Studies on the utilization of useful genes isolated from the wild plant for control of virus diseases

克隆并测定了无花果核糖体失活蛋白(Cf-RIP)的基因序列。该基因全长包含一个开放阅读框架，编码286个氨基酸，与核糖体失活蛋白(RIPs)的保守残基相同。Cf-RIP与其他葫芦科的4个RIP序列同源性为54.8-64.5%。RT-PCR分析表明，Cf-RIP mRNA在无花果叶片和茎组织中均有表达。从无花果叶片中分离纯化了一种抗病毒蛋白，命名为Figaren，并对其进行了部分鉴定。柱层析、SDS-PAGE和高碘酸-席夫染色表明，该蛋白是一种糖蛋白，相对分子质量为23 kDa。N-末端氨基酸序列分析表明，该序列含有S等位基因相关核糖核酸酶的保守区。凝胶内核糖核酸酶分析表明，无花果能消化酵母菌RNA。荧光原位杂交显示，1μg/ml的Figaren和核糖核酸酶(牛肉胰酶和核糖核酸酶T1)对C…的抑制作用相似豌豆叶片中MV侵染较多。5-500 ng/mlFigaren和RNase对CMV的局部病斑形成有相似的抑制作用。在接种CMV前6小时或0小时，用Figaren处理的烟草叶片中CMV的积累被抑制。用无花果处理烟株上部叶，10min后接种CMV，多数烟株花叶症状延迟5~7d，部分植株仍无症状。在接种CMV和随后接种无花果的甜瓜原生质体中，CMV RNA和CMV抗原的数量随着时间的推移而增加，对照的酶联免疫吸附试验和局部病斑检测表明，在接种CMV前5~10min用无花果处理的烟草、甜瓜、豌豆和藜菜叶片上表面的CMV感染几乎完全被完全抑制。菲格伦几乎完全抑制了未处理的烟草叶片上的CMV感染。无花果还诱导烟草植株产生局部获得性抗性。较少

A cDNA coding a putative ribosome-inactivating protein(CF-RIP) from Cucunis figarei was cloned and sequenced. The entire cDNA contains an ORF encoding a protein of 286 amino acids which share conserved residues for ribosome-inactivating proteins(RIPS). CF-RIP share 54.8-64.5% identity with four RIPs from other cucurbits. RT-PCR analysis indicated that CF-RIP mRNA was expressed in leaf and stem tissues of Cucumis figarei.An antiviral protein, designated figaren, was purified from leaves of C.figarei and partially characterized. Column chromatography, SDS-PAGE and periodic acid-Schiff staining revealed that figaren is a glycoprotein with a molecular weight of 23 kDa. N-terminal amino acid sequencing indicated that figaren contained the conserved region for the S-allele-associated ribonucleases(RNases). In-gel RNase assay showed that figaren digested yeast RNAs. Fluorescence in situ lybridization revealed that figaren and Rnases(beef pancrease and RNase T1)at 1μ g/ml similarly inhibited C … More MV infection in cowpea leaves. Figaren and Rnases at 5-500ng/mlhad similar inhibitory effect on local lesion formation by CMV.CMV accumulation detected by ELISA in tobacco leaves treated with figaren 6 or 0 hr before inoculation with CMV was suppressed. When upper leaves of tobacco plants were treated with figaren and inoculated 10 min later with CMV, mosaic symptoms were delayed for 5-7 days on most of tobacco plants, and some plants remained asymptomatic. The amount of CMV RNAs and CMV antigen in melon protoplasts inoculated with CMV and subsequently inoculated with figaren similarly increased with time as did that the control ELISA and local lesion assays indicated that CMV infection on the upper surfaces of the leaves of tobacco, melon, cowpea and Chenopodium amaranticolor whose lower surfaces had been treated with figaren 5-10 min before CMV inoculation was almost completely inhibited. Figaren almost completely inhibited CMV infection on the untreated halves of leaves of tobacco. Figaren also induced localized acquired resistance in tobacco plants. Less