【作者】 邓丛良；

【导师】 李怀方；

【作者基本信息】 中国农业大学 ， 植物病理学， 2004， 博士

【摘要】 通过RT-PCR技术从我国北方地区显花叶症状的白草植株上获得白草花叶病毒两个分离物(PenMV-B和PenMV-C)，两个分离物基因组全序列共9611个核苷酸，5′-末端和3′-末端的非翻译区序列分别为172和241个核苷酸，中间为9198个核苷酸的开放读框，编码3065个氨基酸，分子量约为350kD；10个多肽间的切割位点顺次为：Y/A，G/G，E/H，O/S，Q/G，Q/G，E/A，Q/G和Q/s，其中P3/6K1处的E/H蛋白切割位点序列与Potyvirus其它病毒均不相同，为该病毒所仅有。预期的Potyvirus病毒一些重要的、功能性的保守基序都存在于该病毒的基因组序列中。Potyvirus病毒多聚蛋白氨基酸序列的系统进化树分析表明PenMV和MDMV、SrMV、SCMV有较近的亲缘关系。根据ICTV最新的马铃薯Y病毒属分类标准，这两个分离物属于PenMV病毒，进一步确立了PenMV在Potyvirus SCMV亚组新成员的地位。 PenMV-B(AY642590)和PenMV-C的全基因组核菅酸序列同源性为88.5％，氨基酸序列同源性为96.8％，两个分离物间的NIb核苷酸序列同源性最低，为85.0％，3′-NTR核苷酸序列同源性最高，为96.3％。PenMV-B、PenMV-C和PenMV-A(AY172336)三个分离物之间NIb核苷酸序列同源性为85.0％～88.4％，氨基酸序列同源性为93.1％～95.8％；CP的核苷酸和氨基酸序列同源性分别为92.1％～93.0％和98.3％～98.7％；3′-NTR序列同源性为96.3％～99.2％。三个分离物的3′-NTR序列保守性很高，而NIb基因保守性很差，同一病毒不同分离物之间的NIb基因保守性如此低，在Potyvirus病毒中从没有报道过(通常认为NIb基因是Potyvirus病毒最保守的区域)。 对SCMV亚组病毒和分离物基因组各区域作多重序列比较，结果表明PenMV只有在P1、NIb和CP区域分别与我国报道的SCMV-SD和SrMV-YH亲缘关系最近，在5′-NTR和3′-NTR区域则分别与MDMV-BG和ZeMV亲缘关系最近，在NIa-Pro区域与SCMV-A亲缘关系最近，而其全基因组和其它各区域核苷酸序列与SrMV-H具有较近的亲缘关系。分别构建SCMV亚组病毒的基因组全序列和各区域核苷酸序列系统进化树，结果PenMV分离物在各区域存在异常成簇。推测该病毒在远缘进化过程中可能与MDMV、SrMV、SCMV和ZeMV发生基因重组，传入我国后和我国的SCMV玉米分离物在NIb区域发生基因重组，和SrMV在CP区域发生基因重组，同时，该病毒基因组为了适应新的环境压力有可能发生了高频率的基因突变。 将PenMV-B分离物的外壳蛋白(CP)基因克隆到表达质粒pET22b(+)中，构建其在大肠杆菌中的表达载体pET-PenMV CP。SDS-PAGE和Western blot检测结果表明，这一表达载体在大肠杆菌BL21(DE3)中经IPTG诱导可表达分子量为36kDa的特异性融合蛋白，并具有免疫学活性。以Ni′亲合柱层析纯化这一融合蛋白，作为抗原免疫德国大白兔制备了特异性较高，效价为1／8192的抗血清(ACP-ELISA检测)，可以直接用于PenMV侵染植株的检测。更多还原

【Abstract】 Two Pennisetum mosaic virus isolates (PenMV-B and PenMV-C) from perennial whitegrass (Pennisetum centrasiaticum Tzvel) in North China were characterized at the molecular level. The complete nucleotide (nt) sequences of the two isolates have been determined. The viral genome of both isolates comprised 9611 nts excluding the 3’-terminal poly (A) sequence, potentially encoding a single polyprotein of 3065 amino acids with a calculated Mr of 349,575 Da, flanked by 5’- and 3’-NTRs with 172 and 244 nucleotides respectively. Nine putative cleavage sites were identified by a sequence comparison of this viral polyprotein with those of other potyvimses. The result showed that the putative cleavage site between P3 and 6K.1 for this virus was E/H, which is different from those of any other potyvimses. Several specific motifs were also present in the polyprotein of PenMV. Sequence comparisons of the complete nucleotides and amino acids suggested that the vims was closely related to some monocot-infecting potyvimses such as Sorghum mosaic virus (SrMV), Sugarcane mosaic virus (SCMV) and Maize dwarf mosaic virus (MDMV). Sequence comparison and phylogenetic analysis confirmed our previous report that PenMV is a distinct potyvirus within the SCMV subgroup.The complete sequences of PenMV-B and PenMV-C share identities of 88.5% and 96.8% at the nucleotide and amino acid levels, respectively; However, the nucleotide sequences in the NIb-coding region was only 85.0% identical, which was the lowest level of identity observed in comparison to other regions of two PenMV isolates. The nucleotide sequence alignment showed that PenMV-B, PenMV-C and PenMV-A shared identities of 92.1% to 93.0% in the CP gene and 96.3% to 99.2% in the 3’ NTR, respectively; but only shared identities of 85.0% to 88.4% in the NIb region, which is distinct from other potyvimses where the NIb was among the most conserved regions.GenBank/EMBL search using BLAST and phylogenetic analyses were performed separately for the nucleotide sequences of each functionally distinct part of the genome. These sequences of two PenMV isolates and other vimses and isolates grouped inconsistently for each part of the genome in these analyses. The alignment results indicated that the isolates were most closely related to MDMV-BG for the 5’-NTR and ZeMV for the 3’-NTR; however, they were most closely related to SrMV-YH in the P1 gene SCMV-A in the NIa gene, SCMV-SD in the NIb gene and SrMV-YH in the CP gene, though they were more closely related to SrMV-H in the whole genome than to any other vimses and isolates. One could speculate that this was the result of one or more possible recombination events between the ancestors of SCMV, MDMV and SrMV or ZeMV in the distant past. Moreover, a high mutation rate during the evolution of this viral genome could have occurred following various selective pressures.The expression vector pET-PenMV CP containing coat protein (CP) gene of Pennisetum mosaic virus was constructed by cloning PenMV-B CP gene into the plasmid pET22b(+) and being transferred into E. coli BL21(DE3). The results of SDS-PAGE and Western blot showed that the expression of specific fusion protein of about 36 kDa was enhanced by IPTG induction and the protein was highly immunogenic. The specific fusion protein was purified by Ni2+-affinity chromatography and wasinjected into rabbit to raise antiserum. The antiserum titer was 1/8192 by antigen coating plate-ELISA (ACP-ELISA) and has been proved useful for identification of PenMV in plant materials.更多还原