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组学解析凡纳滨对虾早期发育相关重要生物学过程的分子机制
隗健凯1,2
学位类型博士
导师相建海
2015-05-21
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生物学
关键词凡纳滨对虾 早期发育 转录组 蛋白质组 幼体变态 食性转变 免疫体系发生 Toll基因家族
摘要对虾作为甲壳动物的典型代表,具有其独特的发育模式。对虾的早期发育包括胚胎发育和幼体发育两个阶段。胚胎发育从受精卵开始,经历卵裂期、囊胚期、原肠期、肢芽幼体期发育至膜内无节幼体期。而幼体发育则通过无节幼体期、溞状幼体期、糠虾幼体期和仔虾期四个阶段的变态过程实现自身的组织器官重建。在此过程中随着个体形态的变化,其生理习性和身体机能也发生着巨大变化,比如运动能力的增强、食性的转变、免疫体系的完善等。开展对虾早期发育的研究对于进一步了解甲壳动物的变态发育模式和对虾在进化和系统发育中的地位具有重要意义,同时对于保障对虾育苗产业的健康发展也具有重要的指导作用。目前关于对虾早期发育的研究多集中在形态学和幼体营养学方面,对其发育机制仍知之甚少。组学技术为从整体水平上了解对虾早期发育过程中基因和蛋白的表达变化,解析相关生物学过程提供了新的思路。本论文以凡纳滨对虾(Litopenaeus vannamei)为研究对象,基于转录组和蛋白质组技术,结合对虾基因组数据,开展对虾早期发育的组学特征分析和生物学过程解析。论文的主要研究结果如下:
1. 对凡纳滨对虾五个早期发育时期进行了高通量转录组测序。共拼接得到66,815条unigenes,其中有37,292条在Nr、Nt、Swiss-Prot、GO、COG、KEGG等数据库中得到了注释。通过对相邻发育时期比较所得差异表达基因的富集分析发现,从胚胎发育至无节幼体,差异基因大量富集在激素诱导的细胞程序性死亡过程;从无节幼体发育至溞状幼体,差异基因大量富集在消化腺分泌和物质代谢通路;从溞状幼体发育至糠虾幼体,差异基因在肌肉组成蛋白的组织和装配过程中显著富集;从糠虾幼体发育至仔虾,差异表达基因在几丁质代谢过程显著富集,这些信息为解析相关生物学过程提供了很好的思路。对凡纳滨对虾20个早期发育阶段进行了高通量表达谱测序,获得了各功能基因在整个发育历程中的表达水平数据;同时通过层级聚类、主成分分析和相关性分析将20个发育样品分为三个时期,其中由膜内无节幼体和无节幼体组成的时期是发育过程中重要的基因表达转换期。
2. 采用2D-DIGE分离MOLDI-TOF-MS/MS鉴定以及iTRAQ标记LC-ESI-MS/MS鉴定两种方法开展了对虾早期发育的差异蛋白质组研究。其中通过2D-DIGE的方法实现了受精卵、囊胚、原肠胚、肢芽幼体、无节幼体5个时期的差异蛋白点分析,并鉴定了其中90个差异蛋白。而基于iTRAQ标记的方法共鉴定到2560个蛋白,实现了受精卵、囊胚、原肠胚、肢芽幼体、无节幼体、溞状幼体、糠虾幼体和仔虾8个时期的差异蛋白分析。
3. 将获得的早期发育功能基因数据集与对虾EST数据以及其他三种节肢动物果蝇、家蚕和水溞的功能基因数据集进行了比较;将发育转录组数据与已有凡纳滨对虾基因组序列进行了比对,初步分析了功能基因的结构特征,为研究早期发育相关功能基因的基因结构、基因排列以及可变剪接事件打下了基础。
4. 依据组学数据从肌肉发育和表皮形成两个角度分析了凡纳滨对虾幼体变态的分子基础。调控肌肉发育的关键信号twist和mef2在肢芽期表达。而附肢肌肉的重要组分Projectin蛋白则在膜内无节幼体、溞状幼体和糠虾幼体三次启动表达。分析了蜕皮相关调控因子的表达,发现幼体执行蜕皮的几丁质酶和组成外骨骼的表皮蛋白均具有高度的多样性,并根据钙化表皮蛋白的表达模式推测表皮的钙化过程发生于仔虾期。
5. 依据组学数据对凡纳滨对虾幼体的食性转变开展了研究。通过生物信息学分析发现有296条unigene注释为16种不同的消化酶。对其中糖酶、肽酶和脂肪酶在发育过程中的基因表达动态模式和多样性进行了分析。胚胎期和无节幼体期消化酶表达量保持在相对较低的水平,溞状幼体期消化酶的表达剧烈上升,而发育至糠虾幼体期蛋白酶表达有所下降,到仔虾期则表达趋于稳定。通过对注释为同种消化酶的不同unigene进行分析发现,随着发育的进行,消化酶基因表达种类的多样性也逐渐显现。
6. 依据组学数据对凡纳滨对虾免疫体系的形成和Toll家族进行了分析。根据免疫相关分子的表达模式将其分为三个不同的启动期:受精卵中表达的多为母源免疫分子,肢芽幼体期自身免疫通路启动,溞状幼体期免疫分子大量出现。鉴定了对虾中GATA家族成员Serpent调控的造血分化途径,根据其表达推测胚胎造血自肢芽幼体期启动。而根据血蓝蛋白的表达推测幼虫造血自溞状幼体期启动。鉴定了对虾早期发育表达的10个Toll基因,并对其结构域特征和基因结构进行了分析。根据各Toll基因在早期发育中的表达模式,结合成体组织分布和成体WSSV感染前后的表达变化,对不同Toll基因的功能进行了讨论,找到了可能参与免疫应答的Toll基因。
其他摘要Penaeid shrimp, as a typical representative of crustacean, has a distinctive pattern for early development. During embryo stage, it gets through the journey from zygote to cleavage embryo, blastula, gastrula, limb bud embryo and larva in membrane. After hatching from membrane, it steps into larva stage which includes nauplius, zoea, mysis and postlarvae. Both its morphological and physiological features change dramatically along with metamorphosis in this period. The researches on early development of penaeid shrimp will provide insight into crustacean evolution and development modularity and also will have considerable significance for shrimp larvae aquaculture. For now, many studies are focused on nutriology of larvae shrimp while little is known about the molecular mechanism of shrimp development. Omics technologies provide a brand new way for acquiring the expression patterns of genes and proteins comprehensively. In this thesis, we use genome-wide transcriptome and proteome analysis for understanding the biological processes during the early development of Pacific white shrimp Litopenaeus vannamei. The main progresses are as follows:
1. The transcriptomes of embryo, nauplius, zoea, mysis and postlarvae were sequenced with Illumina sequencing technology. The reads were assembled and clustered into 66,815 unigenes. 37,292 of which have annotations in Nr, Nt, Swiss-Prot, GO, COG or KEGG databases. The differentially expressed genes (DEGs) between adjacent developmental stages were identified. GO term and KEGG pathway  enrichment analysis showed that DEGs from embryo to nauplius were significantly enriched in hormone induced programmed cell death process which related to histolysis and reconstruction; DEGs from nauplius to zoea were significantly enriched in pancreatic secretion and nutrient metabolic pathway which related to food digestion and absorption; DEGs from zoea to mysis were significantly enriched in myosin Ⅱ filament assembly and organization which related to muscle development; DEGs from mysis to postlarvae were significantly enriched in chitin or polysaccharide metabolic process which related to exoskeleton reconstruction. Twenty early developmental samples of L. vannamei were also sequenced for acquiring a more precise gene expression pattern. By hierarchical clustering analysis and principal components analysis, 20 samples were divided into 3 groups. Group 2 which was composed of larva in membrane and nauplius was inferred as a crucial transition period for development.
2. Two quantitative proteomic approaches were applied to analyze the proteins related to early development of L. vannamei. Using a 2D-DIGE system, we compared the proteome differences at five stages: zygote, blastula, gastrula, limb bud embryo and nauplius. 90 differentially expressed protein spots were identified according to MALDI-TOF-MS/MS. Based on the iTRAQ method, a total of 2560 proteins were detected. The differentially expressed proteins across eight early developmental stages were also analyzed.
3. Alignment and comparative analysis were conducted among the acquired gene set from our study, EST data set from L. vannamei and functional gene sets from other arthropods. We then mapped our Illumina sequencing data to the genome, and analyzed the characteristics of functional genes. A series of exon junctions and gene arrangement information were also obtained.
4. The molecular basis of morphological change during early development of L. vannamei were analyzed from the perspective of muscle development and cuticle reconstruction. For muscle development, the vital regulatory signals twist and mef2 were expressed from limb bud stage, while constitutive proteins projectin were expressed from larva in membrane stage. For cuticle reconstruction, we found that chitinases which are responsible for molting and cuticle proteins which constitute the exoskeleton were highly diversified. We also inferred that the calcification of exoskeleton occurred at postlarvae stage according to the expression pattern of calcified cuticle proteins.
5. Using RNA-Seq data, the types and expression characteristics of the digestive enzyme genes were analyzed during early development of L. vannamei. Among the obtained 66,815 unigenes, 296 were annotated as 16 different digestive enzymes including five types of carbohydrase, seven types of peptidase and four types of lipase. Such a diverse suite of enzymes illustrated the capacity of L. vannamei to exploit varied diets to fit their nutritional requirements. The analysis of their dynamic expression patterns during development also indicated the importance of transcriptional regulation to adapt to the diet transition. Most of the digestive enzymes kept a relatively low expression level at embryo and nauplius stages. Their expression increased sharply at zoea stage. After the expression of peptidase decreased at mysis stage, the expression of digestive enzymes kept stable at postlarvae stage. Their diversity appeared along with development and multiple alignments of amylase  genes inferred the different expression preference between larvae and adults.
6. The formation of shrimp immune system were also discussed according to our RNA-Seq data. We found three initiating periods according to the expression pattern of immune related genes: the first initiating stage was zygote which inherit from maternal immunity; the second was limb bud embryo when immune-related pathway became activated; the third was zoea when massive immune effectors appeared. The regulatory pathway for hematopoietic differentiation was identified. The expression pattern showed that embryo hematopoiesis happened at limb bud embryo stage, while larva hematopoiesis happened at zoea stage. We identified ten Toll genes according to the early developmental transcriptomes of L. vannamei and analyzed their domain and structure. Their tissue distribution and expression pattern when infected with WSSV were analyzed in adult shrimp.
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/23246
专题实验海洋生物学重点实验室
作者单位1.中国科学院海洋研究所实验海洋生物学重点实验室
2.中国科学院大学
第一作者单位实验海洋生物学重点实验室
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隗健凯. 组学解析凡纳滨对虾早期发育相关重要生物学过程的分子机制[D]. 北京. 中国科学院大学,2015.
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