Study On Construction Of Transfer Vector Of Fibroin Gene Of Antheraea Pernyi And Transgenic Chinese Oak Silkworms

Silk is a proteinaceous polymer secreted by specialized exocrine glands in several groups of arthropods. Fibroin of Antheraea pernyi is secreted by the 5th larva. There is difference on structure of fibroin between Bombyx mori and A. pernyi, the former consists of two chains (H-fibroin and L-fibroin), which are linked by a disulfide bond, whereas the later consists of a single chain. Two transfer-expressing vectors of fibroin gene were constructed and experimented on sperm-mediated transformation method that based on its characteristic.The 5' flanking fragment of fibroin gene of A. pernyi was amplified by PCR. It consists of CAAT box, TATA box, prim transcript, start code ATG, partial sequence of structure gene and the first intron. Compared with Japanese Oak silkworm (Antheraea yamamai), three high homology regions have been observed with 91.6%, 95%, and 95%, respectively. Compared with Japanese Oak silkworm and B. mori on the homology of CAAT box, TATA box and prim transcript, the former are higher than that of the latter. The TATA box located at the upstream -25bp and CAAT box at -70bp from the prim transcript, which is similar as the character of eukaryotic promoters.The total RNA was extracted from the gland of silkworm, A. pernyi. The transcription initiation site of fibroin gene of A. pernyi was identified by RLM-RACE. The GFP gene was inserted into the vector, pGFP-N2/Fib. The results of visual screening under fluorescent inverted microscope and western blot analysis indicated that the GFP gene was expressed in the primary cells of ovary origins from A. pernyi.A 1.4Kb DNA fragment containing 3' flanking sequence of fibroin gene of silkworm, A. pernyi, was obtained from the silk gland's mRNA of 5th larva. Analysisof this sequence with another A. pernyi fibroin (accession No. D83241) revealed that it is consisted of a completely open reading frame (ORF), which includes 14 polyalanine-containing units (motifs) and lOObp 3'-UTR. The sequence of the predicted amino acid reveals the highest level of overall identity (90%) with D83241. It was found that it contains 5 Chi-like sequences and it loses a repeat region at the upstream of TAA codon.The transcription initiation site of A. pernyi was identified and targeting vector was constructed. The green fluorescent protein (GFP) gene was inserted into this vector under the control of the fibroin promoter. This recombinant vector was used to target the GFP gene to the fibroin region of silkworm genome. The DNA was injected into the testes during the pupae and was also transferred into the egg via sperm during fertilization. The analysis showed that the GFP gene had integrated into the fibroin gene on the genome by homologous recombination and was expressed in the silk gland.