Luan Hexiang, Liao Wenlin, Song Yingpei, Niu Haopeng, Hu Ting, Zhi Haijian
National Center for Soybean Improvement, Nanjing Agricultural University, Nanjing, 210095, Jiangsu, China.
College of Life Sciences, Institute of Plant Genetic Engineering, Qingdao Agricultural University, Qingdao, 266109, China.
AMB Express. 2020 Apr 6;10(1):62. doi: 10.1186/s13568-020-00997-6.
Soybean mosaic virus (SMV) is one of the most destructive viral diseases in soybean and causes severe reduction of soybean yield and destroys the seed quality. However, the production of SMV resistant plants by transgenic is the most effective and economical means. Based on our previous yeast two-hybrid assay, the GmVma12 was selected as a strong candidate gene for further function characterization. Here we transformed soybean plants with a construct containing inverted repeat of-GmVma12 sequence to analyze the role of GmVma12 during SMV invasion. Totals of 33 T and 160 T plants were confirmed as positive transgenic plants through herbicide application, PCR detection and LibertyLink strip screening. Based on the segregation ratio and Southern Blot data, T lines No. 3 and No. 7 were selected to generate T plants. After SMV-SC15 inoculation, 41 T and 38 T plants were identified as highly resistant, and their quantification disease levels were much lower than non-transformed plants. The transcript level of GmVma12 in T plants decreased to 70% of non-transformed plants. The expression level of SMV-CP transcript in T transgenic plants was lower than that in non-transformed plants and SMV CP protein in T plants could not be detected by Enzyme-linked Immunosorbent assay, which indicated that SMV production would be inhibited in transgenic plants. Moreover, coat mottles of T seeds were obliterated significantly. In conclusion, inverted repeat of the hairpin structure of GmVma12 interfered with the transcription of GmVma12, which can induce resistance to SMV in soybean. This research lays the foundation for the mechanism of SMV pathogenesis, and provides new ideas for SMV prevention and control.
大豆花叶病毒（SMV）是大豆中最具破坏性的病毒病害之一，会导致大豆产量严重下降并破坏种子质量。然而，通过转基因培育抗SMV植株是最有效且经济的手段。基于我们之前的酵母双杂交试验，选择GmVma12作为进一步功能鉴定的有力候选基因。在此，我们用含有GmVma12序列反向重复的构建体转化大豆植株，以分析GmVma12在SMV侵染过程中的作用。通过除草剂处理、PCR检测和LibertyLink试纸条筛选，共确认33株T₀和160株T₁植株为阳性转基因植株。根据分离比例和Southern杂交数据，选择3号和7号T₁株系来培育T₂植株。接种SMV - SC15后，41株T₂和38株T₃植株被鉴定为高抗，其定量病害水平远低于未转化植株。T₂植株中GmVma12的转录水平降至未转化植株的70%。T转基因植株中SMV - CP转录本的表达水平低于未转化植株，且酶联免疫吸附测定法检测不到T₂植株中的SMV CP蛋白，这表明转基因植株中SMV的产生会受到抑制。此外，T₂种子的种皮斑驳明显消失。总之，GmVma12发夹结构的反向重复干扰了GmVma12的转录，从而可诱导大豆对SMV产生抗性。本研究为SMV致病机制奠定了基础，并为SMV的防治提供了新思路。
