Screening, Cloning, Identifying And Function Analyzing Of Differently Expressed Genes In Mammary Gland Of Xinong Saanen Goats At Early And Peak Lactation Stages

Mammary gland, taking the function of synthesizing and secreting milk, which undergoes pregnancy-associated proliferation, differentiation, and involution during a lactation cycle of diary goat. During each cycle, mammary epithelial cells experience the change of proliferation, invasion, differentiation and cell apoptosis, and this normal physiological changes of mammary gland and epithelial cells directly effects or determines the length of lactation period, the change of milk composition and even the levels of milk yield. With further accumulation of knowledge on gene structure and function, people gradually recognized that the underlying mechanism depends on the related genes of mammary gland differently expressed. Thus we collected mammary gland samples of Xinong saanen goats at early and peak lactation stages to construct forward and reverse subtracted cDNA libraries of mammary gland, further identify and clone the differently expressed genes, and predict their structure and function by bioinformatics, and analyze their expression pattern by real-time reverse transcriptase-polymerase chain reaction, and talk about the preliminary function of serum amyloid A 3 protein (SAA3) which is one of the different expression genes. The main results were showed as following:1. Suppression subtractive hybridization technology is used to construct forward and reverse subtracted cDNA libraries of mammary gland at early and peak lactation stages called E-P (mRNA of mammary gland at early stage as tester, and that at peak as driver) and P-E (mRNA of mammary gland at peak stage as tester, and that at early as driver) subtracted cDNA library. The subtraction efficiency was estimated by a housekeeping gene named GAPDH, and the results showed that GAPDH was subtracted efficiently at 210 and 25 folds for E-P and P-E subtracted cDNA library respectively which demonstrated that differentially expressed genes were also enriched at the same folds.2. 56 and 87 clones were isolated from E-P and P-E subtracted cDNA library, and 30 clones selected randomly from each library which had different insert fragments were sequenced. The results showed that there exist 8 goat ESTs in E-P subtracted cDNA library,and 3 of them are known in goat, 5 are unknown in goat but have more than 95% homology with other species, they represent immune related genes (CD36 antigen, Melanoma-associated antigen), bone formation related gene (osteoglycin), and milk protein related genes (prealpha-lactalbumin, alpha-S1-casein, as2-casein, beta-casein, kappa casein); in P-E subtracted cDNA library there exist 18 goat ESTs in P-E subtracted cDNA library, and 1 of them is known in goat, 16 are unknown in goat but have more than 87% homology with bovine, and 1 of them is the sequence of goat mitochondrion, these differentially gene mainly include 5 groups: cell proliferation and differentiation related genes: SAA3, ATP-binding cassette, sub-family G, member 2 (ABCG2), H3 histone, prothymosin, alpha, laminin receptor 1), fat metabolism related genes: heart fatty acid-binding protein (HFABP), xanthine dehydrogenase (XDH), transcription complex related genes : ribosomal protein S11, S12, S16, S19, R19), cell energy supplement related genes: cytochrome c oxydase subunit 4, and Vb, and 4 unknown genes.3. Real-time reverse transcriptase-polymerase chain reaction was used to analyse the change of mRNA expression level of SAA3, ABCG2, HFABP and XDH in early and peak lactation stage selected form P-E subtracted cDNA library. The results showed that they had 17.0, 7.6, 16.0 and 5.02 folds higher in peak lactation stage than that in early lactation stage. We designed primers based on the high homology sequence in gene coding region among different species and existed sequence for goat, and successfully cloned the coding region of SAA3, ABCG2 and XDH of goat for the first time, namely, Capra hircus serum amyloid A3 protein (ORF 396bp, GenBank No: DQ904356), Capra hircus ATP-binding cassette sub-family G member 2 (ORF 1977bp, GenBank No: DQ839400) and Capra hircus xanthine oxidoreductase (ORF 4002bp, GenBank No: EF151286), which laied a foundation for further functional research.4. The structure and function of SAA3, ABCG2, HFABP and XDH were compared among goat, bovine, human, and mouse using bioinformatics software. We found the ORF and amino acid of each of the 4 genes in different species had high homology; the protein structure and potential function domain of each gene among the 4 species were similar. SAA3 whose sequence are 9 aa longer than that of homo and mus is a secretable amphipathic protein, which result in one N-glycosylation site, 2 phosphorylation functional site of goat SAA3 more than that in homo and mus corresponding; ABCG2 is a transmembrane protein which can transport the ATP-dependent translocation of a variety of lipophilic substrates to the outside of cell and have many functional domains, such as ATP-binding cassette, ABC transporter, ABC-2 type transporter,P-loop nucleotide binding motif, while goat ABCG2 had another IMP dehydrogenase / GMP reductase domain in transmembrane site which maybe have tightly relationship with the fatty acid's property of goat milk; HFABP which binds and transports fatty acid is a lipoprotein with ringent reticulate spatial structure; XDH which is a hydrophobic protein with complex space has a dehydrogenase active center including 1 Mo, 1 FAD domain, and 2 Fe/S cluster domains.5. The mRNA expression patterns of SAA3, ABCG2, XDH and HFABP were analyzed in different lactation stages (early, peak, middle, late and end) with normalized by GAPDH by real-time reverse transcriptase-polymerase chain reaction. The results showed that the expression trends of three genes including ABCG2, HFABP and XDH were similar with goat lactation curve, they had significant increase from early to peak lactation stage and then reached the max values, followed by a mild decrease from peak to late stage and a drop to the lowest values at the end lactation stage, we speculated they were important regulatory factors in the change of secretion ability of mammary gland during a lactation cycle; SAA3 showed the significant increase after early stage and remained a plateau expression level during peak and mid stages before reached the highest level in late stage followed by a sharp drop to the lowest level in the end stage of lactation which support the multi-functions associated with cell proliferation and differentiation, lipid metabolism, and protection of mammary gland against infection and inflammation.6. The coding region of SAA3 was constructed into pET-32a(+) vector for proeukaryotic expression analysis, into green fluorescent protein expressive vector pEGFP-C1 for cell location, into eukaryotic expressive vector pcDNA3.1/myc-His(-) A for cell positioning, MTT experiment and the change of fatty acid, and the results showed that the 35 kD recombinant protein with histidine tags was expressed successfully in BL21 E. coli, and the expression condition was optimized at 30℃which was induced for 3h by 1 mmol/L IPTG.SAA3 was located in the cytoplasm and cytomembrane of human breast cancer cell (MCF-7) cells; the growth suppression trends were observed in MCF-7 cells cultured in normal conditions after 24 h transfected Ubi-d4; the content of C14:0 had 3.73 folds higher in MCF-7 cells transfected pcDNA3.1/myc-His(-) A-SAA3 than MCF-7 cells transfected by pcDNA3.1/myc-His(-) A, but the content of other fatty acid (C16:0, C18:0, C18:1 and C18:2) were lower than the control group. So we concluded SAA3 had function in regulating mammary epithelial cell proliferation and involving in fatty acid metabolism.