马铃薯卷叶病毒中国株(PLRV-Ch)复制酶基因结构研究
作者：张鹤龄 梁成罡 张彤 哈斯阿古拉 赵国芬
单位：内蒙古大学生物系，呼和浩特 010021
关键词：马铃薯卷叶病毒；依赖RNA的RNA聚合酶；基因克隆；序列分析
中国病毒学000309摘要：用设计合成的两对特异性引物，以马铃薯卷叶病毒中国分离株PLRV-Ch RNA为模板，经反转录PCR扩增，将复制酶基因3′端0.6 kb和5′端1.2 kb，克隆于pUC19中，分别构建了重组质粒pLR3和pLR5，并分五段进行了序列分析。将获得的核苷酸序列及氨基酸序列与国外报导的四个PLRV分离株的相应区段的序列同源性进行了比较。结果表明具有高度同源性。文中对核苷酸序列中存在的可能移码序列和其下游的茎环结构或假节结构、以及特征性三次重复区及氨基酸序列中复制酶蛋白N端的碱性氨基酸序列以及C端区域中包括GDD在内的8个特征序列进行了讨论。作者发现移码序列上游的三次重复的核苷酸序列可以形成连续折叠的互补双链区和发夹结构，这一结构可能和转译移码有关。此外PLRV复制酶蛋白N端部分氨基酸序列易变，而C端氨基酸序列十分保守，可能和复制酶功能有更重要关系。
, http://www.100md.com
中图分类号：S435.32 文献标识码：A
文章编号：1003-5125(2000)03-0255-09
Study on the Replicase Gene Structure of
Potato Leafroll Virus Chinese Isolate
ZHANG He-ling，LIANG Cheng-gang，ZHANG Tong，HASI Agula，ZHAO Guo-fen
(Department of Biology, Inner Mongolia University, Hohhot, 010021, China)
Abstract: According to the genomic sequence of PLRV-S, two pairs of specific primers were designed and synthesized. The cDNA of the replicase gene of PLRV Chinese isolate (PLRV-Ch) was synthesized and amplified by RT-PCR using the viral RNA as a template. The synthesized 3′ and 5′ cDNA fragments of PLRV-Ch replicase gene were inserted into pUC 19 and cloned in E.coli JM109 and sequenced respectively. The homology of nucleotide sequences between PLRV-Ch and PLRV-S (Scotland, UK), PLRV-N (Netherlands), PLRV-C (Canada), PLRV-A (Australia) are 98.8%, 97.7%, 97.8% and 96.2%, and the homology of putative amino acid sequences are 98.9%, 97.4%, 97.6% and 95.8% respectively. In 5′ region of replicase gene a slippery sequence (heptanucleotide motif) for -1 frameshift and its down stream “stem—loop” or “pseudoknot”, and upstream nucleotide sequence repeats were found. Authors suggested that the nucleotide repeat sequences characteristic for PLRV could form a tight successively folded complementary double stranded regions and hairpins. This structure possibly has something to do with -1 frameshift. The amino acid sequence of C terminus region was very conservative unlike the N terminus region in which the amino acid sequence is more diversified. The amino acid domains: motifs Ⅰ-Ⅷ in the C-terminus and the basic amino acid sequence region in the N-terminus of RNA-dependent RNA polymerase of PLRV were discussed.′ and 5′ cDNA fragments of PLRV-Ch replicase gene were inserted into pUC 19 and cloned in E.coli JM109 and sequenced respectively. The homology of nucleotide sequences between PLRV-Ch and PLRV-S (Scotland, UK), PLRV-N (Netherlands), PLRV-C (Canada), PLRV-A (Australia) are 98.8%, 97.7%, 97.8% and 96.2%, and the homology of putative amino acid sequences are 98.9%, 97.4%, 97.6% and 95.8% respectively. In 5′ region of replicase gene a slippery sequence (heptanucleotide motif) for -1 frameshift and its down stream “stem—loop” or “pseudoknot”, and upstream nucleotide sequence repeats were found. Authors suggested that the nucleotide repeat sequences characteristic for PLRV could form a tight successively folded complementary double stranded regions and hairpins. This structure possibly has something to do with -1 frameshift. The amino acid sequence of C terminus region was very conservative unlike the N terminus region in which the amino acid sequence is more diversified. The amino acid domains: motifs Ⅰ-Ⅷ in the C-terminus and the basic amino acid sequence region in the N-terminus of RNA-dependent RNA polymerase of PLRV were discussed.
, http://www.100md.com
Key words: Potato leafroll virus; RNA dependent RNA polymerase (RdRp); Gene cloning; Gene structure
基金项目：内蒙自然科学基金资助项目(9610E28)
作者简介：张鹤龄(1931年-)，男，藉贯：辽宁沈阳，植物病毒学教授，研究方向为病毒分子生物学
参考文献
[1] Murphy FA, Fauquet CM, Bishop DHL, et al (eds). Virus Taxonomy. Classification and nomenclature of viruses. Sixth Report of the International Commitee [R]. Springer Wiew New York. Archives of Virology, 1995(suppl.10)
, http://www.100md.com
[2] Mayo MA, Robinson DJ, Jolly CA et al. Nucleotide Sequence of Potato leafroll luteovirus RNA [J]. J Gen Virol, 1989,70:1037-1051
[3] Van der Wilk F, Huisman MJ, Cornelissen BJC et al. Nucleotide sequence and organization of potato leafroll virus genomic RNA [J]. FEBS Letters, 1989,245.51-56
[4] 张鹤龄.马铃薯卷叶病毒基因组研究进展[J].中国病毒学，1996，11(1)：1-8
[5] Koonin EV. The phylogeny of RNA-depenent RNA polymerases of positive-strand RNA Virnses [J]. Gen Virol, 1991,72:2197-2206
, 百拇医药
[6] Prufer D, Tacke E, Schmitz J et al. Ribozomal frameshifting in plants: a novel signal directs the -1 frameshifting in the synthesis of the putative viral replicase of potato leafroll luteoviruses [J], EMBO J, 1992,11(3):1111-1117
[7] Kujawa AB, Drugron G, Hulanicka D et al. Structural requirement for efficient translational frameshifting in the synthesis of the putative viral RNA-depedent RNA oplymerase of potato lefroll virus [J]. Nucleic Acids Res, 1993,21(9):2165-2171
, 百拇医药
[8] 哈斯阿古拉，施一，张鹤龄.The cDNA synthesis ,molecular cloning and uncleotide sequence of potato leafroll virus coat protein gene [J]. Proceedings of Asia-Pacific Conference on Agriculture Biotechnology, August 20-24, 1992,Beijing, China, 153-156
[9] 张荣信，哈斯阿古拉，张鹤龄.马铃薯卷叶病毒56 kd蛋白基因及3′端非编码区克隆和序列分析[J].病毒学报，1997，13(3)：247-254
[10] 董江丽，哈斯阿古拉，张鹤龄.马铃薯卷叶病毒基因间隔区克隆和序列分析[J].中国病毒学，1996(2)：144-148
[11] 梁成罡，哈斯阿古拉，张鹤龄.马铃薯卷叶病毒复制酶基因3′端克隆和序列分析[J].病毒学报，1997，13(3)：278-282
, 百拇医药
[12] 孟清，张鹤龄.马铃薯卷叶病毒的提纯[J].病毒学报，1987，3(2)，151-155
[13] 萨姆布鲁克J.费里奇EF，曼尼阿蒂斯T著.金冬雁，黎孟枫等译.《分子克隆—实验指南》[M].第二版，北京：科学出版社，1992
[14] Kesse P, Martin RR, Kawchuk LM， et al. Nucleotide sequence of an Australian and Canadian isolate of potato leafroll luteovirus and their relationships with two European isolates [J]. Gen Virol, 1990,71:719-724
[15] Kaniewski W, Lawson C, Loveless J, et al. Expression of Potato leafroll virus (PLRV) replicase genes in Russet Burbank potatoes provide field immunity to PLRV [C]. Scientific Program and Abstracts. American Society for Virology. 13th Annual Meeting, Madison, Wisconsin, July 9-13, 1994,P107
收稿日期：1999-04-07，修回日期：1999-10-25, 百拇医药
单位：内蒙古大学生物系，呼和浩特 010021
关键词：马铃薯卷叶病毒；依赖RNA的RNA聚合酶；基因克隆；序列分析
中国病毒学000309摘要：用设计合成的两对特异性引物，以马铃薯卷叶病毒中国分离株PLRV-Ch RNA为模板，经反转录PCR扩增，将复制酶基因3′端0.6 kb和5′端1.2 kb，克隆于pUC19中，分别构建了重组质粒pLR3和pLR5，并分五段进行了序列分析。将获得的核苷酸序列及氨基酸序列与国外报导的四个PLRV分离株的相应区段的序列同源性进行了比较。结果表明具有高度同源性。文中对核苷酸序列中存在的可能移码序列和其下游的茎环结构或假节结构、以及特征性三次重复区及氨基酸序列中复制酶蛋白N端的碱性氨基酸序列以及C端区域中包括GDD在内的8个特征序列进行了讨论。作者发现移码序列上游的三次重复的核苷酸序列可以形成连续折叠的互补双链区和发夹结构，这一结构可能和转译移码有关。此外PLRV复制酶蛋白N端部分氨基酸序列易变，而C端氨基酸序列十分保守，可能和复制酶功能有更重要关系。
, http://www.100md.com
中图分类号：S435.32 文献标识码：A
文章编号：1003-5125(2000)03-0255-09
Study on the Replicase Gene Structure of
Potato Leafroll Virus Chinese Isolate
ZHANG He-ling，LIANG Cheng-gang，ZHANG Tong，HASI Agula，ZHAO Guo-fen
(Department of Biology, Inner Mongolia University, Hohhot, 010021, China)
Abstract: According to the genomic sequence of PLRV-S, two pairs of specific primers were designed and synthesized. The cDNA of the replicase gene of PLRV Chinese isolate (PLRV-Ch) was synthesized and amplified by RT-PCR using the viral RNA as a template. The synthesized 3′ and 5′ cDNA fragments of PLRV-Ch replicase gene were inserted into pUC 19 and cloned in E.coli JM109 and sequenced respectively. The homology of nucleotide sequences between PLRV-Ch and PLRV-S (Scotland, UK), PLRV-N (Netherlands), PLRV-C (Canada), PLRV-A (Australia) are 98.8%, 97.7%, 97.8% and 96.2%, and the homology of putative amino acid sequences are 98.9%, 97.4%, 97.6% and 95.8% respectively. In 5′ region of replicase gene a slippery sequence (heptanucleotide motif) for -1 frameshift and its down stream “stem—loop” or “pseudoknot”, and upstream nucleotide sequence repeats were found. Authors suggested that the nucleotide repeat sequences characteristic for PLRV could form a tight successively folded complementary double stranded regions and hairpins. This structure possibly has something to do with -1 frameshift. The amino acid sequence of C terminus region was very conservative unlike the N terminus region in which the amino acid sequence is more diversified. The amino acid domains: motifs Ⅰ-Ⅷ in the C-terminus and the basic amino acid sequence region in the N-terminus of RNA-dependent RNA polymerase of PLRV were discussed.′ and 5′ cDNA fragments of PLRV-Ch replicase gene were inserted into pUC 19 and cloned in E.coli JM109 and sequenced respectively. The homology of nucleotide sequences between PLRV-Ch and PLRV-S (Scotland, UK), PLRV-N (Netherlands), PLRV-C (Canada), PLRV-A (Australia) are 98.8%, 97.7%, 97.8% and 96.2%, and the homology of putative amino acid sequences are 98.9%, 97.4%, 97.6% and 95.8% respectively. In 5′ region of replicase gene a slippery sequence (heptanucleotide motif) for -1 frameshift and its down stream “stem—loop” or “pseudoknot”, and upstream nucleotide sequence repeats were found. Authors suggested that the nucleotide repeat sequences characteristic for PLRV could form a tight successively folded complementary double stranded regions and hairpins. This structure possibly has something to do with -1 frameshift. The amino acid sequence of C terminus region was very conservative unlike the N terminus region in which the amino acid sequence is more diversified. The amino acid domains: motifs Ⅰ-Ⅷ in the C-terminus and the basic amino acid sequence region in the N-terminus of RNA-dependent RNA polymerase of PLRV were discussed.
, http://www.100md.com
Key words: Potato leafroll virus; RNA dependent RNA polymerase (RdRp); Gene cloning; Gene structure
基金项目：内蒙自然科学基金资助项目(9610E28)
作者简介：张鹤龄(1931年-)，男，藉贯：辽宁沈阳，植物病毒学教授，研究方向为病毒分子生物学
参考文献
[1] Murphy FA, Fauquet CM, Bishop DHL, et al (eds). Virus Taxonomy. Classification and nomenclature of viruses. Sixth Report of the International Commitee [R]. Springer Wiew New York. Archives of Virology, 1995(suppl.10)
, http://www.100md.com
[2] Mayo MA, Robinson DJ, Jolly CA et al. Nucleotide Sequence of Potato leafroll luteovirus RNA [J]. J Gen Virol, 1989,70:1037-1051
[3] Van der Wilk F, Huisman MJ, Cornelissen BJC et al. Nucleotide sequence and organization of potato leafroll virus genomic RNA [J]. FEBS Letters, 1989,245.51-56
[4] 张鹤龄.马铃薯卷叶病毒基因组研究进展[J].中国病毒学，1996，11(1)：1-8
[5] Koonin EV. The phylogeny of RNA-depenent RNA polymerases of positive-strand RNA Virnses [J]. Gen Virol, 1991,72:2197-2206
, 百拇医药
[6] Prufer D, Tacke E, Schmitz J et al. Ribozomal frameshifting in plants: a novel signal directs the -1 frameshifting in the synthesis of the putative viral replicase of potato leafroll luteoviruses [J], EMBO J, 1992,11(3):1111-1117
[7] Kujawa AB, Drugron G, Hulanicka D et al. Structural requirement for efficient translational frameshifting in the synthesis of the putative viral RNA-depedent RNA oplymerase of potato lefroll virus [J]. Nucleic Acids Res, 1993,21(9):2165-2171
, 百拇医药
[8] 哈斯阿古拉，施一，张鹤龄.The cDNA synthesis ,molecular cloning and uncleotide sequence of potato leafroll virus coat protein gene [J]. Proceedings of Asia-Pacific Conference on Agriculture Biotechnology, August 20-24, 1992,Beijing, China, 153-156
[9] 张荣信，哈斯阿古拉，张鹤龄.马铃薯卷叶病毒56 kd蛋白基因及3′端非编码区克隆和序列分析[J].病毒学报，1997，13(3)：247-254
[10] 董江丽，哈斯阿古拉，张鹤龄.马铃薯卷叶病毒基因间隔区克隆和序列分析[J].中国病毒学，1996(2)：144-148
[11] 梁成罡，哈斯阿古拉，张鹤龄.马铃薯卷叶病毒复制酶基因3′端克隆和序列分析[J].病毒学报，1997，13(3)：278-282
, 百拇医药
[12] 孟清，张鹤龄.马铃薯卷叶病毒的提纯[J].病毒学报，1987，3(2)，151-155
[13] 萨姆布鲁克J.费里奇EF，曼尼阿蒂斯T著.金冬雁，黎孟枫等译.《分子克隆—实验指南》[M].第二版，北京：科学出版社，1992
[14] Kesse P, Martin RR, Kawchuk LM， et al. Nucleotide sequence of an Australian and Canadian isolate of potato leafroll luteovirus and their relationships with two European isolates [J]. Gen Virol, 1990,71:719-724
[15] Kaniewski W, Lawson C, Loveless J, et al. Expression of Potato leafroll virus (PLRV) replicase genes in Russet Burbank potatoes provide field immunity to PLRV [C]. Scientific Program and Abstracts. American Society for Virology. 13th Annual Meeting, Madison, Wisconsin, July 9-13, 1994,P107
收稿日期：1999-04-07，修回日期：1999-10-25, 百拇医药