【作者】 徐杰；

【导师】 刘红梅；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2014， 硕士

【摘要】 Meq基因是马立克氏病病毒的主要致瘤基因,MEQ蛋白N端为碱性亮氨酸拉链结构域,C端为富含脯氨酸结构域,在马立克氏病病毒的增殖和致病中起重要作用。农杆菌瞬时侵染分析表明,MEQ能明显增强植物瞬时侵染介导的RNA沉默,而RNA沉默是植物抵抗病毒侵染的主要防御策略,对于MEQ沉默增强机制的研究,可以使我们了解RNA沉默途径及机制的更多细节,同时为利用沉默增强基因获得植物广谱病毒抗性提供依据。为了揭示MEQ增强RNA沉默的分子机制,针对亮氨酸拉链和转录激活结构域分别构建了缺失突变体和点突变体,分析沉默增强作用的关键位点和功能域；荧光定量PCR检测Meq基因对沉默途径相关基因表达的影响,探讨其介入RNA沉默途径的可能方式；利用叶盘法和花序侵染法将Meq基因分别转化烟草和拟南芥,接种病毒进行抗性分析。主要的研究结果如下：(1)Meq基因能够显著增强瞬时侵染介导的局部和系统性RNA沉默。构建Meq基因植物表达载体,转化农杆菌GV3101,以GFP转基因本生烟16c为材料,农杆菌瞬时侵染鉴定其对RNA沉默的影响,长波紫外灯下持续观察绿色荧光蛋白的表达。结果表明,侵染后第3d (3dpi), MEQ与GFP共侵染处理已无绿色荧光,而GFP单独侵染仍有较强绿色荧光,Northern blot检测表明,GFP mRNA的积累水平与观察结果一致,而MEQ与GFP共侵染区GFP siRNA的积累水平显著高于GFP基因的单独侵染。侵染15dpi, MEQ与GFP共侵染与GFP单独侵染相比,植株上部有更多新生叶片变红,GFP mRNA的积累水平更低,发生了更强的系统性RNA沉默。(2)MEQ蛋白包含399个氨基酸,1-117位为DNA结合结构域,118-399位为转录激活结构域,其中1-28位为富含Pro/Gln区,有与转录辅阻遏物CtBp互作的位点,28-78位包含2个富含碱性氨基酸残基的DNA结合区及核定位信号,79-117位为亮氨酸拉链区。突变分析表明,△1-28突变体不影响沉默增强功能,而△1-78突变体却丧失了沉默增强功能,说明28-78之间的DNA结合区对MEQ的沉默增强功能是必须的。△118-399的突变使MEQ沉默增强能力丧失,而△175-399突变和△302-399突变都未对沉默增强功能产生影响,说明MEQ的转录激活结构域中118-174是发挥RNA沉默增强功能的核心区。将MEQ亮氨酸拉链中的亮氨酸突变掉2个或3个(L92AL99A和L92AL99AL106A)时其沉默增强功能明显减弱,而仅有1个亮氨酸突变(L92A)不影响其沉默增强功能,这说明亮氨酸拉链结构对MEQ蛋白发挥作用非常关键。亚细胞定位分析表明,与GFP融合表达的MEQ主要分布于细胞核中,而沉默增强功能需要MEQ的DNA结合结构域和转录激活结构域共同起作用,因此推测,MEQ可能以转录因子的方式通过调控其它基因的表达发挥RNA沉默增强功能。(3)为了进一步了解MEQ影响沉默途径的方式,选取本生烟中20个已知的沉默途径相关基因,通过荧光定量PCR检测MEQ基因瞬时侵染对这些基因表达的影响。结果显示,在所检测的基因中,有7个表达量明显上调,AGO10和HEN1基因的表达上调最为明显,其次为MET1、NRPD2a、SGS3、AGOla和DRM3基因。其中HEN1、MET1、NRPD2a、SGS3和DRM3基因与DNA甲基化有关,其余2个基因属于AGO蛋白家族,是RNA沉默途径的重要蛋白,由此推测,Meq基因可能通过影响AGO基因的表达介入RNA沉默途径,同时对DNA的甲基化有影响,但二者之间是否存在联系还需要进一步研究验证。(4)Meq基因农杆菌介导法转化烟草和拟南芥,经卡那霉素抗性筛选PCR检测获得转基因株系若干。选取4个转基因烟草株系扩繁,分别接种黄瓜花叶病毒(CMV)、马铃薯X病毒(PVX)和马铃薯Y病毒(PVY)进行抗病性分析,ELISA检测和Northen blot结果表明,4个转基因烟草株系对CMV表现不同抗性,而对PVX.PVY无明显抗性,CMV的抗病程度与Meq基因表达水平之间未见明显相关性。转基因拟南芥的抗病性分析还在进行中。更多还原

【Abstract】 The Meq gene is the major oncogenic gene of Marek’s disease virus (MDV), which plays an important role in the multiplication and pathogenicity of MDV. The N-terminal and the C-terminal of MEQ protein are the basic leucine zipper domain and prolines-rich domain respectively. The Agrobacterium transient coinfiltration assays shows that MEQ can significantly enhance the transient coinfiltration-mediated RNA silencing which is the main defense strategy against virus infection. The molecular mechanism analysis on the silencing enhancement of MEQ would help to understand RNA silencing better, as well as provide a new way to acquire the broad-spectrum viral resistance. In order to reveal the key amino acids and functional domain of MEQ involved in RNA silencing enhancement, the deletion mutations and point mutations of Meq were constructed for functional analysis. To further explore the possible mode by which Meq gene participates in the silencing pathway, the real-time fluorescence quantitative PCR was performed to test the expression of the genes which are related to silencing pathway in the case of Meq gene expressing. In additional, the virus resistance of the Meq transgenic tobacco and Arabidopsis were analyzed through virus artificial inoculation. The main results are as follows:Meq gene could prominently enhance both local and systemic RNA silencing induced by transient coinfiltration. The Meq gene was constructed into binary plant expression vector pBI121and transformed into Agrobacterium tumefaciens strain GV3101. RNA silencing enhancement activity of Meq gene was identified in green fluorescent protein (GFP) transgenic Nicotiana benthamiana line16c using Agrobacterium-mediated transient expression system and the expression of green fluorescent protein was observed persistently under a long wave UV lamp. The results indicated that the green fluorescent couldn’t be seen in the patches coinfiltrated with35S-MEQ and35S-GFP at3days post infiltration (dpi), while slight GFP fluorescent was observed in35S-GFP alone infiltration. Northern blot analysis indicated that the accumulation of GFP mRNA levels was much higher in the35S-GFP expressing tissues than that of35S-GFP plus35S-MEQ coinfiltration. While the accumulation of GFP siRNA in the patches coinfiltrated with35S-MEQ and35S-GFP was higher than that of35S-GFP infiltration. At15dpi,35S-MEQ and35S-GFP coinfiltrated plants were observed much more red new emerging leaves than that of35S-GFP infiltrated plants under UV light. It is suggested that Meq gene also accelerated the systemic RNA silencing.The MEQ protein comprised of399amino acids. The first1-117amino acids are the DNA-binding domain and the118-399amino acids are transcriptional activation domain. Among the first117amino acids, the1-28Pro/Gln-rich region can interact with the transcriptional corepressor CtBp; the28-78region contains two positively charged amino acid-rich DNA-binding domains and the nuclear localization signal; the79-117amino acids are the leucine zipper. The mutation analysis of MEQ suggested that the A1-28mutation could enhance RNA silencing but the A1-78mutation not, suggesting the DNA-binding region between28-78amino acids were necessary to silencing enhancement. A118-399mutation lost silencing enhancement activity while A175-399and A302-399mutations had no effect on the enhancement activity, which showed that118-174were critical for enhancement function of MEQ. The alanine substitution of two or three leucines (L92AL99A and L92AL99AL106A) weakened the silencing enhancement activity of MEQ while one site substitution (L92A) did not affect the enhancement activity, which demonstrated that the leucine zipper region was the key functional domain of MEQ on RNA silencing enhancement. The sub-cellular localization analysis turned out that the MEQ-GFP fusion protein mainly distributed in the nucleus, and the enhancement activity needed the DNA-binding domain and transcriptional activation domain to work together. Therefor it is presumable that MEQ acts as a transcription factor to enhance RNA silencing by regulating the expressing of other genes.In order to further understand the mechanisms on MEQ involved in silencing pathway, twenty genes that were proved to relat to RNA silencing pathway in N. benthamiana were selected to test their expression levels in the case of Meq expressing using real-time fluorescence quantitative PCR. The results showed seven genes had a significant increase in the expression level, especially the AGO1and HEN1genes, MET1, NRPD2a, SGS3, AGO1a and DRM3genes were next. The five genes HEN1、MET1、NRPD2a、SGS and DRM3were related to DNA methylation and the remaining two genes belonged to the AGO gene family which were the important proteins participating in RNA silencing. It’s speculate that the Meq gene might involve in RNA silencing pathway by increasing the expressing of AGO genes. And whether there was a link between RNA silencing enhancement and DNA methylation need to be further studied.To analyze the virus resistance of Meq transgenic tobacco, four transgenic lines were obtanied using Agrobacterium-mediated method which exhibited kanamycin resistance and positive results in PCR tests. Cucumber mosaic virus (CMV)、Potato virus X (PVX) and Potato virus Y (PVY) were inoculated in the transgenic plants respectively, and the ELISA results and Northern blot analysis revealed that the transgenic lines presented significant resistance in CMV but no obvious resistance to PVX and PVY. Moreover, there was no significant correlation between the CMV resistance and the expression level of Meq gene. And the virus resistance analysis on transgenic Arabidopsis is going on.更多还原