Rt-Lamp Detection Of Bean Pod Mottle Virus In Soybean Seeds And Construction Of Soybean Mosaic Virus Expression Vector

Soybean is an important leguminous vegetable of the pea family that consists of large amounts of protein, carbohydrates, and oil and excellent amounts of dietary fiber, vitamins, and minerals. Therefore, soybean is an important source of the feed, oil and protein and it is also an important crop for raising soil and maintaining the ecological balance. Around the soybean, this study carried out on the research of rapid detection technology of soybean virus disease and the construction of plant virus expression vector for the disease preventive and gene function research.RT-LAMP detection of BPMV:Bean pod mottle virus (BPMV) is a catastrophic virus easily causing huge economic losses and it widely distributes in many soybean-producing areas, and it is a quarantine virus in China. This study established a one-step RT-LAMP detection technology that could rapidly detect the BPMV in soybean seeds by the combination of RT-LAMP and rapid RNA extraction. This technique included the primers and the optimized reaction system. It could make a rapid, specific and accurate detection of the BPMV in soybean seeds and without cross reaction. The optimum operating temperature of this technology was62.5-65CC. The templates range of this technology was0.2-1.8μL/10μL. This technology was1000times more sensitive than BPMV RT-PCR. So this technology has its potential applications in inspection and quarantine work, as well as prevention of BPMV pandemic.Construction of SMV expression vector:Although several expression vectors of different strains of Soybean mosaic virus (SMV) have been reported, the genomes of these SMV are mainly about9585bp, there is no SMV expression vector about the recombinant SMV strains with the genome of about9994bp. The recombinant SMV strain was identitied in Nanjing in2011. The5’ terminus sequence of genome of the recombinant SMV is a reorganization of Bean common mosaic virus (BCMV) and SMV. Therefore, there is a significantly difference in the5’terminus and a few difference in the3’terminus between the recombinant SMV and the common SMV. This study constructed a chimeric recombinant SMV expression vector by recombining the5’ terminus of the recombinant SMV with the3’terminus of the common SMV. When the β-glucuronidase gene (GUS) was inserted between the PI and the HcPro of the SMV vector, GUS staining was specifically detected on the system leaves of the soybean inoculated with vector plasmid after30days. Meanwhile, GUS staining was also specifically detected on the system leaves of the Nicotiana benthamiana injected by agrobacterium with the SMV vector. Therefore, the chimeric recombinant SMV expression vector constructed in this study can be used as a technology of gene function study and play its potential value in gene function studies of soybeans, Nicotiana benthamiana and their pathogens, and obtaining foreign proteins.This study has successfully established the one-step BPMV RT-LAMP detection technology and SMV expression vector system that provided new techniques and tools for the soybean disease prevention and the gene function study.