Sequence and bioinformatic characterization of expressed sequence tags originated from Gloydius shedaoensis shedaoensis venom gland.

ABSTRACT

First, a cDNA library from Gloydius shedaoensis shedaoensis venom gland (GSSG) was constructed using SMART technique. The total RNA in a pair of GSSGs was separated and the mRNA was further isolated from it. The first-strand cDNA was synthesized through PowerScript(™) reverse transcriptase by a CDS III/3' PCR primer. A SMART IV(™) -oligonucleotide was used as the template for the synthesis of double-strand cDNA and amplified by long-distance polymerase chain reaction (LD-PCR). LD-PCR product was purified, digested, fractionated, and finally ligated to pDNR-LIB vector. The recombinant DNA was transformed into Escherichia coli DH10B. The titer of this library was 8 × 10(7) cfu/mL. Most inserts were > 1,000 bp with great recombination efficacy. 216 GSSG-cDNAs were randomly analyzed and obtained 211 expressed sequence tags (ESTs) in high quality. A total of 84 ESTs were known functional genes showing significant similarities to known function proteins, 29 ESTs were unknown functional genes showing significant similarities to unknown function proteins, and 98 ESTs were novel genes with no match in any database, which account for 39.8%, 13.7%, and 46.5% of obtained ESTs. We successfully constructed a cDNA library and characterized partial ESTs of GSSG, which provides a solid base for cloning and expressing target genes and studying the biological functions of target proteins from GSS.