A Method for Radioactive Labelling of Hebeloma cylindrosporum to Study Plant-fungus Interactions
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Author:
Date:
2017-10-20
[Abstract] In order to quantify P accumulation and P efflux in the ectomycorrhizal basidiomycete fungus Hebeloma cylindrosporum, we supplied 32P to mycelia previously grown in vitro in liquid medium. The culture had four main steps that are 1) growing the mycelium on complete medium with P, 2) transfer the mycelia into new culture solution with or without P, 3) adding a solution containing 32P and 4) rinsing the mycelia before incubation with or without plant. The main point is to rinse very carefully the mycelia after 32P supply in order to avoid ...
[摘要] 为了量化外生菌根担囊菌真菌Hebeloma cylindrosporum中的P积累和P流出,我们向以前在体外生长的菌液提供了 32 P 中。 培养物有四个主要步骤:1)在具有P的完全培养基上培养菌丝体,2)将菌丝体转移到具有或不具有P的新培养溶液中,3)加入含有32和32的溶液 )在与或不与植物孵育之前冲洗菌丝体。 要点是在 32 P供应之后非常仔细地冲洗菌丝体,以避免过高估计P P。 【背景】众所周知,菌根真菌和植物之间的关联改善了宿主植物的P营养(由Smith和Read,2008; Plassard和Dell,2010; Cairney,2011; Smith等人,2015)。这种积极的作用主要是由于真菌细胞探索远离根部的土壤的磷酸盐(Pi)吸收,允许探索大量的土壤超过主要吸收根部周围形成的耗尽区(Smith and Read,2008; Cairney,2011; Smith ,2015)。然而,为了受益于宿主植物,吸收的Pi必须从探测土壤的真菌细胞转移到与宿主细胞紧密接触的细胞中。在外生菌根共生中,这些交流被认为发生在外生菌根内的“Hartig网”领域(Smith and Read,2008; ...
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Establishing a Symbiotic Interface between Cultured Ectomycorrhizal Fungi and Plants to Follow Fungal Phosphate Metabolism
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Author:
Date:
2017-10-20
[Abstract] In ectomycorrhizal plants, the fungal cells colonize the roots of their host plant to create new organs called ectomycorrhizae. In these new organs, the fungal cells colonize the walls of the cortical cells, bathing in the same apoplasm as the plant cells in a space named the ‘Hartig net’, where exchanges between the two partners take place. Finally, the efficiency of ectomycorrhizal fungi to improve the phosphorus nutrition of their host plants will depend on the regulation of phosphate transfer from the fungal cells to plant cells in the Hartig net through as yet unknown mechanisms. In ...
[摘要] 在外生菌根植物中,真菌细胞定植于其宿主植物的根部,以产生称为外生菌根的新器官。在这些新器官中,真菌细胞定居在皮质细胞的壁上,与称为“Hartig网”的空间中的植物细胞在同一质粒中沐浴,两地之间的交流发生。最后,外源菌根真菌提高其宿主植物的磷营养的效率将取决于通过尚未知的机制调节从真菌细胞到Hartig网中植物细胞的磷酸转移。为了研究这些机制,我们开发了一种体外实验装置,模拟外生菌根(Hartig网)的常见质粒来研究外生菌根真菌中的磷代谢。 当真菌细胞与寄主植物“Pinus pinaster”的植物细胞相关联时。该装置可用于监测从先前与 32 P-正磷酸盐一起培养的真菌的磷酸盐外排。 【背景】已知菌根真菌和植物之间的关联可以改善植物P营养(由Smith和Read,2008; Plassard和Dell,2010; Cairney,2011; ...
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Generation of Caenorhabditis elegans Transgenic Animals by DNA Microinjection
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Author:
Date:
2017-10-05
[Abstract] Microinjection is the most frequently used tool for genetic transformation of the nematode Caenorhabditis elegans, facilitating the transgenic expression of genes, genome editing by the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system, or transcription of dsRNA for RNA intereference (RNAi). Exogenous DNA is delivered into the developing oocytes in the germline of adult hermaphrodites, which then generate transgenic animals among their offspring. In this protocol, we describe the microinjection procedure and the subsequent selection of transgenic progeny.
[摘要] 显微注射是线虫秀丽隐杆线虫遗传转化中最常用的工具,促进基因的转基因表达,通过聚集的定期散布的短回文重复序列(CRISPR)-Cas9系统的基因组编辑或转录 dsRNA用于RNA干扰(RNAi)。 外源DNA被递送到成年雌雄同株的种系中的发育中的卵母细胞中,然后在它们的后代中产生转基因动物。 在该方案中,我们描述了显微注射程序和随后的转基因后代选择。 【背景】在C.通过显微注射的DNA转化通常用于产生过表达或异位表达可以与标签(例如,绿色荧光蛋白[GFP])融合的基因的转基因动物,允许突变体的表型拯救和/或蛋白质的定位和功能的分析(Carter等人,1990; Chalfie等人,1994; Mello和Fire,1995)。聚集的定期散布的短回文重复(CRISPR)-Cas9系统的出现需要显微注射以通过引入点突变或插入/缺失突变来实现高度特异性的基因组编辑(概述于Dickinson和Goldstein,2016)。此外,该技术被应用于dsRNA的可诱导和/或组织特异性转录以便于遗传性RNA干扰(RNAi)(Tavernarakis等人,2000) ...
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