This study was conducted to determine the appropriate seeding dates by verifying the difference in winter survival and productivity of alfalfa according to fall sowing dates in the central area of South Korea. The experiment was conducted for 2 years (2020 and 2021) at the field in the Department of Animal Resources Development, NIAS located in Cheonan. Sowing dates started from September 18 to November 8 with 10 days of intervals during 2020 and 2021; SO1 (September 18), SO2 (September 28), SO3 (October 8), SO4 (October 18), SO5 (October 28), and SO6 (November 8). After sowing, the winter survival rate was measured in the spring of the following year, and the dry matter yield was measured by harvesting at 10% flowering and harvesting five times a year. SO6 failed to winter survival, and SO5 also had a lower winter survival rate than SO1~4 (p<0.05). The average annual dry matter yield of alfalfa linearly decreased with delaying sowing dates (p<0.05). The feed value did not differ in the same year by delaying the sowing date in the same year. These results suggest that sowing date should be started before October 18 to increase winter survival and productivity of alfalfa in the central area of South Korea.

This peptide has antibacterial activity against several Gram-positive and Gram-negative bacteria. BmCecB1 is antimicrobial peptides from Bombyx mori and belongs to cecropin family. Antimicrobial peptides are important components of the innate immune systems in all living organism. To produce the BmCecB1 antimicrobial peptide, we constructed transgenic silkworm that expressed BmCecB1 gene under the control BmA3 promoter using piggyBac vector. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor vector and helper vector were micro-injected into 600 eggs of bivoltin silkworms, Baegokjam. In total, 49 larvae (G0) were hatched and allowed to develop into moths. The resulting G1 generation consisted of 22 broods, and we selected 2 broods containing at least 1 EGFP-positive embryo. The rate of successful transgenesis for the G1 broods was 11%. We identified 9 EGFP-positive G1 moths and these were backcrossed with wild-type moths. With the aim of identifying a BmCecB1 as antimicrobial peptide, we investigated the Radical diffusion Assay (RDA) and then demonstrated that BmCecB1 possesses high antibacterial activities against Gram-negative bacteria.

Silkworm transgenesis is now a routine method leading to a satisfactory yield of transformed animals and the reliable expression of transgenes during multiple successive generations. However, the screening of G1 transgenic individuals from numerous progeny has proved to be difficult and time-consuming work. Previously, we characterized the promoter of heat shock protein 70 from Bombyx mori (bHsp70), which is ubiquitously expressed in all tissues and developmental stages. To investigate the utilization of the bHsp70 promoter to screen transgenic individuals, the EGFP marker gene was inserted into the piggyBac vector under the control of the bHsp70 promoter. Mixtures of the donor and helper vectors were micro-injected into 3,060 eggs of bivoltine silkworms (Keomokjam). EGFP fluorescence was observed in 17 broods of transgenic silkworms under a florescence stereomicroscope. Interestingly, this fluorescent marker protein was detected not only in parts of the embryo segments on the seventh day of the G1 embryonic developmental stage but it was also detected in a part of the body of G1 hatched larvae, in the middle silk gland of G1 fifth instar larvae, and in the wings of seven-day-old G1 pupae and G1 moths. Therefore, we suggest that the bHsp70 promoter can be used for the rapid and simple screening of transgenic silkworms.

To product the blue fluorescent protein (AmCyan) expressed cocoon, we were fused AmCyan cDNA to the heavy chain gene and injected the gene into a silkworm. AmCyan was one of the existing violet fluorochromes and originally derived from the fluorescent protein amFP486. AmFP486 was cloned from the sea anemone Anemonia majano (GenBank accession number AF168421), and belongs to the family of fluorescent proteins (FPs) isolated from coral reef organisms. The AmCyan fusion protein, each with N- and C- terminal sequences or the fibroin H-chain, were designed to be secreted into the lumen of the posterior silk glands. The expression of the AmCyan/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3 EGFP as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor and helper vectors were micro-injected into 300 eggs of bivoltine silkworms (Baegokjam). EGFP fluorescence was observed in 3 broods of transgenic silkworms under a florescence stereomicroscope. The cocoon was displayed strong blue fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the AmCyan gene expressed cocoon will be enable the production of the novel biomaterials based on the transgenic silk.

To product the blue fluorescent protein (AmCyan) expressed cocoon, we were fused AmCyan cDNA to the heavy chain gene and injected the gene into a silkworm. AmCyan was one of the existing violet fluorochromes and originally derived from the fluorescent protein amFP486. AmFP486 was cloned from the sea anemone Anemonia majano (GenBank accession number AF168421), and belongs to the family of fluorescent proteins (FPs) isolated from coral reef organisms. The AmCyan fusion protein, each with N- and C- terminal sequences or the fibroin H-chain, were designed to be secreted into the lumen of the posterior silk glands. The expression of the AmCyan/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3 EGFP as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor and helper vectors were micro-injected into 300 eggs of bivoltine silkworms (Baegokjam). EGFP fluorescence was observed in 3 broods of transgenic silkworms under a florescence stereomicroscope. The cocoon was displayed strong blue fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the AmCyan gene expressed cocoon will be enable the production of the novel biomaterials based on the transgenic silk.

BmCecB1 are antimicrobial peptides from Bombyx mori and belongs to cecropin family. Antimicrobial peptides are important components of the innate immune systems in all living organism. This peptide has antibacterial activity against several Gram-positive and Gram-negative bacteria. To produce the BmCecB1 antimicrobial peptide, we constructed transgenic silkworm that expressed BmCecB1 gene under the control BmA3 promoter using piggyBac vector. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor vector and helper vector were micro-injected into 600 eggs of bivoltin silkworms, Baegokjam. In total, 49 larvae (G0) were hatched and allowed to develop into moths. The resulting G1 generation consisted of 22 broods, and we selected 2 broods containing at least 1 EGFP-positive embryo. The rate of successful transgenesis for the G1 broods was 11%. We identified 9 EGFP-positive G1 moths and these were backcrossed with wild-type moths. With the aim of identifying a BmCecB1 as antimicrobial peptide, we investigated the Radical diffusion Assay (RDA) and then demonstrated that BmCecB1 possesses high antibacterial activities against Gram-negative bacteria.

Immune-inducible antimicrobial peptides were produced using transgenic silkworms that expressed Rel family transcription factor, truncated BmRelish1 (BmRelish1t) genes under the control of the BmA3 promoter using the piggyBac vector. BmRelish1t gene contains all domains of Bmrelish: a Rel homolog domain (RHD), nuclear localization signal (NLS), acidic and hydrophobic amino acids (AHAA) rich region except the Ankyrin repeat domain (ANK) and the death domain (DD). (1:1) Mixtures of the donor vector (pG-3xP3EGFP-BmA3BmRelish1t) and helper vector were micro-injected into 1,800 eggs of bivoltin silkworms, Baegokjam and EGFP-induced fluorescence was observed for 25 broods of transgenic silkworms under a florescence stereomicroscope. Analysis by real-time PCR indicated that transgenic silkworms expressing BmRelish1t recombinant proteins displayed higher mRNA expression levels of the Bombyx mori antimicrobial peptides such as lebocin, moricin, and nuecin than the normal silkworms. Moreover, transgenic silkworms expressing BmRelish1t showed antibacterial activity against Escherichia coli. We suggest that transgenic expression of BmRelish1t may find useful applications for the production of various antimicrobial peptides at the same time in transgenic silkworms.

Silkworm transgenesis is now a routine method leading to a satisfactory yield of transformed animals and the reliable expression of transgenes during multiple successive generations. However, the screening of G1 transgenic individuals from numerous progeny has proved to be difficult and time-consuming work. Previously, we characterized the promoter of heat shock protein 70 from Bombyx mori (bHsp70), which is ubiquitously expressed in all tissues and developmental stages. To investigate the utilization of the bHsp70 promoter to screen transgenic individuals, the EGFP marker gene was inserted into the piggyBac vector under the control of the bHsp70 promoter. Mixtures of the donor and helper vectors were micro-injected into 3,060 eggs of bivoltine silkworms (Keomokjam). EGFP fluorescence was observed in 17 broods of transgenic silkworms under a florescence stereomicroscope. Interestingly, this fluorescent marker protein was detected not only in parts of the embryo segments on the seventh day of the G1 embryonic developmental stage but it was also detected in a part of the body of G1 hatched larvae, in the middle silk gland of G1 fifth instar larvae, and in the wings of seven-day-old G1 pupae and G1 moths. Therefore, we suggest that the bHsp70 promoter can be used for the rapid and simple screening of transgenic silkworms.

Attacin is a well-studied glycine-rich antibacterial protein in insect immune response, which has limitary antibacterial effect to some Gram-negative bacteria. A cDNA encoding the attacin gene was screened and isolated from the immunized larvae of the swallowtail butterfly, Papilio xuthus. The complete P. xuthus attacin cDNA is 949 nucleotides encoding a 250 amino acid precursor that contains a putative 18-residue signal peptide, a common 42-residue propeptide sequence and a presumed 190-residue mature protein with a theoretical mass of 19904.01 and a pI of 9.13. The putative mature protein of P. xuthus attacin showed 48%~52% and 24%~30% identity in amino acid sequences with that of lepidopteran and dipteran insects, respectively. The attacin transcript was induced at significant level after injection with bacterial lipopolysaccharide (LPS). Recombinant attacin was highly expressed in E. coli BL21 (DE3) cells by fusing with an N-terminal S-tag/thrombin cleavage site configuration protein to avoid the cell death during induction. The expressed fusion protein was purified by Ni-NTA immobilized metal affinity chromatography (IMAC). After desalting and cleavage with thrombin, the recombinant attacin was released and showed considerably antibacterial activity against Gram-negative bacteria, E. coli ML 35. Our results proved that this protein family with a potent antibacterial activity may play a role in the immune response of butterflies.