Cytochrome P450 Genes Expressed in Phasmatodea Midguts

In June 2021, representative sequences for insect cytochrome P450s were downloaded from NCBI, limiting the search to those in the UniProtKB database. The resulting 111 sequences were used as a query to mine the above transcriptomes using tblastn with an expect value threshold of e-10. These were manually annotated by removing truncated sequences, using the ExPASy online translation tool to obtain the complete amino acid sequences, removing duplicates using the sRNAtoolbox webserver, and confirming that the sequences were cytochrome P450s by identifying them using blastp against the NCBI database. The resulting sequences were combined with the representatives from NCBI, aligned using the Clustal W program built into the software MEGA version X. Any sequences missing the heme-binding domain FXXGXXXCXG/A, which is a signature motif for CYPs, were deleted. The sequences were also checked for the presence and absence of other four signature motifs from insect CYPs: helix C (WxxxR), helix I (GxE/DTT/S), helix K (ExLR), and PERF (PxxFxPE/DRE/F).2021年6月，我们从NCBI（美国国家生物技术信息中心，National Center for Biotechnology Information）下载了昆虫细胞色素P450（cytochrome P450，CYP）的代表性序列，并将检索范围限定于UniProtKB（通用蛋白质知识库，Universal Protein Knowledgebase）数据库中的条目。将得到的111条序列作为查询序列，使用tblastn工具、以e-10作为期望值阈值挖掘上述转录组数据。随后通过以下步骤开展人工注释：去除截短序列；使用ExPASy在线翻译工具获取完整的氨基酸序列；借助sRNAtoolbox在线服务器去除重复序列；通过blastp工具比对NCBI数据库进行序列鉴定，确认所得序列为细胞色素P450。将最终得到的序列与NCBI来源的代表性序列进行合并，随后使用内置在MEGA X软件中的Clustal W程序进行多序列比对。删除缺失血红素结合结构域FXXGXXXCXG/A（细胞色素P450的特征基序）的序列。同时对这些序列进行检查，确认其是否包含昆虫细胞色素P450的其余4种特征基序：螺旋C（WxxxR）、螺旋I（GxE/DTT/S）、螺旋K（ExLR）以及PERF（PxxFxPE/DRE/F）。