Identification Of Differentially Expressed Genes In Mammary Glands Between Mid And Late Lactation Stage Of Xinong Saanen Goat And Function Analysis Of GBTN1A1 Gene

Mammary glands are unique organs in the mammalian. They repeat cycles of development, lactateion, and involution for each pregnancy, at the same time, milk yield and milk component are also varied in the different lactation stages. Therefore, identification, cloning and functional analysis of the differentially expressed genes between mid and late lactation stages will contribute to analyzing changes of milk yield and components, and explaining molecular mechanism of mammary gland involution. Considering above-mentioned reasons, some studies were taken basing on the following aspects: (1) SSH was used to screen the differentially expressed genes in mammary gland between mid and late lactation of XINONG Saanen goat. The mRNA abundance of differentially expressed genes was analyzed by real-time RT-PCR; (2) The cDNA sequence of gBTN1A1 gene was cloned by RT-PCR and RACE from total RNA of the goat mammary gland. The putative signal sequences, transmembrane domain, secondary and tertiary structure of BTN1A1 protein were predicted by way of bioinformatics. (3) The mRNA abundance of gBTN1A1 gene in different lactation stage (early, peak, mid, late and end lactation stages) was analyzed by real-time PCR, and the green fluorescence protein expression vector was constructed to investigate the cellular localization of gBTN1A1 in the MCF-7 cells. (4) The prokaryotic expression vector of gBTN1A1 was constructed, the optimal expression condition of gBTN1A1 in prokaryotic cells was achieved, and the gBTN1A1 recombination protein was detected by Western-blot. (5) The eukaryotic expression vector of gBTN1A1 was constructed to analyze the constituent variance of fatty acids in MCF-7 cells. MTT was used to check its effect on the cell growth. (6) The promoters of gBTN1A1 gene were cloned from the goat genomic DNA,and the CAT recombination vector was constructed to analyze the activities of promoters. The activity variation of promoters was studied in MCF-7 cells stimulated by different doses of prolactin. The sequence similarity and transcriptional factor binding sites of BTN1A1 gene promoters were analyzed among goat, bovine, human and mouse. The results as following:(1) M-L (mid Vs late) and L-M (late Vs mid) subtracted libraries were constructed, and the subtracted efficiencies were 25 and 210, respectively. After sequencing partial mono-clones, we found that the M-L library mainly included 3 groups of functional genes: milk fat related genes (BTN1A1,XDH and AFABP); milk protein genes(αs2,βandК-casein,α-lactalbumin) and transcription complex genes(ribosomal protein S12 and S27a). L-M library mainly included 6 groups functional genes: apoptosis related genes(Ubi-d4,FeHc,stromal cell derived factor 4); lipid metabolism related genes(SCD and SAA3); antioxygen related genes(MGST ), transcription initiation genes(ribosomal protein L17 and L35a) and energy metabolism related genes(smooth muscle myosin heavy chain 11 isoform SM1). The relative contents of four caseins were different between mammary gland and milk, especially toК-casein, which was 3 times higher in mammary gland than in milk.(2) The whole sequence of gBTN1A1 gene was gained, and it included 7 exons and 6 introns. The open reading frame was composed of 1581bp, which encoded a polypeptide of 526 amino acids with a putative signal peptide of 26 amino acids and a mature protein of 500 amino acids. The homologies of cDNA nucleotide sequence of gBTN1A1gene with the counterparts of bovine (NM₁74508), human (NM₀01732) and mouse (AK145168) were 95%, 88% and 84%,and the homologies of peptide sequence were 96%, 84% and 70%, respectively. gBTN1A1 was mono-transmembrane protein and the transmembrane domain located at 243-269 peptide. Random coil,α-helico and extend strands were the major in the secondary structure of gBTN1A1 protein. gBTN1A1 protein included 3 functional domains of IgV, IgC and B30.2. The structure of gBTN1A1 were similar with the counterparts in bovine, human and mouse, so we concluded gBTN1A1 gene could be also essential for milk fat globule secretion in goat mammary gland.(3) The expression profile of gBTN1A1 gene during the whole lactation period was analyzed by real-time quantitative PCR. The expression level showed the significant increase from early to peak lactation stage with the highest level in peak stage followed by a relative constant expression level during mid lactation stage before gradual decrease in late and end lactation stages, which reflected the correlation of BTN1A1gene expression to secretion of milk fat globule. However, the expression level in early stage was remarkably higher than that in end lactation stage, which could be the indication of early expression of BTN1A1 in late-pregnancy period before lactating start. The expression profile of goat BTN1A1 was same as the milk yield curve, which indicted gBTN1A1 gene could be regulated by milk hormones. gBTN1A1 gene was located in the cytoplasma in MCF-7 cells, as was our prediction, and this indicated cytoplasma could be the main function place for gBTN1A1gene. (4) The whole protein of gBTN1A1 could not be expressed in E. coli BL21 induced by IPTG, and the amount was very low because several rare codons existing in the gBTN1A1. E. coli Rosetta could add the tRNA of rare codons. gBTN1A1 was successfully expressed in the E. coli Rosetta and showed antigenicity proved by Western-blot. The concentration of IPTG did not affect the protein amount, but the time and temperature may affect the protein amount. The amount reached maximum at 30℃induced for 2 h, then decreased, but increased at 4.5 h again.(5) The content of C14:0,C18:0,C18:1 was higher in MCF-7 cells transfected by pcDNA 3.1/myc-His(-)A-gBTN1A1 than MCF-7 cells transfected by pcDNA 3.1/myc-His(-) A, and the C16:0 and C18:2 were lower than the control. It indicated that the C-temini of gBTN1A1 gene may bind the fatty acid synthase (FAS) and form a complex, which decreased the activity of FAS and affected the synthesis of endogenous fatty acid (C16:0). gBTN1A1 could also bind the linoleic acid, secreted into supernatant of MCF-7 cells, and it would inhibit the MCF-7 cells growth.(6) The activity of BTNP1 promoter was higher than BTNP2 promoter by detecting the CAT report gene expression amount. The expression amount of CAT was increased in MCF-7 cells transfercted pCAT3-Basic-BTNP1 and stimulated by prolactin. Prolactin might up-regulate the gBTN1A1 gene. The similarities of gBTN1A1 gene promoter with the counterparts of bovine, human and mouse were 65%, 36% and 19%, respectively. BTN1A1 gene in four species lacked typical promoter elements such as Inr, TATA box, DPE, and so on, which indicated the BTN1A1 gene had a special element for transcript start. The transcriptal factor binding sites were very similar between goat and bovine BTN1A1 gene, although they were greatly different from others.