【作者】 王恒；

【导师】 章孝荣； 刘洪瑜；

【作者基本信息】 安徽农业大学 ， 动物遗传育种与繁殖， 2015， 博士

【摘要】 牛肉肌间脂肪的沉积和分布(即大理石花纹等级)是评价高档牛肉的重要指标。micro RNA(mi RNA)是一类重要的非编码小RNA分子,其通过对靶基因转录后调控实现对动植物生理功能的调节,进而影响机体的生产性能。目前国内外学者开展了有关肉牛脂肪沉积及肌肉发育相关mi RNA的鉴定及功能等方面的工作,但在主要肉牛品种间mi RNA差异、性别引起的脂肪沉积差异mi RNA等方面研究较少。开展调控肉牛脂肪沉积的mi RNA筛选及鉴定工作,将有助于认识mi RNA调控脂肪沉积的分子机制,为肉牛分子育种及优秀肉牛品种利用奠定分子生物学基础。为进一步揭示mi RNA在不同肉牛品种、不同肉牛性别引起的脂肪沉积的差异,阐明其对牛脂肪沉积的调控作用,本课题进行了调控肉牛脂肪沉积的mi RNA筛选及鉴定方面的研究。本研究主要分为两大部分。第一部分,不同肉牛品种肌间脂肪差异mi RNA的筛选及鉴定。选取安格斯牛、西门塔尔牛和日本和牛6月龄公犊牛各3头,在相同营养水平及饲养管理条件下育肥至24月龄,进行了肉牛品种肌间脂肪的小RNA高通量测序。(1)分析了三个肉牛品种肌间脂肪小RNA分布及序列特征;(2)分析了三个肉牛品种肌间脂肪mi RNA转录组特征,得到了三个肉牛品种的mi RNA表达谱及基因组信息,为后继的功能分析提供参考;(3)通过两两比较的方法,揭示三个肉牛品种肌间脂肪mi RNA差异表达情况,并随机选取5个mi RNA的定量验证。第二部分,肉牛性别差异导致的脂肪沉积差异mi RNA的筛选与鉴定。选取相同月龄的日本和牛和皖南牛杂交一代公、母牛犊牛各5头,24月龄时屠宰,进行了公、母牛肌间脂肪的Affymetrix micro RNA 3.0基因芯片分析。揭示公、母牛肌间脂肪mi RNA差异表达情况。并采用生物信息学方法进行了差异表达mi RNA的靶基因分析,同时采用定量PCR对芯片试验结果进行了验证。本研究主要结果:(1)成功构建了安格斯牛、西门塔尔牛和日本和牛公牛肌间脂肪组织的mi RNA文库,在三个文库中分别获得11,873,367;11,588,623和11,843,883条纯净序列(clean reads),分别占总文库的99.41%,97.04%和99.19%。三个文库的小RNA片段长度均主要分布在20~24nt之间,其中22nt长度片段所占总的片段长度数量分别为49.17%,45.47%和50.41%。(2)安格斯牛有9,135,368条总纯净序列可以对比到牛的基因组上,占总数的76.94%,归结的唯一序列有128,405条可以对比到牛的基因组上,占总唯一序列的45.41%;西门塔尔牛有9,168,676条总纯净序列可以对比到牛的基因组上,占总数的79.12%,归结的唯一序列有112,138条可以对比到牛的基因组上,占总唯一序列的30.27%;日本和牛有9,385,045条总纯净序列可以对比到牛的基因组上,占总数的79.24%,归结的唯一序列有85,957条可以对比到牛的基因组上,占总唯一序列的34.48%。(3)在总的纯净序列里面,mi RNA在三个牛小RNA文库中分别占70.63%,62.98%,71.22%。(4)通过两两比较的方法,对比三个文库中总纯净序列和唯一序列中的共同和特有的序列。安格斯牛和西门塔尔牛相比较,有97.46%的纯净序列是相同的,而与日本和牛相比较,有98.11%的序列是相同的,西门塔尔牛与日本和牛相同的序列占97.77%。在唯一序列中,安格斯牛和日本和牛共有的序列占了17.82%,而安格斯牛和西门塔尔牛只有15.49%是相同的。(5)在安格斯牛、西门塔尔牛和日本和牛的三个牛肌间脂肪文库中分别检测到347个,347个和319个mi RNA;55个,48个和53个mi RNA-5p;51个,50个和48个mi RNA-3p。通过Mireap软件预测,在安格斯牛的文库中,发现了55个潜在的mi RNA,在西门塔尔牛的文库中发现了30个潜在的mi RNA,在日本和牛的文库中,发现了43个潜在的mi RNA。(6)在安格斯牛和西门塔尔牛的mi RNA文库中,差异表达的mi RNA有129个,其中上调表达的49个,下调表达的80个;在安格斯和日本和牛的mi RNA文库中个,差异表达的mi RNA有103个,其中上调表达的24个,下调表达的79个;在西门塔尔牛和安格斯的mi RNA文库中,差异表达的mi RNA有55个,其中上调表达的12个,下调表达的43个。(7)bta-mi R-1281和bta-mi R-148b是日本和牛与皖南牛杂交F1代公母牛个体脂肪沉积的差异表达的mi RNA。(8)利用靶基因预测软件Target Scan和Pic Tar,预测牛mi R-148b的潜在靶基因,两个数据库共同的可能调控靶基因有189个。(9)SOX5基因是mi R-148b的一个潜在靶基因,而SOX5基因是调控动物脂肪沉积的一个重要基因。因此,下一步可深入研究mi R-148b通过SOX5调控动物脂肪沉积的机理。更多还原

【Abstract】 The intermuscular fat deposition and distribution in beef cattle,usually being categerized into different degree of marbling,is an important index for evaluation of high-grade beef.Micro RNAs(mi RNAs)are a group of small noncoding RNAs,playing regulatory roles in many aspects of development in animals and plants by post-transcriptional regulation of target genes,thereby affecting the production performance of animals and plants.Currently,although identification and functional analysis of mi RNAs closely related to intermuscular fat development have been well studied,researches on differences of mi RNAs among major beef cattle breeds or differentially expressed mi RNAs attributed to different genders are still scarce.Therefore,screen and identification of mi RNAs potentially regulating intermuscular fat deposition will better our understanding of molecular mechanisms underlying intermuscular fat deposition regulated by mi RNAs,and will provide molecular basis for breeding and utilization of excellent beef cattle.To explore differentially expressed mi RNAs and their potential roles in fat deposition in beef cattle of different breeds or genders,and lay foundation for clarification of such mi RNAs’ role in the regulation of bovine intermuscular fat deposition,the present study was conducted to screen and identify mi RNAs potentially involving in the regulation of fat deposition in beef cattle.The research mainly includes two parts.The first part was to screen and identify mi RNAs of different beef cattle breeds.Six-month-old bull calves of Angus,Simmental and Wagyu,with 3 of each breeds,were respectively selected,raised at the same nutrition level and management conditions,and fattened to 24 months of age,the small RNAs extracted from intramuscular fat in aformentioned three breeds were sequenced using high-throughput sequencing methods.(1)the distribution and sequence characteristics of small RNAs in intramuscular fat of three breeds were analyzed;(2)the transcriptomic characteristics of mi RNAs in intramuscular fat of three breeds were analyzed,and the expression profile and genomic information of such mi RNAs of three breeds were then obtained,which represents a reference for subsequent functional analysis;(3)By pairwise comparison,we revealed differences of mi RNAs expressed in intermuscular fat among three cattle breeds,and quantitative real-time PCR also validated sequencing results of five randomly selected mi RNAs.The second part was to screen and identify differentially expressed mi RNAs involving in intermuscular fat deposition in beef cattle of different genders.One bull calf and five heifers of F1 crossbred Wagyu-Wannan cattle were selected at the same age and subjected to slaughter at 24 months of age,and mi RNAs in intramuscular fat were then arrayed with Affymetrix micro RNA 3.0 gene chip.Differential expression of mi RNAs in intermuscular fat from bull calves and heifers was determined.The results from gene chip were verified by quantitative real-time PCR,and the target gene of differentially expressed mi RNAs were also predicted.The detail results and conclusions are as follows:(1)The mi RNAs libraries of intramuscular fat tissue from Angus,Simmental and Wagyu beef bulls were successfully constructed.In three libraries,we obtained 11873367,11588623 and 11843883 clean reads,which accounted for 99.41%,97.04% and 99.19% of total libraries,respectively.The small RNAs fragments in three libraries were dominantly distributed in 20~24nt,and the fragments of 22 nt length accounted for 49.17%,45.47% and 50.41% of the fragments of all length,respectively.(2)There are 9135368 total clean reads that can be matched to the bovine genome of Angus cattle,accounting for 76.94% of the total,and only 128405 unique reads which can be matched to the bovine genome,accounting for 45.41% of the total unique reads;There are 9168676 total clean reads that can be matched to the bovine genome of Simmental cattle,accounting for 79.12% of the total,and only 112138 unique reads which can be matched to the bovine genome,accounting for 30.27% of the total;There are 9385045 of total clean reads which can be matched to the bovine genome of Wagyu cattle,accounting for 79.24% of the total,and only 85957 unique reads that can be matched to the bovine genome,accounting for total 34.48% of the total unique reads.(3)In total clean reads,mi RNAs represented 70.63%,62.98%,and 71.22% of three small RNAs libraries,respectively.(4)Comparison of common and specific sequence of total clean reads and unique reads in three libraries was acomplished by using pairwise comparison method.As for clean reads,Angus shared 97.46% in common with Simmental,while had 98.11% in common with Wagyu,and Simmental shared 97.77 % in common with Wagyu.As for unique reads,Angus engaged 17.82% in common with Wagyu,while Angus and Simmental only shared 15.49 % common unique sequence.(5)In the intramuscular fat libraries of Angus,Simmental and Wagyu,we detected 347,347 and 319 mi RNAs,respectively;55,48 and 53 mi RNA-5p,respectively;51,50 and 48 mi RNA-3p,respectively.Using Mireap software,we predicted 55 potential mi RNAs in Angus library,30 potential mi RNAs in Simmental library and 43 potential mi RNAs in Wagyu library.(6)In mi RNAs libraries of intramuscular fat in Angus and Simmental breeds,we found 129 differentially expressed mi RNAs,of which 49 up-regulated and 80 downregulated;In mi RNAs libraries of intramuscular fat in Angus and Wagyu,we observed 103 differentially expressed mi RNAs,of which 24 up-regulated and 79 down-regulated;In mi RNAs libraries of intramuscular fat in Simmental and Wagyu,we revealed 55 differentially expressed mi RNAs,of which 12 up-regulated and 43 down-regulated;(7)bta-mi R-1281 and bta-mi R-148 b were differentially expressed mi RNAs involving in fat deposition in F1 Wagyu-Wannan bull calves and heifers.(8)Using Target Scan software and Pic Tar software,189 possible common target genes of mi R-148 b were predicted.(9)Of note,SOX5 gene was predicted as one potential candidate target gene of mi R-148 b,shown already that it is an important regulation gene in animal fat deposition.Therefore,if mi R-148 b regulates intermuscular fat deposition of beef cattle through SOX5 requires further study.更多还原