• Journal of Sericultural and Entomological Science
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• v.39 no.1
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• pp.22-29
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• 1997

Ultraviolet weavelength (UV) of 366 nm produced clearer fluorescent dolor than that of 254 nm for the inspection of silkworm cocoons. Fluorescent color of silkworm cocoons varied in color, appears no relationship with the natural color under the normal light. Uniformity of fluorescent color was improved by selection of blue or yellow line from wild types. Blue and yellow, located at the opposite poles on the color solid and L*a*b* color system, confirmed as pure standard of fluorescent color in the silkworm races for commercial white cocoons. the cocoons with blue fluorescence occupied as high as 1.7 to 8.6 times than those with yellow in the Japanese silkworm races. Fluorescence of silkworm cocoon was not affected by forced flow dry at 70$^{\circ}C$ for 6 hrs. While the Japanese races revealed no sexual difference in fluorescent color, sex-dependence of the color was common in the Chinese races for commercial white cocoon. The fluorescence of cocoon shell of Chinese races showed clear separation of blue of median color. Silkworm strain of Dc20 and Fc24 were sexualy segregated 98.8${\pm}$1.20%, 99.0${\pm}$1.00% by cocoon fluorescence, as that of 99.3${\pm}$0.44% by typical larval marking of sex-limited inheritance. Specific expression of cocoon fluorescence, applicable to breeding of simple discrimination of sex for Chinese races, inspected thoroughly on the surface and inner layer of cocoon shell.

• International Journal of Industrial Entomology and Biomaterials
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• v.34 no.1
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• pp.11-15
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• 2017

We constructed the fibroin H-chain expression system to produce enhanced cyan fluorescent proteins (ECFP) in transgenic silkworm cocoon. Fluorescent cocoon could be made by fusing ECFP cDNA to the heavy chain gene and injecting it into a silkworm. The ECFP fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the ECFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworms. The EGFP fluorescence became visible in the ocelli and in the central and peripheral nervous system on the seventh day of embryonic development. A mixture of the donor and helper vector was micro-injected into 1,020 Kumokjam, bivoltin silkworm eggs. We obtained 6 broods. The cocoon was displayed strong blue fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the ECFP fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.

• Journal of Sericultural and Entomological Science
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• v.30 no.1
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• pp.33-39
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• 1988

In the case of white cocoon, the fluorescence colors are classified as a yellowish fluorescence cocoon(Y.F.C.) and a violet fluorescence cocoon(V.F.C.) by exposing to ultra-violet ray. Accordingly, experiments were carried out to investigate the difference of sericin behaviors between Y.F.C. and V.F.C. by measuring the sericin solubility, surface tension and viscosity of the sericin solution. Also, the reelability of two different type of cocoons was investigated in the silk reeling process. The results were summarized as follows; 1. The sericin solubility of Y.F.C. shell is higher than that of V.F.C. shell with the dissolution temperature and time. It is shown that the sericin solubility curves of Y.F.c. and V.F.C. are similar in shape, but the difference of sericin solubility between Y.F.C. and V.F.C. is more significant at higher bath temperature. 2. The initial sericin dissolution curves of Y.F.C. and V.F.C. cocoon shell can be divided by four parts within the range of dissolving time from 5 minutes to 60 minutes. The initial dissolution velocity of Y.F.C. shell is faster than that of V.F.C. but the velocity difference is negligible after 30 minutes of dissolving time. 3. The gelation of V.F.C. sericin solution is faster than that of Y.F.C. at early stage(in the range of 15 minutes to 60 minutes). 4. In the silk reeling process, the reelability of Y.F.C. is better than that of V.F.C. with about 11%. This is mainly due to the higher sericin solubility in Y.F.C. followed by the fast dissolution velocity.

• Journal of Sericultural and Entomological Science
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• v.50 no.2
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• pp.87-92
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• 2012

We constructed the fibroin H-chain expression system to produce Discosoma sp. red fluorescent protein variant2 (DsRed2) in transgenic silkworm cocoon. Fluorescent cocoon could be made by fusing DsRed2 cDNA to the heavy chain gene and injecting it into a silkworm. The DsRed2 fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the DsRed2/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworms. The EGFP fluorescence became visible in the ocelli and in the central and peripheral nervous system on the seventh day of embryonic development. A mixture of the donor and helper vector was micro-injected into 1,020 Kumokjam, bivoltin silkworm eggs. We obtained 6 broods. The cocoon was displayed strong red fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the DsRed2 fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.

• Journal of Sericultural and Entomological Science
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• v.51 no.2
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• pp.153-158
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• 2013

To express green fluorescent protein in the cocoon of silkworm, we constructed the fibroin H-chain expression system to produce enhanced green fluorescent protein (EGFP) in the cocoon of transgenic silkworms. The EGFP fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the EGFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3-driven DsRed2 cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. A mixture of the donor and helper vector was micro-injected into 1,200 eggs of bivoltin silkworms, Baegokjam. We obtained 8 broods. The cocoon displayed strong green fluorescence, proving that the fusion protein was present in the cocoon. Also, the presence of fusion proteins in cocoons was demonstrated by SDS-PAGE and immunoblotting. Accordingly, we suggest that the EGFP fluorescence silk will enable the production of the novel biomaterial based on the transgenic silk.

• International Journal of Industrial Entomology and Biomaterials
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• v.45 no.2
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• pp.56-69
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• 2022

Silk is a unique natural biopolymer with outstanding biocompatibility, high mechanical strength, and superior optical transparency. Due to its excellent properties, silk has been widely reported as an ideal biomaterial for several biomedical applications. Recently, fluorescent silk protein, a variant of native silk, has been reported as a biophotonic material with the potential for bioimaging and biosensing. Despite the realization of fluorescent silk, the traditional degumming process of fluorescence silk is crude and often results in fluorescence loss. The loss of fluorescent properties is attributed to the sensitivity of silk fibroin to temperature and solvent concentration during degumming. However, there is no comprehensive information on the influence of these processing parameters on fluorescence evolution and decay during fluorescent silk processing. Therefore, we conducted a spectroscopic study on fluorescence decay as a function of temperature, concentration, and duration for fluorescent silk cocoon degumming. Sodium carbonate solution was tested for degumming the fluorescent silk cocoons with different concentrations and temperatures; also, sodium carbonate solution is combined with Alcalase enzyme and triton x-100 to find optimal degumming conditions. Additionally, we conducted a molecular dynamics study to investigate the fundamental effect of temperature on the stability of the fluorescent protein. We observed degumming temperature as the prime source of fluorescent intensity reduction. From the MD study, fluorescence degradation originated from the thermal agitation of fluorescent protein Cα atoms and fluctuations of amino acid residues located in the chromophore region. Overall, degumming fluorescent silk with sodium carbonate and Alcalase enzyme solution at 25 ℃ preserved fluorescence.

• Journal of Sericultural and Entomological Science
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• v.52 no.1
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• pp.25-32
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• 2014

We produced the transgenic silkworm that expressed the enhanced blue fluorescent protein (EBFP) in the cocoon of silkworms. The EBFP fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the EBFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the $3{\times}P3$-driven DsRed2 cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. A mixture of the donor and helper vector was micro-injected into 300 eggs of silkworms, Baegokjam. We obtained 5 broods. The cocoon displayed blue fluorescence, proving that the fusion protein was present in the cocoon. Also, the presence of fusion proteins in cocoons was demonstrated by SDS-PAGE and western blot analysis. Accordingly, we suggest that the EBFP fluorescence silk will enable the production of the silk-based biomaterials.

• Journal of Sericultural and Entomological Science
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• v.52 no.2
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• pp.117-122
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• 2014

On previous studies, we constructed a transgenic silkworm which produces the chimeric silk fused green fluorescent protein (EGFP), but the transgenic silkworm has decreased commercial feasible traits such as convenience of breeding and productivity of silk. In this study, we performed cross fertilization between green fluorescent silk transgenic silkworm and colored cocoon silkworm descents to make the transgenic the transgenic silkworm producing improved fluorescence cocoon. In the result, we found out a bit valuable cross fertilization manners ($female{\times}male$) in respect of silk productivity such as $T59B{\times}Jam26$, $Jam329{\times}T59W$, $T59W{\times}Jam329$, and $T59W{\times}Jam178$. The color-difference of offspring cocoons were measured according to different cross manners using by CIE Lab-based formulae with a X-rite VS450. In the result, the depth of green color of cocoons was a little high at cross manners as $Jam329{\times}T59W$, $T59W{\times}Jam178$. Meanwhile, the depth of yellow clolor of cocoons was remarkable at cross manners as $Jam178{\times}T59W$, $T59W{\times}Jam178$, respectively.

• Journal of Sericultural and Entomological Science
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• v.51 no.2
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• pp.142-146
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• 2013

The fluorescent proteins are generally denatured by heat treatment and thus lose their color. The normal reeling method includes processing by drying and cooking the cocoons near $100^{\circ}C$ before reeling. Therefore, the usual processing method cannot be used for making colored fluorescent silks. To develop a method that is applicable to producing transgenic silk without color loss, we develop reeling methods adequate for a recombinant fluorescence cocoons. It was found that the fluorescence cocoons keep their native color when dried at temperatures lower than $60^{\circ}C$ for 15 h. Also, a new cooking method to soften the fluorescent cocoons was developed: the cocoons were soaked in a solution of 0.2% sodium carbonate ($Na_2CO_3$)/0.1% nonionic surfactant (Triton X100) at $60^{\circ}C$ and then placed under vacuum. The repeated vacuum treatments enabled complete penetration of the solution into the cocoons, and the cocoons were thus homogenously softened and ready for reeling. In this state, the cooked cocoons can be reeled by an automated reeling machine. Our results suggest that drying and cooking of the cocoons at low temperature enables the subsequent reeling of the colored fluorescent silks by an automatic reeling machine without color loss and can produce silks that can be used for making higher value-added silk materials.