植物VIGS技术及其在林业科学中的研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2020-1452

摘要/Abstract

摘要：

随着基因组序列信息的激增,生物科学研究进入大数据时代,但如何对基因组信息进行功能注释成为了一个重要的研究目标。病毒诱导的基因沉默（virus-induced gene silencing,VIGS）技术作为一种强大的工具,可以针对缺乏稳定遗传转化体系的林木物种进行基因功能分析,已经被用来研究了多个植物生长发育过程中的关键基因。该技术利用植物先天抗病毒防御机制,通过把目的基因片段插入病毒载体中进而在植物体内产生小干扰RNA,使该植物内源基因的转录物成为被降解的靶标,从而导致目的基因的表达量下调。系统综述了VIGS技术的优势、局限性、以及相应的解决方案,同时还讨论了VIGS在林木科学研究中的应用,最后探究了VIGS作为探究和表征植物基因功能的有力工具的最新改进方案和未来前景。

关键词: 病毒诱导的基因沉默, 功能基因组学, 病毒载体, 林业

Abstract:

With the explosion of genomic sequence information,biological science has entered the era of big data. Functional annotation of genomic information has become an important research goal. Virus-induced gene silencing（VIGS）is a powerful tool in analysis of gene function for forest species that are lack of stable genetic transformation system. VIGS has been used to study multiple key genes in plant growth and development. VIGS works as such way,a fragment of target gene is inserted into a viral vector to generate small interfering RNAs（siRNAs）based on the mechanism of natural innate antiviral defense in plant,then the transcript of endogenous gene in the plant becomes the degraded target,resulting in down-regulation of target gene expressions. Here we systematically review the advantages and limitations of VIGS,as well as corresponding solutions. Meanwhile we also discuss the application of VIGS system in forest science. Finally,we explore the latest improving solutions and future prospects while VIGS as a powerful tool for assessing and characterizing gene functions in plant.

Key words: virus-induced gene silencing, functional genomics, virus vectors, forestry

高鹏飞, 席飞虎, 张泽宇, 胡凯强, 陈凯, 魏文桃, 丁家治, 顾连峰. 植物VIGS技术及其在林业科学中的研究进展[J]. 生物技术通报, 2021, 37(5): 141-153.

GAO Peng-fei, XI Fei-hu, ZHANG Ze-yu, HU Kai-qiang, CHEN Kai, WEI Wen-tao, DING Jia-zhi, GU Lian-feng. Research Progress of Plant VIGS Technology and Its Application in Forestry Science[J]. Biotechnology Bulletin, 2021, 37(5): 141-153.

图/表 4

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软件名称 Software name	软件介绍 Software introduction	参考文献 Reference
SGN	面向用户快速友好的交互式Web工具,在计算机中模拟细胞中发生的VIGS过程。帮助研究人员更快地设计VIGS构建载体,使用SGN工具可帮助选择沉默区域,识别最可能的目标和非目标位置,还可以进行预测沉默效率 http://vigs.solgenomics.net/	[44]
pssRNAit	利用全基因组数据设计特异性siRNA,可以进行全基因组脱靶评估是用于设计植物RNAi的有效的网络服务器工具。开发了针对RNAi途径的每个步骤的计算模型,有助设计沉默构建体 https://plantgrn.noble.org/pssRNAit/Home.gy	[45]
siRNA-Scan	具有多个集成组件的搜索环境,包括序列相似性搜索,以预测载体序列是否会潜在脱靶和脱靶效率。可以搜索数据库中的其他脱靶基因然后选择特异性序列,用来设计具有最小脱靶概率的VIGS载体 http://bioinfo2.noble.org/RNAiScan/RNAiScan.htm	[46]
DESIR	设计siRNA的工具,基于siRNA反义链位置特异性核苷酸序列位置的线性模型进行设计。可得出siRNA设计结果以及预测沉默结果 http://biodev.extra.cea.fr/DSIR/DSIR.html	[47]
DesiRm	用于设计能够高效沉默基因的siRNA,可以提交mRNA以找出针对其的有效siRNA,以及提交siRNA及其靶序列以通过核苷酸错配设计更有效的siRNA http://www.imtech.res.in/raghava/desirm/	[48]
i-Score	对基因沉默RNA折叠剪切后所产生的siRNA进行效率检测,结果由高到低进行排序 http://www.med.nagoya-u.ac.jp/neurogenetics/i_Score/i_score.html	[49]
DEQOR	对基因区域能够发生较高沉默的区域进行预测,通过所选物种的转录组和基因组进行BLAST搜索,评估输入的靶基因位置的沉默效率 http://cluster-1.mpi-cbg.de/Deqor/deqor.html	[50]
P-SAMS	web应用程序设计人工microRNA,有助于特异性的靶向不同的双子叶和单子叶植物物种中的一个以及多个基因 http://p-sams.carringtonlab.org/	[51]
WMD3	基于web应用程序可用于设计人工microRNA构建基因沉默表达载体,可用目标搜索找到潜在的靶基因 http://wmd3.weigelworld.org/cgi-bin/webapp.cgi	[52]
PhieCBEs	新型,高效,广靶向的植物编辑系统,是水稻和其他植物（作物）的单碱基编辑有效实用的工具,可在基因功能筛选,大规模饱和突变,编辑调控元件,RNA可变剪接,基因沉默等研究中广泛应用	[53]

软件名称 Software name	软件介绍 Software introduction	参考文献 Reference
SGN	面向用户快速友好的交互式Web工具,在计算机中模拟细胞中发生的VIGS过程。帮助研究人员更快地设计VIGS构建载体,使用SGN工具可帮助选择沉默区域,识别最可能的目标和非目标位置,还可以进行预测沉默效率 http://vigs.solgenomics.net/	[44]
pssRNAit	利用全基因组数据设计特异性siRNA,可以进行全基因组脱靶评估是用于设计植物RNAi的有效的网络服务器工具。开发了针对RNAi途径的每个步骤的计算模型,有助设计沉默构建体 https://plantgrn.noble.org/pssRNAit/Home.gy	[45]
siRNA-Scan	具有多个集成组件的搜索环境,包括序列相似性搜索,以预测载体序列是否会潜在脱靶和脱靶效率。可以搜索数据库中的其他脱靶基因然后选择特异性序列,用来设计具有最小脱靶概率的VIGS载体 http://bioinfo2.noble.org/RNAiScan/RNAiScan.htm	[46]
DESIR	设计siRNA的工具,基于siRNA反义链位置特异性核苷酸序列位置的线性模型进行设计。可得出siRNA设计结果以及预测沉默结果 http://biodev.extra.cea.fr/DSIR/DSIR.html	[47]
DesiRm	用于设计能够高效沉默基因的siRNA,可以提交mRNA以找出针对其的有效siRNA,以及提交siRNA及其靶序列以通过核苷酸错配设计更有效的siRNA http://www.imtech.res.in/raghava/desirm/	[48]
i-Score	对基因沉默RNA折叠剪切后所产生的siRNA进行效率检测,结果由高到低进行排序 http://www.med.nagoya-u.ac.jp/neurogenetics/i_Score/i_score.html	[49]
DEQOR	对基因区域能够发生较高沉默的区域进行预测,通过所选物种的转录组和基因组进行BLAST搜索,评估输入的靶基因位置的沉默效率 http://cluster-1.mpi-cbg.de/Deqor/deqor.html	[50]
P-SAMS	web应用程序设计人工microRNA,有助于特异性的靶向不同的双子叶和单子叶植物物种中的一个以及多个基因 http://p-sams.carringtonlab.org/	[51]
WMD3	基于web应用程序可用于设计人工microRNA构建基因沉默表达载体,可用目标搜索找到潜在的靶基因 http://wmd3.weigelworld.org/cgi-bin/webapp.cgi	[52]
PhieCBEs	新型,高效,广靶向的植物编辑系统,是水稻和其他植物（作物）的单碱基编辑有效实用的工具,可在基因功能筛选,大规模饱和突变,编辑调控元件,RNA可变剪接,基因沉默等研究中广泛应用	[53]

树种Species	病毒载体来源 Viral vector source	参考文献 Reference
胡杨（Populus euphratica）	TRV	[56]
银灰杨（Populus canescens）	TRV	[56]
麻风树（Jatropha curcas）	TRV	[57]
苹果（Malus domestica）	ALSV	[58]
木薯（Manihot esculenta）	ACMV	[59]
梨（Pyrus betulaefolia）桃（Prunus persica）	ALSV,TRV TRV	[58,60] [61]
樱桃（Prunus avium）	TRV	[62]
荔枝（Litchi chinensis）	TRV	[63]
欧洲越橘（Vaccinium myrtillus）	TRV	[64]
凤丹（Paeonia ostii）	TRV	[65]
橄榄（Oleaceae hoffmanns）	TRV	[66]
喜树（Camptotheca acuminata）	TRV	[67]
紫薇（Lagerstroemia indica）	TRV	[68]

树种Species	病毒载体来源 Viral vector source	参考文献 Reference
胡杨（Populus euphratica）	TRV	[56]
银灰杨（Populus canescens）	TRV	[56]
麻风树（Jatropha curcas）	TRV	[57]
苹果（Malus domestica）	ALSV	[58]
木薯（Manihot esculenta）	ACMV	[59]
梨（Pyrus betulaefolia）桃（Prunus persica）	ALSV,TRV TRV	[58,60] [61]
樱桃（Prunus avium）	TRV	[62]
荔枝（Litchi chinensis）	TRV	[63]
欧洲越橘（Vaccinium myrtillus）	TRV	[64]
凤丹（Paeonia ostii）	TRV	[65]
橄榄（Oleaceae hoffmanns）	TRV	[66]
喜树（Camptotheca acuminata）	TRV	[67]
紫薇（Lagerstroemia indica）	TRV	[68]