杂交盘羊尾部抑制性消减文库的构建与转录组初步分析

发布时间：2018-08-19 15:05

【摘要】：绵羊生产中一直有为羔羊断尾的工作,不仅增加了工作量而且操作不当会使羔羊死亡,本实验室羊场有含75%血盘羊,其尾部短小,无需断尾。为探讨盘羊小尾形成的分子机制,本研究以含25%血杂交盘羊和含75%血杂交盘羊尾部组织为研究对象,采用抑制性消减杂交技术(SSH)和高通量转录组测序技术,建立抑制性消减杂交数据库与转录组差异表达数据库,以期找到与尾部发育相关的功能基因,为培育小尾羊新品种奠定基础。抑制性消减杂交实验以含25%血盘羊尾部组织为实验组,以含75%血盘羊尾部组织为对照组,构建正、反向消减杂交文库,在正、反向文库中各挑选200个阳性克隆送去公司测序,发现正向文库中测序成功129个,反向文库中测序成功109个。正向文库内随机选取了44个差异基因,反向随机取了15个差异基因。结果正向文库中FN1和LGALS3和反向文库中TBX2与TBX18可能与骨骼发育有关。高通量转录组测序实验通过Hiseq2000平台完成,测序结果通过NR基因功能注释、GO功能分类分析和KEGG通路富集分析,结果表明25%血和75%血杂交盘羊尾部组织Unigene分别为133,542个和136,318个;GO功能分类注释到生物过程的Unigene有195,878个、分子功能相关Unigene为45,376个和细胞成分相关Unigene有26,200个;KEGG通路富集分析得到所有基因通路注释36,503个,其中差异基因通路注释有4,807个。25%血与75%血杂交盘羊的差异表达基因共有8,515个,其中以75%血为参照,上调基因3,474个,下调基因5,041个。上调最显著的三个基因是TPM3(78.1979)、MYH1(57.2913)和TNNT1(34.8466),下调幅度最大基因是MHC(-15.3141)、RNASET2(-14.3305)和KRTAP10-8(-13.9062)。在转录测序中还发现了T-box家族基因(TBX2、TBX3、TBX15、TBX18和TBX19),其中TBX3、TBX15和TBX18是关于骨骼生长发育,很有可能在小尾形成机制中起关键作用。最终我们选定TBX18和TBX15作为绵羊小尾性状的候选基因。
[Abstract]:In sheep production, the work of cutting off the tail of the lamb has always been done, which not only increases the workload but also leads to the death of the lamb by improper operation. The sheep farm in our laboratory contains 75% blood sheep, the tail of which is short and does not need to be cut off. In order to investigate the molecular mechanism of small tail formation in Panyang sheep, the tail tissues of the sheep with 25% blood and 75% blood were studied by suppression subtractive hybridization (SSH) and high-throughput transcriptome sequencing. The database of suppression subtractive hybridization and transcriptome differential expression was established in order to find the functional genes related to tail development and to lay a foundation for the breeding of new breeds of small tail sheep. In the suppression subtractive hybridization experiment, the positive and reverse subtractive hybridization libraries were constructed by using 25% blood sheep tail tissue as experimental group and 75% blood pan sheep tail tissue as control group. 200 positive clones were selected from the positive and reverse libraries to be sent to the company for sequencing. It was found that 129 positive libraries and 109 reverse libraries were sequenced successfully. 44 genes were randomly selected in the forward library and 15 genes were randomly selected in reverse. Results FN1 and LGALS3 in forward library and TBX2 and TBX18 in reverse library may be related to bone development. The high-throughput transcriptome sequencing experiment was carried out by Hiseq2000 platform. The sequencing results were analyzed by functional classification analysis of NR gene and KEGG pathway enrichment. The results showed that the Unigene of tail tissue of 25% and 75% crossbred sheep was 133542 and 136318 Unigene respectively. The molecular functional correlation (Unigene) was 45376 and the cellular component related Unigene had 26200 KEGG pathway enrichment analysis. All the gene pathway annotations were 36503, and the differential gene pathway annotated 4807 .25% blood and 75% blood crossbred sheep, there were 8515 differentially expressed genes. Among them, 75% blood was used as reference, 3474 genes were up-regulated and 5041 genes were down-regulated. The three most up-regulated genes were TPM3 (78.1979) MYH1 (57.2913) and TNNT1 (34.8466), and the most down-regulated genes were MHC (-15.3141), RNASET2 (-14.3305) and KRTAP10-8 (-13.9062). In transcriptional sequencing, T-box family genes (TBX2, TBX3, TBX15, TBX18 and TBX19) were also found. TBX3, TBX15 and TBX15 are related to bone growth and development, which may play a key role in the mechanism of small tail formation. Finally, we selected TBX18 and TBX15 as candidate genes for small tail traits in sheep.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

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