鸡VDAC1、VDAC2和MOSPD1基因在前等级卵泡中的时空表达及VDAC基因与产蛋性状的关联
发布时间：2019-05-22 04:43
【摘要】：开产日龄、产蛋量和蛋重等产蛋性状是优质蛋鸡育种中的重要性状,而鸡前等级卵泡的发育程度以及同时被募集和选择的优势卵泡数目是决定鸡产蛋能力的关键因素。作为VDAC家族(VDAC1、VDAC2和VDAC3)中广泛存在的重要蛋白,关于VDAC1基因和VDAC2基因的研究较深入,主要包括该基因在细胞能量转化和细胞凋亡中发挥的重要作用[2]。MOSPD1位于人和鼠的X染色体上,是跨膜成年精子蛋白家族(major sperm protein,MSP)的4个成员之一,编码N末端主要精子蛋白域和两个C末端跨膜区域[1],在某些间质细胞和上皮细胞转化过程发挥关键作用,如成骨细胞、成肌细胞和脂肪瘤细胞。MOSPD1在密集细胞群中表达量较高,参与卵巢癌的过程。目前国内未见VDAC1基因、VDAC2基因和MOSPD1基因在卵泡细胞中表达的相关文献报道。本研究分为三个部分,分别阐述MOSPD1、VDAC1和VDAC2基因在卵巢中的定位和表达水平,对VDAC基因进行SSCP检测,并进行大骨鸡产蛋性状关联。实验的主要内容有:通过半定量RT-PCR和RNA原位杂交技术,以150日龄有规则产蛋周期的海兰褐蛋鸡为素材,检测VDAC1、VDAC2和MOSPD1基因mRNA在该时期卵泡中的表达和定位情况。半定量RT-PCR检测结果表明,VDAC1基因和VDAC2基因mRNA在150日龄海兰褐前等级卵泡、等级卵泡和卵巢间质中均维持着高表达水平,且表达水平的差异均不显著(P0.05)。其中,VDAC1基因mRNA在6-7mm和F3中的表达量较高,在F1中的表达量最低;VDAC2基因mRNA在等级前卵泡(直径小于8mm)中的表达量高于排卵前等级化卵泡(F6、F5、F4、F3、F2和F1)。其中,在4-5mm、6-7mm和7-8mm卵泡中的表达量较高,在F2和F1中的表达量最低。VDAC基因的RNA原位杂交结果显示,鸡VDAC1和VDAC2基因在150日龄海兰褐蛋鸡前等级卵泡的卵母细胞、颗粒细胞和膜层细胞中均表达,与半定量RT-PCR的检测结果一致。其中,VDAC1基因mRNA在前等级卵泡颗粒细胞、膜层细胞和卵母细胞中表达水平无显著差异,VDAC2基因mRNA在颗粒层和膜层细胞中表达水平略低于在卵母细胞中的表达水平。MOSPD1基因mRNA在被检测的120个组织样本中均有表达,且表达水平存在差异。在1-4mm卵泡和F1中mRNA相对表达量较高,分别为1.068705±0.260和1.013716±0.465,差异显著(P0.05)。在F3和F4中表达量较低,分别为0.085935±0.172和0.069848±0.142,差异显著。在表达丰度上,MOSPD1基因的表达低于VDAC1基因和VDAC2基因,对不同时期卵泡发育的调控作用存在剂量依赖性。在本研究条件下,由于原位杂交检测技术的局限性和MOSPD1基因mRNA本身在前等级卵泡中低表达,未检测到该基因在卵泡细胞中的阳性表达结果。综上推断得出,VDAC1、VDAC2和MOSPD1基因可能对鸡前等级卵泡的发育发挥重要作用,但具体的调控机制有待更进一步的研究探讨。二,通过PCR-SSCP和克隆测序技术,以地方性大骨鸡母鸡为实验素材,分别检测VDAC1(NC_006100)基因和VDAC2(NC_006093)基因SNP位点并与大骨鸡产蛋性状进行关联分析。多态性检测结果显示,VDAC1基因共检测到两种基因型,分别为AA和AB,在该群体中未检测到BB单倍基因型。AA基因型频率高于AB基因型频率,等位基因A的基因频率高于等位基因B的基因频率。由此推断,VDAC1基因的AB单倍型为优势基因型。经序列比对结果发现,在第6内含子存在一个突变位点,即G15362448A转换。产蛋性状关联分析结果显示,VDAC1基因的单倍基因型AB型个体具有最高的43w产蛋数和66w产蛋数,分别为89.25±4.32和139.91±3.21,且显著高于AA型个体(P0.05)。VDAC2基因经SSC P共检测到两种基因型,分别命名为GG和GT,其中,GG单倍基因型频率高于G T单倍型,等位基因G的基因频率大于等位基因T。由此推断,VDAC2基因的GT单倍型为优势基因型。序列比对结果发现,在3’调控区存在1个多态位点,即G14420258A转换。产蛋性状关联分析结果显示,VDAC2基因的单倍型GT型个体具有较高的30w产蛋数、43w产蛋数、57w产蛋数和66w产蛋数,分别为18.83±1.62、95.00±4.47、78.00±3.93和122.80±2.26,且显著高于GG型个体(P0.05)。由此得出,VDAC1基因AB单倍型和VDAC2基因GT单倍型为优势基因型,均可作为蛋鸡早期产蛋性状选择的潜在分子标记,为优质蛋鸡优势产蛋性状的分子辅助选择提供理论依据。
[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.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831.2
本文编号：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.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831.2
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