發(fā)布時間：2018-05-22 15:05

  本文選題：獺兔 + 轉(zhuǎn)錄組��； 參考：《浙江師范大學》2015年碩士論文

【摘要】：獺兔,典型的皮用兔,因被毛獨特而倍受人們喜愛。近年來,在生產(chǎn)中發(fā)現(xiàn),某些獺兔會在腹部和四肢以及臀部出現(xiàn)皺褶,稱其為皺襞型獺兔。皺襞型獺兔的個體比非皺襞型稍大,同時具有正常獺兔相同的皮毛品質(zhì),這對于以皮張面積和皮毛品質(zhì)為重要經(jīng)濟效益因素的獺兔來說,意義重大,提供了新的獺兔育種方向。本研究采用RNA-Seq技術,對皺襞型(試驗組)和非皺襞型(對照組)白色獺兔的皮膚樣本進行測序分析。分析測序結(jié)果,篩選出與皮膚生長發(fā)育相關的基因。將樣本的有效reads合并,通過Trinity軟件,使用兩端重復進行de novo拼接。兩組皮膚樣本測序總共獲得377,618條Unigenes, Unigenes的平均長度是680 nt,最長的序列長達20,293 nt。將Unigenes與NR、Swiss-Prot、KEGG和KOG等數(shù)據(jù)庫比對(E-Value10"5),根據(jù)比對的注釋,分析已知Unigene的功能。由于家兔的全基因組信息還不完善,85.5931%序列無法比對到任何數(shù)據(jù)庫上。根據(jù)NR注釋及Blast2GO,總共有42,895條Unigene被映射到不同的GO term上,并按照分子功能、細胞組分及生物過程將其分類；KOG的結(jié)果顯示60,593條Unigenes被分為25類。KEGG數(shù)據(jù)庫比對顯示,13,431條Unigene分別被注釋到399個KEGG pathway中,其中注釋到PI3K-Akt信號通路(ko04151)的基因最多(686,5.108%),該信號通路在皮膚的生長發(fā)育過程中具有重要的意義。兩種不同表型的樣本中,共有308個顯著的差異表達基因(P-Value≤0.05且Iog2|foldchange| 1),其中118個在皺襞型樣品中為上調(diào)基因,190個為下調(diào)基因。這些基因中有99個被注釋到不同的GO Term上,其中只有29個基因被注釋到KEGG不同的代謝途徑中。除此之外的209個基因被稱為新基因,在這些基因中,33個只在非皺襞型個體中表達,56個基因只在皺襞型個體中表達。通過qRT-PCR驗證測序結(jié)果。選擇7個候選基因,使用oligo 7.0設計特異性引物,采用△△Ct法計算皺襞型和非皺襞型樣品的相對表達量。結(jié)果顯示,qRT-PCR的結(jié)果和轉(zhuǎn)錄組測序的結(jié)果相符。說明測序結(jié)果是正確的,據(jù)此初步確定與家兔皮膚發(fā)育相關的基因：MYL12B、LAMBS、PLOW、EIF3E、CEBPA、FAD2, ENPP6等。候選基因MYL12B是細胞骨架的重要組分,作為肌球蛋白的調(diào)節(jié)性輕鏈,可通過調(diào)節(jié)輕鏈的磷酸化與去磷酸化,調(diào)節(jié)細胞的許多動力學過程。本研究克隆了MYL12B基因的CDS序列,并與人、黑猩猩、小鼠等12個物種的CDS序列和氨基酸序列進行比較。發(fā)現(xiàn),除馬外,該基因與哺乳動物CDS相似性高達94%,與鳥類和兩棲類的同源性也較高。生物信息學分析顯示MYL12B編碼的蛋白是一個不穩(wěn)定的、具有一定親水性的結(jié)構(gòu)蛋白,同時富含磷酸化和糖基化位點。同時該基因的CDS序列在皺襞型和非皺襞型的個體中沒有序列的差異,表達量的差異可能是由肌球蛋白作用機制決定的。本研究采用PCR-SSCP技術,設計候選基因LAMB3第5、6、7、8、9和10等外顯子的特異性引物,通過聚丙烯酰胺凝膠電泳檢測,將不同帶型的PCR產(chǎn)物送生物工程公司測序,結(jié)果發(fā)現(xiàn)LAMB3第8外顯子存在1個潛在的突變位點,通過統(tǒng)計分析,這些突變位點在皺襞型和非皺襞型白色獺兔的皮膚組織樣品中具有顯著性差異。本研究首次將轉(zhuǎn)錄組測序技術應用于白獺兔皺襞性狀的探索中,初步確定與皺襞性狀相關的基因,并通過該基因的功能注釋分析,了解該基因在皮膚的生長發(fā)育過程中的作用方式和途徑,為進一步研究該基因在分子和蛋白質(zhì)層面的功能和作用機制提供了依據(jù)。
[Abstract]:Rex Rabbit, a typical skin rabbit, is very popular because of its unique hair. In recent years, it has been found in production that some Rex Rabbits appear wrinkles in the abdomen, limbs and hips. They are called fold Rex rabbits. The individuals of the fold type Rex rabbits are slightly larger than those of non fold fold, and have the same fur quality as normal Rex rabbits, which is for skin area and skin. In this study, RNA-Seq technology was used to analyze the skin samples of the White Rex Rabbits of the fold type (test group) and the non fold type (control group). The results were analyzed and the genes related to the skin growth and development were screened out. The samples were selected. The effective reads combination, through the Trinity software, uses both ends to repeat de novo splicing. Two groups of skin samples are sequenced to obtain 377618 Unigenes, the average length of Unigenes is 680 NT, and the longest sequence is as long as 20293 nt.. The function of Unigene is known. Since the whole genome information of the rabbit is not perfect, the 85.5931% sequence can not be compared to any database. According to the NR annotation and Blast2GO, a total of 42895 Unigene are mapped to different GO term and classified according to the molecular function, cell component and biological process; the result of KOG shows 60593 Unigenes. The 25 types of.KEGG database alignment showed that 13431 Unigene were annotated to 399 KEGG pathway, of which the genes of the PI3K-Akt signaling pathway (ko04151) were most (686,5.108%), and the signaling pathway was significant in the growth and development of the skin. There were 308 significant differences in the samples of the two different phenotypes. The expression gene (P-Value < 0.05 and Iog2|foldchange| 1), of which 118 were up-regulated in the folds, and 190 were down regulated genes, 99 of these genes were annotated to different GO Term, of which only 29 genes were annotated to different metabolic pathways in KEGG. The 209 genes, besides this, were called new genes in these bases. 33 genes were only expressed in non fold individuals and 56 genes were only expressed in plica type individuals. The results were verified by qRT-PCR. 7 candidate genes were selected, specific primers were designed with oligo 7 and delta Ct method was used to calculate the relative apparent amount of folds and non fold samples by delta delta Ct. The results showed that the results of qRT-PCR and the sequence of transcriptional sequence were sequenced. The results are consistent. The results of sequencing are correct. According to this, the genes related to the skin development of rabbits are preliminarily identified: MYL12B, LAMBS, PLOW, EIF3E, CEBPA, FAD2, ENPP6, etc. the candidate gene MYL12B is an important component of the cytoskeleton. As a regulatory light chain of myosin, it can regulate the cell by modulating the phosphorylation and dephosphorylation of the light chain. Many dynamic processes. This study cloned the CDS sequence of the MYL12B gene and compared the CDS sequence and amino acid sequence of 12 species, such as human, chimpanzee, and mice. It was found that the similarity of the gene to mammalian CDS was up to 94%, and the homology of the birds and amphibians was higher than that of the mammal. Bioinformatics analysis showed the MYL12B encoded egg. White is an unstable, hydrophilic structural protein, which is rich in phosphorylation and glycosylation sites, and the CDS sequence of the gene has no sequence differences in folds and non fold individuals, and the difference in expression may be determined by the mechanism of myosin action. This study uses PCR-SSCP technology to design candidates. The specific primers of LAMB3 5,6,7,8,9 and 10 exons were detected by polyacrylamide gel electrophoresis. The PCR products of different bands were sent to bioengineering companies to be sequenced. The results showed that there were 1 potential mutation sites in exons of LAMB3 eighth. By statistical analysis, these mutation sites were in the skin of the fold and non fold White Rex rabbits. There are significant differences in the tissue samples. In this study, the transcriptional sequencing technology was first applied to the exploration of the White Rex folds, and the genes related to the fold traits were preliminarily identified. Through the analysis of the functional annotation of the gene, the function and ways of the gene in the growth and development of the skin were understood to further study the base. It provides a basis for the function and mechanism of action at the molecular and protein levels.
【學位授予單位】：浙江師范大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S829.1

【參考文獻】

相關碩士學位論文 前1條

1 張春園;美系獺兔肉用品質(zhì)特性研究[D];甘肅農(nóng)業(yè)大學;2011年

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本文編號：1922568