Structural and functional alayses of the plant viral RNA polymerases for the breeding of virus-resistant transgenic plants.

1.利用烟草花叶病毒183K蛋白的反义RNA聚合酶Domein，通过亲和层析成功地从侵染TMV的烟草叶提取物中纯化出具有活性和模板特异性的TMV RNA聚合酶。推测该脱辅酶是由126K和183K两个蛋白质分子组成的异源二聚体。通过酵母双杂交系统检测到与TMV RNA聚合酶解旋酶Domein结合的三种不同宿主蛋白。黄瓜花叶病毒(CMV)和花生矮化病毒(PSV)的基因组均由RNA1、RNA2和RNA3三个单链RNA组成，RNA1和RNA2分别编码1a蛋白和2a蛋白是病毒复制所必需的。利用CMV RNAs和PSV RNAs的感染性RNA转录本，重新验证了RNA1和RNA2互换的可行性。当我们将所有的假重组子共同接种到烟草原生质体中时，含有PSVRNA1和CMVRNA2的伪重组体是可复制的，而含有CMVRNA1和PSVRNA2的伪重组体是可复制的更多的2人没有。利用酵母双杂交系统，PSV-1a蛋白的C末端区域不仅与PSV 2a蛋白的N末端区域相互作用，而且与CMV 2a蛋白的N末端区域相互作用。这些结果与原生质体复制的结果相关联。利用小麦土传花叶病毒(SBWMV)RNA1和RNA2的全长感染性克隆，构建了一系列152K和211K复制酶蛋白的RNA1突变体。结果表明，含有甲基转移酶、解旋酶和聚合酶结构域的211K蛋白，在不具有前两个结构域的情况下，足以满足病毒的低水平复制。152K蛋白和211K蛋白一起被证明是高水平RNA复制所必需的。在RNA2的3‘末端编码一个富含半胱氨酸的蛋白是病毒复制必不可少的。对一株美国株进行了感染性克隆的构建。美国RNA1和日本RNA2的重组实验表明，日本RNA1和美国RNA2的组合对小麦植株具有系统侵染性，而美国RNA1和日本RNA2的组合即使对局部病斑寄主藜麦也不具侵染性。较少

1. Active and template-specific tobacco mosaic virus (TMV) RNA polymerase was successfully purified from TMV-infected tobacco leaf extracts through an affinity chlomatography using anti-RNA polymerase domein of TMV 183K protein. The apoenzyme was presumed to be a heterodimer composed of each one molecules of 126K and 183K proteins. While, three different host proteins bound to helicase domein of TMV RNA polymerase were detected by the yeast two-hybrid system.2. The genome of both cucumber mosaic virus (CMV) and peanut stunt virus (PSV) consists of three single-stranded RNAs, RNA 1, 2 and 3. 1a protein and 2a protein encoded by RNA1 and RNA2, respectively, are necessary for the viral replication. Feasibility of exchanges of RNAs 1 and 2 was re-examined by using infectious RNA transcripts for CMV RNAs and PSV RNAs. When we coinoculated all the pseudorecombinants into tobacco protoplasts, pseodorecombinants with PSV RNA 1 and CMV RNA 2 were replicable, but those with CMV RNA 1 and PSV RNA … More 2 were not. Using yeast two-hybrid system, the C terminal region of PSV-1a protein was exhibited to interact with not only the N terminal region of PSV 2a protein but also that of CMV 2a protein. These results were correlated with those of replication in protoplasts.3. Using full-length infectious cDNA clones to RNA 1 and RNA2 of Soil-borne wheat mosaic virus (SBWMV), a series of RNA 1 mutants of 152K and 211K replicase proteins were constructed. Results showed that the 211K protein with methyltransferase, helicase and polymerase domains in the absence of the 152K protein possessing the former two domains is sufficient for virus replication at a low level. The 152K protein together with the 211K protein were shown to be required for the high-level RNA replication. Expression of a cysteine-rich protein encoded in the 3'-terminal region of RNA2 was absolutely required for virus replication. Infectious cDNA clones to a US strain were constructed. Reassortment experiments between US and Japanese strains showed that the combination of Japanese RNA1 and US RNA2 was systemically infectious to wheat plants whereas that of US RNA1 and Japanese RNA2 was not infectious even to Chenopodium quinoa, a local lesion host. Less