Proteomic evidence for the silk fibroin genes of spider mites (order Trombidiformes: family Tetranychidae)

Spider mites are a group of arachnids belonging in the family Tetranychidae, known to produce nano-scale silk fibers characterized by high Young’s modulus. Silk fibroin gene of spider mites has been computationally suggested through genomic analysis of <i>Tetranychus urticae</i>, but it is yet to be confirmed by proteomic evidences. In this work, we sequenced and assembled the transcriptome from two genera of spider mites, <i>Tetranychus kanzawai</i>and <i>Panonychus citri</i>, and combined with the silk proteomics of <i>T. urticae </i>and <i>P. citri</i>to characterize the fibroin genes through comparative genomics and multi-omics analysis. As a result, two fibroins were identified, that are different genes from the previous computational prediction. Amino acid composition and secondary structure suggests similarity to aciniform or cylindrical spidroins of spider silk, which partly mirrors their mechanical property exhibiting high Young’s modulus. Availability of full-length fibroin sequences of spider mites facilitates the study of the evolution of silk genes that emerged in multiple lineages sometimes in convergent manner, and in the industrial application of artificial protein fibers through the study of the amino acid sequence and the resulting mechanical properties of these silks. <br>Sample preservation, RNA extraction, sequencing and assembly have been conducted for <i>T. kanzawai</i>, and <i>P. citri</i>based on methods previously described for spiders [18], with some modification. Briefly, a single specimen of each of the spider mite was preserved in RNAlater stored at -80 ˚C, and RNA was extracted using Direct-zol RNA Microprep Kit (Zymo Research). Illumina library was prepared using KAPA HyperPlus Kit (KAPA Biosystems) targeting 300bp fragments. Sequence library was then sequenced on NextSeq 500 (Illumina) with High Output Mode 300 cycles as paired-end reads. Sequences were basecalled, demultiplexed, and adaptors sequences were removed with bcls2fastq v.2 software (Illumina). Transcriptome assembly was performed using Bridger software with default parameters [19]. Expression levels of transcripts were quantified using kallisto v.0.44 [20]using the assembled transcriptome as well as previously published predicted coding sequences from the genome of <i>T. urticae</i>[16]. Assembly completeness was assessed using BUSCO v.3 [21]with Arthropoda dataset through gVolante server [22].

叶螨（Spider mites）是隶属于叶螨科（Tetranychidae）的一类蛛形纲动物，可产生具备高杨氏模量（Young’s modulus）特性的纳米级丝纤维。此前通过对二斑叶螨（Tetranychus urticae）的基因组分析，学界已通过计算预测出叶螨的丝素蛋白基因，但该结论仍需蛋白质组学证据加以验证。本研究对两个属的叶螨——神泽氏叶螨（Tetranychus kanzawai）与柑橘全爪螨（Panonychus citri）——进行了转录组测序与组装，并结合二斑叶螨（T. urticae）与柑橘全爪螨（P. citri）的丝蛋白蛋白质组数据，通过比较基因组学与多组学分析对丝素蛋白基因开展功能注释与特征解析。本研究最终鉴定得到两种丝素蛋白，二者与此前通过计算预测得到的基因存在差异。氨基酸组成与二级结构分析表明，这些丝素蛋白与蜘蛛丝的腺状蛛丝蛋白（aciniform spidroins）或柱状蛛丝蛋白（cylindrical spidroins）具有相似性，这在一定程度上印证了其具备高杨氏模量的力学特性。叶螨丝素蛋白全长序列的获取，将有助于解析在多个演化支中以趋同方式起源的丝蛋白基因的演化历程，同时通过对这类丝蛋白的氨基酸序列及其力学特性的研究，推动人工蛋白纤维的工业化应用。<br>本研究针对神泽氏叶螨（Tetranychus kanzawai）与柑橘全爪螨（Panonychus citri）的样本保存、RNA提取、测序及组装流程，参考了此前针对蜘蛛的相关研究方法[18]并进行了部分优化。具体而言，将单头叶螨样本保存于RNAlater试剂中，并置于-80℃冰箱冻存；随后使用Direct-zol RNA Microprep试剂盒（Zymo Research公司）提取总RNA。使用KAPA HyperPlus试剂盒（KAPA Biosystems公司）构建插入片段长度约为300bp的Illumina测序文库。将构建好的测序文库置于NextSeq 500测序平台（Illumina公司），采用High Output Mode模式开展300轮双端测序。使用bcls2fastq v.2软件（Illumina公司）完成序列碱基识别、双端拆分以及接头序列切除操作。使用Bridger软件（默认参数）完成转录组组装[19]。使用kallisto v.0.44软件[20]，以本研究组装得到的转录组以及此前公开的二斑叶螨（T. urticae）基因组预测编码序列[16]作为参考，对转录本的表达水平进行定量分析。通过gVolante服务器[22]，使用节肢动物（Arthropoda）数据集的BUSCO v.3工具[21]对转录组组装的完整性进行评估。