Sun Huayu, Wang Sining, Zhu Chenglei, Yang Kebin, Liu Yan, Gao Zhimin
Key Laboratory of National Forestry and Grassland Administration/Beijing for Bamboo & Rattan Science and Technology, Beijing, 100102, China.
Institute of Gene Science and Industrialization for Bamboo and Rattan Resources, International Centre for Bamboo and Rattan, Beijing, 100102, China.
Plant Methods. 2023 Mar 2;19(1):20. doi: 10.1186/s13007-023-00993-4.
Bamboo is a perennial and renewable biomass forest resource and its leaf flavonoid is an antioxidant for biological and pharmacological research. The established genetic transformation and gene editing systems in bamboo are significantly limited by the dependence on bamboo regeneration capability. The way to improve the flavonoid content in bamboo leaves through biotechnology is still not feasible.
Here, we developed an in-planta, Agrobacterium-mediated gene expression method for exogenous genes via wounding and vacuum in bamboo. We demonstrated that the RUBY served as a reporter efficiently expressed in bamboo leaves and shoots, albeit unable to integrate into the chromosome. We have also developed a gene editing system by creating an in situ mutant of the bamboo violaxanthin de-epoxidase (PeVDE) gene in bamboo leaves, with lower NPQ values under the fluorometer, which can serve as a native reporter for gene editing. Furthermore, the bamboo leaves with increased flavonoid content were achieved by knocking out the cinnamoyl-CoA reductase genes.
Our method can be applied for the functional characterization of novel genes in a short time and is helpful for bamboo leaf flavonoid biotechnology breeding in the future.
竹子是一种多年生可再生生物质森林资源，其叶黄酮是用于生物学和药理学研究的抗氧化剂。竹子中已建立的遗传转化和基因编辑系统受到对竹子再生能力依赖的显著限制。通过生物技术提高竹叶中黄酮含量的方法仍然不可行。
在此，我们开发了一种通过在竹子中伤口处理和真空处理，利用农杆菌介导的外源基因在植物体内表达的方法。我们证明，红宝石蛋白作为报告基因在竹叶和竹茎中高效表达，尽管它无法整合到染色体中。我们还通过在竹叶中创建竹子紫黄质脱环氧化酶（PeVDE）基因的原位突变体，开发了一种基因编辑系统，该突变体在荧光计下具有较低的非光化学猝灭（NPQ）值，可作为基因编辑的内源报告基因。此外，通过敲除肉桂酰辅酶A还原酶基因，实现了竹叶中黄酮含量的增加。
我们的方法可在短时间内用于新基因的功能表征，并有助于未来竹叶黄酮生物技术育种。
