雞VDAC1、VDAC2和MOSPD1基因在前等級卵泡中的時空表達及VDAC基因與產(chǎn)蛋性狀的關(guān)聯(lián)
發(fā)布時間：2019-05-22 04:43
【摘要】：開產(chǎn)日齡、產(chǎn)蛋量和蛋重等產(chǎn)蛋性狀是優(yōu)質(zhì)蛋雞育種中的重要性狀,而雞前等級卵泡的發(fā)育程度以及同時被募集和選擇的優(yōu)勢卵泡數(shù)目是決定雞產(chǎn)蛋能力的關(guān)鍵因素。作為VDAC家族(VDAC1、VDAC2和VDAC3)中廣泛存在的重要蛋白,關(guān)于VDAC1基因和VDAC2基因的研究較深入,主要包括該基因在細胞能量轉(zhuǎn)化和細胞凋亡中發(fā)揮的重要作用[2]。MOSPD1位于人和鼠的X染色體上,是跨膜成年精子蛋白家族(major sperm protein,MSP)的4個成員之一,編碼N末端主要精子蛋白域和兩個C末端跨膜區(qū)域[1],在某些間質(zhì)細胞和上皮細胞轉(zhuǎn)化過程發(fā)揮關(guān)鍵作用,如成骨細胞、成肌細胞和脂肪瘤細胞。MOSPD1在密集細胞群中表達量較高,參與卵巢癌的過程。目前國內(nèi)未見VDAC1基因、VDAC2基因和MOSPD1基因在卵泡細胞中表達的相關(guān)文獻報道。本研究分為三個部分,分別闡述MOSPD1、VDAC1和VDAC2基因在卵巢中的定位和表達水平,對VDAC基因進行SSCP檢測,并進行大骨雞產(chǎn)蛋性狀關(guān)聯(lián)。實驗的主要內(nèi)容有:通過半定量RT-PCR和RNA原位雜交技術(shù),以150日齡有規(guī)則產(chǎn)蛋周期的海蘭褐蛋雞為素材,檢測VDAC1、VDAC2和MOSPD1基因mRNA在該時期卵泡中的表達和定位情況。半定量RT-PCR檢測結(jié)果表明,VDAC1基因和VDAC2基因mRNA在150日齡海蘭褐前等級卵泡、等級卵泡和卵巢間質(zhì)中均維持著高表達水平,且表達水平的差異均不顯著(P0.05)。其中,VDAC1基因mRNA在6-7mm和F3中的表達量較高,在F1中的表達量最低;VDAC2基因mRNA在等級前卵泡(直徑小于8mm)中的表達量高于排卵前等級化卵泡(F6、F5、F4、F3、F2和F1)。其中,在4-5mm、6-7mm和7-8mm卵泡中的表達量較高,在F2和F1中的表達量最低。VDAC基因的RNA原位雜交結(jié)果顯示,雞VDAC1和VDAC2基因在150日齡海蘭褐蛋雞前等級卵泡的卵母細胞、顆粒細胞和膜層細胞中均表達,與半定量RT-PCR的檢測結(jié)果一致。其中,VDAC1基因mRNA在前等級卵泡顆粒細胞、膜層細胞和卵母細胞中表達水平無顯著差異,VDAC2基因mRNA在顆粒層和膜層細胞中表達水平略低于在卵母細胞中的表達水平。MOSPD1基因mRNA在被檢測的120個組織樣本中均有表達,且表達水平存在差異。在1-4mm卵泡和F1中mRNA相對表達量較高,分別為1.068705±0.260和1.013716±0.465,差異顯著(P0.05)。在F3和F4中表達量較低,分別為0.085935±0.172和0.069848±0.142,差異顯著。在表達豐度上,MOSPD1基因的表達低于VDAC1基因和VDAC2基因,對不同時期卵泡發(fā)育的調(diào)控作用存在劑量依賴性。在本研究條件下,由于原位雜交檢測技術(shù)的局限性和MOSPD1基因mRNA本身在前等級卵泡中低表達,未檢測到該基因在卵泡細胞中的陽性表達結(jié)果。綜上推斷得出,VDAC1、VDAC2和MOSPD1基因可能對雞前等級卵泡的發(fā)育發(fā)揮重要作用,但具體的調(diào)控機制有待更進一步的研究探討。二,通過PCR-SSCP和克隆測序技術(shù),以地方性大骨雞母雞為實驗素材,分別檢測VDAC1(NC_006100)基因和VDAC2(NC_006093)基因SNP位點并與大骨雞產(chǎn)蛋性狀進行關(guān)聯(lián)分析。多態(tài)性檢測結(jié)果顯示,VDAC1基因共檢測到兩種基因型,分別為AA和AB,在該群體中未檢測到BB單倍基因型。AA基因型頻率高于AB基因型頻率,等位基因A的基因頻率高于等位基因B的基因頻率。由此推斷,VDAC1基因的AB單倍型為優(yōu)勢基因型。經(jīng)序列比對結(jié)果發(fā)現(xiàn),在第6內(nèi)含子存在一個突變位點,即G15362448A轉(zhuǎn)換。產(chǎn)蛋性狀關(guān)聯(lián)分析結(jié)果顯示,VDAC1基因的單倍基因型AB型個體具有最高的43w產(chǎn)蛋數(shù)和66w產(chǎn)蛋數(shù),分別為89.25±4.32和139.91±3.21,且顯著高于AA型個體(P0.05)。VDAC2基因經(jīng)SSC P共檢測到兩種基因型,分別命名為GG和GT,其中,GG單倍基因型頻率高于G T單倍型,等位基因G的基因頻率大于等位基因T。由此推斷,VDAC2基因的GT單倍型為優(yōu)勢基因型。序列比對結(jié)果發(fā)現(xiàn),在3’調(diào)控區(qū)存在1個多態(tài)位點,即G14420258A轉(zhuǎn)換。產(chǎn)蛋性狀關(guān)聯(lián)分析結(jié)果顯示,VDAC2基因的單倍型GT型個體具有較高的30w產(chǎn)蛋數(shù)、43w產(chǎn)蛋數(shù)、57w產(chǎn)蛋數(shù)和66w產(chǎn)蛋數(shù),分別為18.83±1.62、95.00±4.47、78.00±3.93和122.80±2.26,且顯著高于GG型個體(P0.05)。由此得出,VDAC1基因AB單倍型和VDAC2基因GT單倍型為優(yōu)勢基因型,均可作為蛋雞早期產(chǎn)蛋性狀選擇的潛在分子標記,為優(yōu)質(zhì)蛋雞優(yōu)勢產(chǎn)蛋性狀的分子輔助選擇提供理論依據(jù)。
[Abstract]:The egg-laying characters, such as the day-of-birth, the laying amount and the weight of the egg, are important characters in the breeding of high-quality layer-laying hens, and the degree of development of the pre-chicken-grade follicle and the number of the dominant follicles that are both raised and selected are the key factors to determine the egg-laying ability of the chicken. As an important protein in the VAC family (VDAC1, VDAC2 and VDAC3), the research on the VDAAC1 gene and the VDAC2 gene is in-depth, mainly including the important role of the gene in the transformation of the energy and the apoptosis of the cells[2]. MOSPD1 is located on the X-chromosome of human and mouse, one of the four members of the transmembrane adult sperm protein family (MSP), encodes the N-terminal main sperm protein domain and the two C-terminal transmembrane regions[1], plays a key role in the transformation of some of the mesenchymal cells and the epithelial cells, Such as osteoblasts, myoblasts and lipoma cells. MOSPD1 is highly expressed in a dense population of cells and is involved in the process of ovarian cancer. There are no relevant literature reports on the expression of the VDAAC1 gene, the VDAC2 gene and the MOSPD1 gene in the follicular cells at present. This study was divided into three parts: the localization and expression level of MOSPD1, VDAC1 and VDAC2 gene in the ovary were described, and the expression level of VDAC1, VDAC1 and VDAC2 gene was detected by SSCP, and the egg-laying characters of the large bone were associated. The main contents of the experiment were: by semi-quantitative RT-PCR and RNA in situ hybridization, the expression and location of VDAAC1, VDAC2 and MOSPD1 mRNA in the follicle during the period were detected by using a semi-quantitative RT-PCR and an RNA in situ hybridization technique. The results of semi-quantitative RT-PCR showed that the VAC1 and VAC2 mRNA levels maintained a high level of expression in the level of follicle, grade and ovary at 150-day-old, and there was no significant difference in the level of expression (P0.05). Among them, the expression of VAC1 mRNA in 6-7 mm and F3 is high, and the expression in F1 is the lowest; the expression of VAC2 gene mRNA in the pre-ovulation follicle (less than 8 mm) is higher than that of the pre-ovulatory level follicle (F6, F5, F4, F3, F2 and F1). Among them, the expression in the follicles of 4-5 mm,6-7 mm and 7-8 mm is high, and the amount of expression in F2 and F1 is the lowest. In situ hybridization of VAC gene, the results of in situ hybridization showed that the VAC1 and VAC2 genes were expressed in the oocytes, granulosa cells and membrane-layer cells of the first-grade follicle of the 150-day-old Helan brown layer, and the results were consistent with the results of the semi-quantitative RT-PCR. There was no significant difference in the level of expression of VAC1 mRNA in the granulosa cells of the pre-grade, the cell of the membrane and the oocyte, and the level of the expression of the VAC2 mRNA in the cell and the cell of the membrane was slightly lower than that in the oocyte. The mRNA of the MOSPD1 gene was expressed in the 120 tissue samples to be tested, and there was a difference in the expression level. The relative expression of mRNA in the 1-4 mm follicles and F1 was 1.068705-0.260 and 1.013716-0.465, respectively (P0.05). The expression was lower in F3 and F4, 0.085935, 0.172 and 0.069848-0.142, respectively. In the expression abundance, the expression of the MOSPD1 gene is lower than that of the VDAC1 gene and the VDAC2 gene, and there is a dose-dependent effect on the regulation and regulation of the follicular development in different periods. In this study, the positive expression of the gene in the follicular cells was not detected due to the limitations of the in situ hybridization detection technique and the low expression of the MOSPD1 gene mRNA in the pre-grade follicles. It is concluded that the VDAAC1, VDAC2 and MOSPD1 genes may play an important role in the development of the pre-chicken-grade follicle, but the specific regulatory mechanism is still to be further studied. 2. The SNP sites of VDAAC1 (NC _ 006100) and VAC2 (NC _ 006093) were detected by PCR-SSCP and cloning and sequencing. The results showed that the two genotypes were detected by the VAC1 gene, AA and AB respectively, and the BB haplotype was not detected in the population. The frequency of the AA genotype was higher than that of the AB genotype, and the gene frequency of the allele A was higher than that of the allele B. It is concluded that the AB haplotype of the VDAC1 gene is the dominant genotype. It was found that there was a mutation site in intron 6, that is, G15362448A. The results of correlation analysis of egg-laying characters showed that the single-genotype AB-type of the VAC1 gene had the highest 43-w egg-laying number and 66-w egg-laying number, which were 89.25-4.32 and 139.91-3.21, respectively, and were significantly higher than that of the AA-type individuals (P0.05). The VDAC2 gene was co-detected by SSC-P to two genotypes, named GG and GT, respectively, The genotype frequency of the GG haplotype was higher than that of the G-T haplotype, and the gene frequency of the allele G was higher than that of the allele T. It was concluded that the GT haplotype of the VDAC2 gene was the dominant genotype. The results show that there are 1 polymorphic site in the 3 'regulatory region, that is, G14420258A. The results of correlation analysis of laying character showed that the single type GT type of the VAC2 gene had a high 30 w egg number,43 w egg number, 57w egg number and 66 w egg number, 18.83, 1.62, 95.00, 4.47, 78.00, 3.93 and 122.80, 2.26, respectively, and significantly higher than that of the GG type (P0.05). As a result, the genotype of the VDAAC1 gene and the GT haplotype of the VDAC2 gene are the dominant genotype, which can be used as the potential molecular marker of the early egg-laying character selection of the laying hens, so as to provide a theoretical basis for the molecular-assisted selection of the superior egg-laying character of the high-quality layer laying hens.
【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S831.2
本文編號：2482688
[Abstract]:The egg-laying characters, such as the day-of-birth, the laying amount and the weight of the egg, are important characters in the breeding of high-quality layer-laying hens, and the degree of development of the pre-chicken-grade follicle and the number of the dominant follicles that are both raised and selected are the key factors to determine the egg-laying ability of the chicken. As an important protein in the VAC family (VDAC1, VDAC2 and VDAC3), the research on the VDAAC1 gene and the VDAC2 gene is in-depth, mainly including the important role of the gene in the transformation of the energy and the apoptosis of the cells[2]. MOSPD1 is located on the X-chromosome of human and mouse, one of the four members of the transmembrane adult sperm protein family (MSP), encodes the N-terminal main sperm protein domain and the two C-terminal transmembrane regions[1], plays a key role in the transformation of some of the mesenchymal cells and the epithelial cells, Such as osteoblasts, myoblasts and lipoma cells. MOSPD1 is highly expressed in a dense population of cells and is involved in the process of ovarian cancer. There are no relevant literature reports on the expression of the VDAAC1 gene, the VDAC2 gene and the MOSPD1 gene in the follicular cells at present. This study was divided into three parts: the localization and expression level of MOSPD1, VDAC1 and VDAC2 gene in the ovary were described, and the expression level of VDAC1, VDAC1 and VDAC2 gene was detected by SSCP, and the egg-laying characters of the large bone were associated. The main contents of the experiment were: by semi-quantitative RT-PCR and RNA in situ hybridization, the expression and location of VDAAC1, VDAC2 and MOSPD1 mRNA in the follicle during the period were detected by using a semi-quantitative RT-PCR and an RNA in situ hybridization technique. The results of semi-quantitative RT-PCR showed that the VAC1 and VAC2 mRNA levels maintained a high level of expression in the level of follicle, grade and ovary at 150-day-old, and there was no significant difference in the level of expression (P0.05). Among them, the expression of VAC1 mRNA in 6-7 mm and F3 is high, and the expression in F1 is the lowest; the expression of VAC2 gene mRNA in the pre-ovulation follicle (less than 8 mm) is higher than that of the pre-ovulatory level follicle (F6, F5, F4, F3, F2 and F1). Among them, the expression in the follicles of 4-5 mm,6-7 mm and 7-8 mm is high, and the amount of expression in F2 and F1 is the lowest. In situ hybridization of VAC gene, the results of in situ hybridization showed that the VAC1 and VAC2 genes were expressed in the oocytes, granulosa cells and membrane-layer cells of the first-grade follicle of the 150-day-old Helan brown layer, and the results were consistent with the results of the semi-quantitative RT-PCR. There was no significant difference in the level of expression of VAC1 mRNA in the granulosa cells of the pre-grade, the cell of the membrane and the oocyte, and the level of the expression of the VAC2 mRNA in the cell and the cell of the membrane was slightly lower than that in the oocyte. The mRNA of the MOSPD1 gene was expressed in the 120 tissue samples to be tested, and there was a difference in the expression level. The relative expression of mRNA in the 1-4 mm follicles and F1 was 1.068705-0.260 and 1.013716-0.465, respectively (P0.05). The expression was lower in F3 and F4, 0.085935, 0.172 and 0.069848-0.142, respectively. In the expression abundance, the expression of the MOSPD1 gene is lower than that of the VDAC1 gene and the VDAC2 gene, and there is a dose-dependent effect on the regulation and regulation of the follicular development in different periods. In this study, the positive expression of the gene in the follicular cells was not detected due to the limitations of the in situ hybridization detection technique and the low expression of the MOSPD1 gene mRNA in the pre-grade follicles. It is concluded that the VDAAC1, VDAC2 and MOSPD1 genes may play an important role in the development of the pre-chicken-grade follicle, but the specific regulatory mechanism is still to be further studied. 2. The SNP sites of VDAAC1 (NC _ 006100) and VAC2 (NC _ 006093) were detected by PCR-SSCP and cloning and sequencing. The results showed that the two genotypes were detected by the VAC1 gene, AA and AB respectively, and the BB haplotype was not detected in the population. The frequency of the AA genotype was higher than that of the AB genotype, and the gene frequency of the allele A was higher than that of the allele B. It is concluded that the AB haplotype of the VDAC1 gene is the dominant genotype. It was found that there was a mutation site in intron 6, that is, G15362448A. The results of correlation analysis of egg-laying characters showed that the single-genotype AB-type of the VAC1 gene had the highest 43-w egg-laying number and 66-w egg-laying number, which were 89.25-4.32 and 139.91-3.21, respectively, and were significantly higher than that of the AA-type individuals (P0.05). The VDAC2 gene was co-detected by SSC-P to two genotypes, named GG and GT, respectively, The genotype frequency of the GG haplotype was higher than that of the G-T haplotype, and the gene frequency of the allele G was higher than that of the allele T. It was concluded that the GT haplotype of the VDAC2 gene was the dominant genotype. The results show that there are 1 polymorphic site in the 3 'regulatory region, that is, G14420258A. The results of correlation analysis of laying character showed that the single type GT type of the VAC2 gene had a high 30 w egg number,43 w egg number, 57w egg number and 66 w egg number, 18.83, 1.62, 95.00, 4.47, 78.00, 3.93 and 122.80, 2.26, respectively, and significantly higher than that of the GG type (P0.05). As a result, the genotype of the VDAAC1 gene and the GT haplotype of the VDAC2 gene are the dominant genotype, which can be used as the potential molecular marker of the early egg-laying character selection of the laying hens, so as to provide a theoretical basis for the molecular-assisted selection of the superior egg-laying character of the high-quality layer laying hens.
【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S831.2
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