【作者】 肖冬来；

【导师】 谢联辉；

【作者基本信息】 福建农林大学 ， 植物病理学， 2010， 博士

【摘要】 水稻条纹病毒(Rice stripe virus,RSV)是纤细病毒属的代表种,由RSV引起的水稻条纹叶枯病近年来在我国多次爆发流行,给我国的水稻生产造成了严重的经济损失。经过国内外学者多年来的研究,RSV编码蛋白的功能、致病性分化、遗传多样性、病毒与寄主间的互作以及病害的防治都有了一定的认识。为了控制病害的发生,促进我国水稻生产的可持续发展,需要深入地了解病毒与水稻、介体昆虫三者之间的互作关系。本文利用酵母双杂交技术研究了RSV NS2、NS3蛋白与寄主水稻间的互作,获得了一些阳性克隆。为进一步确定获得的阳性克隆基因所编码的全长蛋白是否能与NS2或NS3互作,选取了与NS2互作的5个基因,与NS3互作的3个基因片段进行全长扩增,并构建至酵母表达载体pGADT7,分别与NS2或NS3共转化酵母菌AH109,通过不同的营养缺陷型培养基筛选,确定全长蛋白与NS2或NS3互作情况。结果表明,完整水稻果胶酶蛋白(pectinesterase)、Ras相关蛋白(ras-related protein)、胆碱磷酸胞苷酰转移酶(cholinephosphate cytidylyltransferase)、水稻基因沉默抑制子3 (suppressor of gene silencing 3, SGS3)及一功能未知蛋白均能与NS2互作,完整水稻UBA/TS-N域包含蛋白(UBA/TS-N domain containing protein)、3-磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)能够与NS3互作,而完整蛋白O-甲基转移酶(O-methyltransferase)不能与NS3互作,且水稻SGS3 (OsSGS3)蛋白也能够与NS3互作。水稻SGS3 (OsSGS3)蛋白与拟南芥SGS3蛋白同源,为研究NS2、NS3与水稻SGS3互作的关键区段,根据OsSGS3的保守结构域将其分为3段,分别为包含Zinc结构域的SGS3-1区段、包含XS结构域的SGS3-2区段、包含Coiled coil结构域的SGS3-3区段,并与NS2或NS3共转化酵母菌AH109,通过不同的营养缺陷型培养基筛选互作的区段。结果表明NS2、NS3均可以与这3个区段互作,但NS2与SGS3-3区段的互作较弱。双分子荧光互补研究表明在烟草叶片细胞中NS3与水稻GAPDH (OsGAPDH)、NS2与OsSGS3存在的互作。亚细胞定位研究表明OsGAPDH主要定位于细胞质中,OsSGS3定位于细胞质,而NS2定位于细胞核和细胞膜,且在膜上形成小点结构,在核内能定位于柯浩体。OsSGS3与NS2主要共定位于细胞核,推测NS2改变了OsSGS3的定位,进而干扰其功能。免疫共沉淀结果也表明NS2与OsSGS3存在互作。拟南芥SGS3在小RNA生成过程中发挥作用,为研究NS2与OsSGS3互作的可能功能,首先通过荧光定量PCR检测了相关反式作用小RNA的靶标基因(5个生长素应答因子)表达量变化。结果表明,RSV侵染水稻后5个生长素应答因子表达量上调,表明RSV侵染后水稻反式作用小RNA途径可能受到影响。为此进一步检测了相应反式作用小RNA的表达量变化,结果表明小RNA表达量相对于健康水稻上升了2倍,推测可能是水稻的反馈调节作用造成的。进一步对OsSGS3、反式作用siRNA前体TAS3基因表达量进行了检测,结果表明OsSGS3基因表达量上调了2.7倍,TAS3基因表达量上调了3倍。以灰飞虱(Laodelphax striatellus)mRNA为起始模板,利用Gateway技术构建了灰飞虱酵母双杂交cDNA文库。经过检测表明:构建的初级cDNA文库的库容量为1.85×107cfu;扩增文库滴度为7.7×108 cfu/mL,重组率约为97%;扩增文库插入片段主要集中在1000-1500 bp之间。随机挑取10个克隆,测序后与GenBank数据库比对结果显示7个克隆具有同源序列,其中L2、L9为已公布的灰飞虱序列。灰飞虱酵母双杂交cDNA文库的构建为克隆全长目的基因及研究灰飞虱与其传播的水稻病毒间的互作奠定了基础。构建了包含RSV NS3的酵母表达载体pDB-NS3,以NS3为诱饵筛选了灰飞虱cDNA文库,钓取与之互作的灰飞虱蛋白。首先将酵母表达载体pDB-NS3转化酵母菌MaV203,以含有pDB-NS3的酵母菌制备酵母感受态,采用顺序转化法筛选灰飞虱cDNA文库,转化产物分别涂布不同的营养缺陷型培养基,观察酵母菌生长情况并检测β半乳糖苷酶活性。结果表明,以NS3为诱饵共筛选获得4个阳性克隆,序列比对表明只有一个克隆具有正确读码框,且与一些昆虫的ribosomal protein L4(RPL4)蛋白有较高的同源性。进一步研究表明水稻核糖体蛋白L4也能够与NS3在酵母中互作。更多还原

【Abstract】 Rice stripe virus (RSV), the type member of the genus Tenuivirus, was the causative agent of rice stripe disease which was one of the most economically important pathogens of rice and is repeatedly epidemic in China. The biological functions of virus-encoded proteins, pathogenicity differentiation, genetic diversity of virus, virus-plant host interactions and management of virus were studied during the past years. But RSV genomes, movement, and virus-host interactions, transmission were little known. In order to control the disease better and promote the sustainable development of rice production, it was essential to study the virus-rice interaction, virus-vector interaction in detail.Yeast two-hybrid system was used to detect the interaction between RSV and rice. Some positive clones interacted with RSV NS2 or NS3 were obtained. In order to study whether the full-length clones interacted with NS2 or NS3, five full-length clones interacted with NS2, three full-length clones interacted with NS3 were amplified inspectively and constructed into vector pGADT7. Then, these eight full-length clones were co-transformed into yeast strain AH109 with pGBKT7-NS2 or pGBKT7-NS3 respectively. Different synthetic dropout nutrient medium were detected. The results showed that full-length proteins pectinesterase, ras-related protein, cholinephosphate cytidylyltransferase, suppressor of gene silencing 3 (SGS3) and a function unknown protein interacted with NS2 respectively, and full-length proteins UBA/TS-N domain containing protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and SGS3 interacted with NS3. Full-length proteins O-methyltransferase did not interact with NS3.Rice (Oryza sativa L. ) SGS3 was homologous to Arabidopsis thaliana SGS3 (ATSGS3) protein and was designated as OsSGS3. OsSGS3 possessed the three domains typical of AtSGS3: zinc finger, XS and coiled coil. Three OsSGS3 deletion mutants: OsSGS3-1, which contained the znic finger domain, OsSGS3-2, which contained the XS domain, and OsSGS3-3, which contained the coiled coil domain were constructed. The coding sequences of the mutants were subcloned individually into pGADT7 and cotransformed with pGBKT7-NS2 or pGBKT7-NS3 into yeast strain AH109. All of the transformants were able to grow on SD/-His/-Trp medium and on SD medium lacking Ade, His, Leu, and Trp (SD/-Ade/-His/-Leu/-Trp), but the transformants of NS2 and SGS3-3 interacted weakly.The interaction between NS2 and Oryza sativa L. GAPDH (OsGAPDH) was verified by Bimolecular fluorescence complementation (BiFC). Cellular localization studies showed that OsGAPDH accumulated in cytoplasmic, OsSGS3 accumulated in cytoplasmic foci that perfectly resemble those of AtSGS3 and NS2 located in nucleus and punctate structures in association with cell membrane. We confirmed the interaction between OsSGS3 and NS2 by using co-IP, BiFC, and colocalization. The results of colocalization revealed that a considerable proportion of OsSGS3 was redistributed to nucleus by NS2. We speculate that the redistribution of OsSGS3 by NS2 might disrupt the normal function of OsSGS3. AtSGS3 has been demonstrated to be involved in small RNA production. To explore the possible biological implications of the interaction of RSV NS2 with OsSGS3, we first detected the expression patterns of 5 auxin responsive factors (ARFs) that had been domonstrated to be potential targets of trans acting siRNAs. The results indicated that the overall accumulation levels of the 5 genes increased in RSV infected rice. This suggested that the trans acting siRNA might indeed be affected by RSV infection. Further, we detected TAS3 derived ta-siRNAs of rice. The result indicated that the accumulation level of this small RNA was more than 2 fold higher in RSV infected rice compared with healthy control. One explanation for this observation was that a feedback regulation system of the trans acting small RNA pathway may have compensated for the loss of SGS3 activity in RSV infected rice. To test this possibility, we detected the accumulation of several other genes involved in the generation of trans acting small RNAs including the TAS3 transcript precursor and OsSGS3. The results indicated that the accumulation level of OsSGS3 and the accumulation level of TAS3 was more than 2.7 and 3 fold higher in RSV infected rice respectively compared to healthy control.A yeast two-hybrid cDNA library was constructed using CloneMiner? cDNA Library Construction Kit. The results showed that unamplified library consisted of 1.85×107 independent clones; the titer of amplified cDNA library was 7.7×108 cfu/mL, and the recombinant rate was about 97%. The size of most inserts were 1000-1500 bp. Random sequence analysis of ten clones showed that seven clones had homologous sequences in GenBank. Clones L2 and L9 were the same as the published Laodelphax striatellus. Construction of this yeast two-hybrid cDNA library of Laodelphax striatellus laid the foundation for cloning of functional genes in Laodelphax striatellus and carrying out the research on Laodelphax striatellus-Virus interaction. Bait plasmids pDB-NS3 was constructed and used to screen the yeast two-hybrid cDNA library of Laodelphax striatellus. The transformants were plated on different dropout nutrient mediums andβ-galactosidase activity were detected. One positive clone homologous to ribosomal protein L4 of some insects was obtained. The interaction between NS3 and ribosomal protein L4 of rice was also detected, the results showed that NS3 could either interact with ribosomal protein L4 of rice or ribosomal protein L4 of Laodelphax striatellus in yeast.更多还原