利用转录组测序筛选控制白色獭兔皱襞性状相关基因

发布时间：2018-05-22 15:05

本文选题：獭兔 + 转录组　；参考：《浙江师范大学》2015年硕士论文

【摘要】：獭兔,典型的皮用兔,因被毛独特而倍受人们喜爱。近年来,在生产中发现,某些獭兔会在腹部和四肢以及臀部出现皱褶,称其为皱襞型獭兔。皱襞型獭兔的个体比非皱襞型稍大,同时具有正常獭兔相同的皮毛品质,这对于以皮张面积和皮毛品质为重要经济效益因素的獭兔来说,意义重大,提供了新的獭兔育种方向。本研究采用RNA-Seq技术,对皱襞型(试验组)和非皱襞型(对照组)白色獭兔的皮肤样本进行测序分析。分析测序结果,筛选出与皮肤生长发育相关的基因。将样本的有效reads合并,通过Trinity软件,使用两端重复进行de novo拼接。两组皮肤样本测序总共获得377,618条Unigenes, Unigenes的平均长度是680 nt,最长的序列长达20,293 nt。将Unigenes与NR、Swiss-Prot、KEGG和KOG等数据库比对(E-Value10"5),根据比对的注释,分析已知Unigene的功能。由于家兔的全基因组信息还不完善,85.5931%序列无法比对到任何数据库上。根据NR注释及Blast2GO,总共有42,895条Unigene被映射到不同的GO term上,并按照分子功能、细胞组分及生物过程将其分类；KOG的结果显示60,593条Unigenes被分为25类。KEGG数据库比对显示,13,431条Unigene分别被注释到399个KEGG pathway中,其中注释到PI3K-Akt信号通路(ko04151)的基因最多(686,5.108%),该信号通路在皮肤的生长发育过程中具有重要的意义。两种不同表型的样本中,共有308个显著的差异表达基因(P-Value≤0.05且Iog2|foldchange| 1),其中118个在皱襞型样品中为上调基因,190个为下调基因。这些基因中有99个被注释到不同的GO Term上,其中只有29个基因被注释到KEGG不同的代谢途径中。除此之外的209个基因被称为新基因,在这些基因中,33个只在非皱襞型个体中表达,56个基因只在皱襞型个体中表达。通过qRT-PCR验证测序结果。选择7个候选基因,使用oligo 7.0设计特异性引物,采用△△Ct法计算皱襞型和非皱襞型样品的相对表达量。结果显示,qRT-PCR的结果和转录组测序的结果相符。说明测序结果是正确的,据此初步确定与家兔皮肤发育相关的基因：MYL12B、LAMBS、PLOW、EIF3E、CEBPA、FAD2, ENPP6等。候选基因MYL12B是细胞骨架的重要组分,作为肌球蛋白的调节性轻链,可通过调节轻链的磷酸化与去磷酸化,调节细胞的许多动力学过程。本研究克隆了MYL12B基因的CDS序列,并与人、黑猩猩、小鼠等12个物种的CDS序列和氨基酸序列进行比较。发现,除马外,该基因与哺乳动物CDS相似性高达94%,与鸟类和两栖类的同源性也较高。生物信息学分析显示MYL12B编码的蛋白是一个不稳定的、具有一定亲水性的结构蛋白,同时富含磷酸化和糖基化位点。同时该基因的CDS序列在皱襞型和非皱襞型的个体中没有序列的差异,表达量的差异可能是由肌球蛋白作用机制决定的。本研究采用PCR-SSCP技术,设计候选基因LAMB3第5、6、7、8、9和10等外显子的特异性引物,通过聚丙烯酰胺凝胶电泳检测,将不同带型的PCR产物送生物工程公司测序,结果发现LAMB3第8外显子存在1个潜在的突变位点,通过统计分析,这些突变位点在皱襞型和非皱襞型白色獭兔的皮肤组织样品中具有显著性差异。本研究首次将转录组测序技术应用于白獭兔皱襞性状的探索中,初步确定与皱襞性状相关的基因,并通过该基因的功能注释分析,了解该基因在皮肤的生长发育过程中的作用方式和途径,为进一步研究该基因在分子和蛋白质层面的功能和作用机制提供了依据。
[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.
【学位授予单位】：浙江师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S829.1

【参考文献】