【摘要】：1.利用基因組重測(cè)序數(shù)據(jù)分析中外豬品種馴化過程中的拷貝數(shù)變異在野豬到家豬的馴化和進(jìn)一步形成品種的過程中,遺傳、變異和選擇在不同程度上,對(duì)品種的形成以及品種差異的產(chǎn)生起著一定的作用。通過研究家豬與野豬及家豬不同品種之間基因組序列的差異,可以發(fā)現(xiàn)基因組上的變異。拷貝數(shù)變異(copy number variation,CNV)是指與生物正常的基因序列相比,基因組中所發(fā)生的長(zhǎng)度范圍介于1Kb至數(shù)Mb的變異,其形式包括重復(fù)、缺失及衍生出的復(fù)雜染色體微結(jié)構(gòu)變異。本研究利用豬基因組重測(cè)序數(shù)據(jù),對(duì)中外家豬13個(gè)品種共49個(gè)個(gè)體進(jìn)行CNV分析,并分析拷貝數(shù)變異區(qū)域(CNVR)內(nèi)相關(guān)基因的功能。同時(shí),通過比較中外豬品種在馴化過程中產(chǎn)生的CNV,研究中外豬品種在馴化過程中受到選擇的CNV及其相關(guān)基因,從而發(fā)現(xiàn)中外豬品種表型差異的遺傳基礎(chǔ)。為進(jìn)一步解析豬基因組變異及豬品種改良奠定基礎(chǔ)。主要結(jié)果如下:1.1利用生物信息學(xué)方法分析家豬馴化過程中的CNV。以本實(shí)驗(yàn)室通城豬基因組個(gè)體重測(cè)序數(shù)據(jù)及公共數(shù)據(jù)庫下載的不同豬品種和野豬共49個(gè)個(gè)體重測(cè)序數(shù)據(jù)為材料,利用CNVseq和CNVnator軟件分別進(jìn)行CNV掃描。發(fā)現(xiàn),從野豬到家豬馴化過程中產(chǎn)生的CNVRs共有3131個(gè),其中拷貝數(shù)增加的區(qū)域有745個(gè)、減少的區(qū)域2364個(gè),增加和減少都存在的區(qū)域有22個(gè);根據(jù)CNVR在基因組上的位置,繪制出豬全基因組CNVs圖譜。1.2利用實(shí)時(shí)熒光定量PCR方法驗(yàn)證CNVRs。利用實(shí)時(shí)熒光定量PCR方法對(duì)隨機(jī)選取的28個(gè)區(qū)域進(jìn)行拷貝數(shù)的驗(yàn)證,結(jié)果24個(gè)CNVRs的拷貝數(shù)與預(yù)測(cè)CNVRs內(nèi)拷貝數(shù)的增加或減少相符合,驗(yàn)證符合率為86%。1.3 CNV的分布特征分析。對(duì)3131個(gè)CNVRs及上下游10Kb區(qū)域中的重復(fù)元件(SINE、LINE和LTR等)的數(shù)量及分布密度進(jìn)行統(tǒng)計(jì)分析,結(jié)果發(fā)現(xiàn)在CNVRs及上下游10Kb的區(qū)域中,不同重復(fù)元件的分布密度均顯著高于基因組中的平均水平,表明CNV常分布在基因組中重復(fù)元件附近,重復(fù)元件對(duì)CNV發(fā)生有重要影響。1.4家豬馴化過程中CNVR相關(guān)基因的功能分析。利用BioMart工具,在家豬馴化過程中產(chǎn)生的3131個(gè)CNVRs中發(fā)現(xiàn)1266個(gè)編碼蛋白的基因,利用DAVID工具對(duì)基因進(jìn)行功能富集分析,發(fā)現(xiàn)這些基因主要參與細(xì)胞粘附、GTP酶活性、細(xì)胞連接、免疫反應(yīng)、嗅覺和MAPK通路等。1.5中外家豬在馴化過程中產(chǎn)生的差異cnvr及相關(guān)基因功能分析。通過分析發(fā)現(xiàn),中國家豬中存在2278個(gè)cnvrs,歐洲家豬中存在1706個(gè)cnvrs。分別特異存在于中外家豬中的cnvrs有129個(gè)和147個(gè),分別對(duì)cnvrs內(nèi)相關(guān)基因進(jìn)行功能富集分析,結(jié)果顯示,中國家豬馴化過程中產(chǎn)生的特異cnvrs內(nèi)相關(guān)基因的功能富集在免疫反應(yīng)及生產(chǎn)性狀上;而歐洲家豬馴化過程中產(chǎn)生的特異cnvrs內(nèi)相關(guān)基因的功能富集在肌肉發(fā)育過程。2.利用轉(zhuǎn)錄組數(shù)據(jù)分析豬基因組多聚腺苷酸化位點(diǎn)多聚腺苷酸化是rna轉(zhuǎn)錄后修飾的一個(gè)重要過程,在mrna的轉(zhuǎn)運(yùn)及成熟mrna的翻譯過程中起到關(guān)鍵作用。一個(gè)基因序列上多聚腺苷酸化位點(diǎn)(polyadenylationsite,pas)的數(shù)量以及每個(gè)pas的利用程度不同會(huì)引起選擇性多聚腺苷酸化(alternativepolyadenylation,apa)的形成,從而導(dǎo)致同一個(gè)基因產(chǎn)生多個(gè)轉(zhuǎn)錄本,對(duì)基因的表達(dá)及功能的發(fā)揮產(chǎn)生重要影響。本研究利用豬轉(zhuǎn)錄組數(shù)據(jù),從全基因組水平挖掘豬的pas,通過研究pas與基因表達(dá)量的關(guān)系,進(jìn)一步研究pas對(duì)性狀的影響。主要結(jié)果如下:2.1基于大規(guī)模轉(zhuǎn)錄組數(shù)據(jù)挖掘豬的多聚腺苷酸化位點(diǎn)。利用本實(shí)驗(yàn)室感染藍(lán)耳病病毒前后的通城豬和大白豬中肺泡巨噬細(xì)胞的轉(zhuǎn)錄組數(shù)據(jù)及公共數(shù)據(jù)庫下載的豬的轉(zhuǎn)錄組數(shù)據(jù),包括12種組織、細(xì)胞及精子等的轉(zhuǎn)錄組數(shù)據(jù),共計(jì)120億個(gè)reads,,其中有194萬個(gè)含有poly(a)或poly(t)的reads成功比對(duì)到基因組上,對(duì)這些reads進(jìn)行pas挖掘,共得到28363個(gè)pass。2.2對(duì)pas位置進(jìn)行注釋。依據(jù)目前豬基因組注釋文件中基因的位置信息,對(duì)本研究得到的28363個(gè)pass進(jìn)行位置注釋,共發(fā)現(xiàn)13033個(gè)(47%)pass位于7403個(gè)基因中,其中有7900個(gè)pass(61%)位于基因的3’utr,3441個(gè)pass(26%)位于基因的內(nèi)含子區(qū)域,2187個(gè)pass(17%)位于基因的orf區(qū)域;利用所有轉(zhuǎn)錄組數(shù)據(jù)對(duì)豬的新轉(zhuǎn)錄本進(jìn)行預(yù)測(cè),并對(duì)剩余的15330個(gè)pass進(jìn)行位置注釋,結(jié)果表明,有6806個(gè)(24%)pass位于預(yù)測(cè)的新轉(zhuǎn)錄本內(nèi)部。即:利用基因組注釋文件和預(yù)測(cè)新的轉(zhuǎn)錄本信息,共發(fā)現(xiàn)19839個(gè)pass(70%)位于基因內(nèi)部區(qū)域,8524個(gè)pass(30%)位于基因間區(qū)域。2.3pas在基因組和不同組織中的分布特性�；谪i基因組注釋文件中基因的位置信息,對(duì)基因及其3’utr內(nèi)的pas分布進(jìn)行分析,結(jié)果顯示,近41%的基因中存在至少兩個(gè)以上的PASs,這些PAS可促使同一基因產(chǎn)生多個(gè)轉(zhuǎn)錄本;而對(duì)基因內(nèi)及其3’UTR中相鄰PASs間的距離分析發(fā)現(xiàn),大多數(shù)PASs(45%)間的距離很近(1Kb);對(duì)3’UTR中的PAS與終止子間的距離分析發(fā)現(xiàn),PAS在3’UTR上的位置具有較大差異,該距離的中值為307nt;通過對(duì)肝臟及睪丸組織中PAS進(jìn)行挖掘,分別得到12777和14375個(gè)PASs,而兩個(gè)組織相同的PAS僅有4752個(gè),占總數(shù)量的21%,并且,兩個(gè)組織中相同PAS的利用率差異很大,說明PAS具有組織特異性。2.4利用Pearson方法對(duì)PAS和基因表達(dá)量進(jìn)行相關(guān)性分析。本研究利用源于不同雄性激素水平的睪丸和肝臟組織的轉(zhuǎn)錄組數(shù)據(jù),對(duì)每個(gè)數(shù)據(jù)中基因的表達(dá)量、相應(yīng)基因內(nèi)PAS的數(shù)量及覆蓋的reads數(shù)進(jìn)行統(tǒng)計(jì),利用Pearson方法對(duì)PAS與基因表達(dá)量進(jìn)行相關(guān)性分析。結(jié)果表明,基因內(nèi)PAS數(shù)量與基因表達(dá)量呈中度正相關(guān)(0.4r0.6,p0.01),PAS覆蓋的reads數(shù)與基因表達(dá)量呈強(qiáng)正相關(guān)(0.6r0.8,p0.01)。2.5 PAS利用率對(duì)雄性激素水平及在細(xì)菌感染機(jī)體過程中的作用分析。依據(jù)睪丸和肝臟組織中雄性激素水平的高低,對(duì)不同數(shù)據(jù)中挖掘的PAS進(jìn)行差異利用率分析,結(jié)果表明,肝臟中有272個(gè)PASs在低雄性激素組中的利用率顯著高于在高雄性激素組中利用率(p0.05,|log2FC|≥1),對(duì)差異利用率PAS所在的109個(gè)基因進(jìn)行功能富集分析發(fā)現(xiàn),這些基因參與到了固醇及脂肪酸的代謝、甾類激素的合成和細(xì)胞色素P450的代謝等過程(p0.05);在睪丸中,有260個(gè)PASs的利用率具有極顯著差異的(p0.05,|log2FC|≥1),相應(yīng)的基因有163個(gè),基因功能富集分析表明,很多基因同時(shí)參與精子形成和細(xì)胞周期等過程(p0.05)。對(duì)感染沙門氏菌前后的轉(zhuǎn)錄組數(shù)據(jù)進(jìn)行PAS分析,發(fā)現(xiàn)38個(gè)PASs在感染后有較高的利用率,相關(guān)基因有28個(gè),而41個(gè)PAS在感染前有較高的利用率,相關(guān)基因有26個(gè)(p0.05,|log2FC|≥1)。分別對(duì)感染前后高利用率PAS的相關(guān)基因進(jìn)行功能富集分析,結(jié)果顯示,感染后高利用率PASs的相關(guān)基因參與免疫應(yīng)答和細(xì)胞因子的調(diào)控等過程,而感染前高利用率PASs的相關(guān)基因參與翻譯等過程,與免疫反應(yīng)無直接關(guān)系的過程(p0.05)。
[Abstract]:1. * using genome sequencing data to analyze copy number variation in domestication of Chinese and foreign pig breeds during * * domestication of wild boars and domesticated pigs, genetic variation and selection in different degrees play a certain role in the formation of varieties and the variety differences. Genomic variations can be found in genomic sequences among the same species. Copy number variation (CNV) refers to variations between 1Kb and Mb in length that occur in the genome in the form of repetition, deletion and derivation of complex chromosomal microstructural variations, as compared with normal biological gene sequences. This * * is to carry out CNV analysis of 49 individuals from 13 domestic and foreign pig breeds, and to analyze the functions of the related genes in the copy number variation region (CNVR). * by comparing the CNV produced by domestication and domestication of pig breeds, the CNV and related genes selected in domestication of Chinese and foreign pig breeds are studied. This * * * found the genetic basis of phenotypic differences between Chinese and foreign pig breeds. This lays the foundation for further analysis of pig genome variation and pig breed improvement. The main results are as follows: 1.1 bioinformatics method is used to analyze CNV. in domestication of pigs domestically. CNVRs were scanned by CNVseq and CNVnator software in 49 individuals of species and wild boar. A total of 3131 CNVRs were detected during domestication from wild boar to domestic boar, of which 745 were increased, 2364 were decreased, and 22 were increased or decreased according to CNVR. The whole genome CNVs map of pig was drawn up *.1.2 was validated by real-time fluorescence quantitative PCR. CNVRs. was validated by real time fluorescence quantitative PCR for 28 selected regions. The results showed that the copy number of 24 CNVRs was consistent with the increase or decrease of copy number in CNVRs, and the rate of verification was 86%.1.3 CNV. Distribution characteristics were analyzed. The number and density of repetitive elements (SINE, LINE and LTR) in 3131 CNVRs and 10 Kb upstream and downstream regions were statistically analyzed. The results showed that the distribution density of different repetitive elements in CNVRs and 10 Kb upstream and downstream regions was significantly higher than the average level in the genome, indicating that CNV was normally distributed in the genome. Functional analysis of CNVR-related genes during domestication in pigs. Using BioMart tools, 1266 genes encoding proteins were found in 3131 CNVRs produced during domestication in domestic pigs. The DAVID tools were used to enrich and analyze the functions of these genes. The results showed that these genes were mainly involved in fineness. Differential CNVRs and related gene functions in domestic and foreign pigs during domestication were analyzed. 2 278 CNVRs were found in Chinese pigs and 1 706 CNVRs were found in European pigs. The results showed that the functions of the genes related to CNVRs produced during domestication in Chinese pigs were enriched in immune response and production traits, while those of the genes related to CNVRs produced during domestication in European pigs were enriched in muscle development. Group data analysis * polyadenylation of porcine genome polyadenylation is an important process in post transcriptional modification of RNA. It plays a key role in mRNA translocation and maturation of mRNA translation. The number of polyadenylation sites (polyadenylationsite, PAS) on a gene sequence and the utilization level of each PAS will be different. The formation of alternativepolyadenylation (APA), which results in the production of multiple transcripts of the same gene, has an important effect on the expression and function of the gene. This * * * uses pig transcriptome data to dig pig PAS from the whole genome level, and further studies the relationship between PAS and gene expression. The effects of PAS on traits were studied. The main results are as follows: 2.1 * based on large-scale transcriptome data, the polyadenylation sites of pigs were extracted. * * the transcriptome data of alveolar macrophages in Tongcheng pig and large white pig before and after infection with the blue ear disease virus and the transcriptome data of pigs downloaded from the public database, including 12 tissues and cells, were used. The transcriptome data of spermatozoa and 12 billion reads, 1 million 940 thousand of which have poly (a) or poly (T) reads were successfully compared to the genome, and PAS mining for these reads, a total of 28363 pass.2.2 were selected to annotate the position of PAS * according to the location information of the gene in the genome of the pig genome, 28363 of this study was obtained. Annotated by pass, 13033 (47%) pass were found in 7403 genes, including 7900 pass (61%) located at 3 'UTR of the gene, 3441 pass (26%) located in the intron region of the gene, 2187 pass (17%) located in the ORF region of the gene, * using all transcriptome data to predict the new transcript of the pig, and the remaining 15330 pass. Location annotation showed that 6806 (24%) pass were located within the predicted new transcript. That is, using genomic annotation files and predicting new transcript information, 19839 pass (70%) located in the inner region of the gene were found, and 8524 pass (30%) located in the intergenic domain.2.3pas in the genome and in different tissues. Location information of genes in genomic annotation files was analyzed for the distribution of PAS in genes and their 3'utr. The results showed that nearly 41% of genes contained at least two or more PASs, which could induce multiple transcripts of the same gene. Distance analysis of the genes and their adjacent PASs in 3'UTR revealed that most of the PASs (45%) were between. The distances between PAS and terminators in 3'UTR were very close (1Kb); the median of the distances between PAS and terminators was 307nt, and 12777 and 14375 PASs were obtained respectively from liver and testis tissues, while only 4752 PAS were found in the same tissues, accounting for 21% of the total. In this study, we used transcriptome data from testicular and liver tissues with different levels of androgen to analyze the expression of PAS and the number of PAS in each data. Pearson method was used to analyze the correlation between PAS and gene expression. The results showed that there was a moderate positive correlation between PAS and gene expression (0.4r0.6, p0.01), and a strong positive correlation between PAS and gene expression (0.6r0.8, p0.01). According to the level of androgen in testis and liver tissues, the utilization rate of PAS in low androgen group was significantly higher than that in high androgen group (p0.05, | log2FC | 1), and the difference was significant. Functional enrichment analysis of 109 PAS genes showed that these genes were involved in steroid and fatty acid metabolism, steroid hormone synthesis and cytochrome P450 metabolism (p0.05); in the testis, 260 PASs utilization rates were significantly different (p0.05, | log2FC | 1), 163 corresponding genes, genes. Functional enrichment analysis showed that many genes were involved in both spermatogenesis and cell cycle (p0.05). PAS analysis of the transcriptome data before and after Salmonella infection showed that 38 PASs had higher utilization rate after infection, 28 related genes, 41 PAS had higher utilization rate before infection, 26 related genes (p0.05, | lo) G2FC | 1. Functional enrichment analysis of PAS-related genes before and after infection showed that PAS-related genes were involved in immune response and cytokine regulation, while PAS-related genes before infection were involved in translation, which had no direct relationship with immune response (p0.05). .05).
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S828

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