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Abstract：
The multicellular green alga Volvox carteri has emerged as a valuable model organism for investigating various aspects of multicellularity and cellular differentiation, photoreception and phototaxis, cell division, biogenesis of the extracellular matrix and morphogenetic movements. While a range of molecular tools and bioinformatics resources have been made available for exploring these topics, the establishment of cell type-specific promoters in V. carteri has not been achieved so far. Therefore, here, we conducted a thorough screening of transcriptome data from RNA sequencing analyses of V. carteri in order to identify potential cell type-specific promoters. Eventually, we chose two putative strong and cell type-specific promoters, with one exhibiting specific expression in reproductive cells (gonidia), the PCY1 promoter, and the other in somatic cells, the PFP promoter. After cloning both promoter regions, they were introduced upstream of a luciferase reporter gene. By using particle bombardment, the DNA constructs were stably integrated into the genome of V. carteri. The results of the expression analyses, which were conducted at both the transcript and protein levels, demonstrated that the two promoters drive cell type-specific expression in their respective target cell types. Transformants with considerably diverse expression levels of the chimeric genes were identifiable. In conclusion, the screening and analysis of transcriptome data from RNA sequencing allowed for the identification of potential cell type-specific promoters in V. carteri. Reporter gene constructs demonstrated the actual usability of two promoters. The investigated PCY1 and PFP promoters were proven to be potent molecular tools for genetic engineering in V. carteri.
摘要：
多细胞绿藻Volvoxcarteri已成为研究多细胞和细胞分化各个方面的有价值的模型生物，光接收和趋光性，细胞分裂,细胞外基质的生物发生和形态发生运动。虽然已经提供了一系列分子工具和生物信息学资源来探索这些主题，到目前为止，尚未在V.carteri中建立细胞类型特异性启动子。因此,在这里，我们对来自V.carteriRNA测序分析的转录组数据进行了彻底筛选,以鉴定潜在的细胞类型特异性启动子.最终,我们选择了两个假定的强启动子和细胞类型特异性启动子，在生殖细胞（gonidia）中表现出特异性表达，PCY1启动子，另一个在体细胞中，PFP启动子。克隆两个启动子区域后，它们被引入荧光素酶报告基因的上游。通过使用粒子轰击，DNA构建体稳定整合到V.carteri的基因组中。表达分析的结果，在转录本和蛋白质水平上进行，证明了两个启动子在它们各自的靶细胞类型中驱动细胞类型特异性表达。具有相当不同的嵌合基因表达水平的转化体是可鉴定的。总之,通过对来自RNA测序的转录组数据进行筛选和分析,可以鉴定V.carteri中潜在的细胞类型特异性启动子.报告基因构建体证明了两个启动子的实际可用性。研究的PCY1和PFP启动子被证明是V.carteri基因工程的有效分子工具。
