【作者】 刘涛；

【导师】 于嘉林；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2003， 博士

【摘要】 利用RT-PCR对来自新疆、黑龙江、宁夏、甘肃、河北省甜菜丛根病病田的各BNYVV分离物中RNA5组分进行检测，在来自于新疆、宁夏和黑龙江的分离物中发现有RNA5的存在，说明在我国发生的BNYVV分离物中广泛存在RNA5组分。对新疆、宁夏和黑龙江分离物中的RNA5进行克隆和序列分析后，与已报道的BNYVV日本D5分离物、法国的F72分离物以及我国内蒙古发现的包头分离物和呼和浩特分离物的RNA5的核苷酸序列和推导氨基酸序列进行同源性比较。结果表明，不同分离物RNA5核苷酸序列的同源性为93.0％～98.7％，变异主要集中在编码蛋白启始密码子AUG前100个核苷酸的区域内。所有RNA5基因组都只包含一个开放阅读框架(ORF)，除法国的F72(编码232个氨基酸)外，其它6个分离物都编码228个氨基酸的多肽。各分离物的氨基酸序列同源性为89.7％～98.7％。 构建了RNA5编码蛋白的原核表达载体pETHu26，经IPTG诱导后在大肠杆菌中得到表达，表达的融合蛋白包含载体编码的45个氨基酸和RNA5编码的26kDa蛋白。用融合蛋白免疫家兔，制备了RNA5编码蛋白的特异性抗血清，Western blot分析表明该血清可以用于BNYVV接种番杏叶片后RNA5编码26kDa蛋白的检测。 分别构建了T7启动子和35S启动子控制下的全长RNA5侵染性cDNA克隆，Northern blot检测表明它们都具有侵染性。利用T7控制下的全长RNA5侵染性cDNA克隆pUCHu3的体外转录物与不含RNA5的BNYVV突变株，BNYVV-Hu0和BNYVV-Hu3的RNAs混合接种番杏和甜菜，结果表明RNA5影响病毒侵染后寄主的症状表达和病毒在寄主内的积累，并且和甜菜丛根病的发病程度及甜菜产量有关。这些结果表明除了RNA3外，RNA5是另外一个与BNYVV致病性相关的因素。 在全长RNA5侵染性克隆pUCHu3的基础上构建了4个RNA5编码区缺失突变体和1个点突变体的侵染性克隆。经体外转录后与BNYVV-Hu3的RNAs混合接种番杏，Northern blot检测表明这些突变体的体外转录物都具有侵染性。用编码区翻译起始密码(AUG)突变体侵染性克隆pUCHu3△ATG体外转录物接种番杏叶片，经对发病植物的检测，说明RNA5编码蛋白的表达与否不影响BNYVV在番杏上的症状表现，即RNA5对于BNYVV致病力的影响可能是在核酸水平上的作用。这些突变体对RNA5的功能的影响正在进一步研究中。更多还原

【Abstract】 BNYVV isolates from Ningxia, Xinjiang, Heilongjiang, Gansu and Hebei provinces were screened by RT-PCR and the RNA5 component was identified in the isolates from Ningxia, Xinjiang and Heilongjiang among them, indicating that the BNYVV isolates containing RNA5 component were widely distributed in sugarbeet growing areas of China. The RNA5 components were cloned into pUCm-T vector and transformed in E. coli strain DH-5 a . Sequence analysis showed that the RNA5 components in the isolates from Ningxia, Xinjiang and Heilongjiang have 1347nts, 1348nts, 1346nts respectively. Comparing with the RNA5s in D5, F72, Bao and Hu isolates reported previously, identities shared between them were varied from 93.0%-98.7% in nucleotide sequences and 89.7%-98.7% in deduced amino acid sequences.The bacteria) expression vector of pET-Hu26 containing the coding region of BNYVV RNA5 was constructed and transformed into E.coli strain JM109 (DE3). With induction of IPTG, a fusion protein was over-expressed and used for prepation of antisum, which specifically binds to a protein of 26 kDa encoded by BNYVV RNA5 in Western blot.Full-length cDNAs of BNYVV RNA-5 for in vitro and in vivo transcription were synthesized by cloning into plasmid of pUC18 driven by a bacteriophage T7 promoter or pRT103 adjacent to a Cauliflower mosaic virus 35S promoter, respectively. Infectivities of the transcripts were determined by RT-PCR and Northern blotting after inoculation of plants.In vitro transcripts of RNA5 were co-inoculated to Tetragonia expansa and sugar beet seedlings with RNAs extracted from two BNYVV mutants of HuO containing RNA1 and 2 only, and Hu3 containing RNA1, 2 plus 3. By assay of symptom and viral RNA, it was indicated that the virus infection and accumulation in the hosts can be improved by presence of the RNA5. Cooperating with RNA3, RNAS seems to cause more severe symptom and yield losses in sugar beet. These results suggest that, besides of RNA3, RNA5 is another important element in BNYVV associated with its pathogenicity.In order to identify the motif responsible for the RNA5, four internal deletions and a point mutation at start codon of ATG within the coding region of RNA5 were constructed. In vitro transcripts of these mutants were co-inoculated to T. expansa together with the RNAs of Hu3 mutant It was demonstratedthat all these transcripts were infectious and protein expression encoded by RNA5 was not essential forBNYVV infection. Further experiments are being conducted to test the effects of these mutants on root yield and sugar content in sugar beet.更多还原