| 张秀春，吴坤鑫，赵平娟，刘志昕.人工合成靶序列快速构建多靶标RNAi 表达载体…[J].广东农业科学,2016,43(7):81-86 |
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人工合成靶序列快速构建多靶标RNAi 表达载体… |
| Rapid construction of multiple target RNAi expression vector from synthesized fragment |
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| DOI：10.16768/j.issn.1004-874X.2016.07.013 |
| 中文关键词: 人工合成靶序列 番木瓜环斑病毒 番木瓜叶扭曲花叶病毒 RNAi 表达载体 |
| 英文关键词: sythesized fragment papaya ringspot virus papaya leaf-distortion mosaic virus RNAi expression vector |
| 基金项目:国家自然科学基金（31201264） |
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| 摘要点击次数: 1520 |
| 全文下载次数: 650 |
| 中文摘要: |
| 番木瓜环斑病毒（PRSV）正引起番木瓜毁灭性严重病害，番木瓜叶扭曲花叶病毒（PLDMV）也在威胁着目前转基因番木瓜的推广和应用。通过人工合成包含PRSV 和PLDMV 两种病毒的NIb 和Hc-Pro 基因多个保守区域的靶序列作为模板，扩增两条长短不同但大部分重叠的靶序列，并通过引物设计时
添加的特定酶切位点两个靶序列反向连接，形成以长靶序列的5’一部分序列作为发夹结构的环，不需要另外插入内含子序列的反向重复发夹结构的RNAi 表达载体pPTN-LS。该方法构建的RNAi 表达载体，不仅具有快速、稳定性高等优点，还能同时靶标PRSV 和PLDMV 两种病毒的NIb 和Hc-Pro 基因多个保守区域的靶序列，能有效提高沉默效率，为利用RNAi 技术培育广谱、高效、稳定且安全性高，同时抗PRSV 和
PLDMV 病毒病的转基因番木瓜新种质的奠定基础。 |
| 英文摘要: |
| Papaya production is seriously limited by Papaya ringspot virus（ PRSV） worldwide and Papaya leaf-distortion mosaic virus（ PLDMV） in Eastern Asia. To develop transgenic papaya lines with robust resistance to both of PRSV and PLDMV viruses， we took advantage of a sythesized fragment which included several conserved segments of Nib and Hc-Pro gene of PRSV and PLDMV to constructe pPTN-LS RNAi expression vector. The pPTNLS RNAi expression vector was constructed by two fragments with different length and inverted repeat（ IR） deriving from the sythesized fragment. The IR part served as stem while the extending part of the longer fragment served as loop
of the hairpin structure of pPTN-LS respectively. Since it didn’t need to insert additional intron，the method of RNAi
expression vector construction mentioned above was fast and the result was stable as well. The transgenic Papaya engineering with pPTN-LS should be robust resistant to PRSV and PLDMV at the same time，because the sythesized fragment was derived from the highly conserved regions of Nib and Hc-Pro gene of PRSV and PLDMV. The study maypave the way to develop transgenic papaya lines with robust resistance to both of PRSV and PLDMV viruses. |
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