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

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 (Cula11 and Cula08) from Chinese fir (C. lanceolate) 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 in vivo molecular biology studies in its homologous system, we compared the activities of Cula11 and Cula08 with that of the commonly used promoters in genetic engineering of monocots or dicots, such as CaM35S, CmYLCV, and ZmUbi, and our results revealed that Cula11 and Cula08 promoters have stronger activities in Chinese fir protoplasts. Furthermore, the vector containing Cas gene driven by Cula11 promoter and sgRNA driven by the newly isolated CulaU6b polyIII 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, Cula11 promoter has much stronger activity than CaM35S promoter in transgenic poplar, and ZmUbi 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

Low-density lipoprotein receptor promotes crosstalk between cell stemness and tumor immune microenvironment in breast cancer: a large data-based multi-omics study

Abstract Background Tumor cells with stemness in breast cancer might facilitate the immune microenvironment’s suppression process and led to anti-tumor immune effects. The primary objective of this study was to identify potential targets to disrupt the communication between cancer cell stemness and the immune microenvironment. Methods In this study, we initially isolated tumor cells with varying degrees of stemness using a spheroid formation assay. Subsequently, we employed RNA-seq and proteomic analyses to identify genes associated with stemness through gene trend analysis. These stemness-related genes were then subjected to pan-cancer analysis to elucidate their functional roles in a broader spectrum of cancer types. RNA-seq data of 3132 patients with breast cancer with clinical data were obtained from public databases. Using the identified stemness genes, we constructed two distinct stemness subtypes, denoted as C1 and C2. We subsequently conducted a comprehensive analysis of the differences between these subtypes using pathway enrichment methodology and immune infiltration algorithms. Furthermore, we identified key immune-related stemness genes by employing lasso regression analysis and a Cox survival regression model. We conducted in vitro experiments to ascertain the regulatory impact of the key gene on cell stemness. Additionally, we utilized immune infiltration analysis and pan-cancer analysis to delineate the functions attributed to this key gene. Lastly, single-cell RNA sequencing (scRNA-seq) was employed to conduct a more comprehensive examination of the key gene’s role within the microenvironment. Results In our study, we initially identified a set of 65 stemness-related genes in breast cancer cells displaying varying stemness capabilities. Subsequently, through survival analysis, we pinpointed 41 of these stemness genes that held prognostic significance. We observed that the C2 subtype exhibited a higher stemness capacity compared to the C1 subtype and displayed a more aggressive malignancy profile. Further analysis using Lasso-Cox algorithm identified LDLR as a pivotal immune-related stemness gene. It became evident that LDLR played a crucial role in shaping the immune microenvironment. In vitro experiments demonstrated that LDLR regulated the cell stemness of breast cancer. Immune infiltration analysis and pan-cancer analysis determined that LDLR inhibited the proliferation of immune cells and might promote tumor cell progression. Lastly, in our scRNA-seq analysis, we discovered that LDLR exhibited associations with stemness marker genes within breast cancer tissues. Moreover, LDLR demonstrated higher expression levels in tumor cells compared to immune cells, further emphasizing its relevance in the context of breast cancer. Conclusion LDLR is an important immune stemness gene that regulates cell stemness and enhances the crosstalk between breast cancer cancer cell stemness and tumor immune microenvironment

Image_2_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.jpg

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 (Cula11 and Cula08) from Chinese fir (C. lanceolate) 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 in vivo molecular biology studies in its homologous system, we compared the activities of Cula11 and Cula08 with that of the commonly used promoters in genetic engineering of monocots or dicots, such as CaM35S, CmYLCV, and ZmUbi, and our results revealed that Cula11 and Cula08 promoters have stronger activities in Chinese fir protoplasts. Furthermore, the vector containing Cas gene driven by Cula11 promoter and sgRNA driven by the newly isolated CulaU6b polyIII 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, Cula11 promoter has much stronger activity than CaM35S promoter in transgenic poplar, and ZmUbi 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

Image_6_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.jpg

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 (Cula11 and Cula08) from Chinese fir (C. lanceolate) 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 in vivo molecular biology studies in its homologous system, we compared the activities of Cula11 and Cula08 with that of the commonly used promoters in genetic engineering of monocots or dicots, such as CaM35S, CmYLCV, and ZmUbi, and our results revealed that Cula11 and Cula08 promoters have stronger activities in Chinese fir protoplasts. Furthermore, the vector containing Cas gene driven by Cula11 promoter and sgRNA driven by the newly isolated CulaU6b polyIII 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, Cula11 promoter has much stronger activity than CaM35S promoter in transgenic poplar, and ZmUbi 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

Image_3_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.jpg

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 (Cula11 and Cula08) from Chinese fir (C. lanceolate) 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 in vivo molecular biology studies in its homologous system, we compared the activities of Cula11 and Cula08 with that of the commonly used promoters in genetic engineering of monocots or dicots, such as CaM35S, CmYLCV, and ZmUbi, and our results revealed that Cula11 and Cula08 promoters have stronger activities in Chinese fir protoplasts. Furthermore, the vector containing Cas gene driven by Cula11 promoter and sgRNA driven by the newly isolated CulaU6b polyIII 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, Cula11 promoter has much stronger activity than CaM35S promoter in transgenic poplar, and ZmUbi 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

Image_4_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.jpg

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 (Cula11 and Cula08) from Chinese fir (C. lanceolate) 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 in vivo molecular biology studies in its homologous system, we compared the activities of Cula11 and Cula08 with that of the commonly used promoters in genetic engineering of monocots or dicots, such as CaM35S, CmYLCV, and ZmUbi, and our results revealed that Cula11 and Cula08 promoters have stronger activities in Chinese fir protoplasts. Furthermore, the vector containing Cas gene driven by Cula11 promoter and sgRNA driven by the newly isolated CulaU6b polyIII 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, Cula11 promoter has much stronger activity than CaM35S promoter in transgenic poplar, and ZmUbi 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

Image_5_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.jpg

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 (Cula11 and Cula08) from Chinese fir (C. lanceolate) 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 in vivo molecular biology studies in its homologous system, we compared the activities of Cula11 and Cula08 with that of the commonly used promoters in genetic engineering of monocots or dicots, such as CaM35S, CmYLCV, and ZmUbi, and our results revealed that Cula11 and Cula08 promoters have stronger activities in Chinese fir protoplasts. Furthermore, the vector containing Cas gene driven by Cula11 promoter and sgRNA driven by the newly isolated CulaU6b polyIII 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, Cula11 promoter has much stronger activity than CaM35S promoter in transgenic poplar, and ZmUbi 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

Table_1_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.xlsx

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 (Cula11 and Cula08) from Chinese fir (C. lanceolate) 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 in vivo molecular biology studies in its homologous system, we compared the activities of Cula11 and Cula08 with that of the commonly used promoters in genetic engineering of monocots or dicots, such as CaM35S, CmYLCV, and ZmUbi, and our results revealed that Cula11 and Cula08 promoters have stronger activities in Chinese fir protoplasts. Furthermore, the vector containing Cas gene driven by Cula11 promoter and sgRNA driven by the newly isolated CulaU6b polyIII 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, Cula11 promoter has much stronger activity than CaM35S promoter in transgenic poplar, and ZmUbi 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

DataSheet_1_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.docx

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 (Cula11 and Cula08) from Chinese fir (C. lanceolate) 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 in vivo molecular biology studies in its homologous system, we compared the activities of Cula11 and Cula08 with that of the commonly used promoters in genetic engineering of monocots or dicots, such as CaM35S, CmYLCV, and ZmUbi, and our results revealed that Cula11 and Cula08 promoters have stronger activities in Chinese fir protoplasts. Furthermore, the vector containing Cas gene driven by Cula11 promoter and sgRNA driven by the newly isolated CulaU6b polyIII 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, Cula11 promoter has much stronger activity than CaM35S promoter in transgenic poplar, and ZmUbi 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

Image_1_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.jpg

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 (Cula11 and Cula08) from Chinese fir (C. lanceolate) 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 in vivo molecular biology studies in its homologous system, we compared the activities of Cula11 and Cula08 with that of the commonly used promoters in genetic engineering of monocots or dicots, such as CaM35S, CmYLCV, and ZmUbi, and our results revealed that Cula11 and Cula08 promoters have stronger activities in Chinese fir protoplasts. Furthermore, the vector containing Cas gene driven by Cula11 promoter and sgRNA driven by the newly isolated CulaU6b polyIII 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, Cula11 promoter has much stronger activity than CaM35S promoter in transgenic poplar, and ZmUbi 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