LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes,fibH, fibL and fhx,in the silk gland of the silkworm,Bombyx mori

In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-homeodomain protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms. The genes for Ldb and Lcaf were expressed ubiquitously in various tissues, while the gene for Awh was expressed strictly specific in PSG of the wild type silkworms. Misexpression of Awh in transgenic silkworms induced ectopic expression of fibL and fhx as well as fibH in MSG. Coincidently with the induction of fibL and fhx by Awh, binding of SGF-2 to the promoter of fibL and fhx was detected in vitro, and SGF-2 binds directly to the fhx core promoter. Ectopic expression of the fibroin genes was observed at high levels in the middle part of MSG. Moreover, fibL and fhx were induced in the anterior silk gland (ASG) of the transgenic silkworms, but fibH was not. These results indicate that Awh is a key activator of all three fibroin genes, and the activity is probably regulated in conjunction with additional factors.     

An optimized sericin-1 expression system for mass-producing recombinant proteins in the middle silk glands of transgenic silkworms

The middle silk gland (MSG) of silkworm is thought to be a potential host for mass-producing valuable recombinant proteins. Transgenic MSG expression systems based on the usage of promoter of sericin1 gene (sericin-1 expression system) have been established to produce various recombinant proteins in MSG. However, further modifying the activity of the sericin-1 expression system to yield higher amounts of recombinant proteins is still necessary. In this study, we provide an alternative modification strategy to construct an efficient sericin-1 expression system by using the hr3 enhancer (hr3 CQ) from a Chongqing strain of the Bombyx mori nuclear polyhedrosis virus (BmNPV) and the 3′UTRs of the fibroin heavy chain (Fib-HPA), the fibroin light chain (Fib-LPA), and Sericin1 (Ser1PA) genes. We first analyzed the effects of these DNA elements on expression of luciferase, and found that the combination of hr3 CQ and Ser1PA was most effective to increase the activity of luciferase. Then, hr3 CQ and Ser1PA were used to modify the sericin1 expression system. Transgenic silkworms bearing these modified sericin1 expression vectors were generated by a piggyBac transposon mediated genetic transformation method. Our results showed that mRNA level of DsRed reporter gene in transgenic silkworms containing hr3 CQ and Ser1PA significantly increased by 9 fold to approximately 83 % of that of endogenous sericin1. As the results of that, the production of recombinant RFP increased by 16 fold to 9.5 % (w/w) of cocoon shell weight. We conclude that this modified sericin-1 expression system is efficient and will contribute to the MSG as host to mass produce valuable recombinant proteins.     

Spatio-temporal expression of wnt-1 during embryonic-, wing- and silkgland development in Bombyx mori

A homologue of the segment polarity gene wnt-1 from Bombyx mori (Bmwnt-1) has been characterized. The segmentally reiterated pattern of Bmwnt-1 transcrip9t distribution in B. mori embryos suggested its segment polarity function. Maximal levels of Bmwnt-1 RNA during embryonic development were reached by stage 21A. In the larval stages, Bmwnt-1 was expressed in the fore- and hindwing discs, ovaries, testes and gut, reminiscent of the expression domains in Drosophila. Bmwnt-1 was expressed in the wing-margin area of both the fore- and hindwing discs. The pattern of wnt-1 expression in the hindwing discs was similar to that in the butterfly Precis coenia but subtle differences existed in forewing discs of the two species, which correlated well with the absence of proximal bands of pigmentation in the adult Bombyx wings. In addition, Bmwnt-1 was expressed in the silkglands and the expression was confined to the anterior sub-compartment within the middle silkglands throughout development from the embryonic to late larval stages. This domain of Bmwnt-1 expression overlapped with those of Cubitus interruptus (BmCi) and sericin-2 but excluded the Engrailed expression domain viz. the middle and posterior sub-compartments of middle silkglands. Bmwnt-1 expression was detected only during the intermoults and not in the moulting periods.     

带有丝素重链信号肽序列的家蚕丝胶蛋白启动子驱动DsRed的瞬时分泌表达

为了探讨家蚕Bombyx mori丝素蛋白重链信号肽序列在中部丝腺组织中是否具有功能活性,根据家蚕丝蛋白基因的启动子活性高、丝蛋白具有高效分泌的特性,构建了带有丝素重链基因fib-H信号肽的家蚕丝胶-1(ser-1)启动子(ser-HS),用ser-HS驱动DsRed基因构建了分泌型瞬时表达载体pSK-SerHS-DsRed-polyA。转染细胞实验显示,该载体能在家蚕BmN细胞中瞬时表达DsRed。家蚕注射载体后,可在中部丝腺腔中检测到红色荧光,表明瞬时表达的DsRed已分泌到丝腺腔内。据此提出克隆的fib-H信号肽序列在家蚕中部丝腺组织中具有信号肽的功能。     

Novel enhancer and promoter elements indispensable for the tissue-specific expression of the sericin-1 gene of the silkworm Bombyx mori

Sericins are glue proteins produced specifically in the middle silk gland (MSG) of the silkworm Bombyx mori, while the silk fiber protein, fibroin, is produced in the posterior silk gland (PSG). These silk proteins are expected to be useful biomaterials in medical technology as well as biotechnology. In this study, we analyzed promoter elements of the sericin-1 gene (ser1) in vivo by introducing reporter constructs into silk glands via gene gun technology. The region from −1602 to +47 was sufficient to induce MSG-specific expression. The 5′ deletion mutants showed a three-step decrease in promoter activity with the key sequences located between −1362 and −1250, −201 and −116, and −115 and −37. We detected a tissue- and stage-specific factor complex (MSG-intermolt-specific complex: MIC) bound to the sequence elements around the −1350, −320, −180, and −70 regions. A mutation in the −70 region, which inhibits MIC-binding, diminished almost all promoter activity, while another mutation that did not inhibit MIC-binding showed no effect on promoter activity. The results suggest that the binding of MIC to the above elements is intrinsic for the spatiotemporal specificity of ser1 in vivo.