香蕉枯萎病菌内源报告基因比卡菌素聚酮合酶编码基因Bik1的鉴定及应用

Identification and Feasibility Analysis the Endogenous Reporter Gene of bikaverin PKS-encoding gene(Bik1)in Fusarium oxysporum f.sp.cubense race 4

香蕉枯萎病是由尖孢镰刀菌古巴专化型(Fusarium oxysporum f.sp.cubense,Foc)引起的香蕉毁灭性土传病害,其中4号生理小种(Foc4)能感染几乎所有的香蕉品系,危害最严重。本研究克隆鉴定Foc4比卡菌素聚酮合酶编码基因(bikaverin PKS-encoding gene,Bik1)Foc4Bik1,编码一个由2036个氨基酸组成的多功能酶,具有I型PKS聚酮合酶的保守结构域,包含酰基转移酶功能域[acyl-carrier protein(ACP)transacylase,SAT],β-酮脂酰合成酶功能域(β-ketoacyl synthase,KS),丙二酰酰基转移酶功能域(acyltransferase,AT),脱氢酶(dehydratase,DH)以及硫酯酶(thioesterase,TE)等多个保守蛋白结构域。采用Split-marker同源重组技术获得基因敲除突变体ΔFoc4Bik1,通过比较突变体和野生菌株生长速度、产孢量、致病力等差异,证明ΔFoc4Bik1突变体仅影响次生代谢产物比卡菌素的生物合成,不影响病原菌的其他生理表型及致病力;其次,ΔFoc4Bik1突变体摇培后菌液呈白色,与野生型Foc4的暗红色形成鲜明对比,说明Foc4Bik1基因符合作为内源报告基因的条件。本研究以Foc4Bik1为内源靶标基因,利用水稻恶苗病菌(F.fujikuroi)的基因编辑载体pUC-fFuCas9-HTBNLS-hph,尝试以质粒体内表达Cas9和sgRNA的方式探索Foc4中CRISPR/Cas9编辑的可行性。gRNA序列由在线网站设计,构建的sgRNA162导入质粒构建靶向Foc4Bik1的pUC-fFuCas9-HTBNLS-hph-Foc4Bik1基因编辑载体,与供体质粒pUC19-Foc4Bik1-HDR一起导入原生质体通过同源重组修复(homology directed repair,HDR)的方式进行基因编辑。经过潮霉素抗性筛选获得的ΔFoc4Bik1(HDR)基因编辑后的敲除转化子进行PCR检测和摇培,白色菌液表型与PCR检测阳性的敲除转化子相互对应,证实了Foc4基因编辑的可行性。此外,Foc4Bik1可作为香蕉枯萎菌Foc4内源报告基因并评估探索新的分子生物学技术在香蕉枯萎菌上应用的可能性。

Fusarium oxysporum f.sp.cubense(Foc)causes Fusarium wilt(Panama disease),one of the most devastating diseases of banana(Musa spp.).Foc race 4(Foc4)is currently known as a major concern in global banana production.Previous studies reported that bikaverin PKS-encoding gene Bik1 encoded a polyketide synthase responsible for the biosynthesis of a red pigment bikaverin in F.fujikuroi,and theΔBik1 mutant totally lost the ability to produce pigmented mycelia.In this study,the Bik1 orthologous gene(FOIG_14908)in Foc4 was cloned and identified.The objective of this study was to identify the Foc4Bik1 gene(FOIG_14908)in Foc4 which had potential as an endogenous reporter gene of Foc4,which could serve as a promising target gene for assessing the feasibility of newly developed CRISPR/Cas9 gene editing technique in Foc4.Foc4Bik1 is a typical type I fungal multifunctional polyketide synthase(PKS)composed of 2036 amino acids that contains conserved protein domain.SMART revealed that Foc4Bik1 contained conserved protein structure such as acyl-carrier protein(ACP)transacylase(SAT),β-ketoacyl synthase(KS),acyltransferase(AT),dehy-dratase(DH)and thioesterase(TE).TheΔFoc4Bik1 gene-knockout mutants were obtained by the split-marker homolo-gous recombination technique.After two rounds of PCR amplification,the upstream and downstream recombination fragments required for protoplast transformation were successfully amplified by the split-marker approach.TheΔFoc4Bik1 knockout mutant was obtained via PEG-mediated protoplast transformation,the positive transformants were verified with the PCR method.TheΔFoc4Bik1 gene-knockout mutants showed no significant effects on fungal growth,conidial production,and pathogenicity to banana plantlet(Cavendish,AAA),albeit theΔFoc4Bik1 gene-knockout mu-tants totally lost the ability to produce reddish pigmented mycelia,and thus the mycelia turned white when cultured in liquid PDB media.Therefore,Foc4Bik1 could be selected as an endogenous reporter gene to verify whether the feasi-bility of CRISPR/Cas9 gene editing technology available for the filamentous fungus Foc4 via the plasmid CRISPR/Cas9 system.The F.fujikuroi CRISPR/Cas9 vector pUC-fFuCas9-HTBNLS-hph(Addgene,#121092)was used in this study.Foc4Bik1 gRNA sequence was designed via the online software,and the sgRNA162 sequence was introduced into gene editing plasmid resulted the Foc4 CRISPR/Cas9 gene editing vector,pUC-fFuCas9-HTBNLS-hph-Foc4Bik1,associated with donor plasmid pUC19-Foc4Bik1-HDR served as homology directed repair(HDR)template co-transformation into Foc4 protoplasts.Specifically,ΔFoc4Bik1 mutants totally lost the ability to produce pigmented mycelia appeared white mycelia.Using the white mycelia as the judging phenotype,the candidateΔFoc4Bik1(HDR)gene replacement mutants with white mycelia when grown in PDB liquid medium were selected for subsequent PCR detection.However,the white phenotypes were coupled with the positive PCR amplification bands.It is important that the positiveΔFoc4Bik1(HDR)gene replacement mutants could be picked up according to the white phenotype by eyes without PCR procedures.The results proved that CRISPR/Cas9 system is stable and can efficiently disrupt the genes of interest,and Foc4Bik1 gene can serve as the endogenous reporter gene for further Foc4 molecular biology research.