报道了三种阳电滤膜吸附洗脱法浓缩水中病毒的效果。人工加毒水样分别用Zeta Plus 30s、50s、 Zetapore IMDS滤膜吸附后,用pH9.5、3％牛肉膏洗脱病毒,回收率分别为51±％、62±％、67％,此法有效、简便、经济。与目前国外报道...报道了三种阳电滤膜吸附洗脱法浓缩水中病毒的效果。人工加毒水样分别用Zeta Plus 30s、50s、 Zetapore IMDS滤膜吸附后,用pH9.5、3％牛肉膏洗脱病毒,回收率分别为51±％、62±％、67％,此法有效、简便、经济。与目前国外报道回收效果基本一致。展开更多
Large amount of disease-specific protein(SP) accumulated in the rice plant cells infected by rice grassy stunt virus(RGSV). It was deduced that the protein was encoded by NS6 gene on genomic vRNA6 and thus referred to...Large amount of disease-specific protein(SP) accumulated in the rice plant cells infected by rice grassy stunt virus(RGSV). It was deduced that the protein was encoded by NS6 gene on genomic vRNA6 and thus referred to as NS6 protein.But its function is unknown. In an effort to prove the above deduction and to elucidate the function of NS6 protein of RGSV, we constructed a bacterial expression plasmid pGTNS6 producing a fusion protein of glutathione S-transferase (GST) and NS6 protein, and a plant expression vector pCBTNSv6 containing NS6 gene. A recombinant plasmid pTNSv 6 containing the coding region of NS6 gene and the non-coding region at its 5’ terminus, cloned by RT-PCR from purified RNAs of Shaxian isolate of RGSV, was used as the start point. Western blot analysis showed that the fusion protein reacted strongly with antisera raised against RGSV-SP, which served as evidence of the deduction.EHA105 of Agrobacterium tumefasciens containing pCBTNSv6 has been obtained and the transformation of rice is underway.展开更多
Based on the known RNA sequence of rice grassy stunt virus , IRRI isolate (RGSV IR), the cDNA of nucleocapsid protein (NCP) gene was obtained by RT PCR, with genomic RNAs of Shaxian isolate (RGSV SX) as template. The ...Based on the known RNA sequence of rice grassy stunt virus , IRRI isolate (RGSV IR), the cDNA of nucleocapsid protein (NCP) gene was obtained by RT PCR, with genomic RNAs of Shaxian isolate (RGSV SX) as template. The cDNA was then cloned into pGEM T vector and sequenced. The results showed that the nucleotide sequence between the two isolates was 99.4% identical. A bacterial expression plasmid pGTNCP which produced a fusion protein with molecular weight of about 62?kD was constructed using the cDNA clone and vector pGEX 2T. Western blot analysis showed that the fusion protein reacted strongly with antibodies raised against RGSV particles.展开更多
【目的】对水稻草状矮化病毒(Rice grassy stunt virus,RGSV)侵染寄主水稻后其叶片的差异表达蛋白进行筛选、鉴定和生物信息学分析,为进一步研究RGSV和寄主水稻互作的分子机制提供线索。【方法】采用双向荧光差异凝胶电泳(Two-dimension...【目的】对水稻草状矮化病毒(Rice grassy stunt virus,RGSV)侵染寄主水稻后其叶片的差异表达蛋白进行筛选、鉴定和生物信息学分析,为进一步研究RGSV和寄主水稻互作的分子机制提供线索。【方法】采用双向荧光差异凝胶电泳(Two-dimensional fluorescence difference in gel electrophoresis,2D-DIGE)和Image Master 2D platinum 7.0分析软件寻找差异表达蛋白,MALDI-TOF-MS(Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry)鉴定差异蛋白,利用BioTools软件搜索NCBI数据库,寻找匹配的相关蛋白质和功能查询。采用GOminer软件对差异蛋白进行GO(Gene ontology)聚类分析,KEGG(Kyoto encyclopedia of genes and genomes)数据库分析差异蛋白所参与的生物通路。【结果】建立了RGSV侵染与未侵染水稻的叶片双向荧光差异凝胶电泳图谱,RGSV病株与健株相比,差异倍数大于1.5的差异蛋白质点有173个,其中表达丰度升高的点有72个,表达丰度下降的点有101个,质谱鉴定了44个差异蛋白质点,25个获得成功鉴定,分属于24种蛋白质,包括RGSV的2种蛋白20.6K非结构蛋白和P5蛋白,水稻中的蛋白推定的核酮糖-1,5-二磷酸羧化酶/加氧酶大亚基、景天庚酮糖-1,7-二磷酸酶前体、推定的酪氨酸磷酸酶、HAD超家族水解酶-亚族IA蛋白变体3蛋白、水稻核酮糖-1,5-二磷酸羧化酶/加氧酶小亚基结合2-羧基阿拉伯糖醇-1,5-二磷酸复合体、类似C1结构域蛋白以及其他一些功能未知蛋白和假定蛋白。对已鉴定蛋白的生物信息分析显示,差异蛋白涉及氮素固定、氮循环代谢过程、含氮化合物代谢过程、单一生物体代谢过程、代谢过程和生物过程等6个生物学过程,在生物功能上分属7类,包括分子功能、酸胺连接酶活性、酰胺连接酶活性、催化活性、谷氨酸氨连接酶、连接酶活性和形成C-N键的连接酶活性,在细胞组件上,差异蛋白分布于不同的细胞位置,涉及细胞组分、细胞器、细胞、膜结合细胞器、囊(泡)、细胞部分、膜结合囊(泡)、胞内、胞内部分、胞内细胞器、细胞质、胞内膜结合细胞器、细胞质部分、质体、细胞质囊(泡)和细胞质膜结合囊(泡)。KEGG通路分析显示,差异蛋白参与了代谢途径、光合生物固碳反应、碳代谢、乙醛酸和二羧酸代谢、丙氨酸、天门冬氨酸和谷氨酸代谢、氨基酸生物合成、丙酮酸盐代谢、精氨酸和脯氨酸代谢、谷胱甘肽代谢和氮代谢途径等10个KEGG代谢通路。【结论】RGSV侵染诱导了水稻差异蛋白质表达,获得了一些与RGSV侵染水稻相关的蛋白质分子,差异蛋白涉及到多个功能类别。其中与氮有关的生物学过程和光合作用过程变化较为明显。展开更多
文摘Large amount of disease-specific protein(SP) accumulated in the rice plant cells infected by rice grassy stunt virus(RGSV). It was deduced that the protein was encoded by NS6 gene on genomic vRNA6 and thus referred to as NS6 protein.But its function is unknown. In an effort to prove the above deduction and to elucidate the function of NS6 protein of RGSV, we constructed a bacterial expression plasmid pGTNS6 producing a fusion protein of glutathione S-transferase (GST) and NS6 protein, and a plant expression vector pCBTNSv6 containing NS6 gene. A recombinant plasmid pTNSv 6 containing the coding region of NS6 gene and the non-coding region at its 5’ terminus, cloned by RT-PCR from purified RNAs of Shaxian isolate of RGSV, was used as the start point. Western blot analysis showed that the fusion protein reacted strongly with antisera raised against RGSV-SP, which served as evidence of the deduction.EHA105 of Agrobacterium tumefasciens containing pCBTNSv6 has been obtained and the transformation of rice is underway.
文摘Based on the known RNA sequence of rice grassy stunt virus , IRRI isolate (RGSV IR), the cDNA of nucleocapsid protein (NCP) gene was obtained by RT PCR, with genomic RNAs of Shaxian isolate (RGSV SX) as template. The cDNA was then cloned into pGEM T vector and sequenced. The results showed that the nucleotide sequence between the two isolates was 99.4% identical. A bacterial expression plasmid pGTNCP which produced a fusion protein with molecular weight of about 62?kD was constructed using the cDNA clone and vector pGEX 2T. Western blot analysis showed that the fusion protein reacted strongly with antibodies raised against RGSV particles.
文摘【目的】对水稻草状矮化病毒(Rice grassy stunt virus,RGSV)侵染寄主水稻后其叶片的差异表达蛋白进行筛选、鉴定和生物信息学分析,为进一步研究RGSV和寄主水稻互作的分子机制提供线索。【方法】采用双向荧光差异凝胶电泳(Two-dimensional fluorescence difference in gel electrophoresis,2D-DIGE)和Image Master 2D platinum 7.0分析软件寻找差异表达蛋白,MALDI-TOF-MS(Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry)鉴定差异蛋白,利用BioTools软件搜索NCBI数据库,寻找匹配的相关蛋白质和功能查询。采用GOminer软件对差异蛋白进行GO(Gene ontology)聚类分析,KEGG(Kyoto encyclopedia of genes and genomes)数据库分析差异蛋白所参与的生物通路。【结果】建立了RGSV侵染与未侵染水稻的叶片双向荧光差异凝胶电泳图谱,RGSV病株与健株相比,差异倍数大于1.5的差异蛋白质点有173个,其中表达丰度升高的点有72个,表达丰度下降的点有101个,质谱鉴定了44个差异蛋白质点,25个获得成功鉴定,分属于24种蛋白质,包括RGSV的2种蛋白20.6K非结构蛋白和P5蛋白,水稻中的蛋白推定的核酮糖-1,5-二磷酸羧化酶/加氧酶大亚基、景天庚酮糖-1,7-二磷酸酶前体、推定的酪氨酸磷酸酶、HAD超家族水解酶-亚族IA蛋白变体3蛋白、水稻核酮糖-1,5-二磷酸羧化酶/加氧酶小亚基结合2-羧基阿拉伯糖醇-1,5-二磷酸复合体、类似C1结构域蛋白以及其他一些功能未知蛋白和假定蛋白。对已鉴定蛋白的生物信息分析显示,差异蛋白涉及氮素固定、氮循环代谢过程、含氮化合物代谢过程、单一生物体代谢过程、代谢过程和生物过程等6个生物学过程,在生物功能上分属7类,包括分子功能、酸胺连接酶活性、酰胺连接酶活性、催化活性、谷氨酸氨连接酶、连接酶活性和形成C-N键的连接酶活性,在细胞组件上,差异蛋白分布于不同的细胞位置,涉及细胞组分、细胞器、细胞、膜结合细胞器、囊(泡)、细胞部分、膜结合囊(泡)、胞内、胞内部分、胞内细胞器、细胞质、胞内膜结合细胞器、细胞质部分、质体、细胞质囊(泡)和细胞质膜结合囊(泡)。KEGG通路分析显示,差异蛋白参与了代谢途径、光合生物固碳反应、碳代谢、乙醛酸和二羧酸代谢、丙氨酸、天门冬氨酸和谷氨酸代谢、氨基酸生物合成、丙酮酸盐代谢、精氨酸和脯氨酸代谢、谷胱甘肽代谢和氮代谢途径等10个KEGG代谢通路。【结论】RGSV侵染诱导了水稻差异蛋白质表达,获得了一些与RGSV侵染水稻相关的蛋白质分子,差异蛋白涉及到多个功能类别。其中与氮有关的生物学过程和光合作用过程变化较为明显。
