Identification of the Genes that Regulate Silk Production in Spiders: A Computational Biology Approach

The molecular basis of spider silk production is of broad interest because of its possible mechanical applications. For instance, dragline silk, which is produced in the major ampullate gland of certain spiders, has been found to be tougher than nylon and Kevlar®. However, even though there is research on the mechanical and structural properties of spider silk, the gene expression and regulation responsible for spider silk production remains largely unexplored. In this project, we tried to identify the genes that regulate spider silk production by analyzing 8 RNAseq libraries from silk glands of male and female Dysdera spiders. Using a reference transcriptome, a differential expression analysis is done to identify statistically relevant expressed genes. The programs BowTie2 and TopHat were used to perform alignment to the reference transcriptome. To perform differential expression analysis, tuxedo tool suite of programs (CuffLinks, CuffMerge, CuffCompare), HTSeq, and DESeq, were used to determine statistically significant differentially expressed genes. This analysis will provide insights regarding the genes that are either upregulated or downregulated during silk production in Dysdera spiders. Introduction Spider silk has become an important topic in numerous fields due to the potential applications that can be derived from its mechanical properties (reviewed in Ayoub et al. 2007). However, spiders possess more than one type of silk producing glands. It has been shown that Spiders can produce up to six to seven different types of silk from different types of glands, which can vary in tensile strength and elasticity (Lewis, 2006; Fu et al., 2009). One of the spider silk producing glands, the major ampullate gland, has been the focus of spider silk research due to its abundance and ease of access (Xu & Lewis, 1990). Dragline silk, which is produced from the major ampullate gland, is known for its extremely high tensile strength and toughness (Ayoub et al., 2007). Its mechanical properties are often compared to synthetic fibers, which are known to have high modulus and strength. On the other hand, dragline silk, which possesses average strength, are significantly tougher than both nylon and Kevlar (Fu et al., 2009; Xu & Lewis, 1990). These properties are often associated with the proteins structures that constitutes spider silk. The majority of research regarding spider silk proteins revolve around major ampullate spidroins 1 and 2 (MaSp1 and MaSp2, respectively) and these proteins are mostly found in silks produced in major ampullate glands (Xu and Lewis, 1990; Hinman and Lewis, 1992). Research has shown that proteins like MaSp2 and MaSp1 have high molecular weight. MaSp1 and MaSp2 is also shown to contain repetitive domains that is glycine-rich and poly-alanine at its end (Gatesy et al., 2001). These proteins contain different amino acid sequence variation in different spidroins are involved on the different strength and toughness of the fibers (Hayashi & Lewis, 1998; Hayashi et al., 1999; Rising et al., 2005; Gaines & Marcotte, 2008). Although its mechanical and structural properties are extensively being studied, we have yet to discover the complete list of genes that are involved in spider silk gland development and its possible and crucial involvement in spider silk production. Studying the genes that are may be responsible for attaining the uniqueness of the gland may provide insight on how spider silk production is regulated. These genes may be involved in the intricate detailing of how spider silk are manufacture within the gland such as the details of the formation of the silk protein’s secondary structure, and folding and assembly of these silk proteins (Fu et al., 2009). In this research, transcript activity from various silk glands of male and female Dysdera spiders will be analyzed in in silico. These collections of transcript activities were sequenced using a next-generation sequencing technique called RNAseq. Differential expression analysis will be done using the pipeline for the tuxedo tools and R programming’s Bioconductor packages. The RNAseq libraries from male and female Dysdera spiders are first subjected to whole genome alignment using the programs BowTie2 and TopHat. The resulting data from TopHat was then subjected to the tuxedo pipeline for differential expression analysis: CuffLinks, CuffMerge, and CuffCompare. The results are then counted and visualized through the programs HTSeq and DESeq (Anders, Pyl and Huber, 2014; Anders and Huber, 2010). Differential expression analysis will be done using the pipeline for tuxedo tools by first performing whole genome alignment using the programs BowTie2 and TopHat, and finally performing differential expression analysis using CuffLinks, CuffMerge, CuffDiff, and CummeRbund. A separate program, an R Bioconductor package named DESeq, will also be used for differential expression analysis. These methodologies will help visualize which genes are utilized during spider silk formation. Methods RNAseq libraries are formed from the obtained mRNA transcript activities of total glands of male and female Dysdera spiders. Due to the limitations of our current technology, sequencing full-length mRNA sequence have yet to be achieved and therefore transcripts are fragmented first before running through RNAseq (Fig 1). RNAseq then generates a library of these fragmented mRNA sequences (via their complementary DNA). Male and female Dysdera spiders were separated into different libraries (Fig 2). Fig. 1. Sequencing of RNA transcript via RNAseq Extract transcript