Data from: Copy number variation and expression analysis reveals a non-orthologous pinta gene family member involved in butterfly vision

Vertebrate (CRALBP) and Drosophila (PINTA) proteins with a CRAL-TRIO domain transport retinal-based chromophores that bind to opsin proteins and are necessary for phototransduction. The CRAL-TRIO domain gene family is composed of genes that encode proteins with a common N-terminal structural domain. While there is an expansion of this gene family in Lepidoptera, there is no lepidopteran ortholog of pinta. Further, the function of these genes in lepidopterans has not yet been established. Here we explored the molecular evolution and expression of CRAL-TRIO domain genes in the butterfly Heliconius melpomene in order to identify a member of this gene family as a candidate chromophore transporter. We generated and searched a four tissue transcriptome and searched a reference genome for CRAL-TRIO domain genes. We expanded an insect CRAL-TRIO domain gene phylogeny to include H. melpomene and used 18 genomes from 4 subspecies to assess copy number variation. A transcriptome-wide differential expression analysis comparing four tissue types identified a CRAL-TRIO domain gene, Hme CTD31, upregulated in heads suggesting a potential role in vision for this CRAL-TRIO domain gene. RT-PCR and immunohistochemistry confirmed that Hme CTD31 and its protein product are expressed in the retina, specifically in primary and secondary pigment cells and in tracheal cells. Sequencing of eye protein extracts that fluoresce in the ultraviolet identified Hme CTD31 as a possible chromophore binding protein. Although we found several recent duplications and numerous copy number variants in CRAL-TRIO domain genes, we identified a single copy pinta paralog that likely binds the chromophore in butterflies.

携带CRAL-TRIO结构域（CRAL-TRIO domain）的脊椎动物CRALBP蛋白与果蝇PINTA蛋白，可转运视网膜类生色团（retinal-based chromophores）；此类生色团能够结合视蛋白（opsin），是光转导（phototransduction）过程不可或缺的组分。CRAL-TRIO结构域基因家族由一类编码带有共同N端结构域的蛋白质的基因组成。尽管鳞翅目（Lepidoptera）中该基因家族发生了扩张，但目前尚未发现pinta的鳞翅目直系同源基因（ortholog）。此外，这类基因在鳞翅目昆虫中的功能仍未得到阐明。本研究以红带袖蝶（Heliconius melpomene）为研究材料，对其CRAL-TRIO结构域基因的分子进化与表达模式展开系统性探究，旨在筛选出该家族中可作为候选生色团转运蛋白的基因成员。我们构建并分析了四种组织的转录组（transcriptome），同时通过参考基因组（reference genome）检索CRAL-TRIO结构域基因。我们将昆虫CRAL-TRIO结构域基因的系统发育树（phylogeny）扩展至包含红带袖蝶，并利用4个亚种的18个基因组样本评估了拷贝数变异（copy number variation）情况。转录组水平的差异表达分析（differential expression analysis）对比了四种组织的基因表达谱，结果发现CRAL-TRIO结构域基因Hme CTD31在头部组织中显著上调，提示该基因可能在视觉通路中发挥功能。逆转录聚合酶链反应（RT-PCR）与免疫组织化学（immunohistochemistry）实验证实，Hme CTD31及其编码蛋白在视网膜（retina）中特异性表达，具体定位于初级色素细胞、次级色素细胞以及气管细胞（tracheal cells）。对紫外（ultraviolet）荧光眼部蛋白提取物的测序分析显示，Hme CTD31是一种潜在的生色团结合蛋白。尽管我们在CRAL-TRIO结构域基因中发现了多处近期复制事件与大量拷贝数变异，但最终筛选到一个单拷贝的pinta旁系同源基因（paralog），该基因极有可能在蝴蝶体内介导生色团的结合过程。