马铃薯Y病毒辅助成分蛋白酶与加工番茄OPA3-like蛋白结合及其定位研究

The Research on Combination and Its Location between Helper Component Proteinase of Potato Virus Y and Processing Tomato OPA3-like Protein

为了研究加工番茄OPA3-like蛋白与马铃薯Y病毒(Potato virus Y,PVY)的辅助成分蛋白酶(helper component proteinase,HC-Pro)的相互作用,进行了2种蛋白的结合及其定位研究。通过PCR的方法从加工番茄中克隆OPA3-like基因,同时利用Blast方法对其分析,进行了OPA3-like蛋白基因氨基酸序列同源性分析。通过构建双分子荧光互补载体pSPYCE-35S-OPA3-like和pSPYNE-35S-HC-Pro,利用农杆菌介导法共侵染洋葱表皮细胞60 h后,在共聚焦显微镜下观察洋葱表皮细胞中的黄色荧光。结果显示:加工番茄OPA3-like基因ORF长510 bp,编码169个氨基酸,编码蛋白属OPA3蛋白家族。激光共聚焦显微镜下可观察到沿细胞膜呈小泡状分布黄色荧光,说明PVY的HC-Pro蛋白与加工番茄OPA3-like蛋白结合并且共定位在细胞膜附近。这为进一步研究PVY的HC-Pro蛋白与加工番茄OPA3-like蛋白的相互作用机制奠定了一定的基础。

To study interaction between OPA3-1ike protein in processing tomato and helper component proteinase （HC-Pro） of Potato virus Y （PVY）,the combination of the two proteins and their cell locations were studied in this research.The OPA3-like gene from processing tomato was cloned by PCR method,and the deduced amino acid sequence was analyzed by blast. Bimolecular fluorescence eomplementation （BiFC） vectors pSPYCE-35S-OPA3-like and pSPYNE-35S-HC-Pro were constructed, and then transformed into Agrobacterium tumefaciens.After onion epidermal cells were infected with the Agrobacterium- mediated method for 60 h,the yellow fluorescence was detected by confocal laser microscopy.The results showed that OPA3- like gene has a 510 bp open reading frame （ORF） which encoding 169 amino acids.Phylogenetic analysis showed this homology belonged to OPA3 protein family.The yellow fluorescence was observed in some vesicles distributed along the cell membrane in onion epidermal cells.Our research revealed that HC-Pro of PVY could combine with OPA3-like protein in processing tomato,and the sites of combination were located near the cell membrane.This result will lay the foundation for the further studying proteins interaction mechanisms of the HC-pro and OPA3.