Report on construction of gene‐targeting vector for homologous recombination and transformation in silkworm, Bombyx mori L.

Abstract

This paper reports the methods of construction of gene-targeting vector for transformation of silkworm, Bombyx mori L. The genomic DNA was isolated from the posterior silk gland of the fifth-instar silkworm larvae. The short fragment (0.5 kb) and long fragment (5 kb) of the fibroin light-chain gene were obtained by polymerase chain reaction (PCR) analysis with special primers and genome DNA as templates and then recombined with pBlueselect vector into pBs-FS-FL. The target green flourescent protein (GFP) gene, was derived from pGEP-1 vector and recombined with pUC19 vector into pUCG vector. GFP was recovered after cutting with restriction endonucleases, PstI and BamHI. Finally, GFP was recombined with pBs-FS-FL into gene-targeting vector, pBs-FS-GFP-FL.