桑树PDS基因VIGS转化体系的构建与鉴定
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摘要
【目的】建立桑树的病毒诱导基因沉默(VIGS)转化体系,为桑树功能基因的分析研究提供技术支持。【方法】以丰驰桑为试验材料,克隆含Bam H I和Xba I酶切位点的八氢番茄红素脱氢酶(PDS)基因片段,经Xba I和Bam H I双酶切后与烟草脆裂病毒(TRV)连接构建重组病毒载体p TRV2-PDS,转化农杆菌GV3101后通过压迫注射方式侵染桑树幼苗叶片。【结果】克隆获得的桑树PDS基因干扰片段346 bp,与改造后的TRV质粒连接可成功构建携带桑树PDS基因的重组病毒载体p TRV2-PDS。桑树叶片被侵染15 d后,接种重组病毒载体p TRV2-PDS桑树的第二轮真叶开始出现叶片白化现象,侵染20 d后,白化现象沿叶脉向四周逐渐扩散,叶片呈现明显的白化表型。实时荧光定量PCR检测结果显示,重组病毒载体p TRV2-PDS侵染的桑叶PDS基因相对表达量仅为阴性对照(p TRV2)的58%,二者差异极显著(P<0.01),表明桑树PDS基因表达被有效抑制。【结论】利用TRV构建的桑树VIGS体系能有效沉默PDS基因,使桑树叶片呈现明显的白化症状,即可用于后续的桑树功能基因鉴定。
【Objective】The present study was conducted to establish mulberry virus-induced gene silencing(VIGS)transformation system and provide technical support for the analysis and research of the mulberry functional genes.【Method】In this research,Fengchi mulberry was used as experimental material,fragment of phytoene desaturase(PDS)gene with the Xba I and Bam H I restriction enzyme sites were cloned. After Xba I and Bam H I double digestion,the recombinant virus vector p TRV2-MPDS was obtained by ligating them into the tobacco rattle virus(TRV). Then p TRV2-PDS was transformed into Agrobacterium tumefaciens GV3101 and then transformed into mulberry leaves by pressure injection.【Result】The PDS interference fragment with the length of 346 bp was obtained by cloning and then the recombinant virus vector p TRV2-PDS containing mulberry gene PDS was obtained by connection with the modified TRV plasmid.After 15 d of infection,there were albino phenotypes in the second round of mulberry leaves that inoculated with recombinant virus vector p TRV2-PDS,and the albinism gradually spread along the veins when mulberry plants were infected 20 d,and the leaves showed obvious albino phenotypes. Real-time quantitative PCR detection results showed that the expression of gene PDS in leaves of recombinant virus vector p TRV2-PDS inoculated plant dropped to 58% compared with the negative control(recombinant virus vector),there was extremely significant difference between them(P<0.01),which showed that mulberry gene PDS was effectively silenced.【Conclusion】Gene PDS can be effectively silenced by using mulberry VIGS system constructed by TRV and then the mulberry leaves appeared obvious albino phenotypes. The VIGS system can be used for subsequent functional identification of mulberry genes.
【Objective】The present study was conducted to establish mulberry virus-induced gene silencing(VIGS)transformation system and provide technical support for the analysis and research of the mulberry functional genes.【Method】In this research,Fengchi mulberry was used as experimental material,fragment of phytoene desaturase(PDS)gene with the Xba I and Bam H I restriction enzyme sites were cloned. After Xba I and Bam H I double digestion,the recombinant virus vector p TRV2-MPDS was obtained by ligating them into the tobacco rattle virus(TRV). Then p TRV2-PDS was transformed into Agrobacterium tumefaciens GV3101 and then transformed into mulberry leaves by pressure injection.【Result】The PDS interference fragment with the length of 346 bp was obtained by cloning and then the recombinant virus vector p TRV2-PDS containing mulberry gene PDS was obtained by connection with the modified TRV plasmid.After 15 d of infection,there were albino phenotypes in the second round of mulberry leaves that inoculated with recombinant virus vector p TRV2-PDS,and the albinism gradually spread along the veins when mulberry plants were infected 20 d,and the leaves showed obvious albino phenotypes. Real-time quantitative PCR detection results showed that the expression of gene PDS in leaves of recombinant virus vector p TRV2-PDS inoculated plant dropped to 58% compared with the negative control(recombinant virus vector),there was extremely significant difference between them(P<0.01),which showed that mulberry gene PDS was effectively silenced.【Conclusion】Gene PDS can be effectively silenced by using mulberry VIGS system constructed by TRV and then the mulberry leaves appeared obvious albino phenotypes. The VIGS system can be used for subsequent functional identification of mulberry genes.
引文
姜上川,梅超,王小芳,张大鹏.2016.拟南芥SOAR1基因响应ABA与渗透胁迫的表达分析[J].河南农业科学,45(2):29-34.[Jiang S C,Mei C,Wang X F,Zhang D P.2016.Expression analysis of Arabidopsis SOAR1 in response to ABA and osmotic stress[J].Journal of Henan Agricultural Sciences,45(2):29-34.]
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王旭,王新果,张朝贤,黄红娟,魏守辉.2016.刺萼龙葵PDS基因VIGS载体的构建[J].植物保护,42(4):137-141.[Wang X,Wang X G,Zhang C X,Huang H J,Wei S H.2016.VIGS vector construction of PDS gene in Solanum rostratum[J].Plant Protection,42(4):137-141.]
Agüero J,Ruiz-Ruiz S,Del Carmen Vives M,Velázquez K,Navarro L,Pe?a L,Moreno P,Guerri J.2012.Development of viral vectors based on Citrus leaf blotch virus to express foreign proteins or analyze gene function in citrus plants[J].Molecular Plant-Microbe Interactions,25(10):1326-1337.
Bennypaul H S,Mutti J S,Rustgi S,Kumar N,Okubara P A,Gill K S.2012.Virus-induced gene silencing(VIGS)of genes expressed in root,leaf,and meiotic tissues of wheat[J].Functional&Integrative Genomics,12(1):143-156.
Bilichak A,Kovalchuk I.2017.Increasing a stable transformation efficiency of Arabidopsis by manipulating the endogenous gene expression using virus-induced gene silencing[J].Methods in Molecular Biology,1456:225-236.
Buhrow L M,Clark S M,Loewen M C.2016.Identification of an attenuated barley stripe mosaic virus for the virusinduced gene silencing of pathogenesis-related wheat genes[J].Plant Methods,12:12.doi:10.1186/s13007-016-0112-z.
Dobnik D,Lazar A,Stare T,Gruden K,Vleeshouwers V G,?el J.2016.Solanum venturii,a suitable model system for virus-induced gene silencing studies in potato reveals St MKK6 as an important player in plant immunity[J].Plant Methods,12:29.doi:10.1186/s13007-016-0129-3.
Geuten K,Viaene T,Vekemans D,Kourmpetli S,Drea S.2013.Analysis of developmental control genes using virus-induced gene silencing[J].Methods in Molecular Biology,975:61-69.
Huang C J,Qian Y J,Li Z H,Zhou X P.2012.Virus-induced gene silencing and its application in plant functional genomics[J].Science China.Life Sciences,55(2):99-108.
Kandoth P K,Heinz R,Yeckel G,Gross N W,Juvale P S,Hill J,Whitham S A,Baum T J,Mitchum M G.2013.A virusinduced gene silencing method to study soybean cyst nematode parasitism in Glycine max[J].BMC Research Notes,6:255.doi:10.1186/1756-0500-6-255.
Kant R,Sharma S,Dasgupta I.2015.Virus-induced gene silencing(VIGS)for functional genomics in rice using rice tungro bacilliform virus(RTBV)as a vector[J].Methods in Molecular Biology,1287:201-217.
Kjemtrup S,Sampson K S,Peele C G,Nguyen L V,Conkling M A,Thompson W F,Robertson D.1998.Gene silen-cing from plant DNA carried by a Geminivirus[J].The Plant Journal,14(1):91-100.
Kumagai M H,Donson J,della-Cioppa G,Harvey D,Hanley K,Grill L K.1995.Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA[J].Proceedings of the National Academy of Sciences of the United States of America,92(5):1679-1683.
Lee J,Cao V D,Kim J,Pamplona R,Ahn J,Cho S K,Yang SW,Riu K Z,Boo K H.2017.Development of a virus-induced gene silencing(VIGS)system for Spinacia oleracea L[.J].In Vitro Cellular&Developmental BiologyPlant,(2):1-7.
Li C,Yoshikawa N.2015.Virus-induced gene silencing of Ngene in tobacco by apple latent spherical virus vectors[J].Methods in Molecular Biology,1236:229-240.
Li S,Li K,Cao D,Fu D,Zhu H,Zhu B,Luo Y.2016.Genome-wide analysis of tomato NF-Y factors and their role in fruit ripening[J].BMC Genomics,17:36.doi:10.1186/s12864-015-2334-2.
Meng L H,Wang R H,Zhu B Z,Zhu H L,Luo Y B,Fu D Q.2016.Efficient virus-induced gene silencing in Solanum rostratum[J].PLo S One,11(6):e0156228.
Mustafa R,Shafiq M,Mansoor S,Briddon R W,Scheffler BE,Scheffler J,Amin I.2016.Virus-induced gene silencing in cultivated cotton(Gossypium spp.)using tobacco rattle virus[J].Molecular Biotechnology,58(1):65-72.
Nelson D R,Schuler M A,Paquette S M,Werck-Reichhart D,Bak S.2004.Comparative genomics of rice and Arabidopsis.Analysis of 727 cytochrome P450 genes and pseudogenes from a monocot and a dicot[J].Plant Physiology,135(2):756-772.
Pflieger S,Mms R,Blanchet S,Meziadi C,Geffroy V.2013.VIGS technology:An attractive tool for functional genomics studies in legumes[J].Functional Plant Biology,40(12):1234-1248.
Ratcliff F,Martin-Hernandez A M,Baulcombe D C.2000.Technical advance.Tobacco rattle virus as a vector for analysis of gene function by silencing[J].The Plant Journal,25(2):237-245.
Ruiz M T,Voinnet O,Baulcombe D C.1998.Initiation and maintenance of virus-induced gene silencing[J].Plant Cell,10(6):937-946.
Sasaki S,Yamagishi N,Yoshikawa N.2011.Efficient virusinduced gene silencing in apple,pear and Japanese pear using Apple latent spherical virus vectors[J].Plant Methods,7(1):15.doi:10.1186/1746-4811-7-15.
Senthil-Kumar M,Mysore K S.2011.Virus-induced gene silencing can persist for more than 2 years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato[J].Plant Biotechnology Journal,9(7):797-806.
Shao Y,Zhu H L,Tian H Q,Wang X G,Lin X J,Zhu B Z,Xie Y H,Luo Y B.2008.Virus-induced gene silencing in plant species[J].Russian Journal of Plant Physiology,55(2):168-174.
Spitzerrimon B,Can’Ani A,Vainstein A.2013.Virus-aided gene expression and silencing using TRV for functional analysis of floral scent-related genes[J].Methods in Molecular Biology,975:139-148.
Sun Z,Cunningham F X Jr,Gantt E.1998.Differential expression of two isopentenyl pyrophosphate isomerases and enhanced carotenoid accumulation in a unicellular chlorophyte[J].Proceedings of the National Academy of Sciences of the United States of America,95(19):11482-11488.
Zhou X,Huang C.2012.Virus-induced gene silencing using begomovirus satellite molecules[J].Methods in Molecular Biology,894:57-67.
牛义岭,姜秀明,许向阳.2016.番茄GRAS转录因子家族基因SIGLD1的克隆及VIGS载体构建[J].基因组学与应用生物学,35(8):2155-2160.[Niu Y L,Jiang X M,Xu XY.2016.Cloning and VIGS vector construction of GRAStranscription factors family gene SIGLD1 from tomato[J].Genomics and Applied Biology,35(8):2155-2160.]
王旭,王新果,张朝贤,黄红娟,魏守辉.2016.刺萼龙葵PDS基因VIGS载体的构建[J].植物保护,42(4):137-141.[Wang X,Wang X G,Zhang C X,Huang H J,Wei S H.2016.VIGS vector construction of PDS gene in Solanum rostratum[J].Plant Protection,42(4):137-141.]
Agüero J,Ruiz-Ruiz S,Del Carmen Vives M,Velázquez K,Navarro L,Pe?a L,Moreno P,Guerri J.2012.Development of viral vectors based on Citrus leaf blotch virus to express foreign proteins or analyze gene function in citrus plants[J].Molecular Plant-Microbe Interactions,25(10):1326-1337.
Bennypaul H S,Mutti J S,Rustgi S,Kumar N,Okubara P A,Gill K S.2012.Virus-induced gene silencing(VIGS)of genes expressed in root,leaf,and meiotic tissues of wheat[J].Functional&Integrative Genomics,12(1):143-156.
Bilichak A,Kovalchuk I.2017.Increasing a stable transformation efficiency of Arabidopsis by manipulating the endogenous gene expression using virus-induced gene silencing[J].Methods in Molecular Biology,1456:225-236.
Buhrow L M,Clark S M,Loewen M C.2016.Identification of an attenuated barley stripe mosaic virus for the virusinduced gene silencing of pathogenesis-related wheat genes[J].Plant Methods,12:12.doi:10.1186/s13007-016-0112-z.
Dobnik D,Lazar A,Stare T,Gruden K,Vleeshouwers V G,?el J.2016.Solanum venturii,a suitable model system for virus-induced gene silencing studies in potato reveals St MKK6 as an important player in plant immunity[J].Plant Methods,12:29.doi:10.1186/s13007-016-0129-3.
Geuten K,Viaene T,Vekemans D,Kourmpetli S,Drea S.2013.Analysis of developmental control genes using virus-induced gene silencing[J].Methods in Molecular Biology,975:61-69.
Huang C J,Qian Y J,Li Z H,Zhou X P.2012.Virus-induced gene silencing and its application in plant functional genomics[J].Science China.Life Sciences,55(2):99-108.
Kandoth P K,Heinz R,Yeckel G,Gross N W,Juvale P S,Hill J,Whitham S A,Baum T J,Mitchum M G.2013.A virusinduced gene silencing method to study soybean cyst nematode parasitism in Glycine max[J].BMC Research Notes,6:255.doi:10.1186/1756-0500-6-255.
Kant R,Sharma S,Dasgupta I.2015.Virus-induced gene silencing(VIGS)for functional genomics in rice using rice tungro bacilliform virus(RTBV)as a vector[J].Methods in Molecular Biology,1287:201-217.
Kjemtrup S,Sampson K S,Peele C G,Nguyen L V,Conkling M A,Thompson W F,Robertson D.1998.Gene silen-cing from plant DNA carried by a Geminivirus[J].The Plant Journal,14(1):91-100.
Kumagai M H,Donson J,della-Cioppa G,Harvey D,Hanley K,Grill L K.1995.Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA[J].Proceedings of the National Academy of Sciences of the United States of America,92(5):1679-1683.
Lee J,Cao V D,Kim J,Pamplona R,Ahn J,Cho S K,Yang SW,Riu K Z,Boo K H.2017.Development of a virus-induced gene silencing(VIGS)system for Spinacia oleracea L[.J].In Vitro Cellular&Developmental BiologyPlant,(2):1-7.
Li C,Yoshikawa N.2015.Virus-induced gene silencing of Ngene in tobacco by apple latent spherical virus vectors[J].Methods in Molecular Biology,1236:229-240.
Li S,Li K,Cao D,Fu D,Zhu H,Zhu B,Luo Y.2016.Genome-wide analysis of tomato NF-Y factors and their role in fruit ripening[J].BMC Genomics,17:36.doi:10.1186/s12864-015-2334-2.
Meng L H,Wang R H,Zhu B Z,Zhu H L,Luo Y B,Fu D Q.2016.Efficient virus-induced gene silencing in Solanum rostratum[J].PLo S One,11(6):e0156228.
Mustafa R,Shafiq M,Mansoor S,Briddon R W,Scheffler BE,Scheffler J,Amin I.2016.Virus-induced gene silencing in cultivated cotton(Gossypium spp.)using tobacco rattle virus[J].Molecular Biotechnology,58(1):65-72.
Nelson D R,Schuler M A,Paquette S M,Werck-Reichhart D,Bak S.2004.Comparative genomics of rice and Arabidopsis.Analysis of 727 cytochrome P450 genes and pseudogenes from a monocot and a dicot[J].Plant Physiology,135(2):756-772.
Pflieger S,Mms R,Blanchet S,Meziadi C,Geffroy V.2013.VIGS technology:An attractive tool for functional genomics studies in legumes[J].Functional Plant Biology,40(12):1234-1248.
Ratcliff F,Martin-Hernandez A M,Baulcombe D C.2000.Technical advance.Tobacco rattle virus as a vector for analysis of gene function by silencing[J].The Plant Journal,25(2):237-245.
Ruiz M T,Voinnet O,Baulcombe D C.1998.Initiation and maintenance of virus-induced gene silencing[J].Plant Cell,10(6):937-946.
Sasaki S,Yamagishi N,Yoshikawa N.2011.Efficient virusinduced gene silencing in apple,pear and Japanese pear using Apple latent spherical virus vectors[J].Plant Methods,7(1):15.doi:10.1186/1746-4811-7-15.
Senthil-Kumar M,Mysore K S.2011.Virus-induced gene silencing can persist for more than 2 years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato[J].Plant Biotechnology Journal,9(7):797-806.
Shao Y,Zhu H L,Tian H Q,Wang X G,Lin X J,Zhu B Z,Xie Y H,Luo Y B.2008.Virus-induced gene silencing in plant species[J].Russian Journal of Plant Physiology,55(2):168-174.
Spitzerrimon B,Can’Ani A,Vainstein A.2013.Virus-aided gene expression and silencing using TRV for functional analysis of floral scent-related genes[J].Methods in Molecular Biology,975:139-148.
Sun Z,Cunningham F X Jr,Gantt E.1998.Differential expression of two isopentenyl pyrophosphate isomerases and enhanced carotenoid accumulation in a unicellular chlorophyte[J].Proceedings of the National Academy of Sciences of the United States of America,95(19):11482-11488.
Zhou X,Huang C.2012.Virus-induced gene silencing using begomovirus satellite molecules[J].Methods in Molecular Biology,894:57-67.