棉花VIGS病毒载体的构建及其在抗病基因功能鉴定的应用
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摘要
【目的】利用农杆菌介导的VIGS基因沉默技术,研究陆地棉抗病相关基因在棉花抗黄萎病中的功能。【方法】利用分子生物学技术构建VIGS病毒载体,建立VIGS病毒诱导沉默体系;利用此体系将棉花中的一个钙依赖蛋白(CDPK)基因,通过农杆菌介导在陆地棉中沉默,根据此基因沉默植株在病原菌环境下的发病情况研究该基因在棉花抗病中的功能。【结果】利用包含有Gh CPK抗病基因片段的VIGS病毒载体的农杆菌侵染棉花子叶,病毒载体上的目的基因片段由RNA聚合酶识别并合成双链RNA(double stranded RNA,dsRNA),dsRNA由Dicer酶识别并于其它RNA形成RNA诱导的沉默复合体(RNA induced silencing complex,RISC),RISC对内源Gh CPK mRNA进行识别和切割作用,内源Gh CPK mRNA降解而发生基因沉默。Gh CPK基因沉默导致棉苗对黄萎病敏感性增加,证明Gh CPK基因参与调控棉花抗病功能。【结论】病毒诱导的基因沉默技术(VIGS)能够快速有效的研究Gh CPKs基因在棉花抗病中的功能。
【Objective】The purpose is to identify genes in response to Verticillium dahlia by the virus induced gene silencing(VIGS).【Method】With the construction of VIGS virus(virus induced gene silencing)vector by molecular way,we established an Agrobacterium-mediated virus-induced gene silencing(VIGS)assay in cotton. To study function of GhCPK gene family,a GhCPK gene was silenced in cotton using VIGS assay,then the disease index of the gene-silenced cotton seedlings were investigated when they were challenged with Verticillium dahlia. 【Result】By infecting cotton cytoledons with VIGS virus vector containing GhCPK fragment,the GhCPK fragment is identified and catalyzed into dsRNAs. The combination of dsRNAs with other RNA produce RNA induced silencing comlex(RISC) which can target and degrade endogenous GhCPK mRNA and lead to the GhCPK gene silenced. As the result of silencing GhCPK gene in cotton,the GhCPK silenced cottons showed enhanced sensitivity to Verticillium dahlia,which suggested that GhCPK gene may play a role in regulating disease resitance in cotton.【Conclusion】The virus induced gene silencing technology is an efficient way for identifying GhCPKs gene in response to disease stress in cotton.
【Objective】The purpose is to identify genes in response to Verticillium dahlia by the virus induced gene silencing(VIGS).【Method】With the construction of VIGS virus(virus induced gene silencing)vector by molecular way,we established an Agrobacterium-mediated virus-induced gene silencing(VIGS)assay in cotton. To study function of GhCPK gene family,a GhCPK gene was silenced in cotton using VIGS assay,then the disease index of the gene-silenced cotton seedlings were investigated when they were challenged with Verticillium dahlia. 【Result】By infecting cotton cytoledons with VIGS virus vector containing GhCPK fragment,the GhCPK fragment is identified and catalyzed into dsRNAs. The combination of dsRNAs with other RNA produce RNA induced silencing comlex(RISC) which can target and degrade endogenous GhCPK mRNA and lead to the GhCPK gene silenced. As the result of silencing GhCPK gene in cotton,the GhCPK silenced cottons showed enhanced sensitivity to Verticillium dahlia,which suggested that GhCPK gene may play a role in regulating disease resitance in cotton.【Conclusion】The virus induced gene silencing technology is an efficient way for identifying GhCPKs gene in response to disease stress in cotton.
引文
[1]Burch-Smith,T. M.,Anderson,J. C.,Martin,G. B.,&Dinesh-Kumar,S. P.(2010). Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant Journal,39(5):734-746.
[2]Karen S. Century,Allan D. Shapiro,Peter P. Repetti,Douglas Dahlbeck,Eric Holub,&Brian J. Staskawicz.(1997). Ndr1,a pathogen-induced component required for arabidopsis disease resistance. Science,278(5345):1,963-1,965.
[3]Dinesh-Kumar SP,Anandalakshmi R,Marathe R,Schiff M,&Liu Y.(2011). Virus-induced gene silencing. Journal of Hangzhou Normal University(Natural Science Edition),236(236):287-294.
[4] Kawaide,H.,Kuzuyama,T.,Seto,H.,Kamiya,Y.,&León,P.(2000). Analysis of the expression of cla1,a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the2-c-methyl-d-erythritol-4-phosphate pathway in arabidopsis. Plant Physiology,124(1):95-103.
[5]Gao,X.,&Shan,L.(2013). Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced gene silencing. Methods Mol Biol,975:157-165.
[6]Turnage,M. A.,Muangsan,N.,Peele,C. G.,&Robertson,D.(2010). Geminivirus-based vectors for gene silencing in arabidopsis. Plant Journal for Cell&Molecular Biology,30(1):107-114.
[7] Voinnet,O.(2005). Induction and suppression of rna silencing:insights from viral infections. Nature Reviews Genetics,6(3):206-220.
[8]Gao,X.,Li,F.,Li,M.,Kianinejad,A. S.,Dever,J. K.,&Wheeler,T. A.,et al.(2013). Cotton ghbak1 mediates verticillium wilt resistance and cell death. Journal of Integrative Plant Biology,55(7):586-596.
[9]Gao,X.,Wheeler,T.,Li,Z.,Kenerley,C. M.,He,P.,&Shan,L.(2011). Silencing ghndr1 and ghmkk2 compromises cotton resistance to verticillium wilt. Plant Journal,66(2):293-305.
[10]Ludwig,A. A.,Romeis,T.,&Jones,J. D.(2004). Cdpk-mediated signalling pathways:specificity and cross-talk.Journal of Experimental Botany,55(395):181-188.
[11]Harper,J. F.,&Harmon,A.(2005). Plants,symbiosis and parasites:a calcium signalling connection. Nature Reviews Molecular Cell Biology,6(7):555-566.
[12] Shkryl,Y. N.,Veremeichik,G. N.,Bulgakov,V. P.,&Zhuravlev,Y. N.(2011). Induction of anthraquinone biosynthesis in rubia cordifolia cells by heterologous expression of a calcium‐dependent protein kinase gene. Biotechnology&Bioengineering,108(7):1,734-1,738.
[13]Rong J,Bowers JE,Schulze SR,et al.(2005). Comparative genomics of Gossypium and Arabidopsis:unraveling the consequences of both ancient and recent polyploidy. Genome Res,(15):1,198-1,210.
[2]Karen S. Century,Allan D. Shapiro,Peter P. Repetti,Douglas Dahlbeck,Eric Holub,&Brian J. Staskawicz.(1997). Ndr1,a pathogen-induced component required for arabidopsis disease resistance. Science,278(5345):1,963-1,965.
[3]Dinesh-Kumar SP,Anandalakshmi R,Marathe R,Schiff M,&Liu Y.(2011). Virus-induced gene silencing. Journal of Hangzhou Normal University(Natural Science Edition),236(236):287-294.
[4] Kawaide,H.,Kuzuyama,T.,Seto,H.,Kamiya,Y.,&León,P.(2000). Analysis of the expression of cla1,a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the2-c-methyl-d-erythritol-4-phosphate pathway in arabidopsis. Plant Physiology,124(1):95-103.
[5]Gao,X.,&Shan,L.(2013). Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced gene silencing. Methods Mol Biol,975:157-165.
[6]Turnage,M. A.,Muangsan,N.,Peele,C. G.,&Robertson,D.(2010). Geminivirus-based vectors for gene silencing in arabidopsis. Plant Journal for Cell&Molecular Biology,30(1):107-114.
[7] Voinnet,O.(2005). Induction and suppression of rna silencing:insights from viral infections. Nature Reviews Genetics,6(3):206-220.
[8]Gao,X.,Li,F.,Li,M.,Kianinejad,A. S.,Dever,J. K.,&Wheeler,T. A.,et al.(2013). Cotton ghbak1 mediates verticillium wilt resistance and cell death. Journal of Integrative Plant Biology,55(7):586-596.
[9]Gao,X.,Wheeler,T.,Li,Z.,Kenerley,C. M.,He,P.,&Shan,L.(2011). Silencing ghndr1 and ghmkk2 compromises cotton resistance to verticillium wilt. Plant Journal,66(2):293-305.
[10]Ludwig,A. A.,Romeis,T.,&Jones,J. D.(2004). Cdpk-mediated signalling pathways:specificity and cross-talk.Journal of Experimental Botany,55(395):181-188.
[11]Harper,J. F.,&Harmon,A.(2005). Plants,symbiosis and parasites:a calcium signalling connection. Nature Reviews Molecular Cell Biology,6(7):555-566.
[12] Shkryl,Y. N.,Veremeichik,G. N.,Bulgakov,V. P.,&Zhuravlev,Y. N.(2011). Induction of anthraquinone biosynthesis in rubia cordifolia cells by heterologous expression of a calcium‐dependent protein kinase gene. Biotechnology&Bioengineering,108(7):1,734-1,738.
[13]Rong J,Bowers JE,Schulze SR,et al.(2005). Comparative genomics of Gossypium and Arabidopsis:unraveling the consequences of both ancient and recent polyploidy. Genome Res,(15):1,198-1,210.