【作者】 高阳；

【导师】 张德咏；

【作者基本信息】 中南大学 ， 植物病理学， 2012， 硕士

【摘要】 大部分植物病毒通过与其传播介体互作而实现水平传播。烟蚜(Myzus persicae)RPS2蛋白是一种核糖体蛋白,研究证实,RPS2在烟蚜传播马铃薯Y病毒时起了重要作用。利用GenBank查找已报道的烟蚜RPS2蛋白序列,进而得到烟蚜RPS2基因序列,根据实验目的设计引物,通过RT-PCR技术,扩增得到了烟蚜RPS2基因的cDNA全长序列。将烟蚜RPS2基因的cDNA全长序列连接到PGEM-T easy Vector上并转入大肠杆菌(E.coli)NM522菌株中扩增并保存,再以RPS2基因的cDNA全长序列为模板,分别设计用于扩增RPS2基因全长序列的前段、中段和末段三个不同片段的引物,PCR扩增得到三段RPS2基因的部分片段,分别命名为RPS2—1、RPS2-2和RPS2-3。同时为了构建载体在三个片段两端分别引入不同的酶切位点,RPS2-1片段的5’端和3’端分别引入了EcoRI和HindⅢ两个限制性内切酶位点,而RPS2-2和RPS2-3两个片段的5’端和3’端分别引入了EcoRI和BamHI两个限制性内切酶位点。通过酶切、连接等常用的分子实验方法,将三个片段分别连接到LITMUS-28i质粒上,构建成三个含RPS2基因片段的dsRNA原核表达载体。载体通过PCR,酶切以及测序等方法进行验证,结果表明构建的重组载体与预期相同。将三种重组载体分别转化到大肠杆菌(E.coli)HT115(DE3)菌株内,由于LITMUS-28i质粒上存在两个反向的T7启动子,同时大肠杆菌菌株HT115是RNA酶Ⅲ缺陷型菌株,因此经过IPTG诱导,大肠杆菌菌株HT115的细胞内可以自行表达dsRNA并可以通过CTAB法提取到。提取到片段的大小、DNase和RNaseA酶切的结果都能证实,该载体可以表达预期的dsRNA。将这三种体外表达的dsRNA加入烟蚜人工饲料中,证明食用该饲料对烟蚜生长发育并无影响。以上实验为评价这三种dsRNA对烟蚜传播PVY的影响奠定了基础。更多还原

【Abstract】 Plant virus can transform by victor through interaction. The protein RPS2of Myzus persicae is a ribosomal protein, and has been comfirmed that it is useful for Potato Virus Y transmission by Myzus persicae.Using the GenBank to find the protein sequence of RPS2that have been reported in Myzus persicae,and then get the gene sequence of RPS2in Myzus persicae.Primers were designed based on the experimental purp-oses,amplified the complete sequences of RPS2gene by RT-PCR. Conne-cted the complete sequences of RPS2to the PGEM-T easy vector and transformed into Escherichia coli (E. coli) strain named NM522in order to amplify and save it. At the same time, three different couple of primers were designed to amplify the forepart(RPS2-1),the midpiece (RPS2-2) and the end-piece(RPS2-3) of the complete sequences of RPS2by PCR using the complete sequences of RPS2as a template.In order to build the vector,we introducted EcoRl on the5’end and HindⅢ on the3’end of RPS2-1,introducted EcoRl on the5’end and BamHI on the3’end of both RPS2-2and RPS2-3.Three fragments were connected to the LITMUS28i plasmid by digestion, ligation, and other molecular experi-mental methods commonly used.Then,we got three prokaryotic expres-sion vectors which can express dsRNA.The recombinant vector plasmid was consistent with expected results by PCR analysis, restriction enzyme digestion and DNA sequencing.Transformed three recombinant vectors into Escherichia coli (E. coli) strain named HT115(DE3).There are two reverse T7promoter in LITMUS-28i plasmid, while HT115is RNA enzyme Ⅲ-deficient strains, so after IPTG induction, the cells of HT115can express dsRNA and the dsRNA can be extracted by the CTAB method.The size of fragment extracted, DNase and RNaseA restriction results can be confirmed that the three vectors can express the dsRNA as expected.It proved that the artificial diet with these three dsRNAs had no impact on growth and development of Myzus persicae. The experiments above laid the foundation for evaluateing the effect of PVY spread by Myzus persicae.更多还原