Screening Spider Silk Gene And Cloning And Sequence Analysis Of The Part Region Of Araneus Ventricousus Major Ampullate Gland CDNA5′ Flanking

Spider silk is named "biosteel"because of its toughness and elasticity.The spider silk has very board foreground in military ,aviation,architecture,iatrology,and food etc. But it attracts our attention since JohnM. Gosline published the article about the spider silk protein/s mechanismand chemic capability in 1970. Studying spider silk gene was begun untillast tens years. Because of advancing ways and instruments, the study hadabstained good results. At the present time, some kinds of spider silk genewhich including Nephila clavipes ,Araneus bicentenarius ,Araneusdiadematus had been analyzed clearly in some foreign countries. In theaspect of pressing silk gene, the researchers are exploring actively. Thespider silk gene was inserted in plant gene by Udo Conrad, a biologist at theGatersleben graduate school in Germany. In Canada, Xexia biotechs Co.Ltd transferred the spider silk gene into the goat/s galactophore cell andproduced milk included silk protein. In U.N, the researchers at DuPontsynthetic spider dragline silk proteins in Escherichia coli. In Russia ,thescientists implanted the silk gene into the Saccharomyces. Cerevisiae, andproduced the protein resemble as silk protein. In our country the researcheson spider silk had gotten some production but having some disparities withother countries.In this experiment, we do it on the base of the gene AvF1 which wasscreened in our lab. It includes three experiments .First ,the cDNA plaguelibrary of major ampullate gland of Araneus ventricousus was constructedsuccessfully using SMARTTM cDNA Library Construction Kit and its titerwas 1.28×107pfu/mL. Primers were designed according to homogeneity ofAvF1. The probe PROB1 was obtained by PCR. AvF1h cDNA wasscreened by the probe. The length of the cDNA was 720bp and 625bp withthe open reading frame, which encoding 209 amino acid residues andmolecular weight is 18890.9dal. After anlyzing by BLAST and DNASIS,the AvF1h cDNA is one part of AvF1 cDNA. In the second experiment, theFirst-Strand cDNA was synthesized straightly by total RNA and usingrandom primer oligo(dT)15. The TdT added Poly(dC) tail to the 3′end ofthe cDNA , then designing the Anchored primer .The other anti-sense primewas designed by the template of Avf1h which had been screened. AfterPCR, one gene segment AvF1s was abstained . The length of the cDNA was403bp and 402bp with the open reading frame, which encoding 134 aminoacid residues and molecular weight is 19337.22dal. After analyzing byBLAST and DNASIS, AvF1s is the part of the repeated region of the spidermajor ampullate gland silk cDNA 5′flanking. It is a new gene andGenBank accession number is AY940091.In the third experiment, wedesigned the sense-primer and anti-sense primer by the template cDNA 5′end which was published by Cheryl Y.,Hayashi, Randolph V. Lewis. AvFU5was obtained and cloned by PCR. The length of the cDNA was 725bp and708bp with the open reading frame, which encoding 235 amino acidresidues and molecular weight is 22025.88dal. After analyzing by BLASTand DNASIS ,AvFU5 is the part region of the silk gene cDNA 5′flanking .It is new gene and GenBank accession number is AY945306. We predictedthe full length of silk cDNA according the ways which published by CherylY.,Hayashi, Randolph V. Lewis and the three genes which we had abstained.The length of the predicted gene was 10624bp ,which encoding 3478amino...