大麦黄矮病毒GAV与二穗短柄草互作蛋白的筛选鉴定研究

发布时间：2018-06-05 14:43

  本文选题：大麦黄矮病毒 + 二穗短柄草　； 参考：《西北农林科技大学》2016年博士论文

【摘要】：大麦黄矮病毒(Barley yellow dwarf viruses,BYDVs)是一类在世界范围内广泛分布的重要禾谷类作物病毒,仅通过麦蚜传播,能够侵染多种禾本科作物,引起黄矮病,对世界范围内的农业生产造成了严重损失。在我国,BYDVs主要侵染小麦(Triticum aestivum),导致叶片黄化、分蘖减少等症状,其中造成危害最主要的病原之一是BYDV-GAV。因此,为了更好的控制由BYDV-GAV引起的小麦黄矮病,我们需要对病毒和寄主之间的互作关系,以及病毒的致病机制进行更加深入的了解,这对探索新型的病毒防治方法有着重要的意义。但由于小麦基因组非常庞大,且遗传转化体系不太成熟,致使在小麦上开展病毒-寄主互作及病毒分子致病机制的研究非常困难。二穗短柄草(Brachypodium distachyon)是禾本科的模式植物,具有植株矮小,基因组简单且背景清晰,生命周期短和易于培养等优点。更重要的是,它和小麦的农艺性状相似,染色体组也具有很好的共线性。因此,本研究选用二穗短柄草作为模式寄主,探索了BYDV-GAV和寄主之间的互作关系,筛选和鉴定了与BYDV-GAV的互作的寄主因子,主要研究内容如下:(1)建立了二穗短柄草与BYDV-GAV的互作研究体系。通过蚜虫接种的方法,将BYDV-GAV接种到二穗短柄草Bd21-3上,接种21天后植株表现出叶片变红、严重矮化以及根部萎缩等黄矮病症状,并通过RT-PCR和TAS-ELISA的方法确认了BYDV-GAV的侵染。对健康的和感染BYDV-GAV的二穗短柄草的叶片细胞进行透射电镜观察,发现接种植株的筛管伴胞中存在病毒粒子,叶肉薄壁细胞的叶绿体结构也被严重破坏。以BYDV-GAV侵染的二穗短柄草为毒源进行了蚜传实验,表明BYDV-GAV可以在二穗短柄草之间进行传播。将BYDV-GAV同时接种至二穗短柄草和其自然寄主小麦上,观察和比较了接种21天内的症状扩展情况,并通过TAS-ELISA的方法检测了期间病毒含量的变化,结果表明,与BYDV-GAV的自然寄主小麦相比,二穗短柄草发病更早,表现出了更加严重的黄矮病症状,但病毒的积累模式与小麦非常相似。通过以上实验证明了禾本科模式植物二穗短柄草Bd21-3可以被BYDV-GAV成功侵染,侵染过程与小麦比较相似,因此可以作为研究BYDV-GAV的模式寄主。(2)筛选鉴定了与BYDV-GAV互作的寄主蛋白。首先,通过GatewayTM技术中的LR反应将二穗短柄草的入门文库成功转化成用于酵母双杂交筛选的cDNA文库,并通过半固体法对该文库进行了扩增,最终获得了滴度为1.7×107 cfu/mL的cDNA文库,可用于后续的酵母双杂交筛选。其次,通过GatewayTM技术成功构建了BYDV-GAV的外壳蛋白CP、假定的运动蛋白MP及基因沉默抑制子P6的诱饵载体,并对三个诱饵蛋白进行了自激活检测,确定了文库筛选所需的3-AT(3-氨基三唑)浓度为50mM。再次,通过酵母双杂交的方法,用三个诱饵蛋白分别对二穗短柄草的cDNA酵母文库进行筛选。在SC-Leu-Trp-His+50mM 3-AT平板上获得互作子后,通过检测HIS3、URA3、Lac Z三个报告基因的表达情况确定互作后,对互作蛋白的序列进行分析。结果表明,以CP为诱饵蛋白筛到16个与其存在弱相互作用的蛋白,包括2个假定蛋白,5个叶绿体蛋白(叶绿体磷酸果糖激酶、叶绿体转酮醇酶、叶绿体蛋白转运组件、叶绿体二磷酸核酮糖羧化酶/加氧酶活化酶、叶绿素a/b结合蛋白),以及动力蛋白2A、钙调蛋白3、跨膜蛋白147等;以MP为诱饵蛋白筛选到35个强互作蛋白,序列分析后主要为四个蛋白,分别是14-3-3 like蛋白、转录因子VOZ1、富甘氨酸RNA结合蛋白和26S蛋白酶体的一个亚基类似蛋白;以P6为诱饵蛋白没有筛到任何互作子。最后,将筛选到的猎物载体和诱饵载体重新转化酵母细胞验证,最终确定VOZ1与MP存在互作。(3)验证了MP与VOZ1的蛋白互作。首先,通过原核表达分别获得GST-MP和MBP-VOZ1融合蛋白进行GST pull-down实验,确定MP和VOZ1可以在体外互作。其次,通过农杆菌接种的方法,确定了MP和VOZ1在本氏烟中的亚细胞定位。结果表明,MP主要定位在核膜上,少量定位在细胞质中,VOZ1以聚集的形式在细胞质中存在。最后,通过双分子荧光互补实验(BiFC),结合农杆菌接种的方法,确定MP和VOZ1在烟草细胞内可以互作,并通过DAPI染色确定互作位置在细胞质而非细胞核。综上所述,本研究建立了二穗短柄草与BYDV-GAV的互作体系,在此基础上,以二穗短柄草为模式寄主,筛选和鉴定了和BYDV-GAV的互作蛋白,为进一步解析BYDV-GAV致病的分子机理奠定了基础。
[Abstract]:Barley yellow dwarf viruses (BYDVs) is a kind of important cereal crop virus widely distributed worldwide. It can infect a variety of gramineous crops, cause yellow dwarf disease and cause severe damage to agricultural production worldwide. In China, BYDVs mainly infects wheat (Triticum aestiv). UM) causes the symptoms of leaf yellow, tiller reduction and other symptoms, and one of the most important pathogens is BYDV-GAV., so in order to better control the wheat yellow dwarf disease caused by BYDV-GAV, we need to understand the interaction between the virus and host, and the pathogenesis of the virus, which is to explore the new virus. The method of prevention and control is of great significance. But because the genome of wheat is very large and the genetic transformation system is not mature, it is very difficult to study the virus host interaction and the pathogenesis of virus molecules on wheat. Two Brachypodium distachyon is a model plant of Gramineae, with short plants and simple genome. The background is clear, the life cycle is short and easy to be cultivated. More importantly, it is similar to the agronomic traits of the wheat, and the chromosomes have good collinearity. Therefore, this study selects two spikes of short stipe as the model host, explores the interaction relationship between BYDV-GAV and host, and screens and identifies the host that interacts with BYDV-GAV. The main research contents are as follows: (1) a study system for the interaction of two spikes of short stipe and BYDV-GAV was established. By inoculation of aphids, BYDV-GAV was inoculated to Bd21-3 of two spikes of short stalks. After 21 days of inoculation, the plants showed the symptoms of yellow dwarf disease such as leaf redness, severe dwarf and root atrophy, and confirmed by RT-PCR and TAS-ELISA methods. The infection of BYDV-GAV was observed. Transmission electron microscopy was carried out on the leaf cells of two spikes of spikes infected with BYDV-GAV and healthy. It was found that there were virus particles in the cell of the sieve tube of the inoculated plants and the chloroplast structure of the mesophyll parenchyma cells were seriously damaged. The aphid transmission experiment was carried out with the two spikes of BYDV-GAV infected short stalks, indicating that BYDV-GAV It was possible to spread between two spikes of short stalks. BYDV-GAV was inoculated to two spikes of short stipe and its natural host wheat. The symptoms were observed and compared in 21 days, and the changes of virus content were detected by TAS-ELISA method. The results showed that two spikes were compared with the natural host wheat of BYDV-GAV. The early onset of the disease showed a more serious symptom of yellow dwarf disease, but the accumulation pattern of the virus was very similar to that of wheat. Through the above experiments, it was proved that the plant of two spikes of Gramineae plant Bd21-3 could be successfully infected by BYDV-GAV, and the infection process was similar to that of wheat, so it could be used as the model host for the study of BYDV-GAV. (2) screening and identification. The host protein interacted with BYDV-GAV. First, through the LR reaction in the GatewayTM technology, the entry Library of two spikes was successfully converted into a cDNA library for yeast two hybrid screening, and the library was amplified by semi solid method. Finally, a cDNA library with a titer of 1.7 x 107 cfu/mL was obtained, which could be used for subsequent yeast double heterozygosity. Secondly, the shell protein CP of BYDV-GAV was successfully constructed by GatewayTM, the decoy carrier of the hypothetical movement protein MP and the gene silencing suppressor P6, and the three bait proteins were tested by self activation, and the 3-AT (3- amino three azole) concentration needed for the library screening was once again 50mM., and the method of yeast two hybrid was used. Three bait proteins were screened for the cDNA yeast library of two spikes. After obtaining intercropping on the SC-Leu-Trp-His+50mM 3-AT plate, the sequences of the intercropping proteins were analyzed by detecting the expression of three reported genes of HIS3, URA3, Lac Z. The results showed that 16 of them were sieved with CP as bait protein and their existence. Weakly interacting proteins, including 2 hypothetical proteins, 5 chloroplast proteins (chloroplast phosphoric acid fructose kinase, chloroplast transpose enzyme, chloroplast protein transport component, chloroplast two phosphate carboxytransferase / oxygenase Activase, chlorophyll a/b binding protein), and dynamic protein 2A, calmodulin 3, transmembrane protein 147, and MP as bait eggs 35 strong interacted proteins were screened in white. The sequence analysis was mainly four proteins, which were 14-3-3 like protein, transcription factor VOZ1, a subunit of the rich glycine RNA binding protein and 26S proteasome, and P6 as bait protein did not screen any interplant. Finally, the screened prey carrier and bait carrier were reconverted into fermentation. The interaction between VOZ1 and MP was confirmed by the mother cell verification. (3) the interaction between MP and VOZ1 was verified. First, GST-MP and MBP-VOZ1 fusion protein was obtained through the prokaryotic expression for GST pull-down, and the MP and VOZ1 could be interacted in vitro. Secondly, the subcells of MP and VOZ1 were determined by the method of Agrobacterium tumefaciens. The results showed that MP was located mainly on the nuclear membrane and located in the cytoplasm a little, and VOZ1 existed in the cytoplasm in the form of aggregation. Finally, through the double molecular fluorescence complementary experiment (BiFC) and the method of Agrobacterium tumefaciens inoculation, the interaction of MP and VOZ1 in the tobacco cells was determined and the interaction position was determined by DAPI staining in the cytoplasm rather than in the cytoplasm. In summary, the intercropping system of two spikes of short stipe and BYDV-GAV was established in this study. On this basis, two spikes of short stalks were used as model hosts to screen and identify intercropping proteins of BYDV-GAV, which lay a foundation for further analysis of the molecular mechanism of BYDV-GAV.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.12
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本文编号：1982344