小麦黄花叶病毒RNA1和RNA2体外转录物对小麦愈伤细胞侵染.pdfVIP

小麦黄花叶病毒 RNA1 和 RNA2 的体外转录物对小麦愈伤细胞的侵染 In vitro transcription of WYMV full-length genomic cDNAs and infection of wheat cells from cultured calli 董家红，尚巧霞 朱坤 翟亚锋 韩成贵 于嘉林（通讯作者） 中国农业大学农业生物技术国家重点实验室，北京 100094 摘要 小麦黄花叶病毒（Wheat yellow mosaic virus，WYMV ）由两条线性、单链、正义RNA 组成，RNA1 全长约 7.6kb，RNA2 长约 3.6kb，分别编码一个 270kDa 和 100kDa 的多聚蛋 白。为从分子水平上认识小麦黄色花叶病毒的侵染和复制等机制，在已有研究基础上，利用 5′ RACE 方法确认了小麦黄色花叶病毒（WYMV ）基因组的5′末端序列，构建了 T7 启动子 驱动下的病毒基因组全长 cDNA 克隆。体外转录结果表明所获得的 cDNA 克隆可以产生相 应的 WYMV RNA1 和 RNA2 。用电击穿孔法将体外转录的 WYMV RNAs 接种小麦品种鄂恩 1 号（Triticum aestivum L ，cv. Een. No.1 ）愈伤组织细胞。对被接种细胞进行的Western blot 和 Northern blot 检测结果表明，体外合成的 WYMV RNAs 可以在小麦细胞内复制、表达， 由此证明所构建的WYMV cDNA 克隆具有侵染活性。 关键词 小麦黄花叶病毒 体外转录物 小麦愈伤细胞 侵染性 Abstract: Wheat yellow mosaic virus (WYMV) transmitted by Polymyxa graminis widely occurring in the middle and lower valleys of Yangtze and Huai Rivers in China, where it caused severe yield loss of winter wheat in recent years. The genome of WYMV consists of two plus-strand RNA components, among which the RNA1 is 7644 nucleotides encoding a polyprotein of 270 kDa and the RNA2 comprises 3651 nucleotides coding for a 100 kDa polyprotein. After confirmations of the 5’termini of genomic RNA1 and RNA2 of the Huangchuan isolate (WYMV-HC) by 5`RACE, full-length genomic cDNAs driven by T7 promoters were constructed for in vitro transcriptions of RNA1 and RNA2, respectively. The in vitro transcribed WYMV RNAs were used for inoculation of wheat cells from cultured calli by electroporation. Western and Northern blot analysis revealed that these full-length WYMV RNA transcripts were infectious in the inoculated wheat cells, suggesting an established infection system for further definition of the functions of WYMV genes. Keywords: WYMV, full-length cDN