【作者】 许雷；

【导师】 成卓敏；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2001， 博士

【摘要】 将已经测定的GPV株系ORF5部分基因序列与RPV株系的序列进行比较，根据ORF5起始区域的核苷酸序列合成上游引物，以RPV ORF5基因框架3’外侧核苷酸及编码氨基酸序列作为参考，设计3’简并引物，以GPV ORF5基因组RNA为模板，RT—PCR扩增GPV ORF5基因片段，用pGEM-T载体克隆扩增片段。对克隆基因片段进行序列分析结果表明，ORF5全长1326个核苷酸，可编码441个氨基酸、分子量为50kD左右的蛋白质。这是首次获得GPV株系ORF5基因完整序列。 与其他黄症病毒ORF5基因核苷酸序列进行比较的结果显示，GPV与RPV的同源性最高，同源性达68.1%；与马铃薯卷叶病毒（PLRV）和甜菜西方黄化病毒（BWYV）的同源性次之，分别为33.6%和39.8%，与PAV、SGV、GAV、MAV的同源性分别为30.2%、28.8%、28.5%和29%。根据核苷酸序列推测出ORF5基因编码的氨基酸序列，对GPV株系与本组其它病毒的氨基酸序列等进行比较，结果显示GPV与RPV的同源性最高，为72.4%，与PLRV、BWYV的同源性次之，分别为31.4%和40.5%，与PAV、SGV、GAV、MAV的同源性最低分别为、25.6%、22.6%、25.1%、26.5%。根据最新的植物病毒分类研究进展，RPV、PLRV和BWYV均属于黄症病毒科的Polerovirus属，根据ORF5基因的序列分析和基因序列同源性比较结果，初步推测分离自中国小麦黄矮病株上的GPV株系在分类上属于黄症病毒科的Polerovirus属。 对由GPV ORF5和RPV ORF5基因核苷酸序列推测出的氨基酸片段进行氨基酸组成、部分理化性质及计算机模拟的蛋白结构模块进行比较。结果显示尽管GPV ORF5与RPV ORF5存在同源性，但两者在氨基酸片段长度、氨基酸组成、部分理化性质及蛋白结构模块的组成和分布上有较大的差异。 大麦黄矮病毒GPV株系Ohs基因的克隆、表达载体构建及转基因研究 根据测定的基因序列，分别设计合成含有酶切位点的ORFS的5’端 （含Xbl和起始密码AUG）和3’端（含K列）引物，通过RTICR方法扩增Ogys基因片段，将基因片段经过Xbopnl双酶切后插入到pGEM—3Z质粒中，兰白斑筛选重组质粒，提取质粒，Hindlll酶切、ienow酶补平、EcoAI二次酶切，切下带粘平端的ORFS基因片段，插入经Ndel酶切冲平／EcoM二次酶切的原核表达载体 pET6 中，筛选重组质粒，完成Ogys基因片段的非融合原核表达载体的构建。重组质粒转化进受体菌 BL21（DE3），IPTG诱导 GPV ORFS基因的非融合表达，SDS—PAGE分离表达产物，发现诱导后产生一个50KD左右的蛋白组份，与根据基因序列推导出的蛋白质分子量相近，western杂交分析表明，该诱导出的蛋白可与GPV发生血清学反应。 RT—PCR扩增的ORFS基因片段用Xbal／Knl双酶切后插入到带相同双酶切位点的含有Emu启动子和Nos终止子的质粒pEmu－mes－N之中，构建成用于小麦转化的植物表达载体 pPPI（ORFS重组质粒人利用花粉管通道方法，与含有Bar基因的质粒pACH—20进行双质粒共转化，转化品种选自我国小麦黄矮病高发区的高产、优质、不抗病的小麦品种陇鉴127、晋麦47，每个品种转化小花数均在1000个以上，收获种子数分别为74粒和sl4粒。 播种转化种子，提取小麦总DNA，进行PCR检测，结果得到3株阳性的 T。代植株，2株为陇鉴 127、l株为晋麦 47。对阳性植株的 DNA进行PCR、PCRSouthem、Southern分析，均获得阳性的杂交结果。表明转基因小麦后代中存在看己整合进染色体的GPV株系ORFS基因。更多还原

【Abstract】 According to the results of comparison between partial sequence of GPV and the sequences of other Luteoviruses , synthesized the oligoonucleotide which complementation the sequence of GPV ORF5 S抋s upstream primer According to the RNA sequence and encode amino acid sequence of out of ORF5 downstream , designed and synthesized annex downstream primer. Fragment of ORF5 was amplified by RT-PCR with the down-and up-stream primers. Then fragment was inserted into pGEM- T Vector, and the recombinants of pGEM- T 桹RF5 was obtained. Sequence analysis shows that GPV ORF5 contains 1326 nucleotides which can code a protein of 441 amino acids with Mr about 50 kD .Compared with other Luteoviruses, there is a highest homohlogy between GPV and RPV, the similarity is 68.1% . The nucleotide similarity of ORF5 between GPV and another BYDV such as BWLV , PLRV, PAY, SGV, GAV, MAV is 39.8%, 33.6%,30.2%~ 28.8%.~ 28.5% and 29%~According to the sequence of BYDV ORF5 gene, deduced the amino acid. Compare the amino acid of ORF5 gene between GPV and another isolates of Luteoviruses in sequence, compositions character , structuralIII)c~*~4 GPV&~ ORF5~~~ ~block . The results showed that in amino acid sequence of ORF5 there is a highest homology between GPV and RPV, the similarity is 72.4% . The amino acid similarity of ORF5 between GPV and another BYDV such as BWLV , PLRV, PAY, SGV, GAV, MAy is 40.5%, 31.4%, 25.6%~ 22.6 %.. 25.1% and 26.5%.The results also showed that in amino acid composition, character structural block, there are notability different between the GPV ORF5 andRPV ORF5.Based on the sequence of ORF5, primers for ORF5 were designed and the ORF5 gene was amplified by RT-PCR, and cloned into pGEM-3Z. By the restriction enzyme digesting /fill protrudling end! restriction enzyme digesting,the fragment having one blunt end and one protruding end of ORF5, was inserted into prokaryotic expression vector(pET-5a), the ORF5 gene was expressed without a fusion peptide with IPTG induce. The result of SDS-PAGE showed that ORF5 expressed about 5OKD protein. Western blotting indicated that the expression protein of ORF5 could be detected with antiserum of GPV.Plant expression plasmids of pPPI 15(containing ORF5) were obtained by inserting the fragments into pEmu-mcs-N plasmid. Longjian 127 and Jinmai 47 were transformed with pPPI 15 via the pollen tube pathway. The seeds harvested were detected by PCR analysis and southern blot hybridization. Three positive transgenic lines of transgene wheat generation were obtained. PCR and Southern-blotting analysis showed positive result.更多还原