马铃薯龙葵素合成关键SSR2双重基因编辑载体的构建

Construction of Dual Gene Editing Vector Target to Potato Solanine Synthesis SSR2 Gene

马铃薯(Solanum tuberosum Linn)是中国主要的经济作物之一,但是不当储存或生长条件下马铃薯龙葵素含量升高,以及部分新引进和选育品种龙葵素含量较高,由此产生的毒害作用是影响马铃薯产业快速发展和食用安全的关键因素。近年来,CRISPR/Cas9基因组编辑技术发展迅速,被广泛用于植物育种研究。为获得低龙葵素含量的马铃薯品种,以马铃薯龙葵素合成关键甾醇侧链还原酶2基因(SSR2)为靶基因,通过搜索该基因3个拷贝的第一外显子序列,设计2条靶序列,利用重叠区PCR和金门克隆技术,构建了靶向SSR2的双重基因编辑载体,并利用MluⅠ酶切和测序进行了验证。本研究结果将为进一步培育低龙葵素含量的马铃薯提供重要基础。

Potato(Solanum tuberosum Linn)is one of the main economic crops in China.However,the increase of solanine content under improper storage and growth conditions or in some newly selected varieties,leads to deleterious effects which is a key factor affecting the rapid development of the potato industry and food safety.In recent years,CRISPR/Cas9 genome editing technology has developed rapidly and has been widely used in plant breeding research.In order to obtain potato plants with low solanine content,the potato key gene for solanine synthesis,sterol side chain reductase 2 gene(SSR2),was selected as the target,and two sg RNA sequences were designed from the first exons of three copies of SSR2.Afterward,the dual gene editing vector targeting SSR2 was constructed by the overlapping PCR and the Golden gate cloning technology,followed by verified via MluⅠdigestion and sequencing.Summarize,the results of this research provides a reference for the further cultivation of potatoes with low solanine content.