【作者】 翟浩；

【导师】 刘艳；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2011， 硕士

【摘要】 由大麦黄矮病毒(Barley yellow dwarf viruses,BYDVs)引起的小麦黄矮病在世界范围内广泛发生,在我国小麦产区间隙性大发生,威胁小麦的生产。为最终达到防治该病害的目的,各国的科学家们对大麦黄矮病毒的基因表达、进化、传播介体与寄主的相互作用等方面开展了深入的基础研究。筛选鉴定出BYDVs基因组中可能存在的沉默抑制子,可为研究BYDV基因与寄主的互作提供新的证据。本实验室已经初步鉴定出BYDV-GAV的ORF6和BYDV-GPV的P0在局部侵染中具有沉默抑制子活性。在已构建好的质粒载体中筛选出pGD-GPV-P0、pGD-GPV-CP、pGD-GPV-MP、pBin-GAV-CP、pBin-GAV-ORF1和pBin-GAV-ORF6共6个蛋白基因的重组载体,为增加实验的严谨性,本实验新构建了pGD-GPV-UTR-P0重组载体;其次实验通过重复沉默表型观察证明了GPV的P0和GAV的ORF6在局部侵染中确实具有抑制子活性。随后采用半定量PCR对浸润区GFP的mRNA水平进行了检测,发现强抑制子蛋白HC-Pro与pBin-mGFP5-ER共同浸润本生烟后,浸润区GFP的mRNA水平明显比pBin-GAV-ORF6或pGD-GPV-P0与pBin-mGFP5-ER共浸润叶片后GFP mRNA的积累水平要高,其中接种pGD-GPV-P0与pBin-mGFP5- ER共浸润叶片后GFP mRNA的积累水平比pBin-GAV-ORF6与pBin-mGFP5- ER的积累水平高。而作为RNA定量标准的内参基因EF-1-α的mRNA水平在不同浸润组合的植株中都是相同的。结果表明GAV-ORF6抑制子的活性比GPV-P0弱。GFP蛋白的Western blot和GFP mRNA、siRNA的Northern blot分析结果进一步证实了GPV-P0与GAV-ORF6表现出的抑制子活性的差异。虽然GPV-P0、GAV-ORF6的表达与阳性对照HC-Pro一样,能够增加GFP蛋白的表达量和GFP mRNA的累积量,进而增强GFP荧光,但从实验可以看出GPV-P0这种抑制子的GFP蛋白的表达量以及GFP mRNA的累积量明显高于GAV-ORF6。表明GPV-P0是抑制子而GAV-ORF6是弱抑制子。另外,分别将pGD-GPV-P0、pBin-GAV-ORF6与含GFP的植物表达载体pBin-mGFP5- ER对四叶期转基因烟草16c植株共浸润,并以HC-Pro作为对照。在侵染16天后对非浸润叶片进行了Northern blot检测,也证明GPV的P0、GAV的ORF6与PVY的HC-Pro一样能抑制由GFP正单链RNA引起的系统性基因沉默。此外,为研究BYDV的基因组结构中基因突变(插入、缺失、重复等)对病毒复制和表达策略的影响、验证病毒的蛋白功能以及确定重要功能结构域,建立BYDVs侵染性cDNA克隆技术平台尤为重要。本文以PAV-06KM14分离物为对象,成功构建了pTCK303-06KM14 (ubi启动子控制下)和pGD-06KM14全长基因组侵染性克隆载体,分别进行基因枪转染小麦幼胚或农杆菌侵染本生烟以获得具有侵染性的植株。目前实验已获得似BYDV症状的植株,侵染活性验证实验正在进行当中。更多还原

【Abstract】 Wheat yellow dwarf disease caused by barley yellow dwarf viruses (BYDVs) takes place worldwide, and occurs intermittently in china’s main wheat-growing area, threatening wheat production. To achieve the final purpose of preventing and curing this disease, scientists have carried out in-depth researches on gene expression, evolution, and the interaction between transmission vector and host in barley yellow dwarf virus. Screening out silencing suppressors possibly present in BYDV genome can provide new evidence for the study of interaction between BYDV genes and the host.Through preliminary detection, ORF6 in BYDV-GAV and P0 in BYDV-GPV have been identified that they have silencing suppressor activity. On this basis, experimental materials are optimized firstly, and six recombinant vectors P0, CP, MP of GPV with pGD as vector and CP, ORF1, ORF6 of GAV with pBinPLUS as vector are screened out and a new vector pGD-UTR-P0 of GPV is reconstructed to make the experiment more precise and accurate. Besides, through repeating silence phenotype observation, GPV-P0 and GAV-ORF6 is proved to possess silencing suppressor activity in local infection. Then semi-quantitative PCR is used to detect mRNA level of GFP in infiltrating area, and it is found that GFP mRNA accumulation level when strong suppressor protein HC-Pro and pBin-mGFP5-ER are altogether infiltration Nicotiana benthamiana is higher than pBin-GAV-ORF6 or pGD-GPV-P0 and pBin-mGFP5-ER are altogether infiltrated blades, and GFP mRNA accumulation level infiltrated with pGD-GPV-P0 and pBin-mGFP5-ER is higher than that infiltrated with pBin-GAV-ORF6 and pBin-mGFP5- ER. mRNA level of reference genes EF-1-α, quantitative criteria of RNA, is the same in different infiltrating combination of plants. The result indicates that the activity of suppressor GAV-ORF6 is weaker than GPV-P0. The analysis results from Western blot of GFP protein and Northern blot of GFP mRNA and siRNA further confirm the differences in the suppressor activity expressed by GPV-P0 and GAV-ORF6. Though the expression of GPV- P0 and GAV-ORF6, the same as the positive control, can increase the expression and accumulation of GFP protein, the GFP mRNA accumulation, and further strengthens GFP fluorescence. It can also be seen from the experiment that the expression, accumulation and GFP mRNA accumulation of GFP protein of the suppressor GPV-P0 is obviously higher than that of GAV-ORF6. In addition, pGD-GPV-P0, pBin-GAV-ORF6 and the plant expression vector containing GFP (pBin-mGFP5- ER) are respectively used to infiltrate tobacco 16c plant at four-leaf stage with HC-Pro as a control sample. 16 days after the infiltration, Northern blot test to the non-infection leaves proves that GPV-P0, GAV-ORF6, the same as PVY HC-Pro, can restrain the systemic gene silencing caused by single-strand RNA of GFP.In addition, to study the effect of gene mutation (insertion, loss, repetition) of BYDV genome structure on virus replication and its expression strategy, verify the protein function of virus, and confirm the important function domains, it is particularly important to establish BYDVs infectious cDNA clone technology platform. This paper takes isolate PAV-06KM14 as the subject, successfully constructs full-length genome infectious clone vectors pTCK303-06KM14(under control of ubi promoter) and pGD-06KM14, and respectively transfects wheat embryo with Particle gun or infect Nicotiana benthamiana with agrobacterium infiltration to obtain the infective plants. Recently plants with similar BYDV symptoms have been received, and the verification test of the infective activity is underway.更多还原