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为了解大豆蚜(Aphis glycines Matsumura)传播的大豆花叶病毒(soybean mosaic virus,SMV)与其体内共生菌产生的病毒结合蛋白(Gro EL)之间的作用机制,运用RT-PCR技术克隆得到大豆蚜内共生菌groEL基因全序列,把该基因与pET21b载体重组后进行原核表达,经Ni-Agarose His亲和层析得到纯化的蛋白,并利用荧光定量PCR技术分析了饲毒不同时间、不同龄期大豆蚜内共生菌gro EL基因表达量的变化。序列分析表明:大豆蚜内共生菌groEL基因全长序列为1 647 bp,编码548个氨基酸,推测蛋白分子量和pI值分别为69 k Da和5.24,成功构建重组载体pET21bGro EL进行原核表达,Western-blot鉴定确定为目的蛋白,蛋白可溶性分析发现重组蛋白为包涵体。随饲食SMV时间的延长大豆蚜内共生菌groEL的表达量显著增加,且无翅成虫与有翅成虫内共生菌groEL的表达量显著高于其它4个若虫期。大豆蚜内共生菌groEL基因能在原核细胞中稳定、正确表达,并且大豆蚜在饲食SMV后会诱导其内共生菌gro EL的表达,从而增加SMV爆发流行的可能性。
In order to understand the mechanism of action between soybean mosaic virus (SMV) transmitted by Aphis glycines Matsumura and virus-binding protein (Gro EL) produced by symbionts in vivo, soybean aphid was cloned by RT-PCR The endosymbionts groEL gene was sequenced. The recombinant plasmid was prokaryotic expressed after recombination with pET21b vector. The purified protein was purified by Ni-Agarose His affinity chromatography. Fluorescent quantitative PCR (PCR) Changes of gro EL gene expression in soybean aphids. Sequence analysis showed that the full-length sequence of groEL gene of soybean aphid was 1 647 bp encoding a protein of 548 amino acids. The predicted molecular weight and pI were 69 kDa and 5.24, respectively. The recombinant plasmid pET21bGro EL was successfully constructed for prokaryotic expression. Western- blot identification identified as the target protein, protein solubility analysis found that the recombinant protein inclusion body. The expression of groEL in soybean aphid significantly increased with the time of feeding SMV, and the expression of groEL in wingless and winged adults was significantly higher than that in the other 4 nymphs. Soybean aphid endophyte groEL gene in prokaryotic cells stable and correctly expressed, and the soybean aphid feeding SMV will induce its endophyte gro EL expression, thereby increasing the possibility of SMV outbreak epidemic.