Abstract:   【Objective】Soybean mosaic virus (SMV) is one of the most prevalent viral diseases in major soybean growing areas of China and cause significant yield losses and quality deterioration of soybean seeds. Ribonuclease PAC1 was known to bind and degrade the dsRNA molecules generated during plant RNA viruses or viroids replication, thus effectively inhibited replication and accumulation of viruses or viroids in host plants. This feature of PAC1 provides an effective target gene for the creation and cultivation of the broad-spectrum anti RNA virus and viroid transgenic crops. The objective of this study is to introduce a PAC1 derived from Schizosaccharomyces pombe into soybean by transgenic technique, research the effects of over-expression of PAC1 onSMV resistance of soybean, and to provide a basis for the development of SMV-resistant soybean cultivars. 【Method】 The PAC1 was subcloned into the binary expression vector pCAMBIA3300 by restriction enzyme digestion and ligation to generate recombinant construct pCAMBIA3300-PAC1. In the resulting construct, the PAC1 was located between the constitutive promoter CaMV 35S and the terminator NOS. The construct also contained a BAR which confers resistance to the herbicide phosphinothricin (PPT). The soybean cultivar Williams82 was used for Agrobacterium-mediated transformation. Based on the detection of LibertyLink^® strip, PCR and herbicide spray (500 mg·L^-1 Basta), the integration and copy numbers of the exogenous gene in transgenic lines were further analyzed by Southern blot hybridization. Mechanical rub inoculation was performed to evaluate the resistance of the T₂ and T₃ transgenic lines and agronomic traits were analyzed under field conditions. Furthermore, the accumulation of SMV in the transgenic lines 28 days after SMV inoculation was analyzed using qRT-PCR. 【Result】A total of 76 PPT-tolerant plants were generated from  2 600 explants after Agrobacterium-mediated transformation. Among them, 65 regenerated plants were confirmed positive as shown by PCR and LibertyLink^® strip analysis, and the transformation efficiency was 2.48%. The transgenic plants in subsequent generations (T₁ to T₃) were further screened by spraying 500 mg·L^-1 Basta. The transgenic plants showed no visible phenotype change 7 days after treatment, whereas the non-transgenic (NT) plants exhibited symptoms such as chlorosis or necrosis. Southern blot analysis showed that the exogenous gene was integrated into soybean genome with 1-2 copies of T-DNA insertions in the selected transgenic plants. Resistance evaluation by rub inoculation with SMV showed that the NT plants displayed serious mosaic patterns and curling leaves, while only mild mosaic symptom was observed on some of the leaves of the transgenic plants (35 days after inoculation). Moreover, the transgenic lines had significantly lower average disease indices (11.11-22.22) than the non-transformed (NT) control plants (36.81-46.24) and showed enhanced and stable resistance to SMV. The qRT-PCR result further confirmed that the SMV CP expression in the transgenic plants was significantly decreased than that of the NT control 28 days after SMV inoculation. The investigation of agronomic traits showed that there was no significant difference in the leaf shape, flower color, seed color, hilum color, plant height, node number, podding height, maturity period and weight per 100 seeds between the transgenic lines and NT control plants without inoculation SMV. 【Conclusion】The over-expression of PAC1 significantly inhibited the accumulation of SMV and the development of the symptoms, thus enhancing the resistance level to SMV in transgenic soybean plants.

Key words: soybean, Soybean mosaic virus (SMV), PAC1, Agrobacterium-mediated transformation, resistance