A reliable and simple Agrobacterium-mediated transformation system for the unicellular green algae model organism Chlamydomonas reinhardtii has been developed. The protocol has been successfully employed with both neomycin phosphotransferase II (nptII) and the phleomycin resistance (bleI) genes coupled with the selective agents paromomycin and zeocin, respectively. A set of binary vectors were assembled that carry the selectable marker cassettes under control either of the Rbcs2 alone or fused to the HSP270A leader sequence, PsaD, or ß-tubulin2 promoters. The corresponding T-DNA elements also harbored a cassette with a codon-optimized version of yellow fluorescence protein (YFP) under control of the Rbcs2 promoter in which the YFP open reading frame was interrupted with the first intron of Rbcs2 to prevent expression in Agrobacterium tumefaciens. The resultant binary vectors were introduced into A. tumefaciens strain C58C1/pMP90, and the derived transconjugants were used for transformation studies with the walled C. reinhardtii strain CC124. Estimated transformation frequencies ranged from 0.09 to 2.86 colonies per 10⁶ cells inoculated. Molecular characterizations on a subset of the transgenic lineages revealed that most of the transgenic events harbored single locus insertions. Moreover, sequencing of captured junction fragments about the T-DNA insertion site showed that minimal disruption of the C. reinhardtii genome occurred. However, the transgenic lineages often harbored truncated T-DNA regions within the non-selectable marker gene cassettes.

已经开发出用于单细胞绿藻模式生物莱茵衣藻的可靠且简单的农杆菌介导的转化系统。该方案已成功应用于新霉素磷酸转移酶 II ( nptII ) 和腐草霉素抗性 ( bleI ) 基因，分别与选择剂巴龙霉素和博莱霉素结合。组装了一组二元载体，其携带在单独的 Rbcs2 或与 HSP270A 前导序列、PsaD 或 β-tubulin2 启动子融合的控制下的选择性标记盒。相应的 T-DNA 元件还包含一个密码子优化版本的黄色荧光蛋白 (YFP) 盒，受 Rbcs2 启动子控制，其中 YFP 开放阅读框被 Rbcs2 的第一个内含子打断，以防止在根癌农杆菌中表达。将所得二元载体引入根瘤农杆菌菌株 C58C1/pMP90，并将衍生的转接合子用于有壁莱茵衣杆菌菌株 CC124 的转化研究。^{估计的转化频率范围为每10 6 个}接种的细胞0.09至2.86个集落。转基因谱系子集的分子特征表明，大多数转基因事件都含有单基因座插入。此外，对 T-DNA 插入位点附近捕获的连接片段进行测序表明，莱茵衣藻基因组发生了最小程度的破坏。然而，转基因谱系通常在非选择性标记基因盒中含有截短的 T-DNA 区域。