Construction Of Subtracted CDNA Library In Mammary Gland Of Dairy Goat And Study Differently Expression Genes

Mammary gland, which provide nutritious milk for growth and development of newborn mammals, while human life is indispensable to provide dairy products. The cycle of mammary gland has development, lactation and involution of the three processes, during each cycle mammary epithelial cell experience the process of proliferation, differentiation and cell apoptosis. The milk component and milk yield are greatly changed in the different lactation stages of dairy goat. With the study of knowledge on genomics, people gradually recognized that these changes are determined and regulated by a series of functional genes expression during the temporal and spatial in mammary gland. The paper is based of the lactation physiological changes in Xinong Saanen dairy goat. Screening, cloning and functional analysis of differentially expressed genes will elucidate the molecular of mammary gland. Suppression subtractive hybridization(SSH) method is used to isolate differently expressed genes in mammary gland of difference lactation stage stage of the Xinong Saanen goat(Capra hircus),further clone the differently expressed genes and predict their structure and function by bioinformatics,study the function of osteopontin (OPN) gene which is one of the different expression candidate genes. For further study of the molecular mechanisms of lactation, the milk production and milk composition provide a theoretical basis.1. Suppression subtractive hybridization(SSH) technology is used to isolate differently expressed genes in mammary gland of early lactation stage and peak stage of the Xinong Saanen goat(Capra hircus).Some differently expressed genes were sequenced and analyzed.The new genes of dairy goat were searched and studied. Forward and reverse subtracted differently expressed cDNA library (E-P and P-E) were successfully created in mammary gland of different lactation. The subtraction efficiency was confirmed by a housekeeping gene named GAPDH,the results indicated that GAPDH was subtracted efficiently at 25 and 210 folds for E-P and P-E subtracted cDNA library respectively. Partial clone of E-P subtracted cDNA library were sequenced, we found that E-P library mainly included four groups of functional genes:milk protein related genes (αs2,βand K-casein, a-lactalbumin precursors), transcription complex genes(ribosomal protein L10,L23,3B and Cbp/p300),cell energy supplement genes NADH,cell proliferation and differentiation genes (GHITM,OPN and H3F3B).P-E library mainly included three groups of functional genes: milk fat metabolism related genes (HFABP,PPARGC1A),cell growth metabolism related genes (SAA3,SSAT,SPARC and LPO) and transcription related genes (splice factor 3B). Real-time reverse transcriptase-polymerase chain reaction was used to analyse the change of mRNA expression level of OPN and GHITM form E-P subtracted cDNA library. The results showed that they had 5.04 and 4.20 folds higher in early lactation stage than that in peak lactation stage. The change of mRNA expression level of PPARGCIA,SPARC and SSAT form P-E subtracted cDNA library had 3.58,12.13 and 8.0 folds higher in peak lactation stage than that in early lactation stage.2. We designed primers based on the high homology sequence in gene coding region among different species and existed sequence for goat, the CDs region of PPARGC1A,OPN, SPARC,GHITM and SSAT genes were cloned and sequenced from the goat mammary gland by RT-PCR, bioinformatics were employed to predict gene specific structure and function. The structure and function of PPARGC1A,OPN,SPARC,GHITM and SSAT were compared among goat, bovine, human, and mouse using bioinformatics software. The putative signal sequences, transmembrane domain, secondary and tertiary structure of five proteins were predicted by way of bioinformatics. The different species have their characteristics. The peroxysome proliferator-activated receptor gamma coactivator-lalpha(PGCla) is transcriptional coactivators consisting of N-terminal transcriptional activation domains and nuclear hormone receptor-interacting motif (LXXLL) and C-terminal RNA processing motifs that plays a central role in the regulation of cellular energy metabolism. OPN protein is secreted hydrophilic glycoprotein. In different species of OPN have RGD motif, which is correlated with the accumulation and cell adhesion, proliferation and tissue remodeling. Goat OPN had two additional potential binding motifs:cAMP-dependent protein kinase phosphorylation site and agglutination factor V LSPR repeat loci compared to humans'and mice. SPARC is a glycoprotein of small molecule, including N-terminal domain and EF-hand calcium-binding site, which may be related to calcium metabolism in mammary gland. GHITM is a transmembrane protein, there are eight strong transmembrane region, suggesting the gene may be associated with milk-related ingredients transmembrane. SSAT is the key enzyme of the cell metabolic balance and a Gcn5-acetyl-amino-transferase domain is closely related to SSAT of the catalytic effect.3. Using beta-actin gene as the internal control, the SYBR Green quantitative real-time PCR (QPCR) analysis were conducted to determine the mRNA expression of PPARGC1A,OPN, SPARC,GHITM and SSAT genes in mammary gland at 28th,60th,100th,190th,270th and 330th day after kidding. The results showed that the expression trends of PPARGCIA and SSAT genes were similar with goat lactation curve, the dairy goat mammary gland increased milk yield dramatically during the first time of lactation, as evident from the marked increase in expression during the onset of lactation. PGCla mRNA expression reached the highest level on day 100, and subsequently decreased to a very low level on day 330. The expression on day 100 is about 13 folds (P< 0.01) of that on day 330. The results indicated that OPN gene exhibited the higher expression level in early (28 d) and late (190 d) lactation and the lowest level in dry period (330 d), which demonstrated a high-low-high-low pattern.SPARC gene exhibited the highest expression level in early (28 d) and late (190 d) lactation and subsequently decreased to a very low level on day 330.GHITM gene exhibited the highest expression level in early (28 d) subsequently decreased during the lactation of late, peak, end and dry periods.4. Recombinant plasmid of pcDNA3.1-OPN was constructed by inserting the fragment of OPN gene into eukaryotic expression vector pcDNA3.1 and used to transfect the MCF-7 cell line following the restrictive endonuclease cleavage and sequence identification of the target gene segment, the effect of OPN gene on MCF-7 cell proliferation was assessed by MTT analysis. There was a significant difference (P< 0.05) in the proliferation between OPN gene transfect and non-transfect MCF-7 cells, it is suggested that the expression of OPN gene could stimulate the proliferation of MCF-7 cells. The growth rates of the OPN gene transfect is 111.4% while non-transfect MCF-7 cells is 101.3%.