增,获得交配型基因序列,筛选存在差异的序列,预测基因片段的限制性内切酶位点,筛选最佳酶切位点,根据酶切片段的差异鉴定菌株的交配型。结果表明:筛选得到长度为736 bp的基因序列,将其命名为aba1;核型为A1的同核体菌株,采用内切酶EcoRⅤ可以将aba1酶切成大小为257 bp和479 bp的两个片段;核型为A2的同核体菌株,采用内切酶XhoⅠ可以将aba1酶切成大小为327 bp和409 bp的两个片段;如果同时可以被两种限制性内切酶酶切,说明该菌株为异核体。采用基因测序鉴定了5株标记为孢子同核体的菌株和8株标记为原生质体同核体的菌株,测序结果表明:5株标记为孢子同核体的菌株中176P11、176P14和176P17的核型属于A1型,176P12和176P19属于A2型;8株标记为原生质体同核体的菌株中101-1P75和03P75的核型为A1型;152P11、46P23、46P27和152P2为A2型;03P46和176P21属于异核体;同时又采用笔者建立的内切酶方法鉴定了这些菌株,核型与基因测序鉴定结果相同。
Distinguishing homokaryon from heterokaryon has been the bottleneck of hybrid breeding of Agaricus bisporus.This study aimed to establish a quick and accurate method to identify karyotype of A.bisporus based on cleaved amplified polymorphic sequence of mating type gene.First,the mating type genes of A.bisporus W192 homokaryons type A1 and A2 were both amplified from their genomes by 9 different primer pairs to generate different fragments.Each set of two fragments from the same primer pair were then subjected to sequence analysis and screened for specific endonuclease sites as a result of single nucleotide polymorphism(SNP).A primer pair and its products from A1 and A2 were selected if this set of PCR fragments can be digested into different sizes of DNA fragments by restriction enzyme.The results showed that the primer pair ABF1 generated a 736 bp fragment named aba1 that had SNPs in A1 and A2,and thus aba1 of A1 and A2 were able to be distinguished by restriction digestion.The gene aba1 of A1 type was digested by EcoRV to fragments of 257 bp and 479 bp respectively;whereas aba1 of A2 type was cut to fragments of 327 bp and 409 bp by XhoI.A hybrid of A1 and A2 type was able to be cut into two pieces of fragments by either EcoRV orXhoI.Various spore homokaryon and protoplast strains were used as test subjects to validate the reliability of this method by sequencing.Strains 176 P11,176 P14,176 P17,101-1 P75 and 03 P75 were found to be type A1 homokaryon by the restriction method in this study.Strains 176 P12,176 P19,152 P11,46 P23,46 P27 and 152 P2 were found to be type A2 homokaryon.Strains 03 P46 and176 P21 were found to be heterokaryon.These results were all confirmed and validated by sequencing results.
作者
冯志勇
陈辉
汪虹
黄建春
郝海波
王倩
宋晓霞
隽加香
张津京
FENG Zhiyong;CHEN Hui;WANG Hong;HUANG Jianchun;HAO Haibo;WANG Qian;SONG Xiaoxia;JUAN Jiaxiang;ZHANG Jinjing(College of Life Science,Nanjing Agricultural University,Nanjing,Jiangsu 210095,China;Institute of Edible Fungi,Shanghai Academy of Agricultural Sciences,Shanghai Key Laboratory of Agricultural Genetics and Breeding,Key Laboratory of Resources and Utilization of Edible Fungi(South),Ministry of Agriculture,National Engineering Research Center of Edible Fungi,Shanghai 201403,China)
出处
《食用菌学报》
CSCD
北大核心
2019年第4期9-15,共7页
Acta Edulis Fungi
基金
上海市现代农业产业技术体系[沪农科产字(2019)第9号]
关键词
双孢蘑菇
交配型基因
核型
限制性内切酶
Agaricus bisporus
mating type gene
karyotype
restriction endonuclease
