A Targeted In-Fusion Expression System for Recombinant Protein Production in Bombyx mori

The domesticated silkworm, Bombyx mori, is an economically important insect that synthesizes large amounts of silk proteins in its silk gland to make cocoons. In recent years, germline transformation strategies advanced the bioengineering of the silk gland as an ideal bioreactor for mass production of recombinant proteins. However, the yield of exogenous proteins varied largely due to the random insertion and gene drift caused by canonical transposon-based transformation, calling for site-specific and stable expression systems. In the current study, we established a targeted in-fusion expression system by using the transcription activator-like effector nuclease (TALEN)-mediated targeted insertion to target genomic locus of sericin, one of the major silk proteins. We successfully generated chimeric Sericin1-EGFP (Ser-2A-EGFP) transformant, producing up to 3.1% (w/w) of EGFP protein in the cocoon shell. With this strategy, we further expressed the medically important human epidermal growth factor (hEGF) and the protein yield in both middle silk glands, and cocoon shells reached to more than 15-fold higher than the canonical piggyBac-based transgenesis. This natural Sericin1 expression system provides a new strategy for producing recombinant proteins by using the silkworm silk gland as the bioreactor.

The Role of Filippi’s Glands in the Silk Moths Cocoon Construction

Filippi’s glands (FGs), formerly also called Lyonet’s glands, are accessory secretory structures of the labial (silk) glands of lepidopteran caterpillars, which were implicated to play an important role in the maturation of the silk material and the construction of the cocoon. In our previous study, we have identified several species of giant silk moths that completely lack the FGs. Interestingly, the absence of FGs in these species correlates with the construction of a loose cocoon architecture. We investigated the functions of FGs by their surgical extirpation in the last instar larvae of the silkworm, Bombyx mori. We found that the absence of FGs altered the structure of the resulting cocoon, in which the different layers of silk were separated. In further experiments, we found no effects of the absence of FGs on larval cocoon formation behavior or on changes in cocoon mass or lipid content. Differential proteomic analysis revealed no significant contribution of structural proteins from FGs to silk cocoon material, but we identified several low abundance proteins that may play a role in posttranslational modifications of some silk proteins. Proteomic analysis also revealed a difference in phosphorylation of the N-terminal sequence of fibroin-heavy chain molecule. Thus, FGs appear to affect silk stickiness during spinning by regulating posttranslational modifications. This could also explain the link that exists between the absence of these glands and the formation of loose cocoons in some giant silk moth species.

Nuclear matrix associated RNA datasets of posterior silk glands of Bombyx mori during 5th instar larval development

Objectives Bombyx mori is the key contributor to industrial silk production. The maximum production of silk occurs during 5th instar. The posterior silk glands in the larvae are responsible for the production of the main component of silk fibre—fibroin. The expression of genes and their regulation are dependent on the chromatin architecture. The nuclear matrix supports its structure and function by anchoring specific regions to regulate gene expression. The major constituent of the nuclear matrix, crucial to its structural and temporal maintenance, is its RNA. Therefore, the study of nuclear matrix RNA of the posterior silk glands on different days of 5th instar larval development is essential to understand its association to differential expression of genes. Data description The tissue-specific developmental association of nuclear matrix RNA (NuMat RNA) at the genome level has not been done so far for any organism. Bombyx mori, CSR2 X CSR4 is the most popular dihybrid strain in India. The nuclear matrix RNA was isolated from day 1, day 5 and day 7 of 5th instar posterior silk glands of Bombyx mori. The NuMat RNA was sequenced using Illumina platform. The reads obtained were processed and the datasets were deposited in NCBI.

The Filippi’s Glands of Giant Silk Moths: To Be or Not to Be?

The Filippi’s glands (FGs), formerly “Lyonet’s glands”, are paired accessory organs associated with the silk glands. They are unique to Lepidoptera caterpillars and their exact role is not clear. The FGs are thought to be involved in the construction of a silk cocoon in bombycoid moths. FGs can differ in size and shape, therefore, in this study we attempt to find a correlation between FG morphology and phylogenetic position within the Bombycoidea. We use light and electron microscopy to examine the presence and morphology of FGs in a range of wild (giant) silk moths and several related species. Our results confirm that the majority of studied silk moth species have complex type of FGs that continuously increase in size during larval development. We identified several species of giant silk moths and two hawk moth species that completely lack FGs throughout their larval development. Finally, in several hawk moth species in which FGs are well developed during the first larval stage, these glands do not grow and remain small during later larval growth. Our results suggest that FGs are not critical for spinning and that loss of FGs occurred several times during the evolution of saturniids and sphingids. Comparison of FGs in different moths is an important first step in the elucidation of their physiological significance.

A Cluster of Genes at the Terminal Region of Chromosome 1 Contributes to Silk Yield in Bombyx Mori.

Background: The silk yield is the most economically important trait in sericulture. In the domestication of the silkworm, traits related to silk yield are the main selective targets. In previous studies, linkage and association analyses were performed, which narrowed the region of the locus controlling the cocoon shell weight (CSW), an important index of silk yield, to the interval of 0–134 kb on the 1st chromosome. Results In this study, selection pressure analysis showed that most of the loci in this interval were co-selected during silkworm domestication. Association analysis conducted with 95 strains found that there is a 110 kb region that was significantly associated with CSW. An expression profile correlation analysis of eight genes in the region showed that their expression patterns were very similar. Sequence variation analysis detected mutations in the exon regions of BMgn002066 and BMgn002073, leading to premature termination of translation. The sequence variation was significantly correlated with CSW. In addition, another two genes, BMgn002067 and BMgn002071, had significantly different expression levels in the posterior silk glands of 11 high-yield strains and 11 low-yield strains. Therefore, we speculate that the abovementioned four genes and the gene BmAbl1 detected in previous studies may co-regulate CSW. Conclusions This study is the first to identify the gene clusters that affect CSW by forward genetics, thereby providing new evidence for gene clusters that regulate quantitative traits.

Transportation of Chromium (VI) to Bombyx mori L. from mulberry Plant (Morus alba L.) grown at soil irrigated with Chromium (VI) containing effluents

The research was conducted to determine Chromium (VI) toxicity in population Bombyx mori. The synthetic wastewater used to irrigate soil to evaluate the impact of pH (4 to 8) at 100 mg/L and initial Chromium (VI) concentrations (25 mg/L to 300 mg/L) at 5 pH in its bioaccumulation in B. mori foodchain. By using atomic absorption spectrophotometer (AAS) analysis the amount of Chromium (VI) determined in soil, mulberry plants, B. mori larvae, silk glands and silkworm feces. The results showed that local cobalt pollution can be indicated by using B. mori as a template as its body length, body weight and the mortality rate were found to be strongly related to Chromium (VI) concentration. Higher the Chromium (VI) amount in mulberry leaves causes more toxicity to B. mori population. At 300 mg/L Cr (VI) concentration and pH 4 there was maximum deposition of Chromium (VI) in soil, mulberry plants, B. mori larvae, faeces and silk glands from the synthetic effluent. The maximum deposition was 123.5±0.03 mg/kg, 89.76±.031 mg/kg, 23.31±0.019 mg/kg, 41.32±0.069 mg/kg and 35.67±0.04 mg/kg observed respectively.

Shifts in morphology, gene expression, and selection underlie web loss in Hawaiian Tetragnatha spiders

Background A striking aspect of evolution is that it often converges on similar trajectories. Evolutionary convergence can occur in deep time or over short time scales, and is associated with the imposition of similar selective pressures. Repeated convergent events provide a framework to infer the genetic basis of adaptive traits. The current study examines the genetic basis of secondary web loss within web-building spiders (Araneoidea). Specifically, we use a lineage of spiders in the genus Tetragnatha (Tetragnathidae) that has diverged into two clades associated with the relatively recent (5 mya) colonization of, and subsequent adaptive radiation within, the Hawaiian Islands. One clade has adopted a cursorial lifestyle, and the other has retained the ancestral behavior of capturing prey with sticky orb webs. We explore how these behavioral phenotypes are reflected in the morphology of the spinning apparatus and internal silk glands, and the expression of silk genes. Several sister families to the Tetragnathidae have undergone similar web loss, so we also ask whether convergent patterns of selection can be detected in these lineages. Results The cursorial clade has lost spigots associated with the sticky spiral of the orb web. This appears to have been accompanied by loss of silk glands themselves. We generated phylogenies of silk proteins (spidroins), which showed that the transcriptomes of cursorial Tetragnatha contain all major spidroins except for flagelliform. We also found an uncharacterized spidroin that has higher expression in cursorial species. We found evidence for convergent selection acting on this spidroin, as well as genes involved in protein metabolism, in the cursorial Tetragnatha and divergent cursorial lineages in the families Malkaridae and Mimetidae. Conclusions Our results provide strong evidence that independent web loss events and the associated adoption of a cursorial lifestyle are based on similar genetic mechanisms. Many genes we identified as having evolved convergently are associated with protein synthesis, degradation, and processing, which are processes that play important roles in silk production. This study demonstrates, in the case of independent evolution of web loss, that similar selective pressures act on many of the same genes to produce the same phenotypes and behaviors.

Golden orb-weaving spider (Trichonephila clavipes) silk genes with sex-biased expression and atypical architectures

Spider silks are renowned for their high-performance mechanical properties. Contributing to these properties are proteins encoded by the spidroin (spider fibroin) gene family. Spidroins have been discovered mostly through cDNA studies of females based on the presence of conserved terminal regions and a repetitive central region. Recently, genome sequencing of the golden orb-web weaver, Trichonephila clavipes, provided a complete picture of spidroin diversity. Here, we refine the annotation of T. clavipes spidroin genes including the reclassification of some as non-spidroins. We rename these non-spidroins as spidroin-like (SpL) genes because they have repetitive sequences and amino acid compositions like spidroins, but entirely lack the archetypal terminal domains of spidroins. Insight into the function of these spidroin and SpL genes was then examined through tissue- and sex-specific gene expression studies. Using qPCR, we show that some silk genes are upregulated in male silk glands compared to females, despite males producing less silk in general. We also find that an enigmatic spidroin that lacks a spidroin C-terminal domain is highly expressed in silk glands, suggesting that spidroins could assemble into fibers without a canonical terminal region. Further, we show that two SpL genes are expressed in silk glands, with one gene highly evolutionarily conserved across species, providing evidence that particular SpL genes are important to silk production. Together, these findings challenge long-standing paradigms regarding the evolutionary and functional significance of the proteins and conserved motifs essential for producing spider silks.