Cloning And Characterization Of Araneus Ventricosus Masp Gene Based On Fosmid Genomic Library And STS/3D-PCR

Spider silk is an intriguing biomaterial that is extremely strong, elastic, lightweight, radiation hardening, heatproof and biodegradable, and exhibits mechanical properties comparable to the best synthetic fibers produced by modern technology. Based upon the desirable properties of spider silk, scientists have recognized the potential for large-scale production of silk for militarial, biomedical and navigational applications. Dragline silk as a kind of spider silk, also called "biosteel", has received more and more attention.Because spiders are territorial carnivores, domestication of these organisms to create spider farms for silk production is impractical. In addition, one silkworm cocoon yields more fibers than in a complete spider web. Therefore, the best option for moving the application of spider silks forward is the pursuit of biotechnological means of generating source material. But the typical spider-silk proteins, in which lots of consensus motifs are found, such as major ampullate spidroin (MaSp), are coded by genes which lack introns and thus possess only one enormous exon containing long coding sequence. And these are the main obstacles to the polymerase chain reaction (PCR) which is unable to reliably amplify repetitive sequences as found in spider silk genes. As to obtain full-length spider silk gene sequences based on construction and screening of genomic library is a surest way.The largest and most widely distributed genus of spider in China is Araneus ventricosus, of which the silk has received much attention because of the outstanding mechanical properties. So far, the number of reported A. ventricosus partial MaSp gene sequence is limited, only few N terminal sequences which are less than 2.0 Kb have been obtained. To get more A. ventricosus MaSp gene for providing the genetic blueprints for "bionic weave" of spider silk, this thesis has done researches as follow:(1) Preparing for constructing Fosmid library: To construct a Fosmid library with nice integrality, high molecular weight genomic DNA (HMW-gDNA) was extracted from pectoral muscle tissues of A. ventricosus by improved method based on CTAB. This improved method is a simple and less time-consuming method with low cost and high efficiency, and can be used at room temperature without liquid nitrogen. The HMW-gDNA extracted using the improved CTAB method could be over 48.5 Kb in size with high purity (OD₂₆₀/OD₂₈₀ =1.7～1.9), and about 147 ng HMW-gDNA could be obtained from 1 mg spider muscle tissues. On the other hand, for recovering high molecular weight DNA fragments from agarose gels, a miniaturized electroelution cell was developed using laboratory general supplies. With the electroelution cell, HMW-gDNA fragments (about 30～40 Kb in size, meet the demands of Fosmid library) of A. ventricosus were recovered, and the recovery rate was over 75% with OD₂₆₀/OD₂₈₀ ranging from 1.8 to 2.0. A step-by-step fractionation of lambda Hind III DNA molecular weight marker (23.1, 9.4, 6.6 and 4.4 Kb) was also recovered, and only a tiny amount of DNA was lost on elution. Generally, the miniaturized electroelution cell is a general method of recovering target HMW-gDNA fragments from agarose gels with low cost, simplicity, high efficiency and good practicability.(2) Constructing Fosmid library: The HMW-gDNA fragments, obtained by improved CTAB method and miniaturized electroelution cell, were end-repaired and ligated into pCC2FOS^TM Vector. Recombinant molecules were packaged in vitro with the phage Packaging Extract, then transformed into EPI300?-T1^R E coli. A Fosmid library of A. ventricosus was constructed firstly with titer ranging from 6.0×10⁴ to 1.1×10⁵, and gene coverage ratio was up to 7. Approximately 3.9×10⁵ recombinant EPI300^TM-T1^R E coli colonies were picked and arrayed into 1042 384-well plates.(3) STS/3D-PCR screening and characterization of A. ventricosus MaSp gene: According to sequence alignment of reported MaSp amino acid sequence, some degenerate primers were designed and synthesized for amplifying MaSp1/MaSp2 N and C terminal unrepeated gene sequences. By sequencing Degenerate PCR products, MaSp1 N terminal (227 bp in size), MaSp1 C terminal (195 bp in size) and MaSp2 C terminal (171 bp in size) partial gene sequences were obtained and used as sequence tagged site (STS) markers of MaSpl/MaSp2 gene respectively. Based on these STSs, three specific primers used in STS/3D-PCR were also designed. Before STS/3D-PCR screening, 1042 384-well plates were divided into about 26 sets of 40 plates. For each set, primary pools, secondary pools and super pools were constructed and screened by STS/3D PCR respectively. Two MaSp2-positive clones containing the gene of interest were found after screening the library, named FM2-0811 and FM2-1124. Shotgun sequencing of these two positive clones resulted in MaSp2 partial coding sequence (1950 bp in size), named MaSp2-1, and the sequence similarity between MaSp2-1 and previously published partial MaSp2 gene sequences was about 100%. Many typical repeat peptide motifs, such as (A)n, (GA)n and (GGX)n, were found in vary array in amino acid sequence translated from MaSp2-1. In addition, a highly repetitive sequence (977 bp in size) of skeletal muscle protein N terminal coding gene, named AvSmp-1, was also obtained luckily. AvSmp-1 is an especial sequence which has lots of highly conserved exons (～48 bp in size) spaced out by equal quantity of conserved introns(～33 bp in size), and the sequence similarity between AvSmp-1 and the unique skeletal muscle protein N terminal mRNA sequence of Tarantula is over 87%. Additionally, FM2-0811 and FM2-1124 are being full-length sequenced for obtaining full length coding sequence of MaSp.In this paper, a fosmid genomic library in A. ventricosus was constructed with nice integrality, and MaSp2-positive clones containing full-length MaSp2 gene sequences were obtained based on Degenerate PCR and STS/3D-PCR. By shotgun sequencing, new sequences of MaSp conding gene were obtained. The clones sequenced here provide the genetic blueprints for the primary protein components of the major ampullate silk fiber. These designs hold critical information for the mass production of artificial fibers that accurately mimic the spectacular high-performance properties of native spider silk. On the side, a useful method for extracting spider HMW-gDNA was formed based on CTAB method. And for recovering high molecular weight DNA fragments in short times from agarose gels after electrophoresis efficiently, a miniaturized electroelution cell which is simple enough to be used as a routine method with simplicity was developed.