烟草丛顶病毒ORF3的功能分析
植株系统叶片....................................................................................................... 32 2.2.2.11 电镜检测接种GV3101(pGR107-ORF3)植株系统叶片 ...................... 32 2.2.2.12 Western blot检测检测接种GV3101(pGR107-ORF3)植株系统叶片总蛋白....................................................................................................................... 32 2.2.2.13 ELISA检测 ............................................................................................ 34 2.2.3 gfp基因的克隆 ........................................................................................... 34 2.2.3.1 gfp基因的扩增 ........................................................................................ 34 2.2.3.2 PCR扩增pGR208载体上的gfp基因（TaKaRa公司rTaq DNA聚合酶）............................................................................................................................... 34 2.2.3.3 PCR产物割胶回收gfp基因（Axygen公司割胶回收试剂盒） ......... 34 2.2.3.4 克隆载体pMD18-T与gfp基因连接（TaKaRa公司的T4连接酶） 35 2.2.3.5 大肠杆菌感受态细胞DH5α的制备（萨姆布鲁克等译，2002） ...... 35 2.2.3.6 转化及蓝白斑筛选（萨姆布鲁克等译，2002）.................................. 35 2.2.3.7 重组质粒pMD18-GFP提取（TIANGEN高纯度质粒小提试剂盒） 35 2.2.3.8 重组质粒pMD18-GFP的PCR鉴定 ..................................................... 35 2.2.3.9 重组质粒序列测定.................................................................................. 35 2.2.3.10 gfp基因序列分析 .................................................................................. 35 2.2.4 pGR107-GFP-ORF3及pGR107-GFP载体构建 ............................................. 35
2.2.4.1 pGR107-ORF3、pGR107及pMD18-GFP载体的Sal I/Cla I酶切（Takara的Sal I/Cla I酶切体系） .................................................................................... 35 2.2.4.2 分别割胶回收目的片段gfp及pGR107-ORF3、pGR107酶切后载体片段（Axygen公司割胶回收试剂盒） ................................................................. 36 2.2.4.3 pGR107-ORF3、pGR107载体与gfp基因的T4连接(TaKaRa公司) . 36 2.2.4.4 农杆菌GV3101感受态细胞制备(卢圣栋，1999) ............................... 36 2.2.4.5 冻融法转化农杆菌(卢圣栋，1999) ....................................................... 36 2.2.4.6 pGR107-GFP-ORF3及pGR107-GFP质粒提取（TIANGEN高纯度质粒小提试剂盒）....................................................................................................... 36 2.2.4.7 PCR检测pGR107-GFP-ORF3及pGR107-GFP质粒的gfp基因片段36 2.2.4.8 含重组质粒pGR107-GFP-ORF3及pGR107-GFP的农杆菌GV3101注射接种本氏烟....................................................................................................... 36 2.2.4.9 免疫荧光显微镜检测接种pGR107-GFP-ORF3、pGR107-GFP的本氏烟各部位............................................................................................................... 36 2.2.5 免疫胶体金检测烟草丛顶病毒 ....................................................................... 37 2.2.5.1 样品的低温包埋 ............................................................................................ 37
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烟草丛顶病毒ORF3的功能分析
2.2.5.2 胶体金间接标记（在28℃下进行）..................................................... 37 2.2.5.3 电镜观察免疫胶体金颗粒...................................................................... 37 3 结果与分析.............................................................................................................. 38 3.1 烟草丛顶病毒ORF3的克隆及序列分析 ....................................................... 38 3.1.1 烟草丛顶病毒ORF3的RT-PCR扩增结果 ................................................... 38 3.1.2 TBTV ORF3 RT-PCR割胶回收产物............................................................. 38 3.1.3 重组载体pMD18-ORF3的筛选及鉴定 ...................................................... 39 3.1.4 重组载体pMD18-ORF3序列测定及TBTV ORF3序列分析 ...................... 40 3.2 pGR107-ORF3载体构建及检测 ...................................................................... 40 3.2.1 克隆载体pMD18-ORF3及双元载体pGR107 Sal I/Spe I酶切结果 ............ 41 3.2.2 重组载体pGR107-ORF3的PCR鉴定 .......................................................... 42 3.2.3接种pGR107-ORF3植株系统叶片的PCR检测 ........................................... 44 3.2.4 GV3101(pGR107-ORF3)及GV3101(pGR107) 接种植株系统叶片的RT-PCR检测 .............................................................................................................. 45 3.2.5 接种pGR107-ORF3本氏烟发病症状 ............................................................ 45 3.2.6 GV3101(pGR107-ORF3) 接种植株系统叶的电镜检测 ................................ 46 3.2.7pGR107-ORF3接种本氏烟系统叶片总蛋白的Western blot检测 ................. 47 3.2.8 ELISA检测 ....................................................................................................... 50 3.3 gfp基因的克隆及测序 ...................................................................................... 51 3.3.1 PCR扩增pGR208载体上的gfp基因 ............................................................ 52 3.3.2 PCR扩增gfp基因割胶回收 ............................................................................ 52 3.3.3 重组载体pMD18-GFP序列测定及gfp序列分析 ........................................ 53 3.4 pGR107-GFP-ORF3及pGR107-GFP载体构建 ............................................. 54 3.4.1 双元载体pGR107-ORF3、pGR107与克隆载体pMD18-GFP的 Sal I/Cal I双酶切结果 ................................................................................................................. 55 3.4.2 重组载体pGR107-GFP-ORF3、pGR107-GFP的gfp基因PCR鉴定 ........ 56 3.4.3 菌株GV3101（pGR107-GFP-ORF3）、GV3101(pGR107-GFP)挤压接种本氏烟 ............................................................................................................................. 58 3.5接种GV3101(pGR107-GFP-ORF3)、GV3101（pGR107-GFP）本氏烟的荧光显微镜检测 .............................................................................................................. 58 3.6 TBTV ORF3及ORF4编码蛋白在染病烟株细胞中的免疫胶电镜检测 ...... 62 4 讨论.......................................................................................................................... 66 4.1 病毒载体pGR107-ORF3侵染本氏烟检测 .................................................... 66 4.1.1 GV3101(pGR107-ORF3)接种后的PCR及RT-PCR检测 ............................. 66
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烟草丛顶病毒ORF3的功能分析
4.1.2 接种GV3101(pGR107-ORF3)本氏烟发病症状 ............................................ 66 4.1.3 电镜检测接种GV3101(pGR107-ORF3)本氏烟系统叶片 ............................ 67 4.1.4 Western blot检测 .............................................................................................. 67 4.1.5 ELISA检测 ....................................................................................................... 68 4.2 病毒载体pGR107-GFP-ORF3侵染本氏烟荧光显微镜检测 ........................ 68 4.3 免疫胶体金检测 ............................................................................................... 68 4.3.1 免疫胶体金的ORF3检测 ............................................................................... 69 4.3.2 免疫胶体金的ORF4检测 ............................................................................... 69 5 结论.......................................................................................................................... 71 5.1 病毒载体pGR107-ORF3侵染本氏烟检测结论 ............................................ 71 5.2 病毒载体pGR107-GFP-ORF3侵染本氏烟荧光显微镜检测结论 ................ 71 5.3 烟草丛顶病发病烟株免疫胶体金检测结论 ................................................... 71 5.4 小结 ................................................................................................................... 71 参考文献...................................................................................................................... 72 附录A：常用化学试剂和分子生物学试剂 .............................................................. 80 附录B： 常用缓冲液的配制及常用仪器设备 ........................................................ 81 致 谢........................................................................................................................ 84
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烟草丛顶病毒ORF3的功能分析
摘 要
本文主要对烟草丛顶病毒（Tobacco bushy top virus，TBTV）ORF3的功能进行分析，力图探明TBTV ORF3的功能，为TBTV的分子生物学研究及烟草丛顶病的防治提供理论基础。
通过从保山龙陵的烟草丛顶病烟株中提取TBTV RNA，用特异引物RT-PCR扩增TBTV ORF3，克隆测序后，将ORF3连接到双酶切后缺失cp基因的pGR107病毒载体，获得重组病毒pGR107-ORF3。通过农杆菌介导病毒侵染法，将重组病毒pGR107-ORF3转入本氏烟中，运用生物学观察、PCR、RT-PCR、电镜镜检、western blot检测、ELISA检测等手段,综合分析TBTV ORF3的功能。结果发现，pGR107-ORF3侵染本氏烟后表现为斑驳症状，PCR、RT-PCR均可检测到716bp的ORF3片段；而电镜下观察不到pGR107-ORF3病毒粒子，western blot能检测到预期的35KDa蛋白。表明TBTV ORF3可以在接种植物中正常表达，且TBTV ORF3可替代cp基因完成病毒的系统侵染，但ORF3蛋白不具备包裹病毒核酸的功能。
为了进一步观察ORF3在接种本氏烟组织中的分布，在构建好的重组病毒pGR107-ORF3上的多克隆位点插入gfp基因，进一步得到重组病毒pGR107-GFP-ORF3。利用农杆菌介导病毒侵染法，将重组病毒pGR107-GFP-ORF3转入本氏烟，运用荧光电镜观察本氏烟接种叶、系统叶及植株其他部位，可观察到荧光主要分布在叶片、茎部的维管束组织中，从而确定pGR107-GFP-ORF3主要分布在接种本氏烟的维管束组织中，即ORF3与病毒在维管束组织的运输有关，具有帮助病毒进行长距离运输的功能。
通过免疫胶体金标记TBTV ORF3、ORF4抗血清，对TBTV在烟株细胞中进行定位分析。电镜检测显示ORF3、ORF4编码蛋白分布在普通细胞的细胞核、线粒体、叶绿体、细胞质中，而在胞间连丝处只检测到ORF4编码蛋白，在筛管细胞中只检测到ORF3编码蛋白。进一步说明ORF3编码蛋白与TBTV长距离运输相关，ORF4编码蛋白可能与TBTV通过胞间连丝进行胞间移动相关。
关键词：烟草丛顶病毒；ORF3；功能分析；病毒载体pGR107；GFP；Western blot；
荧光电镜检测；免疫胶体金检测
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烟草丛顶病毒ORF3的功能分析
Functional analysis for the ORF3 of Tobacco bushy top virus
Zuo Ruijuan(Plant Pathology) Directed by Prof. Li Fan & Prof. Chen Hairu
ABSTRACT
The function of Tobacco bushy top virus (TBTV) ORF3 was analyzed in this thesis for further studies on the molecular biology and disease control.
Total nucleic acids of TBTV were extracted from diseased tobacco plants from Longling, Baoshan city. TBTV ORF3 was then amplified by RT-PCR with the specific primers, the amplified ORF3 fragment was cloned and sequenced. The cp gene of the virus vector pGR107 was replaced by TBTV ORF3 to construct the recombinant pGR107-ORF3. The Recombinant vector pGR107-ORF3 was inoculated to Nicotiana benthamiana by Agrobactertumtumefaciens, and symptomalogy, molecular biology, electron microscopy, and serology were conducted to identify the function about the ORF3 encoded protein. N. benthamiana showed mottleafter inoculated by pGR107-ORF3. The ORF3 expected 716bp band could be detected by PCR、RT-PCR from the inoculated N. benthamiana. No distinct virus virion could be observed from pGR107-ORF3 inouculated plants by electron microscope. And a 35KDa desired ORF3 protein could be detected of by western blot. The results indicated that TBTV ORF3 could complementary fulfill some function of PVX cp gene for virus systemic infection in the plant.
The distribution of ORF3 encoded protein in infected N. benthamiana was analyzed with the help of the recombinant vector of pGR107-GFP-ORF3, which was constructed by the gfp gene from pGR208 vector with pGR107-ORF3. The Recombinant virus vector of pGR107-GFP-ORF3 was inoculated to N. benthamiana by Agroinfection. The green fluorescence could be detected from vascular bundle of leaves and stem when local leaves, system leaves and other plant parts were tested by fluorescence electron microscope. The results revealed that TBTV ORF3 product distributed in vascular system of the infected plant, and it played important role for virus long distance transportation in the plant.
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