植物VIGS载体构建策略研究与应用进展

doi:10.16420/j.issn.0513-353x.2023-0753

摘要/Abstract

摘要：

病毒诱导的基因沉默（virus-induced gene silencing，VIGS）是一种基于植物抗病毒机制开发的用于基因功能研究的反向遗传学技术。该技术不依赖植物遗传转化体系，具有周期短、操作简单高效、高通量的特点，被广泛应用于植物生长发育、信号转导、代谢途径和抗逆机制等方面的研究。本文中综述了植物VIGS作用机理、应用和存在的问题，重点讨论了载体构建策略和影响因素等方面的内容，旨在为该技术的进一步发展和应用提供参考。

关键词: 病毒诱导的基因沉默, 基因功能, 病毒载体, 沉默载体构建策略

Abstract:

Virus-induced gene silencing（VIGS），a reverse genetic technique based on plant antiviral mechanism，has been widely used in the study of plant growth and development，signal transduction，metabolic pathways and stress resistance due to its advantages of independent of plant genetic transformation system，time-saving，easy and efficient operation，and high-throughput. In this paper，the mechanism，application and problems of VIGS were reviewed，and the vector construction strategies and influencing factors were discussed emphatically，aiming to provide reference for the further development and application of VIGS technology.

Key words: virus-induced gene silencing, gene function, viral vector, silencing vector construction strategy

任恒泽, 李丹莹, 余亚婷, 吕务云, 郝心愿, 王新超, 王玉春. 植物VIGS载体构建策略研究与应用进展[J]. 园艺学报, 2024, 51(7): 1455-1473.

REN Hengze, LI Danying, YU Yating, LÜ Wuyun, HAO Xinyuan, WANG Xinchao, WANG Yuchun. Research Advances of Strategies to Engineer VIGS Vectors and Its Application in Plants[J]. Acta Horticulturae Sinica, 2024, 51(7): 1455-1473.

图/表 2

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病毒类型 Type	病毒名称 Virus name	基因组组分 Numbers of virus segments	载体构建策略 Strategies for vectors construction		参考文献 References
RNA	马铃薯X病毒 Potato virus X（PVX）	1	将靶基因片段插入CP的5′端。 The target gene fragment is inserted at the 5′-end of the CP.		Ruiz et al.，1998
	狗尾草花叶病毒 Foxtail mosaic virus（FoMV）	1	5A突变后不影响病毒复制和侵染，在5A中间加入170 bp FoMV CP亚基因组启动子序列和MCS，将靶基因片段插入MCS。 The mutation of 5A do not affect the replication and infection of virus. The 170 bp subgenomic promoter and MCS used for insertion of target gene fragment are inserted into the middle region of 5A.		Robertson et al.，2000；Liu et al.，2016
	木薯普通花叶病毒 Cassava common mosaic virus（CsCMV）	1	在CP启动子SGP2序列前加入90 bp亚基因组启动子SGP1，SGP1后插入靶基因片段。 A duplicated 90 bp subgenomic promoter（SGP1）is engineered into viral genome at the upstream of CP promoter（SGP2），and the target gene fragment is inserted after SGP1.		Tuo et al.，2021
	马铃薯X病毒 Potato virus X（PVX）	1	在CP的5′端加入PVX亚基因组启动子，在该启动子后插入靶基因片段。 The PVX subgenomic promoter is engineered into the 5′-end of CP，after which a target gene fragment is inserted.		Zhao et al.，2020
	柑橘叶斑驳病毒 Citrus leaf blotch virus（CLBV）	1	在CP的3′或5′端加入PmlI，用于插入靶基因片段；或克隆CP亚基因组启动子并在其后加入PmlI，将该组件构建至CP的3′端或5′端，用于插入靶基因片段。 The PmlI used for insertion of target gene fragment is engineered into the 3′ or 5′-end of CP. Or inserting the CP subgenomic promoter flanked with PmlI into the 3′ or 5′-end of CP.		Agüero et al.，2014
	葡萄病毒A Grapevein virus A（GVA）	1	含有5个ORF，在MP的3′端加入异源移动蛋白亚基因组启动子，在该启动子后插入靶基因片段。 GVA contains 5 ORFs，and a heterogenic movement protein subgenomic promoter is engineered into the 3′-end of MP，after which a target gene fragment is inserted.		Muruganantham et al.，2009
	雀麦花叶病毒 Brome mosaic virus（BMV）	3	在RNA3的CP终止密码子后插入靶基因片段。 A target gene fragment is inserted after the stop codon of CP in RNA3.		Ding et al.，2006，2018
	黄瓜花叶病毒 Cucumber mosaic virus（CMV）	3	第2条链编码2b蛋白，为RNA沉默抑制子，保留2b的N端前81个氨基酸序列，并在其后加入MCS，用于插入靶基因片段。 The second strand of CMV encodes 2b protein, which is an RNA silencing suppressor. The first 81 amino acid sequences lied in the N-terminal of 2b are retained，and MCS is added afterwards for the insertion of target gene fragment.		Wang et al.，2016
	李坏死环斑病毒Prunus necrotic ringspot virus（PNRSV）	3	将靶基因片段插入至RNA3 CP的3′末端，此外，RNA1和RNA2构建至同一个载体上，以便提高侵染效率。 The target gene fragment is inserted into the 3′-end of CP of RNA3. Moreover，the RNA1 and RNA2 are constructed into the same vector to improve infection efficiency.		Cui & Wang，2017；潘佳佳等，2023
RNA	莴苣侵染性黄化病毒Lettuce infectious yellows virus（LIYV）	2	P26紧邻CPm的3′端，在P26的3′端加入CP亚基因启动子，其后用于加入靶基因片段。 The P26 is next to the 3′-end of CPm. The CP subgenomic promoter is inserted after P26，after which a target gene fragment is inserted.	Qiao & Falk，2018；Stewart et al.，2010
	葡萄卷叶伴随病毒-2 Grape vein roll-associated virus（GLRaV-2）	1	在次要外壳蛋白和外壳蛋白中间有亚基因组启动子，在该亚基因组启动子后插入靶基因片段。 The subgenomic promoter is located between minor coat protein and coat protein，after which a target gene fragment is inserted.	Kurth et al.，2012
	柑橘衰退病毒 Citrus tristeza virus（CTV）	1	在p23和CP中间加入酶切位点，用于插入CP亚基因组启动子和靶基因片段。Enzyme restriction site，which is used for inserting subgeno- mic promoter and target gene fragment, is inserted between p23 and CP.	Hajeri et al.，2014
	苦苣菜黄网弹状病毒Sonchus yellow net rhabdovirus（SYNV）	1	位于5'端的N和P蛋白负责病毒的复制，在这两个编码序列中间加入MCS，用于插入靶基因片段。 The N and P proteins are responsible for viral replication. The MCS is added between N and P for insertion of target gene fragment.	Peng et al.，2021
	苹果潜隐病毒 Apple latent spherical virus（ALSV）	2	在RNA2的MP和CP中间MCS，用于插入靶基因片段。 The MCS is added between MP and CP of RNA2 for insertion of target gene fragment.	Igarashi et al.，2009；Gedling et al.，2018
	菜豆荚斑驳病毒 Bean pod mottle virus（BPMV）	2	在MP和L-CP之间加入MCS用于插入靶基因片段。 The MCS is added between MP and L-CP for insertion of target gene fragment.	Zhang & Ghabrial，2006；Zhang et al.，2010
	蚕豆萎蔫病毒2 Broad bean wilt virus 2（BBWV2）	2	在RNA2的CP的终止密码子后加入一个酶切位点，用于插入靶基因片段。在病毒序列3′末端与载体骨架之间添加20个poly (A)和RZ序列。An enzyme restriction site is added after stop codon of CP of RNA2，which is used for insertion of target gene fragment. The 20 poly (A) and RZ sequences are added between the 3′-end of the virus sequence and vector sequence.	Kwak et al.，2016；Choi et al.，2019
	芜菁黄花叶病毒Turnip yellow mosaic virus（TYMV）	1	在CP终止密码子后插入SnaBI酶切位点，用于插入外源基因片段。 The SnaBI is added after stop codon of CP for the insertion of target gene fragment.	Yu et al.，2018
	烟草花叶病毒Tobacco mosaic virus（TMV）	1	在CP编码区的5′端加入靶基因片段，由TMV-U1亚基因组启动子驱动。The target gene fragment is inserted to the 5′-end of CP coding region, driven by the TMV-U1 subgenomic promoter.	Kumagai et al.，1995
	烟草脆裂病毒Tobacco rattle virus（TRV）	2	将RNA2上CP后的29.4 kb和32.8 kb的非结构蛋白替换为MCS用于插入靶基因片段。The 29.4 kb and 32.8 kb non-structural protein sequences are replaced with MCS for insertion of target gene fragment.	Ratcliff et al.，2001
	豌豆早枯病毒 Pea early-browning virus（PEBV）	2	将RNA2上CP后的非结构蛋白2b和2c替换为靶基因片段。 The 2b and 2c non-structural protein sequences of CP of RNA2 are replaced with target gene fragment.	Constantin et al.，2004
	中国小麦花叶病毒Chinese wheat mosaic virus（CWMV）	2	将RNA2上CP的RT部分替换为MCS，用于插入靶基因片段。 The RT of CP of RNA2 is replaced with MCS for insertion of target gene fragment.	Yang et al.，2018
	大麦条纹花叶病毒Barley stripe mosaic virus（BSMV）	3	含有 RNAα、β、γ三组分，在γb下游加入MCS，用于插入靶基因片段。BSMV contains RNAα，β and γ，the MCS is added after γb for insertion of target gene fragment.	Tamilselvan-Nattar-Amutha et al.，2023
DNA	水稻东格鲁杆状病毒Rice tungro bacilliform virus（RTBV）	1	含有ORF I、ORF II、ORF III、ORF IV，保留ORF III和ORF IV，将RTBV 5′端启动子替换为MUP启动子和tRNA binding site，ORF IV终止密码子后加入MCS，用于插入靶基因片段。 RTBC contains ORF I，ORF II，ORF III and ORF IV. The ORF III and ORF IV are retained，and the promoter located in the 5′-end of RTBV is replaced with MUP promoter and tRNA binding site，the MCS is added after stop codon of ORF IV for insertion of target gene fragment.	Purkayastha et al.，2010
	棉花叶皱缩病毒Cotton leaf crumple virus（CLCrV）	2	DNA-A组分中的CP缺失不会影响CLCrV的系统侵染，将其造为MCS用于插入靶基因片段。 The CP of DNA-A，which is dispensable for systematic infection of CLCrV，is replaced with MCS for insertion of target gene fragment.	Tuttle et al.，2008
	白菜曲叶病毒Cabbage leaf curl virus（CaLCuV）	2	DNA-A的外壳蛋白AR1基因对CaLCuV的系统性侵染不是必需的，对其进行改造用于插入amiRNA前体序列。 The coat protein AR1 of DNA-A，which is dispensable for CaLCuV systematic infection，is replaced with amiRNA precursor sequence.	Tang et al.，2013
DNA	番茄金色花叶病毒Tomato golden mosaic virus（TGMV）	2	DNA-A上的编码外壳蛋白的AR1基因序列被替换为靶基因片段。 The AR1 gene sequence of DNA-A is replaced with target gene fragment.		Kjemtrup et al.，1998
	非洲木薯花叶病毒African cassava mosaic virus（ACMV）	2	将DNA-A上部分CP序列（459 bp）改造为MCS，用于插入靶基因片段。The CP sequence（459 bp）of DNA-A is replaced with MCS for insertion of target gene fragment.		Fofana et al.，2004
	白菜曲叶病毒Cabbage leaf curl virus（CaLCuV）	2	AR1是CP蛋白的编码基因，将靶基因片段插入DNA-A的AR1 ORF中。The target gene fragment is inserted into AR1 ORF（encoding CP protein） of DNA-A.		Muangsan et al.，2004；Xiao et al.，2020；Turnage et al.，2002
卫星病毒	竹花叶病毒及其卫星病毒Bamboo mosaic virus（BaMV）and its satellite RNA（satBaMV）		satBaMV的P20基因编码RNA结合蛋白，将P20部分序列替换为靶基因片段。 The part of P20（encoding RNA binding protein）sequence of satBaMV is replaced with target gene fragment.		Liou et al.，2014
	中国番茄黄化曲叶病毒卫星病毒DNAβ Tomato yellow leaf curl China virus DNAβ（TYLCCNV，DNAβ）		DNAβ可导致感病症状产生，将DNAβ的C1 ORF替换为MCS并用于插入靶基因片段，与基因组DNA-A混合接种可发挥沉默作用。 DNAβ causes disease symptoms. The C1 ORF of DNAβ is replaced with MCS for insertion of target gene fragment. Mixed inoculation with genomic DNA-A can silence target gene.		Tao & Zhou，2004；Cai et al.，2007
	贝因迪黄脉花叶病毒卫星DNAβ Bhendi yellow vein mosaic virus β-satellite（BYMV，DNAβ）		将DNAβ的C1 ORF替换为MCS并用于插入靶基因片段。 The C1 ORF of DNAβ is replaced with MCS for insertion of target gene fragment.		Jeyabharathy et al.，2015
	木尔坦棉花曲叶病毒β卫星病毒 Cotton leaf curl Multan betasatellite（CLCuMB）		将CLCuMB的βC1 ORF替换为靶基因片段。 The βC1 ORF of CLCuMB is replaced with target gene fragment.		Kharazmi et al.，2012

病毒类型 Type	病毒名称 Virus name	基因组组分 Numbers of virus segments	载体构建策略 Strategies for vectors construction		参考文献 References
RNA	马铃薯X病毒 Potato virus X（PVX）	1	将靶基因片段插入CP的5′端。 The target gene fragment is inserted at the 5′-end of the CP.		Ruiz et al.，1998
	狗尾草花叶病毒 Foxtail mosaic virus（FoMV）	1	5A突变后不影响病毒复制和侵染，在5A中间加入170 bp FoMV CP亚基因组启动子序列和MCS，将靶基因片段插入MCS。 The mutation of 5A do not affect the replication and infection of virus. The 170 bp subgenomic promoter and MCS used for insertion of target gene fragment are inserted into the middle region of 5A.		Robertson et al.，2000；Liu et al.，2016
	木薯普通花叶病毒 Cassava common mosaic virus（CsCMV）	1	在CP启动子SGP2序列前加入90 bp亚基因组启动子SGP1，SGP1后插入靶基因片段。 A duplicated 90 bp subgenomic promoter（SGP1）is engineered into viral genome at the upstream of CP promoter（SGP2），and the target gene fragment is inserted after SGP1.		Tuo et al.，2021
	马铃薯X病毒 Potato virus X（PVX）	1	在CP的5′端加入PVX亚基因组启动子，在该启动子后插入靶基因片段。 The PVX subgenomic promoter is engineered into the 5′-end of CP，after which a target gene fragment is inserted.		Zhao et al.，2020
	柑橘叶斑驳病毒 Citrus leaf blotch virus（CLBV）	1	在CP的3′或5′端加入PmlI，用于插入靶基因片段；或克隆CP亚基因组启动子并在其后加入PmlI，将该组件构建至CP的3′端或5′端，用于插入靶基因片段。 The PmlI used for insertion of target gene fragment is engineered into the 3′ or 5′-end of CP. Or inserting the CP subgenomic promoter flanked with PmlI into the 3′ or 5′-end of CP.		Agüero et al.，2014
	葡萄病毒A Grapevein virus A（GVA）	1	含有5个ORF，在MP的3′端加入异源移动蛋白亚基因组启动子，在该启动子后插入靶基因片段。 GVA contains 5 ORFs，and a heterogenic movement protein subgenomic promoter is engineered into the 3′-end of MP，after which a target gene fragment is inserted.		Muruganantham et al.，2009
	雀麦花叶病毒 Brome mosaic virus（BMV）	3	在RNA3的CP终止密码子后插入靶基因片段。 A target gene fragment is inserted after the stop codon of CP in RNA3.		Ding et al.，2006，2018
	黄瓜花叶病毒 Cucumber mosaic virus（CMV）	3	第2条链编码2b蛋白，为RNA沉默抑制子，保留2b的N端前81个氨基酸序列，并在其后加入MCS，用于插入靶基因片段。 The second strand of CMV encodes 2b protein, which is an RNA silencing suppressor. The first 81 amino acid sequences lied in the N-terminal of 2b are retained，and MCS is added afterwards for the insertion of target gene fragment.		Wang et al.，2016
	李坏死环斑病毒Prunus necrotic ringspot virus（PNRSV）	3	将靶基因片段插入至RNA3 CP的3′末端，此外，RNA1和RNA2构建至同一个载体上，以便提高侵染效率。 The target gene fragment is inserted into the 3′-end of CP of RNA3. Moreover，the RNA1 and RNA2 are constructed into the same vector to improve infection efficiency.		Cui & Wang，2017；潘佳佳等，2023
RNA	莴苣侵染性黄化病毒Lettuce infectious yellows virus（LIYV）	2	P26紧邻CPm的3′端，在P26的3′端加入CP亚基因启动子，其后用于加入靶基因片段。 The P26 is next to the 3′-end of CPm. The CP subgenomic promoter is inserted after P26，after which a target gene fragment is inserted.	Qiao & Falk，2018；Stewart et al.，2010
	葡萄卷叶伴随病毒-2 Grape vein roll-associated virus（GLRaV-2）	1	在次要外壳蛋白和外壳蛋白中间有亚基因组启动子，在该亚基因组启动子后插入靶基因片段。 The subgenomic promoter is located between minor coat protein and coat protein，after which a target gene fragment is inserted.	Kurth et al.，2012
	柑橘衰退病毒 Citrus tristeza virus（CTV）	1	在p23和CP中间加入酶切位点，用于插入CP亚基因组启动子和靶基因片段。Enzyme restriction site，which is used for inserting subgeno- mic promoter and target gene fragment, is inserted between p23 and CP.	Hajeri et al.，2014
	苦苣菜黄网弹状病毒Sonchus yellow net rhabdovirus（SYNV）	1	位于5'端的N和P蛋白负责病毒的复制，在这两个编码序列中间加入MCS，用于插入靶基因片段。 The N and P proteins are responsible for viral replication. The MCS is added between N and P for insertion of target gene fragment.	Peng et al.，2021
	苹果潜隐病毒 Apple latent spherical virus（ALSV）	2	在RNA2的MP和CP中间MCS，用于插入靶基因片段。 The MCS is added between MP and CP of RNA2 for insertion of target gene fragment.	Igarashi et al.，2009；Gedling et al.，2018
	菜豆荚斑驳病毒 Bean pod mottle virus（BPMV）	2	在MP和L-CP之间加入MCS用于插入靶基因片段。 The MCS is added between MP and L-CP for insertion of target gene fragment.	Zhang & Ghabrial，2006；Zhang et al.，2010
	蚕豆萎蔫病毒2 Broad bean wilt virus 2（BBWV2）	2	在RNA2的CP的终止密码子后加入一个酶切位点，用于插入靶基因片段。在病毒序列3′末端与载体骨架之间添加20个poly (A)和RZ序列。An enzyme restriction site is added after stop codon of CP of RNA2，which is used for insertion of target gene fragment. The 20 poly (A) and RZ sequences are added between the 3′-end of the virus sequence and vector sequence.	Kwak et al.，2016；Choi et al.，2019
	芜菁黄花叶病毒Turnip yellow mosaic virus（TYMV）	1	在CP终止密码子后插入SnaBI酶切位点，用于插入外源基因片段。 The SnaBI is added after stop codon of CP for the insertion of target gene fragment.	Yu et al.，2018
	烟草花叶病毒Tobacco mosaic virus（TMV）	1	在CP编码区的5′端加入靶基因片段，由TMV-U1亚基因组启动子驱动。The target gene fragment is inserted to the 5′-end of CP coding region, driven by the TMV-U1 subgenomic promoter.	Kumagai et al.，1995
	烟草脆裂病毒Tobacco rattle virus（TRV）	2	将RNA2上CP后的29.4 kb和32.8 kb的非结构蛋白替换为MCS用于插入靶基因片段。The 29.4 kb and 32.8 kb non-structural protein sequences are replaced with MCS for insertion of target gene fragment.	Ratcliff et al.，2001
	豌豆早枯病毒 Pea early-browning virus（PEBV）	2	将RNA2上CP后的非结构蛋白2b和2c替换为靶基因片段。 The 2b and 2c non-structural protein sequences of CP of RNA2 are replaced with target gene fragment.	Constantin et al.，2004
	中国小麦花叶病毒Chinese wheat mosaic virus（CWMV）	2	将RNA2上CP的RT部分替换为MCS，用于插入靶基因片段。 The RT of CP of RNA2 is replaced with MCS for insertion of target gene fragment.	Yang et al.，2018
	大麦条纹花叶病毒Barley stripe mosaic virus（BSMV）	3	含有 RNAα、β、γ三组分，在γb下游加入MCS，用于插入靶基因片段。BSMV contains RNAα，β and γ，the MCS is added after γb for insertion of target gene fragment.	Tamilselvan-Nattar-Amutha et al.，2023
DNA	水稻东格鲁杆状病毒Rice tungro bacilliform virus（RTBV）	1	含有ORF I、ORF II、ORF III、ORF IV，保留ORF III和ORF IV，将RTBV 5′端启动子替换为MUP启动子和tRNA binding site，ORF IV终止密码子后加入MCS，用于插入靶基因片段。 RTBC contains ORF I，ORF II，ORF III and ORF IV. The ORF III and ORF IV are retained，and the promoter located in the 5′-end of RTBV is replaced with MUP promoter and tRNA binding site，the MCS is added after stop codon of ORF IV for insertion of target gene fragment.	Purkayastha et al.，2010
	棉花叶皱缩病毒Cotton leaf crumple virus（CLCrV）	2	DNA-A组分中的CP缺失不会影响CLCrV的系统侵染，将其造为MCS用于插入靶基因片段。 The CP of DNA-A，which is dispensable for systematic infection of CLCrV，is replaced with MCS for insertion of target gene fragment.	Tuttle et al.，2008
	白菜曲叶病毒Cabbage leaf curl virus（CaLCuV）	2	DNA-A的外壳蛋白AR1基因对CaLCuV的系统性侵染不是必需的，对其进行改造用于插入amiRNA前体序列。 The coat protein AR1 of DNA-A，which is dispensable for CaLCuV systematic infection，is replaced with amiRNA precursor sequence.	Tang et al.，2013
DNA	番茄金色花叶病毒Tomato golden mosaic virus（TGMV）	2	DNA-A上的编码外壳蛋白的AR1基因序列被替换为靶基因片段。 The AR1 gene sequence of DNA-A is replaced with target gene fragment.		Kjemtrup et al.，1998
	非洲木薯花叶病毒African cassava mosaic virus（ACMV）	2	将DNA-A上部分CP序列（459 bp）改造为MCS，用于插入靶基因片段。The CP sequence（459 bp）of DNA-A is replaced with MCS for insertion of target gene fragment.		Fofana et al.，2004
	白菜曲叶病毒Cabbage leaf curl virus（CaLCuV）	2	AR1是CP蛋白的编码基因，将靶基因片段插入DNA-A的AR1 ORF中。The target gene fragment is inserted into AR1 ORF（encoding CP protein） of DNA-A.		Muangsan et al.，2004；Xiao et al.，2020；Turnage et al.，2002
卫星病毒	竹花叶病毒及其卫星病毒Bamboo mosaic virus（BaMV）and its satellite RNA（satBaMV）		satBaMV的P20基因编码RNA结合蛋白，将P20部分序列替换为靶基因片段。 The part of P20（encoding RNA binding protein）sequence of satBaMV is replaced with target gene fragment.		Liou et al.，2014
	中国番茄黄化曲叶病毒卫星病毒DNAβ Tomato yellow leaf curl China virus DNAβ（TYLCCNV，DNAβ）		DNAβ可导致感病症状产生，将DNAβ的C1 ORF替换为MCS并用于插入靶基因片段，与基因组DNA-A混合接种可发挥沉默作用。 DNAβ causes disease symptoms. The C1 ORF of DNAβ is replaced with MCS for insertion of target gene fragment. Mixed inoculation with genomic DNA-A can silence target gene.		Tao & Zhou，2004；Cai et al.，2007
	贝因迪黄脉花叶病毒卫星DNAβ Bhendi yellow vein mosaic virus β-satellite（BYMV，DNAβ）		将DNAβ的C1 ORF替换为MCS并用于插入靶基因片段。 The C1 ORF of DNAβ is replaced with MCS for insertion of target gene fragment.		Jeyabharathy et al.，2015
	木尔坦棉花曲叶病毒β卫星病毒 Cotton leaf curl Multan betasatellite（CLCuMB）		将CLCuMB的βC1 ORF替换为靶基因片段。 The βC1 ORF of CLCuMB is replaced with target gene fragment.		Kharazmi et al.，2012