Construction Of Araneus Ventricousus Major Ampullate Gland CDNA Library And Screening Spider Silk Gene

The cDNA library of major ampullate glands of Araneus ventricousus was prepared successfully for the first time by using pUC18 vector in the world. Two genes encoding spider silk protein and one gene encoding no-silk protein were cloned by means of randomly picking colony (bird gun). Firstly, the major ampullate glands were got from Araneus ventricousus. Total RNA and mRNA of ampullate glands were isolated and purified. Double strands cDNAs were synthesized in the assistance of AMV-RT and E. coli DNA polymerase I etc. by reverse transcription and replacement synthesis. Fragments shorter than 400bp were removed by a gel chromatography prior to adding EcoR I adaptors to both ends of the double strands cDNAs and phosphorating their 5'ends. At the same time, pUC18 vectors were digested by EcoR I enzyme and their 5;ends were treated by CIAP. Then cDNAs and pUC18 vectors were linked by T4 DNA ligase and transformed into E. coli strain DH5-alpha to generate cDNA library that size is 4.9 l06 recombinants. Two spider silk protein genes (AvFl and AvF2) screened from the library are characterized in a lot of repetitive motifs, a high guanosines or cytidines content, a strong preference for adenosine or thymidine in the third position of a codon and rich residues of glycines or alanines in the proteins translated. AvFl is 1744bp and its region in encoding is 1572bp. Protein encoded by AvFl is 524 amino-acids and molecular weight is 42489.55dal. Its repetitive motif typically is (GGP)nGGX and the homology with Argiope trifasciata fiagelliform silk protein mRNA(AtfF) is 69.3%, which is the maximum comparing with all silk genes known in NCBI GeneBank. AvF2 is 1408bp and 1288bp is in the encoding region. 429 amino-acids are translated by AvF2 and the protein molecular weight is 34070.35dal. Its repetitive motif mainly is (GA)nAm(GA)N and the homology with Araneus diadematus fibroin-1 mRNA(ADF-l) is 69.3% that is themaximum, too. Avgl that is the no-silk protein gene was got. It is 1253bp and encode 334 amino-acids in its 1002bp encoding region. The molecular weight of the protein encoded is 34070.35dal. The high homology genes are not found in all genes known. But the putative conserved domains of peptidase have been detected. In the domains the homology comparing with Callus gallus cathepsin B cDNA is 90%. The objective function of the Avgl must be studied in the future. Construction of Araneus ventricoususampullate gland cDNA library and the finds of two new spider silk genes are the bases of supplying the background of Araneus ventricousus silk genes and further researching in the spider silk proteins.