【作者】 梁文星；

【导师】 范在丰；

【作者基本信息】 中国农业大学 ， 植物病理学， 2004， 硕士

【摘要】 北京地区的紫藤脉花叶病是由马铃薯Y病毒属的一种病毒侵染所致。通过机械接种的方法将此病毒的北京分离物接种到昆诺藜、苋色藜、克利夫兰烟和蚕豆上，分别产生系统褪绿斑、局部褪绿斑、轻微系统褪绿斑和系统褪绿斑等症状。ELISA分析显示此分离物与BCMV和WMV间具有血清学关系。虽然在发病紫藤种子的胚乳中能检测到其外壳蛋白的存在，但此分离物却不能通过种子传播。对提纯的病毒进行SDS-PAGE和Western blot分析，测得其CP亚基的分子量为35.0 kD。在被侵染的蚕豆叶片细胞中观察到了Potyvirus病毒所具有的典型的风轮状内含体。以上生物学特性的研究以及对其CP基因和3′-非翻译区序列的分析证明此病毒为紫藤脉花叶病毒(WVMV)，这是关于此病毒在中国发生的首次报道。 通过7次RT-PCR得到了WVMV北京分离物(WVMV-BJ)基因组的全序列，这是关于此病毒基因组全序列的首次报道。序列分析结果表明：WVMV-BJ全序列共9695个核苷酸，5′-和3′-非翻译区序列分别为164和252个核苷酸，中间为9279个核苷酸的开放读框，编码一个长为3063个氨基酸、分子量约为353.4 kD的多聚蛋白。此病毒基因组及其所编码的多聚蛋白的大小与其它马铃薯Y病毒属病毒较为相近。多聚蛋白含有9个可能的蛋白酶切位点，切割后产生的成熟蛋白产物中具有保守的氨基酸基序，这与其它potyviruses都非常相似。在10个蛋白产物中，CI、NIb和CP相对保守，而P1和P3变异最大。 采用RT-PCR方法自WVMV-BJ的基因组中扩增出其CP基因，连接到原核表达载体pET22b(+)上。获得的重组子pET-WVMVCP转化大肠杆菌BL21(DE3)后，用IPTG进行诱导表达。SDS-PAGE和Western blot分析表明，CP基因在大肠杆菌中获得了高效表达，融合蛋白分子量约为34.4 kD。将融合蛋白纯化后免疫兔子，获得了特异性较高的抗血清。微量免疫沉淀法测定该抗血清的效价为1／1024，酶联法测定效价为1／8192。 蛋白酶抑制剂具有抑制蛋白酶活性的作用，这为通过抑制马铃薯Y病毒属编码的蛋白酶活性从而控制此类病毒提供了新的途径。用RT-PCR方法从新疆杨叶片中分离出一个Kunitz型胰蛋白酶抑制剂(PTI)基因。序列分析表明；PTI基因翻译起点上游具有‘TATA’和‘CCAAT’等转录控制元件，其包含的最大阅读框架能编码一个213aa的多肽。序列比对证明PTI与克隆自欧美山杨的PtTI2和PtTI1同源性最高，分别为95％和80％。将PTI基因以融合蛋白的形式在大肠杆菌中进行表达，纯化后的融合蛋白对胰蛋白酶的活性有抑制作用。Western blotting分析表明融合蛋白与PtTI2特异的抗体之间有明显的血清学反应。更多还原

【Abstract】 The mosaic symptom of wisteria (Wisteria sinensis) in Beijing is caused by a potyvirus. The Beijing isolate of the potyvirus was mechanically transmitted to C. amaranticolor, C. quinoa, N. clevelandii and V. faba, producing local chlorotic lesions, systemic chlorotic lesions, mildly systemic chlorotic lesions and systemic chlorotic lesions, respectively. Although the virus coat protein was present in the endosperms of the seeds of infected wisteria plants by ELASA tests, it is not seed-borne. The virus isolate was serologically related to Bean common mosaic virus (BCMV) and Watermelon mosaic virus (WMV), the molecular weight of its CP subunit was confirmed to be 35.0 kD by SDS-PAGE and Western blot analysis. Cells of V. faba leaves infected with the virus had cytoplasmic pinwheel inclusion bodies typical of potyviruses. The above biological properties and sequence analysis of the coat protein gene and 3’ non-translated region (NTR) showed that this potyvirus is Wisteria vein mosaic virus (WVMV). This is the first report of WVMV occurrence in China.The genomic sequence of WVMV-BJ was determined by sequencing 7 overlapping viral cDNA clones generated by RT-PCR, which represents the first complete sequence for the virus. The sequence is 9695 nucleotides in length excluding the 3’ terminal poly (A) tail and contains a single open reading frame of 9279 nucleotides encoding a large polyprotein of 3092 amino acids with predicted Mr of 353.4 kD. The genome of WVMV-BJ has a 164 nt 5’-non coding and a 252 nt 3’-non coding region. The size of the genome and the encoded polyprotein is in agreement with other potyviruses and contains nine putative proteolytic cleavage sites and motifs conserved in homologous proteins of other potyviruses. The P1 and P3 were the most variable proteins while CI, NIb and CP were relatively conserved.The CP gene of WVMV-BJ was amplified by RT-PCR, and ligated to the expression vector pET22b (+). The recombinant plasmid pET-WVMVCP was transformed into E. coli BL21 (DE3) and then induced by IPTG to express a fusion protein. It was shown by SDS-PAGE and Western blot analysis that the CP gene was expressed at high level. The molecular weight of the fusion protein was about 34.4kD. An antiserum with high specificity was produced after the rabbit was immunized with purified fusion protein, and its titer was determined to be 1/1024 by micro-precipitation, or 1/8192 by ACP-ELISA.To explore novel ways for controlling potyviruses by inhibiting the proteinases encoded by this group of viruses, a proteinase inhibitor gene was cloned and its protein product expressed for test of its inhibitory activity to some proteinases. A Kunitz trypsin inhibitor gene (PT1) was isolated from mechanically wounded poplar (Populus alba var. pyramidalis Bge) leaves by RT-PCR. The longest open reading frame in the PTI gene, which contains conventional ’TATA’ and ’CCAAT’ transcription control elements, potentially encode a peptide of 213 amino acids. Sequence comparisons showed that PTI was most closely related to PtTI2 and PtT11 from trembling aspen, with identities of 95% and 98%,respectively. The PTI gene was expressed in E. coli to produce a fusion protein and the purified product was shown to have inhibitory activity to bovine trypsin in vitro. Western blot analysis showed that the fusion protein of 27.5 kD strongly react with the antibody raised against PtTI2.更多还原