番茄斑萎病毒（TSWV）dsRNA介导的分子免疫调控作用研究
摘要
番茄斑萎病毒(TSWV)是布尼亚病毒科(Bunuyaviridae)唯一侵染植物的番茄斑萎病毒属(Tospovirus)的病毒之一。分布于世界各地,对许多作物及植物造成巨大经济损失,加之难以防治。所以本研究主要从dsRNA介导的分子免疫调控方面来研究防治该病毒的方法。
根据已报道的TSWV基因序列,设计通用引物,通过RT-PCR扩增了TSWV N基因和GN/GC基因的全长序列,并分别克隆到pGEM-T载体上,经PCR筛选和酶切鉴定与预期结果一致,对TSWVN基因和GN/GC基因进行测序,经过与GeneBank所登录的其他株系比对后,结果表明,N基因核苷酸序列与来自South Korea的三个株系的N基因核苷酸序列的一致率最高,达99%；GN／GC基因核苷酸序列与来自Spain的两个株系的GN/GC基因核苷酸序列一致率也高达99%。这说明：本研究中的材料在侵染寄主植物时发生了基因组重组的现象。
根据克隆得到的TSWV N基因的序列,设计新的引物,引入需要的限制性内切酶酶切位点(AscI, SwaI, BamHI, SpeI),分别扩增313bp和536bp的片段。用于构建反向重复片段的植物表达载体。扩增N基因反向片段的引物时,在5’端引入限制性酶切位点SpeI,在3’端引入限制性酶切位点BamHI,扩增N基因正向片段时,在5’端引入限制性酶切位点AscI,在3’端引入限制性酶切位点SwaI,以中间载体pFGC1008自身的一段序列为内含子,构建反向重复dsRNA的载体,以pCAMBIA1304为构建反向重复dsRNA的植物表达载体,通过PCR、酶切、测序等方法验证,均表明构建的重组载体与预期相同。
利用农杆菌介导法将构建好的反向重复dsRNA植物表达载体转化烟草,转化结果有待进一步分析鉴定。
Tomato spotted wilt virus (TSWV) is the type member of the genus tospovirus, the only genus in the family Bunuyaviridae with plant-infecting members. TSWV spreads wide in the world and and caused great economic losses on many crops and plants. what's more, it's difficult to control. Therefore, the author studied TSWV on dsRNA-mediated molecular immunology research to prevent this virus.
According to the reported TSWV gene sequence, designed universal primers, amplified the complete sequences of TSWV N gene and GN / GC gene by RT-PCR, cloned into pGEM-T vector. After PCR analysis and restriction enzyme digestion and gel analysis, the results is consistent with expected results. The author got the complete sequences of TSWV N gene and GN/GC gene by DNA sequencing.Compared with other isolates in the GeneBank, TSWV N gene in this study has higher similarity with three isolates from South Korea: up to 99%. And TSWV GN / GC gene has higher similarity with two isolates from Spain: also up to 99%. This result indicated that the materials in this study had occuring genome rearrangement when they infecting the host plants.
According to the cloned TSWV N gene sequences, introducting restriction enzyme sites (AscI, SwaI, BamHI, SpeI) to design new primers to amplify 313bp and 536bp fragments in order to construct inverted-repeat dsRNA expression vectors.When the author amplified the N gene reverse fragment, introducted Spel on the 5'end and BamHI on the 3'end, When amplified the N gene forward fragment, introducted Ascl on the 5'end and Sawl on the 3'end. The inverted-repeat dsRNA vector---pFGC1008 was using hundreds of its own sequence as the intron, and the inverted-repeat dsRNA expression vector was using pCAMBIA1304 as plant expression vector. The recombinant inverted-repeat dsRNA vector plasmid is consistent with expected results by PCR analysis、restriction enzyme digestion and DNA sequencmg.
The author transformated inverted-repeat dsRNA plant expression vector by agrobacterium-mediated transformation of tobacco, the transforming results need to be further analyzed and identified.
根据已报道的TSWV基因序列,设计通用引物,通过RT-PCR扩增了TSWV N基因和GN/GC基因的全长序列,并分别克隆到pGEM-T载体上,经PCR筛选和酶切鉴定与预期结果一致,对TSWVN基因和GN/GC基因进行测序,经过与GeneBank所登录的其他株系比对后,结果表明,N基因核苷酸序列与来自South Korea的三个株系的N基因核苷酸序列的一致率最高,达99%；GN／GC基因核苷酸序列与来自Spain的两个株系的GN/GC基因核苷酸序列一致率也高达99%。这说明：本研究中的材料在侵染寄主植物时发生了基因组重组的现象。
根据克隆得到的TSWV N基因的序列,设计新的引物,引入需要的限制性内切酶酶切位点(AscI, SwaI, BamHI, SpeI),分别扩增313bp和536bp的片段。用于构建反向重复片段的植物表达载体。扩增N基因反向片段的引物时,在5’端引入限制性酶切位点SpeI,在3’端引入限制性酶切位点BamHI,扩增N基因正向片段时,在5’端引入限制性酶切位点AscI,在3’端引入限制性酶切位点SwaI,以中间载体pFGC1008自身的一段序列为内含子,构建反向重复dsRNA的载体,以pCAMBIA1304为构建反向重复dsRNA的植物表达载体,通过PCR、酶切、测序等方法验证,均表明构建的重组载体与预期相同。
利用农杆菌介导法将构建好的反向重复dsRNA植物表达载体转化烟草,转化结果有待进一步分析鉴定。
Tomato spotted wilt virus (TSWV) is the type member of the genus tospovirus, the only genus in the family Bunuyaviridae with plant-infecting members. TSWV spreads wide in the world and and caused great economic losses on many crops and plants. what's more, it's difficult to control. Therefore, the author studied TSWV on dsRNA-mediated molecular immunology research to prevent this virus.
According to the reported TSWV gene sequence, designed universal primers, amplified the complete sequences of TSWV N gene and GN / GC gene by RT-PCR, cloned into pGEM-T vector. After PCR analysis and restriction enzyme digestion and gel analysis, the results is consistent with expected results. The author got the complete sequences of TSWV N gene and GN/GC gene by DNA sequencing.Compared with other isolates in the GeneBank, TSWV N gene in this study has higher similarity with three isolates from South Korea: up to 99%. And TSWV GN / GC gene has higher similarity with two isolates from Spain: also up to 99%. This result indicated that the materials in this study had occuring genome rearrangement when they infecting the host plants.
According to the cloned TSWV N gene sequences, introducting restriction enzyme sites (AscI, SwaI, BamHI, SpeI) to design new primers to amplify 313bp and 536bp fragments in order to construct inverted-repeat dsRNA expression vectors.When the author amplified the N gene reverse fragment, introducted Spel on the 5'end and BamHI on the 3'end, When amplified the N gene forward fragment, introducted Ascl on the 5'end and Sawl on the 3'end. The inverted-repeat dsRNA vector---pFGC1008 was using hundreds of its own sequence as the intron, and the inverted-repeat dsRNA expression vector was using pCAMBIA1304 as plant expression vector. The recombinant inverted-repeat dsRNA vector plasmid is consistent with expected results by PCR analysis、restriction enzyme digestion and DNA sequencmg.
The author transformated inverted-repeat dsRNA plant expression vector by agrobacterium-mediated transformation of tobacco, the transforming results need to be further analyzed and identified.
引文
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