利用酵母双杂交技术筛选与南方番茄病毒互作的功能基因

发布时间：2018-09-06 19:22

【摘要】：南方番茄病毒(Southern tomato virus,STV)是2009年报道的一种新的dsRNA病毒,但迄今未发现病毒颗粒。本实验室于2012年在新疆加工番茄里格87-5中检测到了该病毒,并通过PCR扩增获得了该病毒的全基因组序列。由于该病毒只能通过种子和花粉传播,不能通过摩擦接种、嫁接等方式传播,已报道STV在不同国家番茄上导致的症状不同,而且田间病株受CMV、PVY、To MV等多种病毒的复合侵染,因此目前尚无法确定STV感染新疆加工番茄后的症状。本实验室前期研究发现,CMV、PVY是新疆番茄上最重要的2种病毒,常与STV复合侵染,危害十分普遍。目的:为了更好的了解STV的生物学特性、致病机制以及症状,本实验以与STV的功能蛋白、CMV和PVY相互作用的宿主因子为突破口,利用酵母双杂交技术筛选与之互作的宿主因子,为后期研究病毒的致病机制以及培育抗病毒的加工番茄新品种奠定基础。同时本实验初步探究了STV感染新疆加工番茄的症状表现。方法:1、通过PCR技术扩增得到STV CP、RdRp基因片段,构建酵母双杂交的诱饵表达载体p Sos-STV CP和p Sos-STV RdRp,转入cdc25H酵母菌株中,验证诱饵蛋白的自激活及毒性作用。2、利用Cyto Trap系统从新疆加工番茄c DNA文库中初步筛选与诱饵蛋白STV RdRp、CMV 2b具有相互作用的宿主因子,并进行测序比对分析。3、通过PCR技术对初步筛选出的宿主因子n进行扩增,重新构建目的基因表达载体p GADT7-n,分别与相应的诱饵基因表达载体(p GBKT7-bait)共转入AH109酵母感受态细胞中,通过GAL4酵母双杂交系统验证诱饵蛋白(STV RdRp、STV CP、CMV 2b、PVY HC-Pro)与目的蛋白的互作关系。4、通过生物学在线工具分析互作因子PT2的生物信息,构建PT2亚细胞定位载体,农杆菌遗传转化烟草,观察PT2的亚细胞定位情况。5、通过一步法RT-PCR检测市场购买的与实验室保存的加工番茄里格87-5种子的带毒情况,并结合杂交育种实验观察STV感染新疆加工番茄的症状表现。结果:1、成功构建了诱饵表达载体pSos-STV CP和pSos-STV RdRp,自激活结果表明,STV CP具有自激活作用,STV RdRp没有自激活作用也没有毒性。2、利用Cyto Trap系统成功筛选出8个与STV RdRp具有相互作用的候选宿主因子,5个与CMV 2b具有相互作用的候选宿主因子,包括核糖体蛋白、受体类蛋白激酶、热休克蛋白、6-磷酸山梨醇脱氢酶、钾离子转运体、RNA结合蛋白、含溴结构域的蛋白、锌脂蛋白以及未知功能的蛋白等。3、成功构建了相应的目的基因表达载体(p GADT7-n)、诱饵基因表达载体(p GBKT7-bait),利用GAL4酵母双杂交系统回转验证,结果表明,CMV 2b可能与Cab-1A具有相互作用。4、PT2的生物信息学分析预测表明,PT2蛋白分子量约87.89 k Da,属于稳定性蛋白,不属于分泌型蛋白,具有13处跨膜区域,亚细胞定位于液泡;成功构建了35S:GFP-PT2亚细胞定位载体,转化农杆菌侵染烟草后获得4个转基因株系,荧光观察PT2可能定位于叶绿体。5、一步法RT-PCR检测(STV、CMV、To MV、PVY)四种病毒表明,市场购买的加工番茄种子中STV和CMV的带毒率高达90.6%和96.9%。而实验室保存的种子带毒率并不高,感染STV与健康的加工番茄相比,叶片、植株没有明显的变化,但4号品种之间的果实形状有明显的差异(感染STV的果实呈卵形,健康的果实呈圆球形),其他品种之间无明显差异。结论:1、STV RdRp、CMV 2b没有自激活作用可以适用于Cyto Trap酵母双杂交系统,STV CP具有自激活作用不适用于Cyto Trap系统,PVY HC-Pro或诱饵融合蛋白对cdc25H有毒性不适用于Cyto Trap系统;而诱饵蛋白STV CP、STV RdRp、CMV 2b和PVY HC-Pro均可适用于GAL4酵母双杂交系统。2、在酵母AH109中CMV 2b可能与Cab-1A具有相互作用,为后续研究奠定了基础;3、成功获得4个35S:GFP-PT2亚细胞定位载体的转基因烟草株系,荧光观察显示PT2蛋白可能定位在叶绿体。4、从STV的检测来看,STV已有加重的趋势,但目前无法确定STV感染加工番茄的症状表型,4号果实形状的差异也许跟品种相关,还需要进一步的研究。
[Abstract]:Southern tomato virus (STV) is a new type of dsRNA virus reported in 2009, but no virus particles have been found so far. The virus was detected in the processing tomato Rig 87-5 in Xinjiang in 2012, and the whole genome sequence of the virus was obtained by PCR amplification. It has been reported that the symptoms of STV in tomatoes from different countries are different, and the field infected by CMV, PVY, to MV and other viruses, so it is not possible to determine the symptoms of STV infection in processing tomatoes in Xinjiang. Objective: In order to better understand the biological characteristics, pathogenesis and symptoms of STV, the host factors interacting with STV, CMV and PVY were screened by yeast two-hybrid technique for the later research. Methods: 1. STV CP and RdRp gene fragments were amplified by PCR, and the bait expression vectors P Sos-STV CP and P Sos-STV RdRp were constructed by yeast two-hybrid, and then transfected into cdc25H. In yeast strains, the self-activation and toxicity of bait proteins were verified. 2. The host factors interacting with the bait proteins STV RdRp and CMV 2B were preliminarily screened from Xinjiang processing tomato C DNA library by Cyto Trap system, and sequenced and analyzed. 3. The host factor N was amplified and reconstructed by PCR technology. To construct the target gene expression vector p GADT7-n and the corresponding bait gene expression vector (p GBKT7-bait) into AH109 yeast receptive cells. The interaction between the bait protein (STV RdRp, STV CP, CMV 2b, PVY HC-Pro) and the target protein was verified by GAL4 yeast two-hybrid system. 4. Biological online tools were used to analyze the interaction factor PT2. Bioinformatics, construction of PT2 subcellular localization vector, Agrobacterium tumefaciens genetic transformation of tobacco, observation of PT2 subcellular localization. 5, through one-step RT-PCR to detect the market purchase and laboratory preservation of processed tomato Rig 87-5 seed virulence, and combined with cross breeding experiment to observe the symptoms of STV infection in Xinjiang processing tomato. Results: 1. The decoy expression vectors pSos-STV CP and pSos-STV RdRp were successfully constructed. The results of self-activation showed that STV CP had self-activation and STV RdRp had neither self-activation nor toxicity. 2. Eight candidate host factors interacting with STV RdRp and five candidate hosts interacting with CMV 2B were successfully screened by Cyto Trap system. Major factors, including ribosomal protein, receptor protein kinase, heat shock protein, 6-phosphate sorbitol dehydrogenase, potassium ion transporter, RNA-binding protein, protein containing bromine domain, zinc lipoprotein and unknown function protein, were successfully constructed corresponding target gene expression vector (p GADT7-n), bait gene expression vector (p GBKT7-bait). The results showed that CMV 2B might interact with Cab-1A. 4. The bioinformatics analysis of PT2 showed that the molecular weight of PT2 protein was about 87.89 K Da, which belonged to stable protein, not secretory protein. It had 13 transmembrane regions and subcellular localization in vacuoles. Four transgenic strains were obtained by transforming Agrobacterium tumefaciens to infect tobacco. PT2 could be localized in chloroplast. Four viruses were detected by one-step RT-PCR (STV, CMV, To MV, PVY). The virulence rates of STV and CMV in processed tomato seeds purchased from the market were as high as 90.6% and 96.9%. Compared with healthy processed tomatoes, STV infection did not change significantly in leaves, but there was a significant difference in fruit shape between varieties 4 (the infected fruit was oval, the healthy fruit was spherical), and there was no significant difference between other varieties. In hybridization system, STV CP is not suitable for Cyto Trap system, PVY HC-Pro or bait fusion protein is not suitable for Cyto Trap system, while bait protein STV CP, STV RdRp, CMV 2B and PVY HC-Pro are all suitable for GAL4 yeast two-hybrid system.2. In yeast AH109, CMV 2B may interact with Cab-1A, which is called Cab-1A. 3. Four transgenic tobacco lines with 35S:GFP-PT2 subcellular localization vectors were successfully obtained. Fluorescence observation showed that PT2 protein might be localized in chloroplast. 4. From the detection of STV, STV had an aggravating trend, but it was not possible to determine the symptoms and phenotypes of processing tomatoes infected with STV at present. Differences in fruit shape of No. 4 might follow the product. Species correlation, further research is needed.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S436.412

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