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
首发时间:2006-11-14
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。
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In vitro transcription of WYMV full-length genomic cDNAs and infection of wheat cells from cultured calli
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 cDNA, in vitro transcription, infection of wheat cells
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