Construction Of CDNA Library Of Xinong Saanen Dairy Goat Mammary Glamd And Cloning And Sequence Analysis Of LPL Gene

For investigating the gene expression and conducting the functional and comparative genomics researches in goat mammary and also promoting the research on the mechanism of the goaty flavor development, in this research, Xinong Saanen dairy goat mammary gland at mid lactation was used to construct a conventional cDNA library. The quality analysis of library was carried on and some clones were selected to be sequenced and analyzed. According to the conservative sequence of LPL gene which plays an important role in goat milk lipolysis, primers were designed and then RT-PCR, 5'-RACE combining with the cDNA library PCR amplification were used to clone full-length cDNA of goat LPL gene. The sequence was analyzed by bioinformatics and its expression pattern in mammary at different lactation stages was analyzed by Real-Time PCR. The results were as follows:1. cDNA library of mammary gland from mid-lactation Xinong Saanen dairy goat was constructed with plasmid vector and the quality of the cDNA libraries was identified. The results showed that the titer of the mammary library was 1.9×10⁶ cfu/mL, the total number of colony-forming units (cfu) was 1.4×10⁷ cfu, the recombinant rate was 96% and the average insert size was longer than 1.1 kb. The library titer, recombinant rate and average insert size all matched quality requirements for cDNA library and this indicated that cDNA libraries of goat mammary was successfully constructed, which will be a valuable resource for functional genomics studies of goat.2. Some clones from the mammary gland cDNA library were randomly collected and sequenced through 5'-end single-pass. 115 sequences were gotten and the success rate of sequencing was 92%. We analyzed the identity of these ESTs in the GenBank nr database and the EST database. The results showed that 115 ESTs could be clustered into two groups, 22 known genes and 29 new discovery clones which could not match any known sequences or had matched sequences of unknown function genes in the GenBank databases.Through cross-species genomic comparison, the five goat gene species that were identified represented five proteins that included beta-lactoglobulin, as2-casein, prealpha-lactalbumin, glycosylation-dependent cell adhesion molecule-1, and fatty acid synthase. Overall, clones homolog to well known function genes represented a wide range of functions including milk and ribosome constituencies, metabolism, immune response and translation protein. The majority of known function clones (59%) coded for milk related proteins including beta-lactoglobulin (28%), beta-casein (20%), as2-casein (5%), kappa-casein (3%) and prealpha-lactalbumin (3%). The 115 ESTs have been submitted to GenBank and gotten the Accession No..3. The primers were designed to clone CDS and 1546-3064 region in 3'-UTR of LPL gene by RT-PCR method. And 5' untranslted region (UTR) was got by 5'-RACE method and 3' end region was gained by PCR amplification from mammary cDNA library. Then these segments were splicing and assembled to form the full-length cDNA of LPL gene, and the cDNA was 3630 bp, with a 1437 bp CDS flanked by a 203 bp 5'-UTR and a 1990 bp 3'-UTR.4. The characteristics of LPL gene among goat, sheep, bovine, human and mouse, were analyzed by bioinformatics. Sequencing results proved Xinong Saanen dairy goat mammary gland LPL gene had a CDS of 1437 bp, coding 478 amino acids, the GenBank number was DQ997818. The homologies of nucleotide of Xinong Saanen goat mammary gland LPL gene with sheep (NM₀01009394), bovine (NM₀01075120), pig(AY559453), human (NM₀00237) and mouse's (NM₀08509) were 98%, 98%, 91%, 89% and 87%, the homologies of peptide sequence were 98%, 98%, 92%, 90% and 88% respectively. The 9 bp (3 amino acid) in goat LPL gene 88-102 nt of code domain was deleted in LPL of human and mouse. Goat LPL had one additional potential motif compare to human's by online motifscan prediction software.5. Real-time fluorescence quantitative RT-PCR was used to determined the expression difference of LPL gene in mammary gland at early, peak, mid, late lactation stages and dry period of Xinong Saanen dairy goat. Results showed the LPL gene expression was highest at early lactation stage, and the next were peak and late lactation stages. At dry period, the expression level of LPL gene was lowest.