草菇菌种退化相关分子标记的筛选
Screening of molecular markers associated with degeneration of Volvariella volvacea strains

【目的】基于SRAP分子标记技术，筛选出与草菇（Volvariella volvacea）菌丝退化性状紧密连锁的分子标记,为进一步研究目标性状基因的克隆与功能奠定基础。【方法】以草菇正常生长菌株V51（对照）与其菌丝退化菌株（VNM1～10）为材料，采用群体分离分析法分别构建2种草菇的近等基因池。利用SRAP分子标记技术，寻找两类菌株的差异片段，回收差异片段后与pMD-18T载体连接，构建重组质粒pMD-VNMDF并转化E.coli DH5α感受态细胞，将筛选的阳性克隆测序。根据差异片段序列设计引物，将其转化成SCAR分子标记，并对草菇正常菌株V51及其菌丝退化菌株VNM进行扩增试验，验证该标记的真实性和可靠性。【结果】81对SRAP引物组合中有2对引物可扩增出稳定差异条带，其中引物对Me8 Em4 PCR扩增出长度约300 bp的差异条带（命名为VNMDF）存在于退化菌株中。对SRAP分子标记片段VNMDF回收、测序并进行序列分析发现，该片段长度为279 bp，其核酸、氨基酸序列与编码26S蛋白酶体亚基的相似性分别达到81%和93%。利用SCAR特异引物对SCAR250可在菌丝退化菌株中稳定扩增出长度约250 bp片段，与预期大小（279 bp）一致，而在正常菌株中未扩增出此片段。【结论】获得了1条可能与草菇菌丝退化性状基因紧密连锁的SRAP分子标记，并将其成功转化为SCAR标记（SCAR250）。
【Objective】The aim of this paper was to screen molecular markers closely linked to hyphae degeneration of Volvariella volvacea using sequence related amplified polymorphism (SRAP) technique and to lay foundation for further cloning and functional analysis of target genes.【Method】Normal Volvariella volvacea V51 strains (CK) and 10 hyphae degenerate strains (VNM) were used to construct isogenic DNA pools by bulked segregation analysis (BAS) method.To find differential fragments between two types of strains,SRAP technique was used.After being recycled,differential fragments were ligated with PMD-18T vector.Then,recombinant plasmids pMD-VNMDF were transformed into E.coli DH5α competent cells and positive clones were sequenced.Based on the identified fragment sequences,special primers were designed to convert target fragment into sequence-characterized amplification region (SCAR) marker.Amplification tests of normal growth strains V51 and mycelium degradation strains were performed to verify the authenticity and reliability of the marker.【Result】From 81 pairs of SRAP primer combinations chosen to screen the markers linked to hyphae degeneration of straw mushroom,two could amplify stable different bands.With the primer pairs of Me8-Em4,a polymorphic fragment of 300 bp (named VNMDF) was found in the degenerate strains gene pool.By recycling,sequencing and sequence analysis,it was found that the similarities of nucleic acid sequences and amino acid sequences between the fragment VNMDF and the fragment encoding the 26S proteasome subunits were 81% and 93%,respectively.A fragment of 250 bp was stably amplified by SCAR specific primers SCAR250 in hyphae degenerate strains,and it was not detected in normal strains.【Conclusion】In this study,a SRAP marker gene fragment closely linked to straw mushroom hyphae degeneration was obtained and successfully transformed into SCAR marker（SCAR250）