CRISPR/Cas9-mediated targeted mutagenesis and function analysis of CPS2 in Nicotiana tabacum

In order to explore the function and regulatory mechanism of CPS2 gene, CRISPR/Cas9 system was used to edit the CPS2 gene in high aroma flue-cured tobacco line 8306. A total of 7 transformed plants with single base A/T/C inserted at the target sites were obtained. The expression of key diterpenoid genes in transformed plants were analyzed by q-PCR. The components in leaf surface exudates and the content (mass fraction)of GGPP were analyzed by GC-MS. The results showed that: 1)The expression levels of CPS2 and ABS decreased significantly in T₀ generation transformed plants, and their contents of cis-abienol and labdenediol decreased significantly. 2) The expression level of DXR significantly increased, and the GGPP content also raised greatly. 3)The expression of CYC-1 and CYP71D16 differed significantly, while the content of cembratrien-diol decreased slightly. 4)The plant height, stalk circumference, leaf spacing, maximum leaf length and maximum leaf width of T₀ generation transformed plants increased. Therefore, the knockout of CPS2 can inhibit the synthesis of labdanoids compounds and contribute to the accumulation of terpenoid synthetic precursor GGPP in the upstream pathway, which might inhibit the accumulation of cembtriene-diol and associate changes in the phenotypic traits.