TMV病毒载体表达重组rPA致受体本氏烟叶片坏死的初步分析

Causes of Leaf Necrosis of Nicotiana benthamiana While Expressing Recombinant rPA by TMV-based Expression Vectors

重组rPA是临床上治疗血栓性疾病的主要溶栓药物。利用烟草花叶病毒(Tobacco mosaic virus,TMV)表达载体在本氏烟(Nicotiana benthamiana)中表达重组rPA会致使接种叶片严重坏死,受体细胞坏死会影响重组产物的积累、定量和下游纯化,剖析坏死症状产生原因可为提高外源基因在植物中表达提供指导。分别从替换TMV病毒载体携带的外源基因和更换TMV病毒表达载体两个方面入手,通过比较叶片接种部位的表型变化和重组产物在叶片中的积累情况,初步解析致接种叶片坏死的主要因素。结果表明,农杆菌菌株和RNA沉默抑制子与本氏烟叶片坏死症状的产生无关联;TMV病毒载体自身不是坏死症状产生的直接原因;外源重组rPA对受体本氏烟细胞无细胞毒性。推测TMV病毒载体与重组rPA相互作用是引起本氏烟接种叶片细胞坏死的主要原因。

Recombinant plasminogen activators(rPA)is the main drug for clinical treatment of thrombotic diseases.Production of rPA by TMV(Tobacco mosaic virus)based expression vectors caused severe necrosis on the inoculated leaves of Nicotiana benthamiana.Leaf necrosis may affect the accumulation,quantification and downstream purification of recombinant rPA.Therefore,exploration of the causes of leaf necrosis may provide guidance for improving the expression of rPA in plants.In this study,the main factors leading to leaf necrosis were preliminarily analyzed by comparing the phenotypic changes of leaf inoculation sites and the accumulation of recombinant products in leaves,starting from the replacement of exogenous genes carried by TMV virus vectors and the replacement of TMV virus expression vectors.The results indicated that Agrobacterium and RNA silencing suppress or were not closely related to leaf necrosis.Otherwise,TMV-based expression vector was not strictly related to necrosis symptoms.Additionally,overexpression of rPA was not accompanied by cytotoxic effects and induced leave senescence.We concluded that interaction between TMV-based vector and recombinant rPA is the main cause of cell necrosis in inoculated N.benthamiana leaves.