苜蓿花叶病毒复制酶基因的克隆、序列分析及其植物表达载体的构建

Molecular Cloning and Nucleotide Sequence of Replicase Gene from Alfalfa Mosaic Virus Chinese Isolate

以侵染苜蓿的苜蓿花叶病毒中国分离株(AlfalfamosaicvirusChineseisolate,A1MV-Ch)RNA2为模板,利用人工合成的特异性引物,进行反转录及PCR扩增,分两段分别扩增了复制酶基因的5′端1.32kb和3′端及其非编码区的1.23kbcDNA序列.用限制性内切酶切割PCR产物后,与pUC19重组,转化大肠杆菌DH5α,筛选重组质粒,用限制性内切酶分析及PCR鉴定,得到分别含有5′端序列和3′端序列的重组质粒,已由上述两种重组质粒构建了含有完整复制酶基因的重组质粒.进行了全序列测定,并与国外报道的A1MV-425株系的相应序列相比较,其复制酶基因编码区核苷酸序列同源性达97.8%,推测的氨基酸序列的同源性达97.6%,3′端非编码区核苷酸序列同源性为98.2%.并将全长复制酶基因与植物表达载体pROK 重组,得植物转化载体pAlMV-FL.

Alfalfa mosaic virus Chinese isolate (AlMVCh) was isolated and purified from infected cultivated alfalfa.The two fragments of the cDNA from AlMVCh replicase (RNA dependent RNA polymerase, RdRp) gene was synthesized respectively by reverse transcription and followed by PCR amplification with synthetic oligonucleotide as primers using AlMVCh RNA2 as a template. The amplified 1.2 kb and 1.3 kb fragments covered the complete replicase gene consisting of 2593 nucleotides.The two fragments were cloned into pUC19 vector respectively.The recombinant plasmids were analyzed by PCR and restriction enzyme mapping.The both end sequences of each cloned cDNA fragment have been determined and compared with the sequence of AlMV Netherlandish isolate published abroad.The homology of the nucleotide and deduced amino acid sequences of the replicase gene between the AlMV Chinese isolate and Netherland isolate are 97.8% and 97.6% respectively.Furthermore,the fulllength replicase gene was recombinated with the plant expression vector pROKⅡ and the plant transforming vector pAlMVFL was obtained.