AUTHOR=Ye Shanwen , Ding Wensha , Bai Weiyuan , Lu Jiaao , Zhou Linying , Ma Xiangqing , Zhu Qiang 

TITLE=Application of a novel strong promoter from Chinese fir (Cunninghamia lanceolate) in the CRISPR/Cas mediated genome editing of its protoplasts and transgenesis of rice and poplar

JOURNAL=Frontiers in Plant Science

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2023.1179394

DOI=10.3389/fpls.2023.1179394

ISSN=1664-462X

ABSTRACT=<p>Novel constitutive promoters are essential for plant biotechnology. Although in angiosperms, a number of promoters were applied in monocots or dicots genetic engineering, only a few promoters were used in gymnosperm. Here we identified two strong promoters (<italic>Cula11</italic> and <italic>Cula08</italic>) from Chinese fir (<italic>C. lanceolate</italic>) by screening the transcriptomic data and preliminary promoter activity assays in tobacco. By using the newly established Chinese fir protoplast transient expression technology that enables <italic>in vivo</italic> molecular biology studies in its homologous system, we compared the activities of <italic>Cula11</italic> and <italic>Cula08</italic> with that of the commonly used promoters in genetic engineering of monocots or dicots, such as <italic>CaM35S</italic>, <italic>CmYLCV</italic>, and <italic>ZmUbi</italic>, and our results revealed that <italic>Cula11</italic> and <italic>Cula08</italic> promoters have stronger activities in Chinese fir protoplasts. Furthermore, the vector containing <italic>Cas</italic> gene driven by <italic>Cula11</italic> promoter and sgRNA driven by the newly isolated <italic>CulaU6b polyIII</italic> promoters were introduced into Chinese fir protoplasts, and CRISPR/Cas mediated gene knock-out event was successfully achieved. More importantly, compared with the commonly used promoters in the genetic engineering in angiosperms, <italic>Cula11</italic> promoter has much stronger activity than <italic>CaM35S</italic> promoter in transgenic poplar, and <italic>ZmUbi</italic> promoter in transgenic rice, respectively, indicating its potential application in poplar and rice genetic engineering. Overall, the novel putative constitutive gene promoters reported here will have great potential application in gymnosperm and angiosperm biotechnology, and the transient gene expression system established here will serve as a useful tool for the molecular and genetic analyses of Chinese fir genes.</p>