ZHU Ting-ting,WANG Chun-sheng,SUN Shan-shan,et al.Improving the Resistance to Phytophthora Root Rot of Soybean by Transforming Broad-Spectrum Disease Resistance Gene NPR1 and CHR3[J].Soybean Science,2021,40(02):168-176.[doi:10.11861/j.issn.1000-9841.2021.02.0168]
转NPR1和CHR3抗病基因提高大豆对疫霉根腐病抗性研究
- Title:
- Improving the Resistance to Phytophthora Root Rot of Soybean by Transforming Broad-Spectrum Disease Resistance Gene NPR1 and CHR3
- Keywords:
- Soybean; NPR1; JL30+GmCHR3; Bivalent gene; Phytophthora root rot disease; Disease resistantance
- 文献标志码:
- A
- 摘要:
- 为检测GmCHR3和广谱抗病基因NPR1双抗基因转化到大豆吉林30中的遗传稳定性和抗病能力，从而为培育出抵抗疫霉根腐病大豆新品种提供有效参考，以大豆品种吉林30转CHR3抗病基因转化品系JL30+GmCHR3为目标受体材料，以吉林30为对照受体材料，利用农杆菌介导法将NPR1导入受体中。利用常规PCR检测基因转化情况，利用Southern杂交和qRT-PCR技术分别鉴定JL30+GmCHR3受体和双抗基因转化株系T1和T2代中两个基因的整合和表达情况，采用下胚轴侵染法鉴定转基因大豆植株对疫霉根腐病的抗性。PCR研究结果显示：转化元件的启动子35s、终止子Nos、筛选标记基因Bar以及目的基因NPR1全部转入到受体基因组中；NPR1基因以单拷贝的形式在转化植株中完成整合；NPR1基因在大豆植株根、茎和叶中均有表达，其中T1代株系在3个部位的相对表达量分别是2.732，1.614和3.316，T2代株系的相对表达量分别是2.936，2.084和3.864；NPR1基因在各组织中的相对表达量为茎＜根＜叶。CHR3和NPR1双抗基因转化株系对疫霉根腐病表现为高抗，NPR1单抗基因转化株系表现为中抗，非转基因吉林30植株表现为感病。结果说明大豆吉林30转入双价抗病基因GmCHR3和NPR1可以增强其对疫霉根腐病的抗性。
- Abstract:
- In order to detect the integration and genetic stability of NPR1 gene in soybean Jilin 30 transgenic CHR3 line, to investigate the disease resistance of transgenic progeny with bivalent gene of CHR3 and NPR1, and to provide an effective reference for breeding new soybean varieties resistant to Phytophthora root rot. In this paper, soybean varieties JL30+GmCHR3 were used as receptor materials, the broad-spectrum resistance gene NPR1 was introduced into the receptor by agrobacteria-mediated. Conventional PCR were used to test the genetic transformation, southern blot hybridization and qRT-PCR were used to identify the integration and expression of the two genes in the T1 and T2 generations of JL30 + GmCHR3 receptor and double-resistant transgenic lines repectively, and hypocotyl infection was used to identify the resistance of transgenic soybean plants to Phytophthora root rot. The results showed that promoter 35s, terminator Nos, screening marker gene Bar and target gene NPR1 were all expressed in receptor genome. Southern blotting showed that the target gene was integrated in soybean plants in a single copy manner. And the qRT-PCR detection results showed that the target gene was expressed in the root, stem and leaf of soybean plant, among them, the average relative expression of roots, stems and leaves of T1 generation were 2.732, 1.614 and 3.316, respectively, the mean relative expression of roots, stems and leaves of T2 generation strains were 2.936, 2.084 and 3.864, respectively. The results showed that the relative expression of the target gene in each tissue was as stem < root < leaf. Transformation lines with dual resistance genes of CHR3 and NPR1 showed high resistance to Phytophthora root rot disease, JL30+NPR1 showed moderate resistance, and non-transgenic plants showed susceptible. The results showed that the transformation of the bivalent resistance genes GmCHR3 and NPR1 into the soybean Jilin 30 could enhance the resistance to Phytophthora root rot.
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备注/Memo
收稿日期：2020-11-03