狍茸顶端组织转录组分析及功能基因克隆检测
发布时间：2018-09-19 13:16
【摘要】：狍(Capreolus pygargus)是我国重要的经济动物,具有广泛的市场应用前景。本研究通过Illumina/Solexa测序平台进行狍茸顶端组织转录组测序,使用自体组装软件Trinity对测序短序列reads进行从头组装,建立狍茸顶端组织转录组数据库。并与蛋白数据库Nr、Swiss-Prot、KEGG和COG进行序列比对、功能注释及代谢通路分析；与已有的梅花鹿鹿茸顶端组织转录组数据库进行简单比较分析；在此基础上,我们选出癌症通路中ANXA-2和PTN功能基因进行克隆测序,获得了包含这两个基因全部编码区的cDNA序列,并与转录组测序数据库中拼接组装得到的这两个Unigene的cDNA序列进行比对,以佐证转录组测序数据库的准确性。从转录组水平开展对狍茸的研究,更有利于揭示狍茸的生长机制,为提高狍茸产量和质量提供理论依据。1、狍茸顶端组织转录组测序共得到的5千多万个高质量短序列reads,采用组装拼接后共获得两端不能再延长的Unigenes 36865个,平均长度932nt,N50值为1579nt,接近98%的序列测序质量值均在Q20(碱基测序错误率为1%)2、在数据库比对及功能基因的注释过程中,发现与Nr数据库比对上的Unigenes共22983条,可以直接确定其CDS区及序列方向。其余序列用ESTscan软件进行编码区预测,结果表明有510条可能为新的蛋白编码序列；在COG功能分类,共有8668条Unigenes被归类到25个功能类别中；通过GO功能分类,共有18273条Unigenes被归类到61个功能类别中：通过KEGG代谢通路分析,共有1 7284条基因注释到258个信号通路中。3、对狍茸转录组数据进行整体筛选分析,按照FPKM值从高到低的顺序排序发现,表达量较高的一类蛋白是胶原蛋白,在胶原蛋白中表达量最高的是COL1A1、 COL1A2, COL16A1、COL9A1、COL27A1。并且发现至少141种与生长相关的基因及受体,生长相关高表达基因中高表达的有TGFB3、IGF、TGFBP、IGF4、CTGFP、 PDGFR等;挑选出至少259种转录因子,这一类与转求相关高表达的基因中,表达量最高的为ATF4、TFAP1、GTFIIF、SNAI2、JunB、TFp65等；挑选出至少384种细胞外基质,大部分的细胞外基质主要集中在胶原类成分。4、克隆包含ANXA-2与PTN基因全部编码区的cDNA序列,分别编码339和168个氨基酸。与转录组数据库所测得序列进行比对,相似性分别为99.7%和99.0%,进一步佐证了本转录组测序结果的准确性。
[Abstract]:Roe deer (Capreolus pygargus) is an important economic animal in China and has a wide market prospect. In this study, the apical tissue transcriptome of roe deer was sequenced by Illumina/Solexa sequencing platform, and the short sequence reads was assembled from the ab initio by using the autologous assembly software Trinity, and the database of the apical tissue transcriptome of roe deer was established. Sequence alignment with protein database Nr,Swiss-Prot,KEGG and COG, functional annotation and metabolic pathway analysis, and a simple comparison with the existing sika deer antler apical tissue transcriptome database were carried out. We selected the ANXA-2 and PTN functional genes in the cancer pathway for cloning and sequencing, obtained the cDNA sequences containing all the coding regions of the two genes, and compared them with the cDNA sequences of the two Unigene assembled in the transcriptome sequencing database. To support the accuracy of transcriptome sequencing database. The study of roe deer from the transcriptional level is more helpful to reveal the growth mechanism of roe deer. In order to provide theoretical basis for improving the yield and quality of roe deer antler, more than 50 million high quality short sequence reads, obtained by sequencing the top tissue of roe deer were assembled and spliced to obtain a total of 36865 Unigenes which could not be extended at both ends. The average length of N50 was 1579nt, and the sequence quality value of nearly 98% was Q20 (the error rate of base sequencing was 1%) 2. In the course of database alignment and functional gene annotation, 22983 Unigenes were found to be compared with Nr database. The CDS region and sequence direction can be determined directly. The remaining sequences were predicted by ESTscan software, and the results showed that 510 sequences might be new protein coding sequences. In COG functional classification, 8668 Unigenes were classified into 25 functional categories. A total of 18273 Unigenes were classified into 61 functional categories: 1 7284 genes were annotated into 258 signaling pathways by KEGG metabolic pathway analysis. According to the order of FPKM value from high to low, it was found that the most expressed protein was collagen, and the highest one was COL1A1, COL1A2, COL16A1,COL9A1,COL27A1.. At least 141 growth-related genes and receptors were found to be highly expressed in growth-related high expression genes, and at least 259 transcription factors were selected. Among these genes, ATF4,TFAP1,GTFIIF,SNAI2,JunB,TFp65 was the most expressed. At least 384 extracellular matrices were selected, most of which were mainly composed of collagen. 4. CDNA sequences containing all coding regions of ANXA-2 and PTN genes were cloned and encoded 339 and 168 amino acids, respectively. Compared with the sequence measured in transcriptome database, the similarity was 99.7% and 99.0, respectively, which further confirmed the accuracy of the sequencing results of the transcriptome.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q953
本文编号：2250233
[Abstract]:Roe deer (Capreolus pygargus) is an important economic animal in China and has a wide market prospect. In this study, the apical tissue transcriptome of roe deer was sequenced by Illumina/Solexa sequencing platform, and the short sequence reads was assembled from the ab initio by using the autologous assembly software Trinity, and the database of the apical tissue transcriptome of roe deer was established. Sequence alignment with protein database Nr,Swiss-Prot,KEGG and COG, functional annotation and metabolic pathway analysis, and a simple comparison with the existing sika deer antler apical tissue transcriptome database were carried out. We selected the ANXA-2 and PTN functional genes in the cancer pathway for cloning and sequencing, obtained the cDNA sequences containing all the coding regions of the two genes, and compared them with the cDNA sequences of the two Unigene assembled in the transcriptome sequencing database. To support the accuracy of transcriptome sequencing database. The study of roe deer from the transcriptional level is more helpful to reveal the growth mechanism of roe deer. In order to provide theoretical basis for improving the yield and quality of roe deer antler, more than 50 million high quality short sequence reads, obtained by sequencing the top tissue of roe deer were assembled and spliced to obtain a total of 36865 Unigenes which could not be extended at both ends. The average length of N50 was 1579nt, and the sequence quality value of nearly 98% was Q20 (the error rate of base sequencing was 1%) 2. In the course of database alignment and functional gene annotation, 22983 Unigenes were found to be compared with Nr database. The CDS region and sequence direction can be determined directly. The remaining sequences were predicted by ESTscan software, and the results showed that 510 sequences might be new protein coding sequences. In COG functional classification, 8668 Unigenes were classified into 25 functional categories. A total of 18273 Unigenes were classified into 61 functional categories: 1 7284 genes were annotated into 258 signaling pathways by KEGG metabolic pathway analysis. According to the order of FPKM value from high to low, it was found that the most expressed protein was collagen, and the highest one was COL1A1, COL1A2, COL16A1,COL9A1,COL27A1.. At least 141 growth-related genes and receptors were found to be highly expressed in growth-related high expression genes, and at least 259 transcription factors were selected. Among these genes, ATF4,TFAP1,GTFIIF,SNAI2,JunB,TFp65 was the most expressed. At least 384 extracellular matrices were selected, most of which were mainly composed of collagen. 4. CDNA sequences containing all coding regions of ANXA-2 and PTN genes were cloned and encoded 339 and 168 amino acids, respectively. Compared with the sequence measured in transcriptome database, the similarity was 99.7% and 99.0, respectively, which further confirmed the accuracy of the sequencing results of the transcriptome.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q953
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