Protoplast transformation as a potential platform for exploring gene function in Verticillium dahliae.
Protoplast transformation as a potential platform for exploring gene function in Verticillium dahliae.
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DOI：
10.1186/s12896-016-0287-4
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发表时间：
2016-07-26
影响因子：
3.5
通讯作者：
Cheng H
中科院分区：
文献类型：
--
作者：
Rehman L;Su X;Guo H;Qi X;Cheng H
大量的努力集中在筛选与大丽轮枝菌的毒力和致病性有关的基因上，大丽轮枝菌是许多植物物种的破坏性真菌病原体，一旦植物被感染就难以控制。虽然农杆菌介导的转化（ATMT）已被广泛用于基因筛选，一直需要一个快速和简单的方法来促进转化。使用driselase（Sigma D-9515）产生在TB 3肉汤（酵母提取物30 g、酪蛋白氨基酸30 g和200 g蔗糖，在IL H2O中）中具有优异再生效率（65%）的高质量原生质体，并使用PEG或电穿孔用GFP质粒或线性GFP盒转化。PEG介导的转化对于线性GFP盒产生600个转化体/微克DNA，对于GFP质粒产生250个转化体/微克DNA;电穿孔对于线性GFP盒产生29个转化体/微克DNA，对于GFP质粒产生24个转化体/微克DNA。为了确定短干扰RNA（siRNAs）是否可以被递送到原生质体中并用于沉默基因，我们通过使用PEG介导的转化将四种不同的siRNAs（siRNA-gfp 1、siRNA-gfp 2、siRNA-gfp 3和siRNA-gfp 4）之一递送到Vd-GFP原生质体中来靶向Vd-GFP（本研究中获得的大丽轮枝菌GFP菌株）的GFP基因。用siRNA-gfp 4获得高达100%的GFP沉默;其他siRNA不太有效（高达10%沉默）。采用相同的方法分别用4种siRNA（siRNA-vta 1、siRNA-vta 2、siRNA-vta 3和siRNA-vta 4）沉默棉花黄萎病菌的粘附转录激活因子（Vta 2）基因;通过菌落直径和定量真实的时间PCR（qRT-PCR）分析，siRNA-vta 1具有最高的沉默效率。本研究建立的快速、简便的转化方法可用于研究与大丽轮枝菌生长、毒力和致病性相关基因的功能。本文的在线版本（doi：10.1186/s12896-016-0287-4）包含补充材料，可供授权用户使用。
Large efforts have focused on screening for genes involved in the virulence and pathogenicity of Verticillium dahliae, a destructive fungal pathogen of numerous plant species that is difficult to control once the plant is infected. Although Agrobacterium tumefaciens-mediated transformation (ATMT) has been widely used for gene screening, a quick and easy method has been needed to facilitate transformation. High-quality protoplasts, with excellent regeneration efficiency (65 %) in TB3 broth (yeast extract 30 g, casamino acids 30 g and 200g sucrose in 1L H20), were generated using driselase (Sigma D-9515) and transformed with the GFP plasmid or linear GFP cassette using PEG or electroporation. PEG-mediated transformation yielded 600 transformants per microgram DNA for the linear GFP cassette and 250 for the GFP plasmid; electroporation resulted in 29 transformants per microgram DNA for the linear GFP cassette and 24 for the GFP plasmid. To determine whether short interfering RNAs (siRNAs) can be delivered to the protoplasts and used for silencing genes, we targeted the GFP gene of Vd-GFP (V. dahliae GFP strain obtained in this study) by delivering one of four different siRNAs—19-nt duplex with 2-nt 3′ overhangs (siRNA-gfp1, siRNA-gfp2, siRNA-gfp3 and siRNA-gfp4)—into the Vd-GFP protoplasts using PEG-mediated transformation. Up to 100 % silencing of GFP was obtained with siRNA-gfp4; the other siRNAs were less effective (up to 10 % silencing). Verticillium transcription activator of adhesion (Vta2) gene of V. dahliae was also silenced with four siRNAs (siRNA-vta1, siRNA-vta2, siRNA-vta3 and siRNA-vta4) independently and together using the same approach; siRNA-vta1 had the highest silencing efficiency as assessed by colony diameter and quantitative real time PCR (qRT-PCR) analysis. Our quick, easy transformation method can be used to investigate the function of genes involved in growth, virulence and pathogenicity of V. dahliae. The online version of this article (doi:10.1186/s12896-016-0287-4) contains supplementary material, which is available to authorized users.