鱼精蛋白PRM基因遗传变异及其对种公牛精液品质的影响

发布时间：2018-05-02 08:28

本文选题：鱼精蛋白 + 遗传变异　；参考：《山东师范大学》2015年硕士论文

【摘要】：种公牛在奶牛群的遗传改良中起着关键性的作用，其精液品质高低不仅是决定种公牛站经济效益的关键因素，，更是影响奶牛受胎率的重要因素。公牛精液品质性状受遗传、环境、营养和饲养管理等多种因素的影响。仅依靠营养、饲养管理及常规育种的手段，来提高公牛的精液品质性状难度很大，且目标周期长、效率低。公牛的精液品质性状属于数量性状，受多基因调控，且精子发生过程复杂，筛选和精液品质性状相关的主效基因或关键调控位点对于提高公牛的精液品质性状显得极为重要。鱼精蛋白在精细胞中是主要的核蛋白，与雄性生殖紧密相关，对精子的发育以及正常功能形成产生了重要的影响。鉴于此，本研究选择鱼精蛋白PRM基因(PRM1和PRM2)为候选基因，从基因转录形成的可变剪切体、基因的启动子活性、功能性SNPs鉴定及其相关性分析等方面来研究这些遗传变异对种公牛精液品质性状的影响，并尝试分析影响公牛精液品质性状的分子调控机理。主要研究结果分述如下： 1.中国荷斯坦公牛PRM2基因多态性研究中国荷斯坦公牛PRM2基因的多态性的筛查是基于DNA直接测序和软件比对技术进行的，在外显子2处检测到一个错义突变的新SNP g.+478AG，在编码氨基酸时将精氨酸（R）错义为甘氨酸（G）。本实验通过PCR-RFLP技术对g.+478AG位点进行基因分型，并将各个基因型与公牛精液品质的关联性进行分析。结果发现，g.478AG位点突变纯合基因型GG的个体的采精量、鲜精活力、冻后活力和鲜精密度显著高于野生纯合型A A和杂合型AG(P＜0.05)，畸形率也显著低于二者(P＜0.05)。因此，GG型个体的精液品质较好，GG可作为有利的分子标记用于高繁殖力公牛的辅助选育。 2.中国荷斯坦公牛PRM2基因可变剪接体研究为了研究PRM2基因可变剪切体在不同组织中的表达模式以及在大小公牛睾丸组织中的差异性表达。本实验采用RT-PCR技术检测了PRM2基因总mRNA在不同组织中的表达，并采用qRT-PCR方法检测PRM2基因两个转录本即全转录本PRM2-CT和可变剪切本PRM2-TV1在大小牛睾丸组织中的相对表达量。RT-PCR结果显示，PRM2-CT和PRM2-TV1在大牛和小牛中均只在睾丸组织中有表达，在心脏组织、肝脏组织、脾脏组织、肺组织和肾脏组织中均不表达。qRT-PCR结果表明PRM2-CT和PRM2-TV1在成年公牛睾丸中的表达量比小牛睾丸中的表达量高，且在大小公牛睾丸组织中均显示PRM2-TV1的表达量显著高于PRM2-CT表达量(P0.05)。此外，该试验也说明PRM2-TV1是转录调控过程中的主转录本。 3.中国荷斯坦公牛PRM1基因启动子和功能性SNP的鉴定本试验通过生物信息学预测到PRM1基因存在一个启动子区，且通过基因扩增发现在该启动子区存在g.-138.A G和g.-111.G A2个SNPs，这两SNPs位点呈现完全连锁，因此可将其看作为一个整体SNP-1（g.-138.A G）进行遗传研究。此外，还发现g.-138.A G位点的突变可以使NF-Zc和H4TF-2转录因子结合位点消失，而以往的研究发现NF-Zc和H4TF-2结合因子具有促进基因转录的功能。通过PRM1基因启动子区缺失片段的克隆，构建出连接不同片段的含荧光素酶报告载体的质粒，然后将这些质粒分别转染进MLTC-1细胞中，根据测定的双荧光素酶的活性来鉴定PRM1启动子的核心区域。结果发现PRM1启动子的核心区域存在于g.-230~g.-89片段之间，且上述两个SNPs恰好均位于该核心启动子区内，同时也验证了SNPs g.-138.A G和g.-111.G A降低了这个核心启动子区的活性。RFLP-PCR对SNP g.-138.A G进行分型并对其与精液品质关联性进行分析研究，发现g.-138.A G和g.-111.GA位点中野生纯合子的鲜精活力、冻精活力和鲜精密度高于突变纯合子和杂合子，畸形率显著比突变纯合子和杂合子低(P 0.05)；这些显著关联表明g.-138.A G-A等位基因和g.-111.G A-G等位基因可以提高鲜精子的活力。因此，这两个SNPs可看作选育中国荷斯坦公牛优质精液品质性状的潜在功能性分子标记。 4.中国荷斯坦公牛PRM1基因可变剪接体研究采用RT-PCR和基因测序方法鉴定PRM1基因是否存在可变剪切体，如有，就对各转录本在不同组织中的表达进行检测；进而采用qRT-PCR方法，检测在成年公牛和小公牛睾丸中不同剪切体的表达量。通过对基因测序结果比对发现了一个新转录本（PRM1-TV1），该转录本缺少包含部分5’UTR和外显子的片段共78bp；RT-PCR结果显示：PRM1mRNA在公牛组织中表达具备组织特异性。在成年公牛组织中，PRM1-CT在各组织中均表达且表达量较高，而PRM1-TV1仅在心脏和睾丸组织中表达且表达量较少；在小牛中，PRM1-CT在各组织中仅有微量的表达，但PRM1-TV1在各组织中几乎不表达。qRT-PCR结果显示：PRM1-CT和PRM1-TV1在两个群体中表达量差异显著，两个转录本在成年公牛睾丸中的表达量比在小牛睾丸中高，并且PRM1-CT的表达量显著高于PRM1-TV1的表达量；在小牛睾丸中，PRM1-TV1转录本几乎不表达，PRM1-CT和PRM1-TV1的表达量差异不显著。这些结果和RT-PCR结果也恰好相符。此外，该试验也说明PRM1-CT是转录调控过程中的主转录本。
[Abstract]:The quality of the semen is not only the key factor in determining the economic benefit of the bull station, but also the important factor affecting the rate of the cow's pregnancy. The quality traits of the bull semen are influenced by many factors, such as heredity, environment, nutrition and feeding management. It is difficult to improve the semen quality traits of the bull, and the target cycle is long and the efficiency is low. The quality traits of the semen of the bulls are quantitative traits, and are regulated by multiple genes, and the process of spermatogenesis is complex. The main factor or key regulatory site related to the quality traits of the semen can be used to improve the semen quality of the bull. Characters appear to be very important.
Protamine is the main nucleoprotein in sperm cells, which is closely related to male reproduction and has an important influence on the development of sperm and the formation of normal function. In view of this, this study chose the protamine PRM gene (PRM1 and PRM2) as a candidate gene, variable shear body from gene transcription, promoter activity of gene, functional SNPs The effects of these genetic variations on the quality traits of the semen of the bulls were studied and the effects of these genetic variations on the quality traits of the semen of the bulls were studied, and the molecular regulation mechanism that affected the quality of the semen of the bulls was analyzed. The main results were as follows:
Study on the polymorphism of PRM2 gene in 1. Chinese Holstein bulls
The polymorphism of the PRM2 gene in the Chinese Holstein bulls was screened based on DNA direct sequencing and software comparison. A missense mutation of a new SNP g.+478AG was detected at exon 2, and the R was misdefined as glycine (G) when the amino acid was encoded. This experiment was genotyping on the g.+478AG site by PCR-RFLP Technology. The relationship between the genotypes and the semen quality of the bulls was analyzed. The results showed that the extraction precision, the fresh sperm vitality, the post freeze vitality and the fresh sperm density of the g.478AG locus homozygous genotype GG were significantly higher than the wild homozygous A A and the heterozygous AG (P < 0.05), and the malformation rate was also lower than that of the two (P < 0.05). Therefore, the semen of the GG individuals Good quality, GG can be used as a favorable molecular marker for the breeding of highly prolific bulls.
Alternative splicing of PRM2 gene in 2. Holstein bulls in China
In order to study the expression patterns of the PRM2 gene variable shear body in different tissues and the differential expression in the testicular tissue of the bulls, the RT-PCR technique was used to detect the expression of the total mRNA of the PRM2 gene in different tissues, and the qRT-PCR method was used to detect the two transcripts of the PRM2 gene, the full transcriptional PRM2-CT and the variable shear book. The relative expression of PRM2-TV1 in the testicular tissue of the calf showed that both PRM2-CT and PRM2-TV1 were expressed only in the testis and in the calves and calves. The expression of PRM2-CT and PRM2-TV1 in the testis of adult bulls was not expressed in the heart, liver, spleen, lung and kidney. The expression of PRM2-CT and PRM2-TV1 in the testis of adult bulls was expressed. The amount of PRM2-TV1 expressed in the testis of calf was higher than that in the testicular tissue of the bulls. The expression of PRM2-CT was significantly higher than that in the testis of bulls (P0.05). In addition, the test also indicated that PRM2-TV1 was the main transcriptional transcript in the process of transcriptional regulation.
Identification of PRM1 gene promoter and functional SNP of 3. Holstein bulls in China
It was predicted by bioinformatics that there was a promoter region of the PRM1 gene and that g.-138.A G and g.-111.G A2 SNPs were found in the promoter region by gene amplification, and the two SNPs loci were completely linked, so they could be considered as a whole SNP-1 (g.-138.A G) for genetic research. Furthermore, g.-138.A G sites were also found. The mutation can make the NF-Zc and H4TF-2 transcription factor binding sites disappear, and previous studies have found that NF-Zc and H4TF-2 binding factors have the function of promoting gene transcription. By cloning the deletion fragment of the PRM1 gene promoter region, the plasmid containing the luciferase reporter carrier of different fragments is constructed, and then these plasmids are transfected into ML respectively. In TC-1 cells, the core regions of the PRM1 promoter were identified based on the activity of the measured double luciferase. The results found that the core regions of the PRM1 promoter existed between the g.-230~g.-89 fragments, and the two SNPs was just located in the core promoter region, and also verified that SNPs g.-138.A G and g.-111.G A reduced this core startup. The subregion's active.RFLP-PCR was typed to SNP g.-138.A G and analyzed its relationship with the semen quality. It was found that the fresh sperm vitality of g.-138.A G and g.-111.GA loci Nakano Oijuriko was higher than that of the mutant homozygote and heterozygote, and the deformity rate was significantly lower than that of the mutant homozygote and heterozygote (P 0.05). The significant correlation shows that g.-138.A G-A alleles and g.-111.G A-G alleles can improve the vitality of fresh sperm. Therefore, these two SNPs can be considered as a potential functional molecular marker for the selection of quality traits of high quality semen of Chinese Holstein bulls.
Alternative splicing of PRM1 gene in 4. Holstein bulls in China
RT-PCR and gene sequencing were used to identify the existence of variable shear bodies in the PRM1 gene. For example, the expression of various transcripts in different tissues was detected, and the expression of different shear bodies in adult bull and bulls' testis was detected by qRT-PCR method. A new transcript was found by comparison of gene sequencing results. PRM1-TV1, the transcriptional transcript lacks a total of 78bp containing partial 5 'UTR and exons; RT-PCR results show that PRM1mRNA is expressed in the bulls. In adult bull tissues, PRM1-CT is expressed in all tissues and is highly expressed, while PRM1-TV1 is only expressed in the heart and testis and is less expressed. In the calf, PRM1-CT had only a trace expression in the tissues, but the expression of.QRT-PCR in the tissues of PRM1-TV1 showed that the expression of PRM1-CT and PRM1-TV1 was significantly different in the two populations. The expression of the two transcripts in the testis of adult bull was higher than that in the small cow's testis, and the expression of PRM1-CT was significantly higher than that of PRM1. The expression of -TV1; in the calf testicles, the PRM1-TV1 transcript was almost not expressed, and the difference in the expression of PRM1-CT and PRM1-TV1 was not significant. These results also coincided with the RT-PCR results. In addition, the test also indicated that PRM1-CT was the main transcriptional transcript in the process of transcriptional regulation.

【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

【参考文献】