摘要
通过氨基酸同源比对(Blast P)以及金针菇冷诱导前后菌丝阶段和原基阶段的转录组数据分析,获得了金针菇中的两个假定G蛋白偶联受体基因Fvgpcr1、Fvgpcr2。对获得的金针菇假定G蛋白偶联受体基因Fvgpcr1、 Fvgpcr2构建了基因组编辑(CRISPR/Cas9)的pCAMBIA0390-hph-Fvcas9-Fvgpcr1-sgRNA1/sgRNA2、pCAMBIA0390-hph-Fvcas9-Fvgpcr2-sgRNA1/sgRNA2等4个表达载体。通过农杆菌介导(ATMT)将表达载体pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA转化金针菇菌丝体,采用潮霉素和头孢毒素低浓度初筛和高浓度复筛,经两段筛选获得金针菇拟转化子。经对拟转化子进行PCR鉴定、RT-qPCR检测和Western杂交验证,结果显示表达载体pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA成功整合进金针菇基因组中,FvCas9蛋白正常表达,但未得到Fvgpcr基因敲除突变体。本研究利用农杆菌介导转化法在金针菇中构建了CRISPR/Cas9敲除体系,对后续目标基因的敲除有着重要意义。
Two putative G protein-coupled receptor genes Fvgpcr1 and Fvgpcr2 in Flammulina filiformis were obtained by amino acid homology alignment(Blast P) and transcriptome data analysis of the fungus in hyphae and primordium stages before and after cold induction. Four expression vectors including p CAMBIA0390-hph-Fvcas9-Fvgpcr1-sg RNA1/sg RNA2 and pCAMBIA0390-hph-Fvcas9-Fvgpcr2-sgRNA1/sgRNA2 were constructed for gene disruption of G-protein coupled receptor gene Fvgpcr1 and Fvgpcr2. The expression vector p CAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA was transformed into the mycelium of F. filiformis by Agrobacterium tumefaciensmediated(ATMT). Transformants harvested from hygromycin and cefotaxime rescreening were identified by PCR, RT-qPCR and Western hybridization. Results showed that the expression vectors pCAMBIA0390-hphFvcas9-Fvgpcr-sgRNA1 and pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA2 were successfully integrated into the genome of F. filiformis and the Fv Cas9 protein was successfully expressed. However, the Fvgpcr deletion mutant was not obtained. This study used the Agrobacterium-mediated transformation method to construct a CRISPR/Cas9 gene disruption system in F. filiformis, which is of great significance for the subsequent gene disruption.
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