本文選題：大麥黃矮病毒 + 二穗短柄草��； 參考：《西北農(nóng)林科技大學(xué)》2016年博士論文

【摘要】：大麥黃矮病毒(Barley yellow dwarf viruses,BYDVs)是一類在世界范圍內(nèi)廣泛分布的重要禾谷類作物病毒,僅通過麥蚜傳播,能夠侵染多種禾本科作物,引起黃矮病,對世界范圍內(nèi)的農(nóng)業(yè)生產(chǎn)造成了嚴(yán)重?fù)p失。在我國,BYDVs主要侵染小麥(Triticum aestivum),導(dǎo)致葉片黃化、分蘗減少等癥狀,其中造成危害最主要的病原之一是BYDV-GAV。因此,為了更好的控制由BYDV-GAV引起的小麥黃矮病,我們需要對病毒和寄主之間的互作關(guān)系,以及病毒的致病機制進行更加深入的了解,這對探索新型的病毒防治方法有著重要的意義。但由于小麥基因組非常龐大,且遺傳轉(zhuǎn)化體系不太成熟,致使在小麥上開展病毒-寄主互作及病毒分子致病機制的研究非常困難。二穗短柄草(Brachypodium distachyon)是禾本科的模式植物,具有植株矮小,基因組簡單且背景清晰,生命周期短和易于培養(yǎng)等優(yōu)點。更重要的是,它和小麥的農(nóng)藝性狀相似,染色體組也具有很好的共線性。因此,本研究選用二穗短柄草作為模式寄主,探索了BYDV-GAV和寄主之間的互作關(guān)系,篩選和鑒定了與BYDV-GAV的互作的寄主因子,主要研究內(nèi)容如下:(1)建立了二穗短柄草與BYDV-GAV的互作研究體系。通過蚜蟲接種的方法,將BYDV-GAV接種到二穗短柄草Bd21-3上,接種21天后植株表現(xiàn)出葉片變紅、嚴(yán)重矮化以及根部萎縮等黃矮病癥狀,并通過RT-PCR和TAS-ELISA的方法確認(rèn)了BYDV-GAV的侵染。對健康的和感染BYDV-GAV的二穗短柄草的葉片細(xì)胞進行透射電鏡觀察,發(fā)現(xiàn)接種植株的篩管伴胞中存在病毒粒子,葉肉薄壁細(xì)胞的葉綠體結(jié)構(gòu)也被嚴(yán)重破壞。以BYDV-GAV侵染的二穗短柄草為毒源進行了蚜傳實驗,表明BYDV-GAV可以在二穗短柄草之間進行傳播。將BYDV-GAV同時接種至二穗短柄草和其自然寄主小麥上,觀察和比較了接種21天內(nèi)的癥狀擴展情況,并通過TAS-ELISA的方法檢測了期間病毒含量的變化,結(jié)果表明,與BYDV-GAV的自然寄主小麥相比,二穗短柄草發(fā)病更早,表現(xiàn)出了更加嚴(yán)重的黃矮病癥狀,但病毒的積累模式與小麥非常相似。通過以上實驗證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,侵染過程與小麥比較相似,因此可以作為研究BYDV-GAV的模式寄主。(2)篩選鑒定了與BYDV-GAV互作的寄主蛋白。首先,通過GatewayTM技術(shù)中的LR反應(yīng)將二穗短柄草的入門文庫成功轉(zhuǎn)化成用于酵母雙雜交篩選的cDNA文庫,并通過半固體法對該文庫進行了擴增,最終獲得了滴度為1.7×107 cfu/mL的cDNA文庫,可用于后續(xù)的酵母雙雜交篩選。其次,通過GatewayTM技術(shù)成功構(gòu)建了BYDV-GAV的外殼蛋白CP、假定的運動蛋白MP及基因沉默抑制子P6的誘餌載體,并對三個誘餌蛋白進行了自激活檢測,確定了文庫篩選所需的3-AT(3-氨基三唑)濃度為50mM。再次,通過酵母雙雜交的方法,用三個誘餌蛋白分別對二穗短柄草的cDNA酵母文庫進行篩選。在SC-Leu-Trp-His+50mM 3-AT平板上獲得互作子后,通過檢測HIS3、URA3、Lac Z三個報告基因的表達情況確定互作后,對互作蛋白的序列進行分析。結(jié)果表明,以CP為誘餌蛋白篩到16個與其存在弱相互作用的蛋白,包括2個假定蛋白,5個葉綠體蛋白(葉綠體磷酸果糖激酶、葉綠體轉(zhuǎn)酮醇酶、葉綠體蛋白轉(zhuǎn)運組件、葉綠體二磷酸核酮糖羧化酶/加氧酶活化酶、葉綠素a/b結(jié)合蛋白),以及動力蛋白2A、鈣調(diào)蛋白3、跨膜蛋白147等;以MP為誘餌蛋白篩選到35個強互作蛋白,序列分析后主要為四個蛋白,分別是14-3-3 like蛋白、轉(zhuǎn)錄因子VOZ1、富甘氨酸RNA結(jié)合蛋白和26S蛋白酶體的一個亞基類似蛋白;以P6為誘餌蛋白沒有篩到任何互作子。最后,將篩選到的獵物載體和誘餌載體重新轉(zhuǎn)化酵母細(xì)胞驗證,最終確定VOZ1與MP存在互作。(3)驗證了MP與VOZ1的蛋白互作。首先,通過原核表達分別獲得GST-MP和MBP-VOZ1融合蛋白進行GST pull-down實驗,確定MP和VOZ1可以在體外互作。其次,通過農(nóng)桿菌接種的方法,確定了MP和VOZ1在本氏煙中的亞細(xì)胞定位。結(jié)果表明,MP主要定位在核膜上,少量定位在細(xì)胞質(zhì)中,VOZ1以聚集的形式在細(xì)胞質(zhì)中存在。最后,通過雙分子熒光互補實驗(BiFC),結(jié)合農(nóng)桿菌接種的方法,確定MP和VOZ1在煙草細(xì)胞內(nèi)可以互作,并通過DAPI染色確定互作位置在細(xì)胞質(zhì)而非細(xì)胞核。綜上所述,本研究建立了二穗短柄草與BYDV-GAV的互作體系,在此基礎(chǔ)上,以二穗短柄草為模式寄主,篩選和鑒定了和BYDV-GAV的互作蛋白,為進一步解析BYDV-GAV致病的分子機理奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S435.12
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本文編號：1982344