Construction Of Araneus Ventricous Spider Genomic DNA Library And Screening Its Dragline Silk Genes With Hybrid System

Spider is the largest member in Arachnoidea, Chelicarata Arthropoda. There are approximately 36,000 kinds of spiders, of which, 2,361 kinds were recorded in our country. All kinds of spiders can produce protein-based silks that are the thinnest silks in natural materials. With special structure, spider silks perform specific mechanical properties in terms of low density, high tensile strength, toughness, reasonable elasticity, and a considerable elongation to break. Due to their wettability, radiation hardening, fire-resistent, heatproof, and be able to bear the low temperature, spider silks have also the high capacity to outperform virtually all other natural and synthetic fibre materials and adapt to environmental changes. Composed with protein polymer in essence, spider silk fibres typically are amiable to environment, as they are able to undergo biodegradation and cyclic regeneration. These diverse and unique biomechanical qualities allow spider silks to be used for a variety of practical purposes ranging from bio-engineering to military equipment as high performance fibers.Because of spider's cannibalism, it is almost impossible for a high density culture. This limits further applications of spider silk fiber protein. So, nowadays, the mass-production of artificial spider silks through transgenic technologies has been a major goal of biology research. Araneus Ventricous is widespread in China, and its dragline silk performance extremely. Although some researchers have studied on this, they have not obtained the complete sequences coding the dragline silks. In order to obtaining dragline silk genes from Araneus ventricosus, This topic establishes tests below:First, we reported a urea lysis method for isolating spiders' genomic DNA. When compared to two existing methods of the CTAB lysis method and TIANamp Genomic DNA Kit, the results showed that the urea lysis method produced spider genomic DNA of higher yields and higher integrity. This urea lysis method is also simpler and less time-consuming, involving only room temperature manipulations, doing without liquid nitrogen and no deposition formed during lysis. Without mixed thoroughly by inverting to avoiding shearing action of fluid shear force to high-molecular-weight DNA, the lysis mixed is suspending. Using this method, we have isolated high quality genomic DNA more than 23 kb in size from spider, and about 150 ng quality genomic DNA can be isolated from 1 mg spider muscle. The isolated genomic DNA is not contaminated by protein.The isolated high-molecular-weight DNA was mechanically sheared through a pipette tip and subsequently treated with End-Repair Enzyme Mix (Epicentre) to produce blunt 5' phosphorylated ends. Approximately 40 kb fragments were gel excised, purified, and ligated into CopyControl pCC2FOS^TMvector (Epicentre). Resulting fosmids were packaged using MaxPlaxTM Lambda Packaging Extracts and transfected into EPI300^TMT1^R E. coli cells. We successfully constructed the unbiased Fosmid library that covered whole genes. The titer of library achieves 8×10⁴ cfu/μg ligated DNA. On the basis of published sequence of the dragline silk gene, we designed and synthesized 3 oligonucleotides sequences to hybridize, which are located separately in the N-terminal, middle repetition area and C-terminal dragline silk gene. These Oligonucleotides were labeled with radioactive [α-³²P]dCTP and detected with chemilluminescent substrate. About 1.3×10³ positive recombinants were screen from approximately 4.8×10⁵ clones using [α-³²P]dCTP-Oligo 2 as probe. Then 82 positive signals were identified from 1.3×10³ colonies using [α-³²P]dCTP-Oligo 3 as probe. At last, 12 positive clones were identified from 82 colonies using [α-³²P]dCTP-Oligo 3 as a probe. Theoretically, the 12 positive clones should include the full-length spider dragline silk genes. Although there is some certain false positive in situ hybridization, they should contain the partially coding sequences at least. Other positive clones also can include partial dragline genes. Through random sequencing in shotgun method, we obtained 4 DNA fragments in which the typical repeat peptide motifs GAGA, GGX and GPGGY of spider dragline silk protein existed. When online Blast, these partial sequences are compared with the previously published silk gene sequences from Araneus ventricosus, the sequence similarity is more than 90%.In this paper, we used the established urea lysis method to solve the difficulty in isolating spider genomic DNA. We successfully constructed Araneus ventricosus Fosmid library that contained any given DNA sequence. We also obtained the new partial sequences and positive clones that conained the dragline genes. These work have laid solid foundation for further research and development of spider silks.