Co-Expression Network and Time-Course Expression Analyses to Identify Silk Protein Regulatory Factors in Bombyx mori.

Yudai Masuoka,Hideki Sezutsu,Kakeru Yokoi,Wei Cao,Hiroki Sakai,Akiya Jouraku

doi:10.3390/insects13020131

Abstract

Simple SummaryPrevious studies have reported how the silk production ability of Bombyx mori can be enhanced, but the mechanism that regulates silk protein genes remains unclear. We performed co-expression network analysis using networkz, an in-house program, which led to the identification of 91 transcription factors were co-expressed with silk protein genes. Of them, 13 transcripts were identified to be novel regulatory factors by time-course expression analysis during the fifth instar larvae stage. Their expression patterns were highly relevant to those of silk protein genes. Our results suggest that the two-step expression screening was robust and highly sensitive to screen relative genes, and a complex mechanism regulates silk protein production in B. mori. The novel candidates that were identified herein can serve as key genes to develop methods to enhance the silk protein production ability of B. mori.Bombyx mori is an important economic insect and an animal model in pharmacomedical research. Although its physiology has been studied for many years, the mechanism via which silk protein genes are regulated remains unclear. In this study, we performed two-step expression screening, namely co-expression network and time-course expression analyses to screen silk protein regulation factors. A co-expression network analysis using RNA-seq data that were obtained from various tissues, including the silk glands of B. mori, was performed to identify novel silk protein regulatory factors. Overall, 91 transcription factors, including some known ones, were found to be co-expressed with silk protein genes. Furthermore, time-course expression analysis during the fifth instar larvae stage revealed that the expression pattern of 13 novel transcription factors was highly relevant to that of silk protein genes and their known regulatory factor genes. In particular, the expression peak of several transcription factors (TFs) was detected before the expression of silk protein genes peak. These results indicated that a larger number of genes than expected may be involved in silk protein regulation in B. mori. Functional analyses of function-unknown transcription factors should enhance our understanding of this system.

Highlights

Silkworms (Bombyx mori) generate silk proteins; they are an economically important insect in sericulture and have proved their value in biotechnology as a bioreactor for the production of recombinant proteins and silk-based biomaterials
To narrow down the candidate regulatory genes, we evaluated the time-course expression pattern of transcription factors (TFs) that were screened by co-expression network analysis in the four silk glands (SGs) regions (A-MSG, middle-middle SG (M-MSG), posterior-middle SG (P-MSG), and posterior SG (PSG)) during the last instar period using quantitative RT-PCR (qRT-PCR) (Figure 2B–D, Figure S1A, Data S2)
20 network modules including 91 TFs were screened by co-expression network analysis using the in-house program networkz, and in the second step, 17 transcripts were screened as silk protein-related genes by time-course expression analysis of the MSG and PSG during the last instar period

Summary

Introduction

Silkworms (Bombyx mori) generate silk proteins; they are an economically important insect in sericulture and have proved their value in biotechnology as a bioreactor for the production of recombinant proteins and silk-based biomaterials. Silk proteins can be broadly classified into sericin and fibroin, which are secreted from the middle and posterior silk glands (SGs), respectively. It has been reported that some transcription factors (TFs), including homeobox genes, regulate the expression of silk protein genes [2,3]. Antennapedia (Antp), a Hox gene that controls leg formation, directly regulates the expression of sericin in the middle SG [4,5]. Silk gland factor-2 (SGF2), a protein complex containing the homeodomain protein Arrowhead (Awh), LIM domain-binding protein, and sequence-specific single-stranded DNA-binding protein, regulates the expression of genes encoding fibroin in the posterior SG [6,7].

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Discussion

Conclusion