狍茸頂端組織轉錄組分析及功能基因克隆檢測
發(fā)布時間：2018-09-19 13:16
【摘要】：狍(Capreolus pygargus)是我國重要的經濟動物,具有廣泛的市場應用前景。本研究通過Illumina/Solexa測序平臺進行狍茸頂端組織轉錄組測序,使用自體組裝軟件Trinity對測序短序列reads進行從頭組裝,建立狍茸頂端組織轉錄組數據庫。并與蛋白數據庫Nr、Swiss-Prot、KEGG和COG進行序列比對、功能注釋及代謝通路分析；與已有的梅花鹿鹿茸頂端組織轉錄組數據庫進行簡單比較分析；在此基礎上,我們選出癌癥通路中ANXA-2和PTN功能基因進行克隆測序,獲得了包含這兩個基因全部編碼區(qū)的cDNA序列,并與轉錄組測序數據庫中拼接組裝得到的這兩個Unigene的cDNA序列進行比對,以佐證轉錄組測序數據庫的準確性。從轉錄組水平開展對狍茸的研究,更有利于揭示狍茸的生長機制,為提高狍茸產量和質量提供理論依據。1、狍茸頂端組織轉錄組測序共得到的5千多萬個高質量短序列reads,采用組裝拼接后共獲得兩端不能再延長的Unigenes 36865個,平均長度932nt,N50值為1579nt,接近98%的序列測序質量值均在Q20(堿基測序錯誤率為1%)2、在數據庫比對及功能基因的注釋過程中,發(fā)現與Nr數據庫比對上的Unigenes共22983條,可以直接確定其CDS區(qū)及序列方向。其余序列用ESTscan軟件進行編碼區(qū)預測,結果表明有510條可能為新的蛋白編碼序列；在COG功能分類,共有8668條Unigenes被歸類到25個功能類別中；通過GO功能分類,共有18273條Unigenes被歸類到61個功能類別中：通過KEGG代謝通路分析,共有1 7284條基因注釋到258個信號通路中。3、對狍茸轉錄組數據進行整體篩選分析,按照FPKM值從高到低的順序排序發(fā)現,表達量較高的一類蛋白是膠原蛋白,在膠原蛋白中表達量最高的是COL1A1、 COL1A2, COL16A1、COL9A1、COL27A1。并且發(fā)現至少141種與生長相關的基因及受體,生長相關高表達基因中高表達的有TGFB3、IGF、TGFBP、IGF4、CTGFP、 PDGFR等;挑選出至少259種轉錄因子,這一類與轉求相關高表達的基因中,表達量最高的為ATF4、TFAP1、GTFIIF、SNAI2、JunB、TFp65等；挑選出至少384種細胞外基質,大部分的細胞外基質主要集中在膠原類成分。4、克隆包含ANXA-2與PTN基因全部編碼區(qū)的cDNA序列,分別編碼339和168個氨基酸。與轉錄組數據庫所測得序列進行比對,相似性分別為99.7%和99.0%,進一步佐證了本轉錄組測序結果的準確性。
[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.
【學位授予單位】：東北林業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q953
本文編號：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.
【學位授予單位】：東北林業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q953
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