利用RNA干扰介导抗病性获得兼抗四种病毒的转基因马铃薯
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摘要
为获得兼抗马铃薯X病毒(Potato virus X,PVX)、马铃薯Y病毒(Potato virus Y,PVY)、马铃薯卷叶病毒(Potato leaf roll virus,PLRV)和马铃薯潜隐花叶病毒(Potato virus S,PVS)4种病毒的转基因马铃薯新材料,分别以这4种病毒全长CP基因为模板,通过设计PCR引物和亚克隆获得4种病毒CP基因相对保守区段的基因片段,并将其拼接成融合基因,以载体pHANNIBAL和pBI121为基础,构建RNA干扰(RNA interference,RNAi)载体,利用农杆菌介导的转基因体系进行马铃薯遗传转化,并对获得的转基因马铃薯进行病毒抗性检测。结果表明,所获得的融合基因片段RH1和RH2,酶切鉴定分别得到长度为1 200 bp的条带,与预期片段相符;构建了含pdk内含子和RH1、RH2融合基因的RNAi植物表达载体,经Bam H I/Sac I双酶切,获得长度约3 200 bp的片段,表明RNAi植物表达载体pBI121-pRH构建成功;转化易感病毒马铃薯品种陇薯11号,PCR检测和PCRSouthern杂交分析表明融合基因已整合到陇薯11号马铃薯基因组中;抗病性检测显示4株转基因马铃薯植株对4种病毒均免疫。表明利用RNAi可筛选出抗多种病毒的转基因马铃薯新种质。
In order to obtain transgenic potato plants resistant to Potato virus X(PVX), Potato virus Y(PVY), Potato leaf role virus(PLRV) and Potato virus S(PVS), specific primers were designed according to the coat protein(CP) genes of PVX, PVY, PLRV and PVS, and then sub-clones of the cDNA fragments were obtained from the conserved regions of CP genes of PVX, PVY, PLRV and PVS, respectively. Then the four cDNA fragments were spliced into a chimeric cDNA. The RNA interference(RNAi)vector pBI121-pRH with the chimeric cDNA was constructed based on the vectors pHANNIBAL and p BI121. Potato transformation was conducted through agrobacterium-mediated system, and then transgenic plants obtained by tissue culture were tested for viral resistance. The fusion gene fragments RH1 and RH2 were obtained, and enzyme digestion resulted in an expected band of 1 200 bp in size, respectively. RNAi vectors containing pdk introns and RH1 and RH2 fusion genes were constructed, and Bam H I/Sac I bi-enzyme was performed to obtain a fragment of about 3 200 bp in length, indicating that the RNAi vector pBI121-PRH was successfully constructed. The infusion genes were integrated into the genome of Longshu 11 by using PCR and PCR-Southern blotting. Four transgenic potato plants were proved immune to the virus infection by the virus infection resistance analysis. This study indicated that multiple virus-resistant potato varieties could be obtained by using RNAi.
In order to obtain transgenic potato plants resistant to Potato virus X(PVX), Potato virus Y(PVY), Potato leaf role virus(PLRV) and Potato virus S(PVS), specific primers were designed according to the coat protein(CP) genes of PVX, PVY, PLRV and PVS, and then sub-clones of the cDNA fragments were obtained from the conserved regions of CP genes of PVX, PVY, PLRV and PVS, respectively. Then the four cDNA fragments were spliced into a chimeric cDNA. The RNA interference(RNAi)vector pBI121-pRH with the chimeric cDNA was constructed based on the vectors pHANNIBAL and p BI121. Potato transformation was conducted through agrobacterium-mediated system, and then transgenic plants obtained by tissue culture were tested for viral resistance. The fusion gene fragments RH1 and RH2 were obtained, and enzyme digestion resulted in an expected band of 1 200 bp in size, respectively. RNAi vectors containing pdk introns and RH1 and RH2 fusion genes were constructed, and Bam H I/Sac I bi-enzyme was performed to obtain a fragment of about 3 200 bp in length, indicating that the RNAi vector pBI121-PRH was successfully constructed. The infusion genes were integrated into the genome of Longshu 11 by using PCR and PCR-Southern blotting. Four transgenic potato plants were proved immune to the virus infection by the virus infection resistance analysis. This study indicated that multiple virus-resistant potato varieties could be obtained by using RNAi.
引文
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Liu Y,Cai H,Liu J,Bai X,Ji W,Zhu YM.2013.Transformation of the GsCRCK gene into Medicago sativa cv.Nongjing No.1 and salt tolerance analysis in transgenic plants.Acta Prataculturae Sinica,22(2):150-157(in Chinese)[刘莹,才华,刘晶,柏锡,纪巍,朱延明.2013.GsCRCK基因转化农菁1号苜蓿及其耐盐性分析.草业学报,22(2):150-157]
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Plasterk RH.2002.RNA silencing:the genome’s immune system.Science,296(5517):1263-1265
Prins M,de Haan PD,Luyten R,van Veller MV,van Grinsven MQV,Goldbach R.1995.Broad resistance to tospovirus in transgenic tobacco plants expressing three-tospoviral gene sequence.Molecular Plant-Microbe Interactions,8(1):85-91
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Smith NA,Singh SP,Wang MB,Stoutjesdijk PA,Green AG,Waterhouse PM.2000.Gene expression:total silencing by intronspliced hairpin RNAs.Nature,407(6802):319-320
Stoutjesdijk PA,Singh SP,Liu Q,Hurlstone CJ,Waterhouse PA,Green AG.2002.HpRNA-mediated targeting of the Arabidopsis FAD2gene gives highly efficient and stable silencing.Plant Physiology,129(4):1723-1731
van den Boogaart T,Wen F,Davies JW,Lomonossoff GP.2001.Replicase-derived resistance against pea early browning virus in Nicotiana benthamiana is an unstable resistance based upon posttranscriptional gene silencing.Molecular Plant-Microbe Interactions,14(2):196-203
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Wu XQ,Wu ZJ,Xie LH,Lin QY.2002.The clone and expression of the coat protein gene of Potato virus S in E.coli.Virologica Sinica,17(3):248-251(in Chinese)[吴兴泉,吴祖建,谢联辉,林奇英.2002.马铃薯S病毒外壳蛋白基因的克隆与原核表达.中国病毒学,17(3):248-251]
Xu L,Song YZ,Wang WJ,Zhu CX,Wen FJ.2010.Different virus-resistance to Potato virus Y infection mediated by different regions of NIb gene.Journal of Plant Protection,37(3):227-233(in Chinese)[徐丽,宋云枝,王文俊,朱常香,温孚江.2010.马铃薯Y病毒复制酶基因不同位置cDNA区段介导对PVY的不同抗性.植物保护学报,37(3):227-233]
Zamore PD.2002.Ancient pathways programmed by small RNAs.Science,296(5571):1265-1269
Zhang HL,Song YR,Peng XX,Li TR,Meng Q,Cui XJ,Hou LL,Zhang LZ,Ma QH.1996.Engineering resistance to mixed PVXand PVY infection in potato cultivars expressing both PVX and PVY coat protein genes.Chinese Journal of Virology,12(4):360-366(in Chinese)[张鹤龄,宋艳茹,彭学贤,李天然,孟清,崔晓江,侯林林,张莉枝,马庆虎.1996.表达马铃薯X病毒、Y病毒双价外壳蛋白基因马铃薯转基因植株的抗病性.病毒学报,12(4):360-366]
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Zhu CX,Song YZ,Wen FJ.2008.Constructing transgenic tobacco for multiple-resistance against Potato virus Y,Cucumber mosaic virus and Tobacco mosaic virus.Scientia Agricultura Sinica,41(4):1040-1047(in Chinese)[朱常香,宋云枝,温孚江.2008.多抗PVY、TMV和CMV转基因烟草的培育.中国农业科学,41(4):1040-1047]
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Bai YF,Guo ZH,Wang XQ,Bai DM,Zhang WF.2008.Acquisition of anti-virus non marker transgenic potato.Progress in Natural Science,18(11):1250-1255(in Chinese)[白云凤,郭志华,王小琦,白冬梅,张维锋.2008.抗双病毒无标记转基因马铃薯的获得.自然科学进展,18(11):1250-1255]
Fuchs M,McFerson JR,Tricoli DM,McMaster JR,Deng RZ,Boeshore ML,Reynolds JF,Russell PF,Quemada HD,Gonsalves D.1997.Cantaloupe line CZW-30 containing coat protein genes of Cucumber mosaic virus,Zucchini yellow mosaic virus,and Watermelon mosaic virus-2 is resistance to these three viruses in the field.Molecular Breeding,3(4):279-290
Gao L,Zhai R,Ding XN,Li K,Liao WL,Zhi HJ.2014.RNAi vector construction of Soybean mosaic virus CP gene and soybean transformation.Journal of Plant Protection,41(4):453-460(in Chinese)[高乐,翟锐,丁雪妮,李凯,廖文林,智海剑.2014.大豆花叶病毒CP基因RNAi载体的构建及大豆遗传转化.植物保护学报,41(4):453-460]
Gao YF,Zhang FF,Zhang JW,Lu YM,Wei GM,Ma TR.2014.Construction of RNA interference vector against Tobacco mosaic virus,Potato virus M and Cucumber mosaic virus and transformation in pepino(Solanum muricatum Ait.).Journal of Nuclear Agricultural Sciences,28(1):44-51(in Chinese)[高宜峰,张菲菲,张金文,陆艳梅,魏桂民,马廷蕊.2014.TMV,PVM和CMV干扰载体构建及对人参果遗传转化.核农学报,28(1):44-51]
Guo ZH,Zhang JW,Chen ZH,Wang LL.2006.The development of virus resistance transgenic potato plants using RNAi technology and laser microbeam puncture technique.Acta Laser Biology Sinica,15(5):525-531(in Chinese)[郭志鸿,张金文,陈正华,王兰岚.2006.利用RNA干涉技术及微束激光转化法培育抗病毒马铃薯.激光生物学报,15(5):525-531]
Helliwell C,Waterhouse P.2003.Constructs and methods for highthroughput gene silencing in plants.Methods,30(4):289-295
Helliwell CA,Waterhouse PM.2005.Constructs and methods for hairpin RNA-mediated gene silencing in plants.Methods in Enzymology,392:24-35
Huang P,He QC,Yan Q.2008.Re-infected virus rate and yield of different gradation virus-free seed potato.Guizhou Agricultural Sciences,36(4):39-40(in Chinese)[黄萍,何庆才,颜谦.2008.马铃薯不同级别脱毒种薯病毒再侵染情况及产量变化.贵州农业科学,36(4):39-40]
Jan FJ,Fagoaga C,Pang SZ,Gonsalves D.2000.A single chimeric transgene derived from two distinct viruses confers multi-virus resistance in transgenic plants through homology-dependent gene silencing.Journal of General Virology,81(Pt8):2103-2109
Jia XX,Qi EF,Wang YH,Wen GH,Gong CW,Wang HM,Li JW,Ma S,Hu XY.2014.Construction of bivalent plant expression vector of DREB1A and Bar genes and studies of genetic transformation of potato.Acta Prataculturae Sinica,23(3):110-117(in Chinese)[贾小霞,齐恩芳,王一航,文国宏,龚成文,王红梅,李建武,马胜,胡新元.2014.转录因子DREB1A基因和Bar基因双价植物表达载体的构建及对马铃薯遗传转化的研究.草业学报,23(3):110-117]
Jia XX,Zhang JW,Wang HN,Ma HY,Liang HG.2009.Construction of plant expression vectors containing two anti-fungal and one anti-herbicide genes and their expression in transgenic tobacco.Acta Prataculturae Sinica,18(1):86-93(in Chinese)[贾小霞,张金文,王汉宁,马怀义,梁慧光.2009.抗真菌和抗除草剂基因多价植物表达载体构建及对烟草遗传转化的研究.草业学报,18(1):86-93]
Kaniewsli W,Lawson C,Sammoms B,Haley L,Hart J,Delannay X,Tumer NE.1990.Field resistance of transgenic Russet Burbank potato to effects of infection by Potato virus X and Potato virus Y.Nature Biotechnology,8:750-754
Lindbo JA,Dougherty WG.1992.Untranslatable transcripts of the Tobacco etch virus coat protein gene sequence can interfere with Tobacco etch virus replication in transgenic plants and protoplasts.Virology,189(2):725-733
Liu Y,Cai H,Liu J,Bai X,Ji W,Zhu YM.2013.Transformation of the GsCRCK gene into Medicago sativa cv.Nongjing No.1 and salt tolerance analysis in transgenic plants.Acta Prataculturae Sinica,22(2):150-157(in Chinese)[刘莹,才华,刘晶,柏锡,纪巍,朱延明.2013.GsCRCK基因转化农菁1号苜蓿及其耐盐性分析.草业学报,22(2):150-157]
Ma XQ,Wang YG,Zhou XJ,Zhao HB,Li KP,Ma JZ.2010.Research advances of potato virus.Science and Technology of Food Industry,31(10):429-434(in Chinese)[马雪青,王永刚,周贤婧,赵虎彪,李昆鹏,马建忠.2010.马铃薯病毒研究新进展.食品工业科技,31(10):429-434]
Plasterk RH.2002.RNA silencing:the genome’s immune system.Science,296(5517):1263-1265
Prins M,de Haan PD,Luyten R,van Veller MV,van Grinsven MQV,Goldbach R.1995.Broad resistance to tospovirus in transgenic tobacco plants expressing three-tospoviral gene sequence.Molecular Plant-Microbe Interactions,8(1):85-91
Qi EF,Liu S,Jia XX,Wen GH,LüHP,Huang W,Gao YP.2018.Investigation and analysis of important potato viral diseases in Gansu Province.Plant Protection,44(4):171-176(in Chinese)[齐恩芳,刘石,贾小霞,文国宏,吕和平,黄伟,高彦萍.2018.甘肃省马铃薯主要病毒病发生情况调查.植物保护,44(4):171-176]
Si HJ,Xie CH,Liu J.2003.An efficient protocol for Agrobacteriummediated transformation with microtuber and the introduction of an antisense class I patatin gene into potato.Acta Agronomica Sinica,29(6):801-805
Smith NA,Singh SP,Wang MB,Stoutjesdijk PA,Green AG,Waterhouse PM.2000.Gene expression:total silencing by intronspliced hairpin RNAs.Nature,407(6802):319-320
Stoutjesdijk PA,Singh SP,Liu Q,Hurlstone CJ,Waterhouse PA,Green AG.2002.HpRNA-mediated targeting of the Arabidopsis FAD2gene gives highly efficient and stable silencing.Plant Physiology,129(4):1723-1731
van den Boogaart T,Wen F,Davies JW,Lomonossoff GP.2001.Replicase-derived resistance against pea early browning virus in Nicotiana benthamiana is an unstable resistance based upon posttranscriptional gene silencing.Molecular Plant-Microbe Interactions,14(2):196-203
Waterhouse PM,Graham MW,Wang MB.1998.Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA.Proceedings of the National Academy of Sciences of the United States of America,95(23):13959-13964
Wu XQ,Chen SH,Wei GB,Wu ZJ,Xie LH.2005.The molecular identification and the distribution of Potato virus S in Fujian Province.Journal of Plant Protection,32(2):133-137(in Chinese)[吴兴泉,陈士华,魏广彪,吴祖建,谢联辉.2005.福建马铃薯S病毒的分子鉴定及发生情况.植物保护学报,32(2):133-137]
Wu XQ,Wu ZJ,Xie LH,Lin QY.2002.The clone and expression of the coat protein gene of Potato virus S in E.coli.Virologica Sinica,17(3):248-251(in Chinese)[吴兴泉,吴祖建,谢联辉,林奇英.2002.马铃薯S病毒外壳蛋白基因的克隆与原核表达.中国病毒学,17(3):248-251]
Xu L,Song YZ,Wang WJ,Zhu CX,Wen FJ.2010.Different virus-resistance to Potato virus Y infection mediated by different regions of NIb gene.Journal of Plant Protection,37(3):227-233(in Chinese)[徐丽,宋云枝,王文俊,朱常香,温孚江.2010.马铃薯Y病毒复制酶基因不同位置cDNA区段介导对PVY的不同抗性.植物保护学报,37(3):227-233]
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