【作者】 杨彩霞；

【导师】 谢联辉；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 双生病毒是一种呈孪生颗粒形态的植物单链环形DNA病毒,至今已在多个国家和地区的多种作物上造成毁灭性危害。自20世纪90年代起,在我国华南地区的作物及杂草上己相继发现了多种双生病毒。本文报告了福建省几种作物和杂草上的双生病毒的分子鉴定结果。从福建福州采集了八份表现矮化症状的普通烟样品,利用双生病毒特异性引物PA/PB扩增得到约500bp的DNA-A部分片段的相似性高于95%。挑选分离物F3进一步进行DNA-A全序列测定,结果显示F3 DNA-A(F3A)全长为2739 nts(EF125190)。利用RCA鉴定出DNA-B分子,F3 DNA-B(F3B)全长是2720 nts(FJ874926)。核苷酸同源性比对发现,F3A与海南报道的苎麻花叶病毒的一个分离物(Ramie mosaic virus,RaMoV,EU596959)相似性最高,为95.1%。因此,我们认为F3是RaMoV的一个分离物。这是首次有关RaMoV自然侵染烟草的报道。为进一步验证RaMoV F3分离物的致病性,我们构建了F3A和F3B的侵染性克隆。农杆菌接种实验发现,F3A单独能完成系统侵染本氏烟、普通烟、珊西烟、心叶烟、三生烟和番茄,但是不能诱导任何症状。混合接种F3 A+B,可在本氏烟、普通烟、珊西烟和三生烟上诱导产生典型的双生病毒侵染症状。从福建福州烟田采集的表现曲叶、耳突、叶脉增厚和矮化症状的普通烟样品YC7中分离出F11和F12两种病毒。利用双生病毒特异性引物PA/PB扩增得到约500bp的DNA-A部分序列,确定YC7为F11和F12不同病毒复合侵染。F11和F12全长分别是2741 nts(FJ869907)和2754 nts(FJ869908)。同源性比对发现,F11与报道的广东番木瓜曲叶病毒的分离物PaLCuGuV-[CN:Gd2:02](AJ558122)的核酸相似性最高,为97.3%;F12与胜红蓟黄脉病毒的台湾分离物AYVVTW-[TW:Tai:99](AF307861)的核酸相似性最高,为90.1%。利用引物β01/02找到F12的伴随卫星DNAβ(F12β),F12β全长1344 nts(FJ869909),与胜红蓟黄脉病毒台湾分离物伴随卫星AYVB-[TW:CHu:02] (AJ542495)相似性最高,96.5%,说明F11和F12分别是PaLCuGuV和AYVV的一个分离物。以样品YC7为毒源,利用粉虱进行传毒,能在普通烟和心叶烟上引起曲叶、耳突、叶脉增厚和矮化症状。PCR检测表明,传毒烟草上存在F11和F12两种病毒和F12β分子。Fp1-Fp4分离物来自福建福州表现轻微的花叶和皱缩症状的圆叶矮牵牛。利用双生病毒特异性引物PA/PB扩增,得到约500bp的DNA片段,它们间的的核苷酸相似性为97.5%,说明它们是同一种病毒的不用分离物。随机选择Fp1进行全序列测定及进一步分析。Fp1全长2828 nts(FJ515896),具有典型的双生病毒DNA-A的基因组结构。Fp1DNA-A全序列与中国江苏报道的番薯曲叶病毒的一个分离物SPLCV-[CN-Js:08](FJ176701)相似性最高,为92.1%,说明Fp1是SPLCV的福建分离物。据我们所知,这是首次报道SPLCV可以自然侵染圆叶矮牵牛。通过构建SPLCV Fp1分离物的侵染性克隆,并进行致病性测定,结果表明,Fp1A不能侵染本氏烟。从福建漳州采集到表现明显黄脉症状的一点红(Fz1-Fz5)和野茼蒿(Fz6)等六个样品。从这些植物的叶片中提取总DNA并采用滚环复制扩增法检测。Fz1-Fz6扩增产物分别用限制性内切酶BamH I、EcoR I、Kpn I、Sac I、Xba I和Sal I处理。Fz1-Fz6酶切片段(EcoR I切下的2.7 kb,Xba I切下的1.3 kb)被测序,部分结果显示这些植物被同一种病毒侵染。选择Fz1进行进一步分析。Fz1全长2725 nts(EU377539),伴随的DNAβ全长1337 nts(FJ869906)。Fz1全序列与印度的夜香牛黄脉病毒分离物VeYVV-[IN:Mad:05] (AM182232)的相似性最高,76.7%。根据双生病毒“种”的分类标准,Fz1是一个双生病毒新种,我们将这个双生病毒新种命名为一点红黄脉病毒(Emilia yellow vein virus,EmYVV)。我们还构建了EmYVV及其伴随DNAβ的侵染性克隆,致病性测定表明,Fz1DNA-A可以单独侵染本氏烟并诱导产生轻微的曲叶症状。Fz1DNA-A+ DNAβ能侵染本氏烟并诱导产生典型的曲叶症状。从福建地区表现黄色花叶症状的大青样品(Fz7-Fz9)上分离到双生病毒。利用简并引物PA/PB对三个分离物进行扩增,获得的约500bp片段的DNA序列,其相似性为99.54%。随机选择分离物Fz7进行病毒全长基因组的扩增和序列测定。结果表明,Fz7 DNA-A全长2776 nts(FJ011668),与重瓣臭茉莉金色花叶病毒(ClGMV)相似性最高,达82%,是一新型的双生病毒,命名为中国大青黄色花叶病毒(Clerodendrum yellow mosaic China virus,ClYMCNV)。从Fz7中发现有DNA-B分子,全长2739 nts(FJ011669),证实ClYMCNV是一个新的双组分双生病毒。ClYMCNV侵染性克隆的构建为后来的致病性测定做好了准备。通过PCR扩增获得了RaMoV八个功能蛋白的基因AV1、AV2、AC1、AC2、AC3、AC4、BV1和BC1基因。将BV1构建到酵母双杂交诱饵载体pGBKT7上,把AV1、AV2、AC1、AC2、AC3、AC4、BV1和BC1构建到捕获载体pGADT7上,通过筛选鉴定获得了重组子pGBK-BV1、pGAD-BV1、pGAD-BC1、pGAD-AC1、pGAD-AC2、pGAD-AC3和pGAD-AC4。将上述重组子分别单独转化到酵母菌AH109中,转化产物涂布不同营养缺陷型培养基,检测八个蛋白对酵母细胞的毒性和自激活活性,结果表明,RaMoV八个蛋白对酵母菌AH109都未表现出毒性和自激活活性。将重组子pGBK-BV1与所有pGAD-X重组子两两共转化到酵母菌AH109中,转化产物涂布不同营养缺陷型培养基,从而检测RaMoV BV1自身互作及BV1与其它七个蛋白之间的互作情况。结果显示, BV1分别与AV2、AC3和BC1之间被检测到互作现象。“本氏烟”文库质量鉴定结果表明:文库初始文库滴度分别为3.1×107 cfu/mL,扩增文库滴度为3.4×107 cfu/mL。文库重组率均大于95%,“本氏烟”文库cDNA插入片段长度集中分布在900-1000 bp之间。该文库可以用于配合实验进行文库筛选。将重组子pGBKT7-BV1单独转化到酵母菌Y187中,转化产物涂布不同营养缺陷型培养基,检测BV1蛋白对Y187细胞的毒性和自激活活性。结果表明,BV1蛋白对酵母菌Y187都未表现出毒性和自激活活性。所以,我们将Y187 ( pGBKT7-BV1 )和AH109 (pGADT7-cDNA)进行配合,配合产物涂布不同营养缺陷型培养基,筛选可能与诱饵蛋白BV1互作的寄主蛋白。结果表明,以BV1为诱饵,从本氏烟文库中筛选到八个阳性克隆。PCR鉴定阳性克隆子中cDNA插入片段的大小,将含有不同长度插入片段的酵母质粒分别转化到大肠杆菌DH5α中,经测序,并在GenBank数据库中对测序结果进行blast比对,根据同源序列的注释信息,发现BV1蛋白与烟草SnRK1和DXR两个蛋白发生互作,其机理和功能有待进一步探讨。更多还原

【Abstract】 Geminiviruses have circular single-stranded DNA genome encapsidated in twinned icosahedral particles. They have caused significant yield loss to many crops worldwide. Several begomoviruses have been reported infecting crops and weeds in south China since 1990s. The current study reported the identification results of begomoviruses in several crops and weeds in Fujian province.Eight leaf samples of Nicotiana tabacum showing stunting symptoms were collected in Fuzhou city of Fujian province in China. The partial DNA-A fragments (500 nucleotide) amplified by PA/PB shared % nucleotide sequence identity. Isolate F3 were selected for determining the complete genome sequence and the complete F3 DNA-A (F3A) was 2739 nts (EF125190). RCA was used to searching the DNA-B molecule and F3 DNA-B (F3B) was determined to be 2720 nts (FJ874926). The complete nucleotide of F3A had the highest sequence identity (95.1%) with an isolate of Ramie mosaic virus (RaMoV, EU596959) from Hainan province. The molecular data showed that F3 was an isolate of RaMoV. This is the first report about RaMoV naturally infencted in N. tabacum. We constructed the infectious clones of F3A and F3B to test the infectivity of F3 in host plants. F3A alone could systemically infect tested plants N. benthamiana, N. tabacum, N. tabacum Xanth, N. glutinosa, N. tabacum cv. Samsun NN/nn and Solanum lycopersicum without inducing symptoms, while F3A+B can induce typical geminiviruses infected symptoms in N. benthamiana, N. tabacum and N. tabacum cv. Samsun NN/nn.Two viruses F11 and F12 was obtained from the same N. tabacum sample YC7 showing leaf curl, enation, vain sticking and stunting symptoms in Fujian province of China. The partial DNA-A sequence (500 nucleotide) amplified by PA/PB showed sample YC7 was mixed infected by viruses F11 and F12. F11 and F12 were determined to be 2741 nts (FJ869907) and 2754 nts (FJ869908), respectively. The complete nucleotide of F11 had the highest sequence identity (97.3%) with PaLCuGuV-[CN:Gd2:02] (AJ558122) , an isolate of Papaya leaf curl Guangdong virus (PaLCuGuV) from Guangdong province. The complete nucleotide of F12 shared the highest sequence identity (90.1%) with AYVVTW-[TW:Tai:99] (AF307861) , an isolate of Ageratum yellow vein virus (AYVV) from Taiwan. The DNAβmolecule associated with the F12 was found with primersβ01/02 (F12β). F12βwas 1344 nts (FJ869909) and it shared the highest sequence identity (96.5%) with AYVB-[TW:CHu:02] (AJ542495) , an isolate of Ageratum yellow vein betasatellite. The molecular data showed that F11 and F12 were another isolate of PaLCuGuV and AYVV, respectively. N. tabacum and N. glutinosa showed leaf curl, enation, vain sticking and stunting symptoms by whitefly transmission when sample as viral source. PCR results showed that these infected plants contained F11, F12 and F12β。Four virus isolates Fp1-Fp4 were isolated from Ipomoea purpurea leaves showing slightly yellow mosaic and crinkled symptoms in Fuzhou city of Fujian province. The 500bp fragments of the isolates amplified by the PA/PB had 97.5% nucleotide sequence identities, suggesting that they were different isolates of the same virus species. Isolate Fp1 was selected for further sequence analysis. Fp1 was 2828 nts, with the typical genomic organization of begomoviral DNA-A (FJ515896). The whole Fp1A sequence showed the highest nucleotide sequence identity (92.1%) with SPLCV-[CN-Js:08] (FJ176701), an isolate of Sweet potato leaf curl virus (SPLCV) from Jiangsu Province of China. The result confirmed that Fp1 was an isolate of SPLCV from Fujian province. To our knowledge, this is the first report of the natural occurrence of SPLCV in I. purpurea. We constructed the infectious clone of Fp1 and test their infectivity in N. benthamiana. The results indicated that Fp1 could not infect N. benthamiana.Six samples of Emilia sonchifolia (Fz1-Fz5) and Crassocephalum crepidioides (Fz6) leaves showing conspicuous yellow veins were collected in Zhangzhou city of Fujian province. Total DNA was extracted from leaves of these plants and tested by rolling circle amplification (RCA). Amplification products of Fz1-Fz6 were digested by the restriction enzyme BamH I, EcoR I, Kpn I, Sac I, Xba I and Sal I, respectively. Restriction product (2.7 kb for EcoR I; 1.3 kb for Xba I) of Fz1-Fz6 were sequenced and the partial sequences indicated that these plants were infected by the same virus. Fz1 was used for further analysis. The complete Fz1 comprised 2725 nts (EU377539) and its associated DNAβcomprised 1337 nts (FJ869906). The complete Fz1 sequence was most closely related to Vernonia yellow vein virus (VeYVV-[IN:Mad:05], AM182232), with 76.7% nucleotide sequence identity. In line with the demarcation criteria for identifying begomovirus species, Fz1 is considered as a distinct begomovirus, for which the name Emilia yellow vein virus (EmYVV) is proposed. We also constructed the infectious clones of Fz1 to test the pathogenicity. Results showed that Fz1DNA-A alone could infected the N. benthamiana with little leaf curl symptom. Fz1 DNA-A + DNAβcould induce sever leaf curl symptom.Virus isolates Fz7-Fz9 were obtained from Clerodendrum cyrtophyllum Turcz plants showing yellow mosaic in Fujian, China. Total 500bp fragments were amplified with the degenerate primers PA/PB, and they shared 99.54% nucleotide sequence identity. The complete Fz7 DNA-A sequence is 2776 nts (FJ011668) and shares the highest nucleotide sequence identity (82%) with ClGMV. The molecular data showed that Fz7-Fz9 were isolates of a new begomovirus for which the name Clerodendrum yellow mosaic China virus (ClYMCNV) is proposed. DNA-B molecule was identified in Fz7 with 2739 nts (FJ011669). The results confirmed ClYMCNV is a novel bipartite begomovirus. Infectious clone of Fz7 was constructed to test the pathogenicity of ClYMCNV next.RaMoV AV1, AV2, AC1, AC2, AC3, AC4, BV1 and BC1 genes were amplified by PCR. Then, BV1 was inserted into yeast two-hybrid system bait vector pGBKT7 and AV1, AV2, AC1, AC2, AC3, AC4, BV1 and BC1 was inserted into prey vector pGADT7. In order to detect the self-activation of the eight proteins and toxicity to yeast cell AH109, all the recombinants were transformed into AH109 individually, and the transformants were plated on the different synthetic dropout nutrient medium. The results showed: all the genes don’t self-activate and have no toxictiy to yeast cell; In order to detect BV1self-interaction, BV1 and other seven proteins, pGBK-BV1 and all pGAD-X were transformed into AH109, individually. Results showed that interaction of BV1 with AV2, BV1 with AC3, BV1 with BC1 were detected.The results of detection showed that the titer of primary“N. benthamiana”cDNA library were 3.1×107 cfu/mL and the titer of amplified library were 3.4×107 cfu/mL; The recombination rates were both above 95%; The size of most inserts were 900-1000bp in the cDNA library. The cDNA library is available for mating screen. In order to detect the self-activation of BV1 and toxicity to yeast cell Y187, the transformants Y187 (pGBKT7-BV1) were plated on the different synthetic dropout nutrient medium. The result showed that BV1 don’t self-activate and have no toxictiy to Y187 yeast cell. Then, the Y187 (pGBKT7-BV1) and AH109 (pGADT7-cDNA) were mated and the products were plated on the different synthetic dropout nutrient medium. Results showed that eight positive clones were acquired by screening“N. benthamiana”cDNA library using RaMoV BV1 as bait protein; According to the function annotation of homologous sequences and reports about the protein function, only three positive clones are meaningful. The mechanism and function of the interaction between viral proteins and three host proteins SnRK1 and DXR were predicted.更多还原