本文選題：魚精蛋白 + 遺傳變異��； 參考：《山東師范大學(xué)》2015年碩士論文

【摘要】：種公牛在奶牛群的遺傳改良中起著關(guān)鍵性的作用，其精液品質(zhì)高低不僅是決定種公牛站經(jīng)濟(jì)效益的關(guān)鍵因素，，更是影響奶牛受胎率的重要因素。公牛精液品質(zhì)性狀受遺傳、環(huán)境、營(yíng)養(yǎng)和飼養(yǎng)管理等多種因素的影響。僅依靠營(yíng)養(yǎng)、飼養(yǎng)管理及常規(guī)育種的手段，來(lái)提高公牛的精液品質(zhì)性狀難度很大，且目標(biāo)周期長(zhǎng)、效率低。公牛的精液品質(zhì)性狀屬于數(shù)量性狀，受多基因調(diào)控，且精子發(fā)生過(guò)程復(fù)雜，篩選和精液品質(zhì)性狀相關(guān)的主效基因或關(guān)鍵調(diào)控位點(diǎn)對(duì)于提高公牛的精液品質(zhì)性狀顯得極為重要。 魚精蛋白在精細(xì)胞中是主要的核蛋白，與雄性生殖緊密相關(guān)，對(duì)精子的發(fā)育以及正常功能形成產(chǎn)生了重要的影響。鑒于此，本研究選擇魚精蛋白PRM基因(PRM1和PRM2)為候選基因，從基因轉(zhuǎn)錄形成的可變剪切體、基因的啟動(dòng)子活性、功能性SNPs鑒定及其相關(guān)性分析等方面來(lái)研究這些遺傳變異對(duì)種公牛精液品質(zhì)性狀的影響，并嘗試分析影響公牛精液品質(zhì)性狀的分子調(diào)控機(jī)理。主要研究結(jié)果分述如下： 1.中國(guó)荷斯坦公牛PRM2基因多態(tài)性研究 中國(guó)荷斯坦公牛PRM2基因的多態(tài)性的篩查是基于DNA直接測(cè)序和軟件比對(duì)技術(shù)進(jìn)行的，在外顯子2處檢測(cè)到一個(gè)錯(cuò)義突變的新SNP g.+478AG，在編碼氨基酸時(shí)將精氨酸（R）錯(cuò)義為甘氨酸（G）。本實(shí)驗(yàn)通過(guò)PCR-RFLP技術(shù)對(duì)g.+478AG位點(diǎn)進(jìn)行基因分型，并將各個(gè)基因型與公牛精液品質(zhì)的關(guān)聯(lián)性進(jìn)行分析。結(jié)果發(fā)現(xiàn)，g.478AG位點(diǎn)突變純合基因型GG的個(gè)體的采精量、鮮精活力、凍后活力和鮮精密度顯著高于野生純合型A A和雜合型AG(P＜0.05)，畸形率也顯著低于二者(P＜0.05)。因此，GG型個(gè)體的精液品質(zhì)較好，GG可作為有利的分子標(biāo)記用于高繁殖力公牛的輔助選育。 2.中國(guó)荷斯坦公牛PRM2基因可變剪接體研究 為了研究PRM2基因可變剪切體在不同組織中的表達(dá)模式以及在大小公牛睪丸組織中的差異性表達(dá)。本實(shí)驗(yàn)采用RT-PCR技術(shù)檢測(cè)了PRM2基因總mRNA在不同組織中的表達(dá)，并采用qRT-PCR方法檢測(cè)PRM2基因兩個(gè)轉(zhuǎn)錄本即全轉(zhuǎn)錄本PRM2-CT和可變剪切本PRM2-TV1在大小牛睪丸組織中的相對(duì)表達(dá)量。RT-PCR結(jié)果顯示，PRM2-CT和PRM2-TV1在大牛和小牛中均只在睪丸組織中有表達(dá)，在心臟組織、肝臟組織、脾臟組織、肺組織和腎臟組織中均不表達(dá)。qRT-PCR結(jié)果表明PRM2-CT和PRM2-TV1在成年公牛睪丸中的表達(dá)量比小牛睪丸中的表達(dá)量高，且在大小公牛睪丸組織中均顯示PRM2-TV1的表達(dá)量顯著高于PRM2-CT表達(dá)量(P0.05)。此外，該試驗(yàn)也說(shuō)明PRM2-TV1是轉(zhuǎn)錄調(diào)控過(guò)程中的主轉(zhuǎn)錄本。 3.中國(guó)荷斯坦公牛PRM1基因啟動(dòng)子和功能性SNP的鑒定 本試驗(yàn)通過(guò)生物信息學(xué)預(yù)測(cè)到PRM1基因存在一個(gè)啟動(dòng)子區(qū)，且通過(guò)基因擴(kuò)增發(fā)現(xiàn)在該啟動(dòng)子區(qū)存在g.-138.A G和g.-111.G A2個(gè)SNPs，這兩SNPs位點(diǎn)呈現(xiàn)完全連鎖，因此可將其看作為一個(gè)整體SNP-1（g.-138.A G）進(jìn)行遺傳研究。此外，還發(fā)現(xiàn)g.-138.A G位點(diǎn)的突變可以使NF-Zc和H4TF-2轉(zhuǎn)錄因子結(jié)合位點(diǎn)消失，而以往的研究發(fā)現(xiàn)NF-Zc和H4TF-2結(jié)合因子具有促進(jìn)基因轉(zhuǎn)錄的功能。通過(guò)PRM1基因啟動(dòng)子區(qū)缺失片段的克隆，構(gòu)建出連接不同片段的含熒光素酶報(bào)告載體的質(zhì)粒，然后將這些質(zhì)粒分別轉(zhuǎn)染進(jìn)MLTC-1細(xì)胞中，根據(jù)測(cè)定的雙熒光素酶的活性來(lái)鑒定PRM1啟動(dòng)子的核心區(qū)域。結(jié)果發(fā)現(xiàn)PRM1啟動(dòng)子的核心區(qū)域存在于g.-230~g.-89片段之間，且上述兩個(gè)SNPs恰好均位于該核心啟動(dòng)子區(qū)內(nèi)，同時(shí)也驗(yàn)證了SNPs g.-138.A G和g.-111.G A降低了這個(gè)核心啟動(dòng)子區(qū)的活性。RFLP-PCR對(duì)SNP g.-138.A G進(jìn)行分型并對(duì)其與精液品質(zhì)關(guān)聯(lián)性進(jìn)行分析研究，發(fā)現(xiàn)g.-138.A G和g.-111.GA位點(diǎn)中野生純合子的鮮精活力、凍精活力和鮮精密度高于突變純合子和雜合子，畸形率顯著比突變純合子和雜合子低(P 0.05)；這些顯著關(guān)聯(lián)表明g.-138.A G-A等位基因和g.-111.G A-G等位基因可以提高鮮精子的活力。因此，這兩個(gè)SNPs可看作選育中國(guó)荷斯坦公牛優(yōu)質(zhì)精液品質(zhì)性狀的潛在功能性分子標(biāo)記。 4.中國(guó)荷斯坦公牛PRM1基因可變剪接體研究 采用RT-PCR和基因測(cè)序方法鑒定PRM1基因是否存在可變剪切體，如有，就對(duì)各轉(zhuǎn)錄本在不同組織中的表達(dá)進(jìn)行檢測(cè)；進(jìn)而采用qRT-PCR方法，檢測(cè)在成年公牛和小公牛睪丸中不同剪切體的表達(dá)量。通過(guò)對(duì)基因測(cè)序結(jié)果比對(duì)發(fā)現(xiàn)了一個(gè)新轉(zhuǎn)錄本（PRM1-TV1），該轉(zhuǎn)錄本缺少包含部分5’UTR和外顯子的片段共78bp；RT-PCR結(jié)果顯示：PRM1mRNA在公牛組織中表達(dá)具備組織特異性。在成年公牛組織中，PRM1-CT在各組織中均表達(dá)且表達(dá)量較高，而PRM1-TV1僅在心臟和睪丸組織中表達(dá)且表達(dá)量較少；在小牛中，PRM1-CT在各組織中僅有微量的表達(dá)，但PRM1-TV1在各組織中幾乎不表達(dá)。qRT-PCR結(jié)果顯示：PRM1-CT和PRM1-TV1在兩個(gè)群體中表達(dá)量差異顯著，兩個(gè)轉(zhuǎn)錄本在成年公牛睪丸中的表達(dá)量比在小牛睪丸中高，并且PRM1-CT的表達(dá)量顯著高于PRM1-TV1的表達(dá)量；在小牛睪丸中，PRM1-TV1轉(zhuǎn)錄本幾乎不表達(dá)，PRM1-CT和PRM1-TV1的表達(dá)量差異不顯著。這些結(jié)果和RT-PCR結(jié)果也恰好相符。此外，該試驗(yàn)也說(shuō)明PRM1-CT是轉(zhuǎn)錄調(diào)控過(guò)程中的主轉(zhuǎn)錄本。
[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.

【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S823

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