A new fungus-mediated RNAi method established and used to study the fatty acid and retinol binding protein function of the plant-parasitic nematode Aphelenchoides besseyi
A new fungus-mediated RNAi method established and used to study the fatty acid and retinol binding protein function of the plant-parasitic nematode Aphelenchoides besseyi
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建立了一种新的真菌介导的RNAi方法，用于研究植物寄生线虫Aphelenchoides besseyi的脂肪酸和视黄醇结合蛋白功能
DOI：
10.1080/15476286.2020.1852779
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发表时间：
2020-12-13
期刊：
影响因子：
4.1
通讯作者：
Xie, Hui
中科院分区：
文献类型：
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作者：
Ding, Shanwen;Wang, Dongwei;Xie, Hui
RNA干扰（RNA interference, RNAi）是植物寄生线虫（plant-parasitic nematodes, PPNs）基因功能分析的有力工具。RNAi包括浸泡在dsRNA溶液和植物方法中，通常应用于ppn基因功能的研究。然而，一些问题限制了这些方法的应用。因此，需要根据不同ppn的生物学特性，建立更便捷有效的RNAi方法。本研究以水稻白尖线虫（Aphelenchoides besseyi， RWTN）的脂肪酸和类维生素a结合蛋白基因Ab-far-1、Ab-far-4以及组合Ab-far-1和Ab-far-4为靶基因，构建了真菌RNAi载体，利用农杆菌介导转化技术，生成了转Ab-far-n dsRNA的灰葡萄孢杆菌（Botrytis cinerea, ARTBn）。RWTN取食ARTBn后，有效地沉默了线虫体内Ab-far-1和Ab-far-4的表达，明显抑制了线虫的繁殖和致病性。Ab-far-1和Ab-far-4的联合RNAi效果优于单独靶向每个基因的效果。此外，rwtn以ARTB1为食诱导的RNAi具有持久性和遗传性。因此，我们建立了一种真菌介导的RNAi检测真菌性ppn的新方法，并被证实是一种有效的、适用于线虫基因功能研究的方法。该技术将为真菌性ppn中多基因co-RNAi的多基因研究提供一种新的方法。本研究也为进一步研究PPNs基因功能提供了理论基础和新思路。缩写：FAR（脂肪酸和视黄醇结合蛋白）；稻白尖线虫；稻白尖线虫；b-far-n （A. basseyi脂肪酸与视黄醇结合蛋白基因）；ARTB1 （Ab-far-1 hpRNA转基因灰葡萄孢）；ARTB4 （Ab-far-4 hpRNA转基因灰葡萄孢）；ARTB1/4（组合Ab-far-1和Ab-far-4 hpRNA转基因绿杆菌）；EVTB（转基因绿杆菌空载体）；GRTB （eGFP hpRNA转基因绿杆菌）；WTB（野生型葡萄球菌）。
ABSTRACT RNA interference (RNAi) is a powerful tool for gene functional analysis of plant-parasitic nematodes (PPNs). RNAi involving soaking in a dsRNA solution and in planta methods is commonly applied in the study of gene function in PPNs. However, certain problems restrict the application of these methods. Therefore, more convenient and effective RNAi methods need to be established for different PPNs according to their biological characteristics. In this study, the fatty acid and retinoid binding protein genes (Ab-far-1, Ab-far-4, and combinatorial Ab-far-1 and Ab-far-4) of the rice white tip nematode (RWTN), Aphelenchoides besseyi, were used as target genes to construct a fungal RNAi vector, and the Ab-far-n dsRNA transgenic Botrytis cinerea (ARTBn) were generated using Agrobacterium-mediated transformation technology. After RWTN feeding on ARTBn, the expression of Ab-far-1 and Ab-far-4 in the nematodes was efficiently silenced, and the reproduction and pathogenicity of the nematodes were clearly inhibited. The Ab-far-1 and Ab-far-4 co-RNAi effects were better than the effects when each gene was individually targeted with RNAi. Additionally, the RNAi induced when RWTNs fed on ARTB1 were persistent and heritable. Thus, a new method of fungus-mediated RNAi was established for fungivorous PPNs and was verified as effective and applicable to the study of nematode gene function. This technique will remove the technological bottlenecks and provide a new method to studying the multiple genes with polygene co-RNAi in fungivorous PPNs. This study also provides a theoretical basis and new thought for further study of the gene function in PPNs. Abbreviations: FAR(Fatty acid and retinol-binding proteins); RWTN (The rice white tip nematode, Aphelenchoides besseyi); Ab-far-n (Fatty acid and retinol binding protein gene of A. besseyi); ARTB1 (Ab-far-1 hpRNA transgenic Botrytis cinerea); ARTB4 (Ab-far-4 hpRNA transgenic Botrytis cinerea); ARTB1/4 (combinatorial Ab-far-1 and Ab-far-4 hpRNA transgenic B. cinerea); EVTB (Empty vector transgenic B. cinerea); GRTB (eGFP hpRNA transgenic B. cinerea); WTB (Wild-type B. cinerea).