生物技术通报 ›› 2021, Vol. 37 ›› Issue (5): 141-153.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1452
高鹏飞1(), 席飞虎1,2, 张泽宇1, 胡凯强1, 陈凯2, 魏文桃1, 丁家治1, 顾连峰1()
收稿日期:
2020-11-27
出版日期:
2021-05-26
发布日期:
2021-06-11
作者简介:
高鹏飞,男,硕士研究生,研究方向:林木遗传育种;E-mail: 基金资助:
GAO Peng-fei1(), XI Fei-hu1,2, ZHANG Ze-yu1, HU Kai-qiang1, CHEN Kai2, WEI Wen-tao1, DING Jia-zhi1, GU Lian-feng1()
Received:
2020-11-27
Published:
2021-05-26
Online:
2021-06-11
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。
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1 VIGS分子机理 (1)病毒载体的构建。(2)侵染进植物细胞。(3)病毒载体在细胞质中转录。(4)通过RDRP合成dsRNA。(5)-(7) Dicer将dsRNA切割成siRNA后在RISC中加载。(8)-(9)激活RISC靶标特异性识别靶基因使宿主mRNA降解,基因表达丧失
Fig.1 Molecular mechanism of VIGS (1) Construction of viral vectors. (2) Infection into plant cells. (3) Viral vectors are transcribed in the cytoplasm. (4) Synthesis of dsRNA by RDRP. (5)-(7) Dicer cut dsRNA into siRNA and then loaded it in RISC. (8)-(9) Activation of RISC target specific recognition of target genes resulted in host mRNA degradation and loss of gene expression
图2 VIGS侵染技术示意图 A: 将靶基因的片段(如PDS)克隆进VIGS载体中。B:农杆菌转化培养PTRV1和PTRV2集菌重悬后等比例混合。C:通过牙签,真空渗透,或注射器注射等方法转化到幼苗期植物中,触发植物防御机制,约3周后观察植物发生的光漂白表型
Fig. 2 Schematic diagram of VIGS infection technology A: The fragment of target gene (e.g. PDS) was cloned into the VIGS vector. B: Agrobacterium transformation culture PTRV1 and PTRV2 were mixed in equal proportion after resuspended. C: The photobleaching phenotype was observed after about 3 weeks after transformation into seedlings by means of toothpick, vacuum osmosis, or syringe injection to trigger plant defense mechanisms
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 | [ |
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 | [ |
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 | [ |
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 | [ |
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 | [ |
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 | [ |
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 | [ |
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 | [ |
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 | [ |
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 | [ |
表1 基因沉默相关设计软件
Table 1 Gene silence related design software
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 | [ |
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 | [ |
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 | [ |
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 | [ |
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 | [ |
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 | [ |
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 | [ |
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 | [ |
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 | [ |
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 | [ |
树种Species | 病毒载体来源 Viral vector source | 参考文献 Reference |
---|---|---|
胡杨(Populus euphratica) | TRV | [ |
银灰杨(Populus canescens) | TRV | [ |
麻风树(Jatropha curcas) | TRV | [ |
苹果(Malus domestica) | ALSV | [ |
木薯(Manihot esculenta) | ACMV | [ |
梨(Pyrus betulaefolia) 桃(Prunus persica) | ALSV,TRV TRV | [ [ |
樱桃(Prunus avium) | TRV | [ |
荔枝(Litchi chinensis) | TRV | [ |
欧洲越橘(Vaccinium myrtillus) | TRV | [ |
凤丹(Paeonia ostii) | TRV | [ |
橄榄(Oleaceae hoffmanns) | TRV | [ |
喜树(Camptotheca acuminata) | TRV | [ |
紫薇(Lagerstroemia indica) | TRV | [ |
表2 VIGS系统在杨树属等林木物种中的研究应用
Table 2 Research and application of VIGS system in poplar and other forest tree species
树种Species | 病毒载体来源 Viral vector source | 参考文献 Reference |
---|---|---|
胡杨(Populus euphratica) | TRV | [ |
银灰杨(Populus canescens) | TRV | [ |
麻风树(Jatropha curcas) | TRV | [ |
苹果(Malus domestica) | ALSV | [ |
木薯(Manihot esculenta) | ACMV | [ |
梨(Pyrus betulaefolia) 桃(Prunus persica) | ALSV,TRV TRV | [ [ |
樱桃(Prunus avium) | TRV | [ |
荔枝(Litchi chinensis) | TRV | [ |
欧洲越橘(Vaccinium myrtillus) | TRV | [ |
凤丹(Paeonia ostii) | TRV | [ |
橄榄(Oleaceae hoffmanns) | TRV | [ |
喜树(Camptotheca acuminata) | TRV | [ |
紫薇(Lagerstroemia indica) | TRV | [ |
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