• International Journal of Industrial Entomology and Biomaterials
• /
• 제6권1호
• /
• pp.49-55
• /
• 2003

We describe the generation of transgenic silkworm that carrying the chimeric fibroin light chain (L-chain) gene. Previously, we have cloned the complete fibroin L-chain gene from the silkworm Baekok-Jam, Bombyx mori, and the complete fibroin gene from the oak silkworm, Antheraea yamamai. The 444 bp repetitive sequence of A. yamamai fibroin gene was inserted into the exon 6 of B. mori fibroin L-chain gene to produce chimeric fibroin L-chain gene. The chimeric fibroin L-chain gene was cloned into the polyhedrin gene site of Autographa californica nuclear polyhedrosis virus (AcNPV) to yield a recombinant baculovirus as a fibroin gene targeting vector, One-day-old fifth instar female silkworm larvae were injected with the recombinant baculovirus and then mated with normal male moths. Genomic DNA from their progenies was extracted and screened for the desired targeting event by using PCR and Southern blot analysis. The analysis showed that the chimeric fibroin gene had intergrated into the L-chain gene on the genome by homologous recombination and was transmitted through generations. The transgenic silkworm carrying the chimeric fibroin gene were approximately 43.2% in $F_2$ generation, and the silkworms synthesized the fusion protein in cocoons layer.

• BMB Reports
• /
• 제41권5호
• /
• pp.394-399
• /
• 2008

Multiple sequence alignments of the Bombyx mori fibroin light chain gene (fib-L) from hybrids and from Chinese and Japanese strains demonstrated that 51.6% of the fib-L third intron is conserved. One of these conserved segments, 41 bp long, contains the sequence CGTTATTATACATATT, which is duplicated in the B. mori Nd-$s^D$ mutant. In the present work, electrophoretic mobility assays and computational analyses revealed a major peak of intrinsic bent DNA within the segment that undergoes breakage in the previously-described Nd-$s^D$ mutation. This result suggested that this intrinsically-curved region might mediate DNA cleavage and enhance recombination events in the third intron of the Bombyx mori fib-L gene.

• 한국잠사곤충학회지
• /
• 제53권2호
• /
• pp.124-129
• /
• 2015

본 실험에서는 누에 피브로인 L-chain 유전자의 인트론 영역을 사용하여 우리나라에서 보존하고 있는 누에 계통중 원산지별 누에 계통 8개를 대상으로 계통 간 유전자다형성을 비교하였다. 실험 결과, 실험에 사용한 누에 계통에 따라서 동일한 계통 내 모든 개체에서 FibL1과 FibL2의 유전자다형성 조합이 일정하게 유지되어 있었으며, 계통 간에는 FibL1과 FibL2의 유전자 다형성 패턴 조합이 다소 상이한 것으로 확인되었다. 따라서, 프라이머 FibL1와 FibL2에 의한 피브로인 L-chain 유전자 다형성 염기서열 정보를 활용한다면 제한된 영역에서 누에 계통 구분을 위한 지표로 활용할 수 있을 것으로 사료된다.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제6권1호
• /
• pp.45-48
• /
• 2003

We have previously cloned and characterized the complete fibroin L-chain gene from one of the silkworm races Baekok-Jam (Bombyx mori) and found two variable regions (I, intron 2 ~ exon 3; II, intron 6) with the primer sets designed to cover these variable regions. We tested the utility of these regions as genetic markers among silkworm races. For the purpose of study, Japanese race (Jam 123), Chinese race (Jam 124) and their F$_1$hybrid Baekok-Jam were used. The PCR product size of region I was 787 bp in Jam 123, 770 bp in Jam 124 and 768 bp in Baekok-Jam. The size of region II was 470 bp in Jam 123, 428 bp in Jam 124 and 429 Up in Baekok-Jam. In the extended experiment, Jam 125 (Japanese race), Jam 126 (Chinese race) and their F$_1$hybrid Daeseong-Jam were also analyzed. The sizes of region I and II in Jam 125, Jam 126 and Daeseong-Jam were similar to those of Jam 123, Jam 124 and Baekok-Jam. DNA sequence divergence between the two geographic races of Jam 123 or Jam 125 and Jam 124 or Jam 126 was substantial. The result suggests that the fibroin L-chain gene of F$_1$hybrids were inherited from Chinese races. These results are concordant with cocoon shapes of tested animals, and suggested that Baekok-Jam or Daeseong-jam is more closely related to Jam 124 or Jam 126 than to Jam 123 or Jam 125. Taken these data together, the primer sets designed from two variable regions of fibroin L-chain gene would be highly useful, as the genetic markers for silkworm races, at least in Japanese and Chinese races, although an extended study including more geographic races is required.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제17권2호
• /
• pp.229-234
• /
• 2008

We used serial analysis of gene expression (SAGE) approach to derive a profile of expressed genes of the posterior silk glands (PSG) and to create a reference for understanding gene cluster related to the mechanism of silk protein synthesis in the silkworm, Bombyx mori. We constructed a 3' SAGE library from the PSG of the fifth instar larvae of the silkworm. In total we obtained 2,406 SAGE tags, of which 682 were unique tags. Sorted by tag count number, 27 (4%) unique tags were significantly more abundant genes (ten or more times), whereas 445 (65%) unique tags were detected as single copies. The annotation of 682 unique SAGE tags revealed that 462 (68%) of the SAGE tag sequences represented known genes, whereas 220 (32%) of the tag sequences had no matches in SAGE map and silkworm EST databases. Of the 682 SAGE tags, the most abundant tag sequences were that of the fibroin light chain gene and the silk protein P25. In addition, we compared two relative abundance results of the SAGE and the EST approaches to verify whether their transcript quantitative aspects are significant or not. The comparative results of relative abundances of the fibroin H-, L- chain and P25 glycoprotein genes indicated that the quantitative approach based on SAGE tags is effective for quantitative cataloging and comparison of expressed genes in same organs. The SAGE tag information reported in this study would be useful for researchers in the field to analyze genes associated with silk processing mechanisms of insects.