雜交盤羊尾部抑制性消減文庫的構(gòu)建與轉(zhuǎn)錄組初步分析
發(fā)布時間：2018-08-19 15:05
【摘要】：綿羊生產(chǎn)中一直有為羔羊斷尾的工作,不僅增加了工作量而且操作不當(dāng)會使羔羊死亡,本實(shí)驗(yàn)室羊場有含75%血盤羊,其尾部短小,無需斷尾。為探討盤羊小尾形成的分子機(jī)制,本研究以含25%血雜交盤羊和含75%血雜交盤羊尾部組織為研究對象,采用抑制性消減雜交技術(shù)(SSH)和高通量轉(zhuǎn)錄組測序技術(shù),建立抑制性消減雜交數(shù)據(jù)庫與轉(zhuǎn)錄組差異表達(dá)數(shù)據(jù)庫,以期找到與尾部發(fā)育相關(guān)的功能基因,為培育小尾羊新品種奠定基礎(chǔ)。抑制性消減雜交實(shí)驗(yàn)以含25%血盤羊尾部組織為實(shí)驗(yàn)組,以含75%血盤羊尾部組織為對照組,構(gòu)建正、反向消減雜交文庫,在正、反向文庫中各挑選200個陽性克隆送去公司測序,發(fā)現(xiàn)正向文庫中測序成功129個,反向文庫中測序成功109個。正向文庫內(nèi)隨機(jī)選取了44個差異基因,反向隨機(jī)取了15個差異基因。結(jié)果正向文庫中FN1和LGALS3和反向文庫中TBX2與TBX18可能與骨骼發(fā)育有關(guān)。高通量轉(zhuǎn)錄組測序?qū)嶒?yàn)通過Hiseq2000平臺完成,測序結(jié)果通過NR基因功能注釋、GO功能分類分析和KEGG通路富集分析,結(jié)果表明25%血和75%血雜交盤羊尾部組織Unigene分別為133,542個和136,318個;GO功能分類注釋到生物過程的Unigene有195,878個、分子功能相關(guān)Unigene為45,376個和細(xì)胞成分相關(guān)Unigene有26,200個;KEGG通路富集分析得到所有基因通路注釋36,503個,其中差異基因通路注釋有4,807個。25%血與75%血雜交盤羊的差異表達(dá)基因共有8,515個,其中以75%血為參照,上調(diào)基因3,474個,下調(diào)基因5,041個。上調(diào)最顯著的三個基因是TPM3(78.1979)、MYH1(57.2913)和TNNT1(34.8466),下調(diào)幅度最大基因是MHC(-15.3141)、RNASET2(-14.3305)和KRTAP10-8(-13.9062)。在轉(zhuǎn)錄測序中還發(fā)現(xiàn)了T-box家族基因(TBX2、TBX3、TBX15、TBX18和TBX19),其中TBX3、TBX15和TBX18是關(guān)于骨骼生長發(fā)育,很有可能在小尾形成機(jī)制中起關(guān)鍵作用。最終我們選定TBX18和TBX15作為綿羊小尾性狀的候選基因。
[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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S826
本文編號：2192019
[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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S826
【參考文獻(xiàn)】
相關(guān)期刊論文 前4條
1 張強(qiáng),毛澤彬,張志文,辛殿旗,郭應(yīng)祿;腎癌相關(guān)基因克隆——腎癌cDNA消減文庫的構(gòu)建[J];生物化學(xué)與生物物理進(jìn)展;2000年03期
2 王慧梅;王延兵;祖元剛;孫蓮慧;;干旱脅迫下黃檗幼苗cDNA消減文庫的構(gòu)建和分析[J];生物工程學(xué)報;2008年02期
3 李玉昌,徐存拴,張?jiān)茲h;應(yīng)用抑制性消減雜交技術(shù)克隆大鼠肝再生過程中特異表達(dá)基因[J];遺傳;2002年02期
4 張志明;趙茂俊;馬永毅;丁海萍;唐云峰;魏秀儉;潘光堂;榮廷昭;;抑制性消減雜交技術(shù)及其在玉米抗病基因研究中的應(yīng)用[J];玉米科學(xué);2006年03期
，本文編號：2192019
本文鏈接：http://sikaile.net/yixuelunwen/dongwuyixue/2192019.html
最近更新
教材專著
