Cloning And Sequence Analysis Of Araneus Ventricosus Dragline Silk Protein Gene

Spider dragline silk with unique characteristics of high strength and elasticity becomes a good natural protein fiber, and was named biosteel in spider's seven different functional silks.Spider dragline silk protein is composed of repeated sequences, high GC and numerous iterations of four different amino acid motifs. It is difficulty to design primers for PCR and RT-PCR, and also it is difficulty to obtain complet gene of spider dragline silk protein because of the competition between templates and primers. At present, there are no reports on the complete spider dragline silk gene and the molecular weight. The gene cloning and expression make progress slowly. Ren Hongling etal constructed the cDNA plague library and only got about 1.7kb Araneus ventricosus dragline silk protein gene. Beckwitt etal only amplified 300bp and 700bp dragline silk protein gene by PCR from Araneus Bicentenarius genome DNA. This paper chose Araneus ventricosus as a research material, and the genome DNA and total RNA were extracted from the special gland named major ampulate (MA) gland. Many primers were designed and synthesed. The dragline silk protein gene was amplified by normal PCR, multiple PCR and hot start PCR etal. PCR and RT-PCR conditions were optimized by primers experiments, MgCl₂ concentration gradient experiments, primer concentration gradient experiments, annealing temperature gradient experiments, extending temperature gradient experiments, touch down cycle experiments etal. The dragline silk protein genes were cloned into pDrive vector and sequenced, expression vector of pET-RS2 was constructed.The clones of Araneus ventricosus dragline silk protein gene AvDS1 and AvRS2 were obtained. AvDS1 composed of no intron was 2199bp and AvRS2 was 951bp. AvDS1 and AvRS2 were presumed to be Araneus ventricosus first fibroin protein gene and another dragline silk protein gene. The recombinant plasmid named pET-RS2 was constructed. This is a prelude to research the structure and expression of Araneus ventricosus dragline silk protein gene and the base of protein artifial filature by applying protein splicing.