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无转基因,基于病毒的基因沉默在植物中通过人工微RNA从最小的前体衍生出来
Adriana E Cisneros1, Tamara Martín-García1, Anamarija Primc1
1Instituto de Biología Molecular y Celular de Plantas (IBMCP), CSIC-Universitat Politècnica de València, Av. de los Naranjos s/n, 46022 Valencia, Spain.
Nucleic acids research
|September 15, 2023
概括
研究人员开发了更短的人工microRNA (amiRNA) 前体,以便在植物中有效地静止基因. 这一突破使得无转基因应用能够使用病毒载体进行作物改进和功能基因组学.
科学领域:
- 植物生物技术 植物生物技术
- 分子生物学分子生物学
- 在RNA干扰过程中,RNA干扰
背景情况:
- 人工微RNA (amiRNA) 是植物功能基因组学和作物改进的宝贵工具.
- 目前的方法需要长的初级miRNA (pri-miRNA) 前体的转基因表达,限制了更广泛的应用.
- 了解最小的前体要求对于开发更高效的amiRNA生产系统至关重要.
研究的目的:
- 确定有效地从Arabidopsis thalianaAtMIR390a pri-miRNA.产生amiRNA的最小结构和序列要求.
- 开发更短,功能性的amiRNA前体,用于植物中的短暂表达和基因沉默应用.
- 探索病毒载体在无转基因amiRNA传递和全植物基因沉默方面的潜力.
主要方法:
- 在Nicotiana benthamiana中对内源基因进行amiRNAs的功能选,使用基于MIR390的缩短前体的构造集合.
- 在N. benthamiana中进行过渡性表达测试,以评估amiRNA的有效性和从最小的前体进行处理.
- 在Arabidopsis thaliana转基因植物中验证最小的amiRNA前体.
- 通过喷射传染性原始提取物来表达最小的amiRNA前体的病毒载体的应用,以实现无转基因基因沉默.
主要成果:
- 高效且精确处理的amiRNAs可以从最短89 nt的仿真前体中产生.
- 最小的amiRNA前体,与全长的不同,产生真实的amiRNA并诱导N. benthamiana的广泛基因沉默.
- 携带最小amiRNA前体的病毒载体可以在通过喷雾应用时实现无转基因,全植物基因沉默.
- 缩短的前体不会影响amiRNA沉默功效.
结论:
- 可以显著减少amiRNA前体的长度,而不会影响基因沉默的有效性.
- 最小的amiRNA前体对于产生真实的amiRNA和实现广泛的基因沉默是有效的.
- 病毒载体介导的最小amiRNA前体的输送为植物基因沉默和作物生物技术提供了一个有前途的无转基因方法.
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