| 茄链格孢 (Alternaria solani) AsRAS和 AsGH28基因功能分析 |
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| 引用本文：付爱华，冯 艳3，4 ，李 清，王红美，李健美，张向东，周 健，张法应，陈 洁，唐 唯.茄链格孢 (Alternaria solani) AsRAS和 AsGH28基因功能分析[J].西北农业学报,2026,(3):510~522 |
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| 基金项目:云南省基础研究重点项目（202301AS070010）；云南师范大学研究生创新基金（YJSJJ23-B156）。 |
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| 中文摘要:旨在通过构建功能基因的沉默和过表达株，探究RAS和 GH28在茄链格孢中的功能。以野生型茄链格孢菌株TA-0410为基础，通过pTOR-mRFP骨架构建沉默和过表达载体，以PEG（Polyethylene glycol）介导的原生质体转化法构建沉默和过表达株，观察菌落形态，测量菌丝生长速度、孢子形态及孢子量；测定和比较菌丝中黑色素含量；利用qRT-PCR分析黑色素合成基因AsPKS表达量；在马铃薯叶片上进行毒性功能验证。结果显示AsRAS和 AsGH28载体介导的平均转化率分别为8.72%和6.61%；挑取两个基因的沉默和过表达菌株各两株进行生物学功能分析，发现沉默菌株生长较慢而相同时间点过表达菌株的菌丝生长较快；沉默菌株分生孢子较小，喙较短、分生孢子数量和WT型TA-0410有极显著差异，过表达菌株分生孢子较TA-0410多，喙较长；相比于TA-0410，AsRAS基因沉默菌株黑色素含量降低， AsGH28基因沉默菌株黑色素含量显著降低；两个基因过表达菌株黑色素含量均显著升高；qRT-PCR表达量分析发现AsRAS和 AsGH28基因沉默菌株黑色素合成基因AsPKS表达量和TA-0410相比存在显著差异；AsRAS和 AsGH28基因过表达菌株AsPKS表达量和TA-0410相比也存在显著性差异；致病性测定结果表明，AsRAS基因沉默株、野生型TA-0410和 AsGH28过表达菌株侵染马铃薯Desiree 72 h后叶片可见明显病斑，然而， AsGH28基因沉默株侵染96 h后才有病斑出现；接种还发现，接种144 h时，与TA-0410相比，沉默菌株致病力均下降，其中 AsGH28基因沉默菌株致病力显著下降，两个基因过表达菌株致病力增大。综上所述，AsRAS和 AsGH28基因正向调控A.solani菌丝生长速度、产孢量及黑色素的积累，在参与A.solani形态发育、代谢及侵染马铃薯过程中发挥重要作用。 |
| 中文关键词:茄链格孢 AsRAS基因 AsGH28基因 产孢量 黑色素 毒性 |
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| Gene Functional Analysis of AsRAS and |
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| Abstract:This study aimed to investigate the function of RAS and GH28 in A.solani by constructing silenced and overexpressed strains.Using the wild-type A.solani TA-0410 as a reference isolate,we constructed silenced and overexpressed strains through pTOR-mRFP vector construction and PEG-mediated (Polyethylene glycol) protoplast transformation.We observed the morphological changes of the isolates and measured and compared their hyphal growth rate,spore morphology and yield,and melanin content in hyphae.We also analyzed the expression level of the melanin synthesis gene AsPKS using qRT-PCR and assessed their pathogenicity on potato leaves.The average transformation rates mediated by AsRAS and AsGH28 vectors were 8.72% and 6.61%,respectively.Two silenced and two overexpressed strains for each of the two genes were selected for biological function analysis.It was found that the silenced strains grew slower,while the mycelial production of the overexpressed strains was faster at the same time points.The conidia of the silenced strains were smaller,with shorter beaks and significantly fewer conidia compared to the wild-type TA-0410,while the overexpressed strains had more conidia and longer beaks than TA-0410.Compared to TA-0410,the melanin content of AsRAS silenced strains decreased,and the melanin content of AsGH28 silenced strains significantly decreased; the melanin content of both overexpressed strains significantly increased.qRT-PCR expression analysis revealed significant differences in AsPKS expression levels,a melanin synthesis gene,between AsRAS and AsGH28 silenced strains and TA-0410; significant differences were also found in AsPKS expression levels between AsRAS and AsGH28 overexpressed strains and TA-0410.Pathogenicity assays showed visible lesions were visible on potato Desiree leaves 72 hours after infection by AsRAS silenced strains,wild-type TA-0410 and AsGH28 overexpressed strains; however,lesions only appeared 96 hours after infection by AsGH28 silenced strains.Inoculation also revealed that at 144 hours post-inoculation,compared to TA-0410,the virulence of all silenced strains decreased,with a significant decrease in virulence of AsGH28 silenced strains,while the virulence of both overexpressed strains increased.To sum up,the two genes (AsRAS and AsGH28) positively regulate the hyphal growth rate,sporulation,and accumulation of melanin in A.solani.They play a crucial role in the morphogenesis,metabolism,and virulence during the infection process of A.solani on potato. |
| keywords:Alternaria solani AsRAS gene AsGH28 gene Sporulation Melanin Virulence |
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