Cloning And Analysis Of Spider Egg Case Silk Gene Based On Fosmid Genomic Library And PCR Screening

Spider silks not only possess variety of molecular structures and ecological functions, but encompass excellent mechanical properties that other high-performance fibers can not be compared with such as their superior strength, elasticity, biocompatibility, moisture retention, and their large initial modulus and breaking energy. Therefore, spider silks have broad potential application prospects in field of textile, aerospace, biomedical and military.However, intraspecific cannibalism and low yield of silk make the gene engineering preferred strategy rather than large-scale fanning spiders. Studies have shown that toughness of egg case silks is extremely high, so this study constructs A. ventricosus gDNA Fosmid library to obtain full-length gene of egg case silks for biomimicking spider silks subsequently.Egg case silks includes aciniform spidroin and tubuliform spidroin, and this project mainly study these two spidrion:1. We successfully isolated aciniform gland and tubuliform gland and extracted total RNA of these two glands, and then obtained partial cDNA of AcSpl and TuSpl through nested PCR. We explored and discussed the molecular mechanism to explain structural pattern and evolution mode which provided theoretical basis for further study of spider silk gene structure and biomimicking of spider silks.2. We successfully obtained partial cDNA of AcSpl including CTR and two complete repeat units through 3'-RACE, which provided reference for structure analysis of AcSpl and theoretical basis of obtaining full-length egg case silk gene and biomimicking spider silks.3. We improved CTAB to extracted HMW-gDNA of A. ventricosus, on the basis of which we successfully constructed unbiased Fosmid library. In this study, after end-repaired the insert DNA(about 30-40kb), ligated in pCC2FOSTM vector, and packaged the CopyControl Fosmid clones with MaxPlaxTM ambda Packaging Extracts and transfected EPI300TM-T1R E. coli, we obtained about 2.99×105 clones which covers 5 times of A. ventricosus genome. 4. Based on plate pool, horizontal pool and vertical pool of 384-well plates, we constructed progressive PCR screening system to screen genomic library, and utilized specific primers to screen the pools to determine the accurate location of positive clone. The progressive PCR screening system is simple and economical, and it also makes up for disadvantages such as long screening cycle, heavy workload and so on.In this study, we successfully obtained egg case silk encoding genes from Argiope amoena and A. ventricosus, and constructed gDNA library of A. ventricosus and developed the progressive PCR screening system, which provided basis for subsequent screening.