水稻齿叶矮缩病毒P8蛋白的抗体制备及其在水稻原生质体内的表达动态

Preparation of polyclonal antibody against P8 protein of rice ragged stunt virus and its expression dynamic trends in rice protoplast

水稻齿叶矮缩病毒(rice ragged stunt virus,RRSV)属于呼肠孤病毒科(Reoviridae)水稻病毒属(Oryzavirus)成员。该病毒引起的水稻齿叶矮缩病在我国南方发病严重,成为我国水稻生产上的重要病害。通过建立水稻原生质体培养体系,对RRSV主要外壳蛋白P8在水稻原生质体内的复制与表达情况进行研究。通过原核表达对P8蛋白进行抗体制备,抗血清经Western印迹检测具有特异性,间接ELISA测得其效价为2 500倍左右。以制备的抗血清为探针,通过Western印迹检测到P8蛋白在病毒侵染原生质体后24h开始表达,48h左右达到最大值,60h后开始下降,但仍保持在较高水平;同时,利用荧光定量PCR技术,对病毒侵染水稻原生质体后的P8RNA含量进行检测,结果表明,P8RNA在8h时开始积累,32h左右达到最大值,48h后表达量开始下降到一个平台期。

Rice ragged stunt virus （RRSV） is the member of Oryzavirus belonging to Reoviridae. Rice ragged stunt disease a-roused by RRSV poses a serious threat to rice production in south China and becomes an important rice disease. By establishing a rice protoplast culture system, the replication and expression of RRSV main outer protein P8 were studied. By prokaryotic ex-pression, polyclonal antibody against P8 protein was prepared, and the specificity was determined by Western blotting analysis, meanwhile, an indirect ELISA measured the titer at about 2 500 times. Using the prepared antiserum as a probe, Western blot-ting analysis shows that P8 gene begins to express at 24 h after virus infection, and reachs the maximum at about 48 h. The ex-pression of P8 protein begins to decline after 60 h, but remains at a high level. Meanwhile, a real time PCR was used to detect the amount of P8 RNA in rice protoplasts after virus infection The results show that P8 RNA begins to accumulate at 8 h, and reachs a maximum at about 32 h. The expression of P8 RNA declines to a platform stage after 48 h.