烟草BBLc基因CRISPR/Cas 9载体构建与遗传转化

Construction and Genetic Transformation of Tobacco BBLc Gene CRISPR/Cas 9 Gene Editing Vector

小檗碱桥状酶在烟碱生物合成的最后一步起到关键催化作用。本研究对小檗碱桥状酶基因家族中的BBLc基因进行研究,利用CRISPR/Cas 9技术构建BBLc基因的编辑载体,通过农杆菌介导法遗传转化烟草,经卡那霉素抗性筛选获得35株抗性苗,利用PCR和RT-qPCR技术鉴定获得7株突变株。观察发现,突变株烟叶褶皱程度较野生型增大,其余农艺性状与野生型相比差别不大。利用HPLC法测定盛花期叶片烟碱含量,突变株烟碱含量较对照平均降低了24.2%;其中突变烟株CB 10的烟碱含量为0.88%,降幅最大(37.6%)。研究表明,BBLc基因在烟草中表达量的降低引起烟碱含量的降低。

Berberine bridge enzyme(BBL) plays a key catalytic role in the last step of nicotinoid biosynthesis. In order to study the function of tobacco BBLc gene of BBL gene family, the CRISPR/Cas 9 gene editing technology were used to construct BBLc knockout vector. 35 resistance seedlings were screened through kanamycin with genetic transformation of tobacco by Agrobaterium-mediated method and 7 mutant seedlings were identified by PCR and RT-qPCR technology. The degree of leaf fold on mutant seedlings was higher than that of wild types, and the other agronomic traits on mutant seedlings were no obvious difference from those of wild types. The nicotine content detected by HPLC of the mutant leaves was decreased 24.2% below average, and the content of mutant CB 10 plant was 0.88%, which had the maximum drop by 37.6%. The resutls showed that the decrease of nicotine content was caused by decrease of BBLc gene expression.