【作者】 原雪峰；

【导师】 于嘉林； 李大伟；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 本论文以甜菜黑色焦枯病毒（Beet black scorch virus,BBSV）侵染性全长cDNA克隆pUBF52为材料,利用体外转录物的核苷酸（nt）突变和绿色荧光蛋白（GFP）标记等技术,对BBSV不同基因的功能和基因组表达策略开展了反向遗传学研究。通过对编码区核苷酸的替换、移码和缺失突变,证实了位于基因组5′端的P22和P82基因所编码的蛋白是参与BBSV复制酶（RdRp）功能的必需组分。通过翻译起始密码子的点突变和引入提前终止密码子,对由计算机推导而来、位于BBSV基因组中部的4个小分子蛋白编码框（P5、P7a、P7b和P5′）分别进行了功能分析。结果发现,其中对P5编码框的突变没有对BBSV侵染苋色藜（Chenopodium amaranticolor）后的枯斑症状,以及病毒RNA在寄主内的积累水平产生任何影响;而对P7a、P7b或P5′编码框的类似突变改造则将导致BBSV接种苋色藜后的局部枯斑症状消失,且体内病毒RNA的积累水平明显降低。 进一步采用GFP替换BBSV外壳蛋白（CP）的标记试验证明,在激光共聚焦显微镜下,对P7a、P7b或P5′中任何一个编码框的突变改造都将导致绿色荧光被局限在单个接种细胞的细胞核内,而野生型BBSV所携带的GFP则可在多个相邻细胞的细胞核及细胞膜中同时得到表达。结合以往关于BBSV CP基因的研究结果,由此确认BBSV基因组可能共计编码6种蛋白质,包括与RdRp相关的P22和P82、协同决定BBSV细胞间运动功能的P7a、P7b和P5′、以及外壳蛋白P24。其中P5′基因是在烟草坏死病毒属（Necrovirus）成员中首次发现。 在此基础上,对BBSV多顺反子基因组的表达策略进行了初步研究。以纯化的BBSV双链RNA为模板,经5′-RACE和体外转录物单核苷酸突变确定了BBSV在复制过程中产生的两条亚基因组RNA（Subgenomic RNA,sgRNA）的转录起始位点。结果表明,BBSV的sgRNA1和sgRNA2分别起始于BBSV基因组2209位或2526位的鸟苷酸（G）,其中由sgRNA1可翻译产生P7a、P7b和P5′三种蛋白,并由此决定了BBSV接种苋色藜后的细胞间运动及枯斑症状;而sgRNA2则负责CP的表达,对病毒侵染寄主后的症状及RNA积累水平没有明显影响。 对亚基因组转录起始点(G²²⁰⁹或G²⁵²⁶)上、下游共9个核苷酸（2204-2213nt或2521-2530nt）的突变分析表明,起始点上游胞苷酸（C）的突变对亚基因组RNA的转录影响较大,即:C²²⁰⁶的突变将导致sgRNA1的转录灭活,而C²⁵²¹、C²⁵²³、或C²⁵²⁴的突变则导致sgRNA2的转录灭活。计算机推导的BBSV基因组二级结构表明,C²²⁰⁶可以在一个“茎-环”结构中与G²¹¹⁸配对;C²⁵²¹、C²⁵²³和C²⁵²⁴则以同样方式与G²³⁸⁰、G²³⁷⁸和G²³⁷⁷位分别配对。对这些碱基对的突变分析发现,维持他们之间的配对关系对相应亚基因组RNA的转录具有重要的作用。进一步对亚基因组转录启动子的缺失突变研究结果表明,sgRNA2的启动子区域可能位于2339nt-2558nt之间,以一种稳定的多重“茎-环”结构存在。鉴于sgRNA1的上游也存在类似的多重“茎-环”结构,由此推断BBSV亚基因组RNA可能是遵循“提前终止机制”复制产生的。 计算机分析发现,BBSV基因组或亚基因组RNA的5′、3′末端之间也存在与其他Necrovirus成员类似的“茎-环”结构,环状区域的核苷酸序列高度保守,并且反向互补。为了认识在这种RNA“茎-环”结构之间可能存在相互作用,我们对其进行了初步地突变分析。结果表明,“茎-环”结构中“环”部核苷酸种类的保守性、以及“茎”部的碱基配对程度对于维持病毒正常复制所必需的RNA结构至关重要。更多还原

【Abstract】 Based on the full-length infectious cDNA pUBF52 of Beet black scorch virus （BBSV）, reverse genetic analysis was carried for gene functions and expression strategy of BBSV by means of in vitro transcription of mutant viral RNAs, GFP labeled expression. By mutation of nucleotide substitution, frame-shift and deletion, it was proven that the P22 and P82 at the 5’ proximal end of BBSV genome were both involved into its replicase （RdRp） essentially. By site-directed mutagenesis of translational start codons or premature, the four small ORFs including P5, P7a, P7b and P5’ that are centrally located in the viral genome by computer prediction were analyzed for their functions during BBSV infection. The results showed that, except the P5, other three ORFs of P7a, P7b and P5’ were coordinately responsible for the local lesion symptoms and viral RNA accumulation after BBSV infection of Chenopodium amaranticolor.For fluorescent expression in plant cells, a GFP gene was fused BBSV RNAs as a substitute of the C? in the each mutant of P7a, P7b and P5’. In contrast to the infection by wild type BBSV labeled with GFP from pBGFP, in which the GFP was observed both on nuclei and cell membranes in the cells around the initially infected cell, GFP expressions of mutants P7a, P7b and P5’ were limited in the nuclei of the original infected cell. Combining with the previous report on BBSV coat protein gene, these results indicated that total six proteins were possibly encoded by BBSV genome, including the RdRp components of P22 and P82, the P7a, P7b and P5’ proteins associated with BBSV movement from cell-to-cell, and the P24 coat protein, among which the P5’ is reported first time in Necroviruses.In further investigation, a strategy for the mRNA expression of BBSV multi-cistron genome was revealed with two subgenomic RNA （sgRNA） in BBSV particles or C. amaranticolor infected by the in vitro transcripts. By 5’ -RACE and mutagenesis of infectious cDNAs, the transcription start sites of sgRNAl and sgRNA2 were determined as G²²⁰⁹ or G²⁵²⁶, respectively. So that, the sgRNAl is responsible for protein expressions of P7a, P7b and P5’ and the sgRNA2 is for the coat protein.Mutations of nucleotides from position 2204 to 2213 nt around G²²⁰⁹ and 2521 to 2530 nt around G²⁵²⁶ showed that mutation of C²²⁰⁶, or any of C²⁵²¹, C²⁵²³ and C²⁵²⁴ would abolish the form of sgRNAl or sgRNA2, respectively. The following mutations of G which complement with these C in the second structure of BBSV and corresponding base pair showed that the base pairing is more important than these C themselves. According to the secondary structure of BBSV RNA by computer analysis, the results from mutations of the base-pairings of C²²⁰⁶-G²¹¹⁸, C²⁵²¹-G²³⁸⁰, C²⁵²³-G²³⁷⁸ and C²⁵²⁴-G²³⁷⁷ indicated that the predicted stem-loop structures upstream from the two transcription start sites played important roles on the RNA transcription. The nucleotide deletions around the sgRNAs start sites provided the further evidences that the promoter region for the sgRNA2 perhaps ranged from 2339 nt to 2558 nt as a stable stem-loop complex form. Since a similar structure also was found in the sgRNAl, itwas speculated that the mechanism of premature termination was employed in transcription of BBSV sgRNAs.Similar to other Necroviruses, the stem-loop structures involved in 5’ -3’ RNA-RNA interaction were also found in the proximal ends of either BBSV genomic RNA or the subgenomic RNAs, in which the nucleotide sequences in the loop regions were highly conserved and reversely complementary. In order to know the possible interaction between these stem-loop structures, mutagenesis analysis was carried out. The preliminary results showed that the conservation of nucleotide sequence in the loop region and the base-pairing in the stem of the stem-loop structures were necessary to keep the correct RNA folding essential for the virus replication.更多还原