【摘要】：南方番茄病毒(Southern tomato virus,STV)是2009年報(bào)道的一種新的dsRNA病毒,但迄今未發(fā)現(xiàn)病毒顆粒。本實(shí)驗(yàn)室于2012年在新疆加工番茄里格87-5中檢測(cè)到了該病毒,并通過PCR擴(kuò)增獲得了該病毒的全基因組序列。由于該病毒只能通過種子和花粉傳播,不能通過摩擦接種、嫁接等方式傳播,已報(bào)道STV在不同國(guó)家番茄上導(dǎo)致的癥狀不同,而且田間病株受CMV、PVY、To MV等多種病毒的復(fù)合侵染,因此目前尚無法確定STV感染新疆加工番茄后的癥狀。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn),CMV、PVY是新疆番茄上最重要的2種病毒,常與STV復(fù)合侵染,危害十分普遍。目的:為了更好的了解STV的生物學(xué)特性、致病機(jī)制以及癥狀,本實(shí)驗(yàn)以與STV的功能蛋白、CMV和PVY相互作用的宿主因子為突破口,利用酵母雙雜交技術(shù)篩選與之互作的宿主因子,為后期研究病毒的致病機(jī)制以及培育抗病毒的加工番茄新品種奠定基礎(chǔ)。同時(shí)本實(shí)驗(yàn)初步探究了STV感染新疆加工番茄的癥狀表現(xiàn)。方法:1、通過PCR技術(shù)擴(kuò)增得到STV CP、RdRp基因片段,構(gòu)建酵母雙雜交的誘餌表達(dá)載體p Sos-STV CP和p Sos-STV RdRp,轉(zhuǎn)入cdc25H酵母菌株中,驗(yàn)證誘餌蛋白的自激活及毒性作用。2、利用Cyto Trap系統(tǒng)從新疆加工番茄c DNA文庫中初步篩選與誘餌蛋白STV RdRp、CMV 2b具有相互作用的宿主因子,并進(jìn)行測(cè)序比對(duì)分析。3、通過PCR技術(shù)對(duì)初步篩選出的宿主因子n進(jìn)行擴(kuò)增,重新構(gòu)建目的基因表達(dá)載體p GADT7-n,分別與相應(yīng)的誘餌基因表達(dá)載體(p GBKT7-bait)共轉(zhuǎn)入AH109酵母感受態(tài)細(xì)胞中,通過GAL4酵母雙雜交系統(tǒng)驗(yàn)證誘餌蛋白(STV RdRp、STV CP、CMV 2b、PVY HC-Pro)與目的蛋白的互作關(guān)系。4、通過生物學(xué)在線工具分析互作因子PT2的生物信息,構(gòu)建PT2亞細(xì)胞定位載體,農(nóng)桿菌遺傳轉(zhuǎn)化煙草,觀察PT2的亞細(xì)胞定位情況。5、通過一步法RT-PCR檢測(cè)市場(chǎng)購買的與實(shí)驗(yàn)室保存的加工番茄里格87-5種子的帶毒情況,并結(jié)合雜交育種實(shí)驗(yàn)觀察STV感染新疆加工番茄的癥狀表現(xiàn)。結(jié)果:1、成功構(gòu)建了誘餌表達(dá)載體pSos-STV CP和pSos-STV RdRp,自激活結(jié)果表明,STV CP具有自激活作用,STV RdRp沒有自激活作用也沒有毒性。2、利用Cyto Trap系統(tǒng)成功篩選出8個(gè)與STV RdRp具有相互作用的候選宿主因子,5個(gè)與CMV 2b具有相互作用的候選宿主因子,包括核糖體蛋白、受體類蛋白激酶、熱休克蛋白、6-磷酸山梨醇脫氫酶、鉀離子轉(zhuǎn)運(yùn)體、RNA結(jié)合蛋白、含溴結(jié)構(gòu)域的蛋白、鋅脂蛋白以及未知功能的蛋白等。3、成功構(gòu)建了相應(yīng)的目的基因表達(dá)載體(p GADT7-n)、誘餌基因表達(dá)載體(p GBKT7-bait),利用GAL4酵母雙雜交系統(tǒng)回轉(zhuǎn)驗(yàn)證,結(jié)果表明,CMV 2b可能與Cab-1A具有相互作用。4、PT2的生物信息學(xué)分析預(yù)測(cè)表明,PT2蛋白分子量約87.89 k Da,屬于穩(wěn)定性蛋白,不屬于分泌型蛋白,具有13處跨膜區(qū)域,亞細(xì)胞定位于液泡;成功構(gòu)建了35S:GFP-PT2亞細(xì)胞定位載體,轉(zhuǎn)化農(nóng)桿菌侵染煙草后獲得4個(gè)轉(zhuǎn)基因株系,熒光觀察PT2可能定位于葉綠體。5、一步法RT-PCR檢測(cè)(STV、CMV、To MV、PVY)四種病毒表明,市場(chǎng)購買的加工番茄種子中STV和CMV的帶毒率高達(dá)90.6%和96.9%。而實(shí)驗(yàn)室保存的種子帶毒率并不高,感染STV與健康的加工番茄相比,葉片、植株沒有明顯的變化,但4號(hào)品種之間的果實(shí)形狀有明顯的差異(感染STV的果實(shí)呈卵形,健康的果實(shí)呈圓球形),其他品種之間無明顯差異。結(jié)論:1、STV RdRp、CMV 2b沒有自激活作用可以適用于Cyto Trap酵母雙雜交系統(tǒng),STV CP具有自激活作用不適用于Cyto Trap系統(tǒng),PVY HC-Pro或誘餌融合蛋白對(duì)cdc25H有毒性不適用于Cyto Trap系統(tǒng);而誘餌蛋白STV CP、STV RdRp、CMV 2b和PVY HC-Pro均可適用于GAL4酵母雙雜交系統(tǒng)。2、在酵母AH109中CMV 2b可能與Cab-1A具有相互作用,為后續(xù)研究奠定了基礎(chǔ);3、成功獲得4個(gè)35S:GFP-PT2亞細(xì)胞定位載體的轉(zhuǎn)基因煙草株系,熒光觀察顯示PT2蛋白可能定位在葉綠體。4、從STV的檢測(cè)來看,STV已有加重的趨勢(shì),但目前無法確定STV感染加工番茄的癥狀表型,4號(hào)果實(shí)形狀的差異也許跟品種相關(guān),還需要進(jìn)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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S436.412

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