| 题名 | 真蛸中枢神经的转录组分析及高温应激相关基因的研究; Transcriptome Analysis of Octopus vulgaris Central Nervous System and Related Genes by High Temperature Stress |
| 作者 | 洪婧妮 |
| 答辩日期 | 2015 ; 2012 |
| 导师 | 唐启升 ; 苏永全 |
| 关键词 | 真蛸 中枢神经转录组 热休克相关基因 抗氧化酶 高温应激 Octopus vulgaris CNS transcriptome heat shock related genes antioxidant enzyme genes high temperature stress |
| 英文摘要 | 真蛸是重要的经济养殖对象，属于暖温性世界广布种，目前，对真蛸的研究主要集中在养殖条件的探索和幼体繁殖等方面，很少涉及分子水平，对逆境下的抗逆分子机制尚未见报道。 本文利用真蛸的中枢神经为模板，建立转录组数据库并进行分析，最终得 到了59859条高质量拼接数据。拼接后的数据用Blastx软件与SWISSPROT/NR蛋白数据库进行比对，分别注释到了8970和1442条序列。与同为软体动物门中的加州海兔(Aplysiacalifornica)和静水椎实螺(Lymnaeastagnalis)的中枢神经系统(CentralNervousSystem,CNS)的cDNA文库比较，分别约有15%和34%能够比对上；而与其他模式生物的CNS文库的比较后，与这个结果可能预示着真蛸的中枢神经系统与鱼类和节肢动物的更为接近。 本文还利用常规PCR、RACE技术，成功克隆了热休克蛋白70(Heatshockprotein70，Hsp70)、热休克转录因子(Heatshocktranscriptionfactor，HSF)和热休克转录因子结合蛋白(Heatshockfactorbindingprotein，HSBP)等3个热休克相关基因和谷胱甘肽过氧化物酶(Glutathioneperoxidase，GPX)和过氧化氢酶(Catalase，CAT)2个抗氧化酶基因全长，并得到超氧化物歧化酶(Superoxidedismutase，SOD)部分序列。通过生物信息学软件对6个基因的功能和结构进行预测，同时，利用这些基因的氨基酸系列进行同源比对和系统进化树构建，探讨了真蛸与其他物种的亲缘关系。 得到的真蛸的热休克蛋白70(OvHsp70)cDNA全长为2222bp，开放阅读框(ORF)为1908bp，共编码635个氨基酸，推测分子量69.8kU，含有HSP70家族3个典型基序和C端特征基序EEMD。功能结构预测出OvHsp70包含ATP结构域、底物肽结合区和C-端结构域。 真蛸的热休克转录因子1(OvHSF1)cDNA全长为2264bp，包含1377bp的ORF区域，共编码458个氨基酸，推测分子量52.1kU，含有HSF家族高度保守的DNA结合域(DNAbindingdomain，DBD)和三聚区域(Trimerizationdomain，TD)，预测到三聚区域中含有2个疏水重复序列(Hydrophobicheptadrepeatarray，HR)—HR-A/B;同时，在C-端发现存在另有一保守的HR-C区域。 获得全长为841bp真蛸热休克转录因子结合蛋白(OvHSBP)cDNA，其中，ORF为273bp，共编码90个氨基酸，推测分子量9.9kU，功能预测发现OvHSBP含有高保守的卷曲螺旋区域，占全序列的60%。 真蛸谷胱甘肽过氧化物酶(OvGPX)的cDNA全长为925bp，得到的ORF为573bp，共编码190个氨基酸，推测分子量21.5kU，推断出的氨基酸序列含有GPX家族2个典型酶活性位点基序和特有的硒半胱氨酸(Selenocysteine,Sec,U)，同时，在OvGPXcDNA序列的3’UTR处预测到1个硒半胱氨酸插入序列(SelenocysteineInsertionSequence,SECIS)。 得到真蛸过氧化氢酶(OvCAT)基因cDNA全长为2109bp，包含1539bp的ORF，共编码512个氨基酸，推测分子量58.3kU，预测到OvCAT氨基酸序列中含有1个酶活性位点和1个酶免疫响应区。 同源比对显示了5个基因都具有高保守性，利用它们的氨基酸序列所构建得NJ系统进化树，体现的物种亲缘关系均与传统分类相符，表明5个基因都能较好反应物种系统进化关系。 利用realtime-PCR技术，分析了真蛸Hsp70、HSF、HSBP和GPX、CAT、SOD等6个基因在正常状态下消化腺、栉鳃、唾液腺、肌肉、中枢神经、心脏、性腺、鳃心和血等9个组织中的mRNA表达量。发现6个基因在各组织都有表达，但表达量存在差异，消化腺、中枢神经和性腺中表达较高，栉鳃中表达量则较少。这6个基因在消化腺中的高表达量可能预示它们在真蛸营养代谢和应激防御中担负重要作用。探索真蛸的3个热休克基因和3个抗氧化基因在不同温度下mRNA表达变化，并对6个基因在热激24h内，mRNA的时空变化规律进行研究，实验结果表明，在热应激前期，真蛸热休克蛋白70的mRNA的表达变化比抗氧化基因明显，推测热休克相关基因对应激响应早于抗氧化基因，热应激基因形成的机体保护系统，可以较快的提高生物体抵御外界刺激的能力。而到热激后期，持续的应激时机体压力加剧，迫使6个基因共同作用以保护机体。将热激24h后的真蛸样品放回常温恢复24h，发现OvHsp70基因的mRNA保持高表达量，可能表明短期应激使真蛸获得较高的热耐受性。 论文首次对真蛸进行了转录组测序，首次克隆并分析了OvHsp70、OvHSF、OvHSBP、OvGPX和OVCAT基因，同时，探索研究vHsp70、OvHSF、OvHSBP、OvGPX、OVCAT和OvSOD等6个基因在不同温度及高温胁迫下不同时间点mRNA表达量的变化规律，初步探讨了热休克相关基因和抗氧化酶基因在真蛸面在热胁迫时的保护机理，研究结果不仅填补了真蛸乃至头足类的空白，也为进一步开展真蛸相关功能基因研究、种质资源保护和抗逆品种的选育提供了可靠的基础理论资料。; Octopus vulgaris have become an important marine species for commercial fisheries in China. While recent studies have focused on the cultured conditions, spawning and hysiological ecology, less attention has been paid to the molecular mechanism. In this paper, we used the template of O.vulgaris central nervous system for transcriptome sequencing. We have generated 59859 high quality contigs from the CNS transcriptome, and there were 8970+1442 contigs were read from blastx and SWISSPROT/NR protein databases. Comparison with three mollusk species A.californica, L.stagnalis and O.vulgaris CNS transcriptome hit 15% and 34%. Then, comparison with CNS sequences database of other model organisms, O.vulgaris CNS transcriptome hit 21%-46% , which may indicated that the CNS of O.vulgaris are closely related with arthropod and fish. We used rapid amplification of cDNA ends (RACE) to obtain full-length sequences of Hsp70、HSF、HSBP、GPX 、CAT and SOD from O.vulgaris. The nucleotide sequences and deduced amino acid sequences of six genes were analyzed and characterized by bioinformatics methods. A 2,222 bp cDNA of full-length of OvHSP70, containing a 1908 bp open reading frame (ORF) that encodes 635 amino acids. The predicted molecular weight was 69.8 kU. OvHSP70 had three conserved amino acid motifs, contained three predicted functional domains, including an ATPase domains, peptide-binding domain and C-teminal domain. The full-length of OvHSF cDNA contained a 1,908 bp ORF, The ORF encoded 458 amino acids, predicting molecular weight of 52.1 kU. The OvHSF amino acids sequence contained a DNA binding domain and a trimerization domain. The obtained full-length of OvHSBP cDNA contained a 273 bp ORF, which encoded 90 amino acids, with molecular weight of 9.9 kU. A predicted high conservative coiled coil region was existed in OvHSBP amino acids sequence. The full-length cDNA of OvGPX is 925 bp, the 573 bp ORF of OvGPX encodes a 190 amino acid protein. It calculated that theory molecular mass is 21.5 kU. OvGPX had a SECIS functional element, selenocysteine (Sec, U) and two GPX family enzymatic activity regions. A 2109 bp cDNA of full-length of OvCAT was obtained, the ORF of OvCAT was 1539 bp, which encodes a 512 amino acid protein. The theory molecular mass of OvCAT amino acids was 58.3 kU. The OvCAT amino acids sequence had active site and immune-responsive domain. OvHsp70, OvHSF, OvHSBP, OvGPX and OvCAT were highly conservative, the phylogenetic analysis of the five genes showed that they conformed to traditional taxonomy. Real-time quantitative PCR was performed to quantify the expression of OvHsp70, OvHSF, OvHSBP, OvGPX, OvCAT and OvSOD, they show similar expression distributionin the organization, they all showed the highest expression level in the digestive gland, gonads and CNS. The highest expresstion of six genes in digestive gland may imply that the six genes played an important role in nutrition metabolism and stress defense. We also investigated the expression patterns of these six genes at different temperatures and time points under high temperature stress in digestive gland and ctenidium. The results showed that the heat shock related genes were more sensitive to high temperature stress than the antioxidant enzyme genes. And the six genes played an important protective effect together when O.vulgaris subjected to persistent temperature stress. OvHsp70 kept high expression when returned to normal temperature, it is suggested that short stress may make the organism obtain a certain tolerance. For the fist time, we analyzed the O.vulgaris CNS transcriptome, and cloned the full-length cDNA of OvHsp70, OvHSF, OvHSBP, OvGPX, OvCAT and OvSOD, then the six genes were analyzed and characterized by bioinformatics analysis. We also explored the expression of six genes in digestive gland and ctenidium against short temperature stress.Through this study, it sets a theoretical foundation for a better understanding of O.vulgaris, and may be beneficial to further in-depth studies on the molecular protective mechanisms, resource protection and breeds selectively of O.vulgaris.; 学位：理学博士; 院系专业：海洋与环境学院_海洋生物技术; 学号：31120090153245 |
| 语种 | zh_CN |
| 出处 | http://210.34.4.13:8080/lunwen/detail.asp?serial=48551 |
| 内容类型 | 学位论文 |
| 源URL | [http://dspace.xmu.edu.cn/handle/2288/81841]  |
| 专题 | 海洋地球－学位论文 |
| 推荐引用方式 GB/T 7714 | 洪婧妮. 真蛸中枢神经的转录组分析及高温应激相关基因的研究, Transcriptome Analysis of Octopus vulgaris Central Nervous System and Related Genes by High Temperature Stress[D]. 2015, 2012. |
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