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基于RNA-Seq技术对齿肋赤藓和银叶真藓HSP70基因家族生物信息学分析和表达模式研究
王明强
学位类型硕士
导师张道远
2015
学位授予单位中国科学院大学
学位授予地点北京
学位专业生物工程
关键词齿肋赤藓 银叶真藓 热激蛋白70 Rna-seq 生物信息学
摘要生物信息学是把基因组DNA序列信息作为分析源头,破译隐藏在DNA序列中的遗传语言,特别是非编码区的实质,同时在发现新的基因信息后进行蛋白质空间结构模拟和预测。植物热激蛋白70(Heat Shock Protein 70, HSP 70)是生物机体应对高温及其他胁迫环境时所产生的一类特定的应激蛋白,它在细胞内的大量表达改善细胞的生存能力,提高生物机体对环境胁迫的耐受性。齿肋赤藓(Syntrichia caninervis)和银叶真藓(Bryum argenteum)是极端耐干藓类植物,形态结构相对简单,但是可以生存在干旱、高温等严酷环境中,并且具有特殊的“干而不死”(可损失体内95%以上的水分)及“死而复生”(在30m内快速恢复光合作用)特点,其独特的耐干胁迫机制已日益引起关注。本研究从齿肋赤藓和银叶真藓转录组数据库中鉴定出HSP70基因家族及具有完整ORF的成员序列进行生物信息学比较分析;在此基础上,克隆具有完整开放阅读框(ORF)的HSP70并进行干燥-复水条件下HSP70基因表达模式研究。研究表明: (1)使用TRINITY对转录组原始reads进行组装和注释后,利用Pfam数据库提供HSP70的HMM概率模型在齿肋赤藓转录组数据库中鉴定出37条长度大于200aa的非冗余ScHSP70s序列,在银叶真藓转录组数据库中鉴定出33条长度大于200aa的非冗余BaHSP70s序列; (2)齿肋赤藓转录组数据库中所有的ScHSP70s序列,不含有完整的开放阅读框ORF。进化分析表明,相似度较高的序列属于同一个亚家族,所有序列分为3支,分别属于DnaK亚家族,Hsc70亚家族,Hsp110/SSE亚家族;银叶真藓转录组数据库中有33条BaHSP70s序列,有两条含有完整ORF。多序列比对表明,120位氨基酸至190为氨基酸序列较保守,含有两个β折叠和一个α螺旋定位于HSP70基因的核酸结合区,对HSP70功能发挥具有重要作用。进化分析表明,相似度较高的序列属于同一个亚家族,所有序列分为3支,分别属于DnaK亚家族,Hsc70亚家族,Hsp110/SSE亚家族; (3)对于鉴定出具有完整ORF的2条银叶真藓BaHSP70s序列(GenBank号为KP087877和KP087878),基于生物信息学的方法从氨基酸组成、保守结构域、理化性质、疏水性/亲水性、信号肽、蛋白质结构、模体的识别及同源性分析等方面进行 了预测和分析。结果表明:2条BaHSP70s基因序列ORF全长分别为2396bp和2356bp,分别编码649aa和650aa;序列模体分析表明BaHSP70s和其它报道的植物HSP70均含有5个相同的模体,并且各模体在蛋白质序列上顺序一致;通过对得到的2条BaHSP70s进行氨基酸序列比对及基因树分析,发现2条序列与雪莲相似度最高; (4)通过RT-qPCR实验表明,银叶真藓热激蛋白70基因家族中不同成员在干旱复水的过程中的表达模式存在差异;其中有7个基因呈现下降趋势,并且5个基因响应复水过程比较强烈。研究表明,CL412.Contig2,CL2786.Contig2,Unigene12388,Unigene32646,Unigene32739基因是在后续抗逆基因克隆研究中良好的候选基因。
其他摘要Bioinformatics sets the organism genome sequence information as the beginning of analysis, it aims to break the DNA genetics information which is hidden in the DNA sequence, especially noncoding area of the chromosome. While, after finding the new gene information, it starts to simulate and predict the protein space structure. Heat shock protein 70 is a kind of particular responsive protein which is induced by high temperature and other stress environments. Its substantial expression can alter the survival ability of organisms, which improve the tolerance of organisms to environmental stress. Syntrichia caninervis and Bryum argenteum are typical desert moss species. Although their morphology is rather simple, they can survive in an extremely drought, high or low temperature environment. Its preeminent genetics resource ensures these species to be a hot research field. Based on RNA-seq technology, we got the global transcriptome of those two mosses during the course of dehydration and rehydration. By utilizing HSP70 Hidden Markov Model probability model provided by Pfam database and HMMER software, we acquired HSP70 gene family member sequence from 2 mosses transcriptome database, and then conducted the bioinformatics analysis and gene expression pattern. The main results were as follows: (1)By using TRINITY software, the transcriptome raw reads were assembled and then annotated. We acquired 37 ScHSP70s and 33 BaHSP70s with more than 200bp length in Syntrichia caninervis and Bryum argenteum separately, from the transcriptome database proposed by HSP70 Hidden Markov Model probability model. (2) All the ScHSP70s sequences didn’t contain complete opening reading frame (ORF). Evolution analysis showed sequences with high similarity belonged to the same sub-family and could be separated into three parts, DnaK Subfamily, Hsc70 Subfamily and Hsp 110/SSE Subfamily. So as to B. argenteum, only 2 of BaHSP70s contained complete ORF. Multiple sequence alignment analysis indicated that the loci from 120aa to 190aa were conserved, and two extended strand and only one alpha helix located in the Nucleotide Binding Domain. Evolution analysis showed sequences with high similarity belonged to the same sub-familyseparated all the sequence into three part, DnaK Subfamily, Hsc70 Subfamily, Hsp 110/SSE Subfamily. (3) We conducted bioinformatics analysis of the two BaHSP 70 with complete ORF from the aspect of amino acid component, conserved domain, physicochemical property, hydrophobicity and hydrophily, signal peptide, protein structure, motif recognition, homologous analysis. The result showed that the length of two BaHSP70s are 649aa and 650aa, respectively. The corresponding transcripts in the transcriptome database is 2396bp and 2356bp. Sequence motif analysis indicates that there were 5 identical motif consistent for all selected species. multiple sequence alignment and homologous analysis demonstrates that BaHSP70s are most closely related to Saussurea, reaching to the identity of 91.2%. (4) Quantitative reverse transcription PCR research experiment shows: the expression pattern of each Bryum argenteum Heat Shock Protein 70 gene family member is distinctive under the environment stress of drought and rehydration. There are seven genes represent downtrend, 5 of them expressed highly in the process of rehydration. The result indicates that CL412.Contig2, CL2786.Contig2, Unigene12388, Unigene32646, Unigene32739 genes are excellent candidate genes in the following research of stress resistance gene cloning.
学科领域生物工程
语种中文
文献类型学位论文
条目标识符http://ir.xjlas.org/handle/365004/14671
专题研究系统_荒漠环境研究室
作者单位中科院新疆生态与地理研究所
推荐引用方式
GB/T 7714
王明强. 基于RNA-Seq技术对齿肋赤藓和银叶真藓HSP70基因家族生物信息学分析和表达模式研究[D]. 北京. 中国科学院大学,2015.
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