Construction Of Soybean Bioreactor Mediated By Soybean Mosaic Virus And Expression Of Herbicide Resistant Protein

Plant bioreactor is the most economical bioreactor,which can provide safe production system and complete eukaryotic protein modification site for exogenous protein expression.However,the experimental period of this system is long and the expression of exogenous protein is low;But plant bioreactor mediated by virus carrier can shorten the test period and increase the expression of exogenous protein at the same time.Soybean Mosaic Virus(SMV)widely spread all over the world,and at present the research of the Virus is mainly concentrated in gene function and disease resistance breeding,etc.,at the same time the SMV gene function and the corresponding protein products are generally clear,but the SMV as the carrier mediated plant bioreactor research are relatively few.In the early stage,the whole gene sequencing of Soybean Mosaic virus northeast No.3 strain was carried out in our laboratory,and the infectious cloning of this strain was constructed.In this experiment,we modified the whole genome infectious clone(SMV3)of Soybean Mosaic virus northeast no.3 strain,in weakened its pathogenicity and did not affect the force of infection at the same time to facilitate the purpose gene insertion and fusion protein expression of the corresponding,and inserted into the restriction enzyme digestion in SMV3 genome site AvrII and Soybean Mosaic Virus protease site,thus build the mediated soybean plant bioreactor used in this experiment the restructuring of the carrier SMV3-2;In addition,because the Bar Gene(Bialaphos Resistance Gene)is one of the commonly used selective marker genes,the PAT protein encoded by this Gene has good herbicidal resistance characteristics and can be used for the detection of subsequent experiments,SMV3-2-bar recombinant vector was constructed in this study and transformed into soybean plants.RT-PCR?ELISA and Western-blot assays were used to detect the expression of the target gene and the corresponding protein respectively,the results showed that the SMV3-2-bar recombinant virus was successfully transferred into soybean plants and PAT protein was expressed;Through the analysis of the PAT protein expression trend,it was found that the PAT expression of the target protein was the highest in the sixth week after transformation;At the same time,the biological test results of soybean showed that PAT protein could give full play to herbicide resistance;The stability analysis results showed that the target gene transformation was accurate and the target protein expression was complete.To sum up,in this experiment,SMV3-2 carrier was constructed and its mediated soybean plant bioreactor was preliminarily explored.Meanwhile,functional verification and stability analysis of the system were conducted.The results showed that the system could not only express the target protein completely,but also had good stability.The results also lay a foundation for the study of gene function and new plant bioreactor.