中国明对虾三倍体性腺发育机理的初步研究

IOCAS-IR > 海洋环流与波动重点实验室

	中国明对虾三倍体性腺发育机理的初步研究
其他题名	Primary Studies on the Regulation Mechanism of Gonad Development in Triploid Shrimp，Fenneropenaeus chinensis
	谢玉素
学位类型	博士
	2009-05-25
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	中国明对虾三倍体联会复合体卵巢抑制性消减杂交技术 Pcna Cas/cse1 Ssrf
摘要	三倍体培育是水产动物遗传改良的重要途径之一，它在提高养殖产量、改良品质方面发挥着重要作用。对虾三倍体在性腺发育和性别比率方面与二倍体之间存在明显差异。本论文对三倍体性腺发育的分子机理进行了初步探讨，为阐明甲壳动物的性腺发育和性别控制机理提供重要依据。本论文取得的主要进展如下：利用联会复合体的分析技术，比较分析了雄性二倍体和三倍体中精母细胞的减数分裂行为。二倍体对虾具有典型的真核生物联会复合体的形态，联会复合体在二价体联会处沿同源染色体长轴分布；未见明显的异型性别染色体；三倍体对虾精母细胞的联会行为复杂，可见二价体、单价体、非同源联会的三价体、同源转换和同源区完全配对的双联会复合体等不同形态；三倍体对虾在晚粗线期普遍表现为三价体同源区完全配对的双联会复合体形态，这种联会行为可能是导致其产生 3n 倍性精子的关键原因。利用抑制性消减杂交技术，建立了对虾二倍体和三倍体卵巢间的2个消减文库；在正向消减文库（以三倍体卵巢作为实验组，二倍体卵巢作为驱动组）中，鉴定到54个基因；在反向消减文库（以二倍体卵巢为实验组，三倍体卵巢为驱动组）中，鉴定到16个基因；选取11个差异表达的基因，利用半定量RT-PCR的方法对其在二倍体和三倍体卵巢间的表达进行了检测，均能很好地与消减结果相吻合；这些差异基因编码多种功能的蛋白，分析表明染色体的三倍化使三倍体卵巢中的基因调控网络受到了影响；为深入揭示维持卵巢正常发育的关键分子调控事件奠定了基础。为进一步分析特定基因对对虾性腺发育的调控机制，选取了在对虾三倍体和二倍体卵巢中差异表达显著的 3 个不同基因，PCNA （proliferating cell nuclear antigen）、CAS/CSE1 （cellular apoptosis susceptibility protein/chromosome segregation 1）和 SSRF （spermatogonial stem-cell renewal factor），进行了相关研究分析，为深入探讨特定基因对对虾性腺发育的调控机制以及三倍体中的基因表达调控机制奠定了基础；中国明对虾PCNA基因在增殖旺盛的性腺组织及造血组织中表达量最高；在二倍体卵巢中的表达水平显著高于三倍体卵巢；在不同病原刺激下的造血组织中的表达模式不同，与对虾对抗不同病原刺激的免疫反应相关；PCNA在序列上的高度保守性，提示了其功能的保守性；利用PCNA基因可以指示细胞的增殖活性的特点，将辅助我们在对虾发育生物学和二倍体、三倍体对虾比较发育生物学的研究；中国明对虾CAS/CSE1基因在二倍体卵巢中高表达；在卵母细胞中，其mRNA大量分布于细胞质及细胞核周围；是早期胚胎发育的母源性因子；在其氨基酸序列的N端具有importin-β 家族蛋白的保守结构，提示其可能通过参与核质运输在发育过程中发挥重要作用；利用原核表达系统成功地对其进行了体外重组表达，为进一步在蛋白水平上的功能研究提供了条件；中国明对虾SSRF（暂时命名）基因在三倍体卵巢中高表达；在正常二倍体对虾的神经组织中表达量最高，提示该基因在神经发育中可能发挥重要作用；在氨基酸序列上与胸苷磷酸化酶（TP）具有最高的相似性；利用原核表达系统成功地对其进行了体外重组表达，为进一步在蛋白水平上的功能研究奠定了基础；对对虾SSRF活性蛋白的酶活及功能验证亟待进行。
其他摘要	Triploidy manipulation is one important approach in genetic improvement for aquaculture animals, which plays important roles in quantity and quality improvement. Gonad development and sex ratio of triploid shrimp show significant differences with that of diploid counterparts. In this study, primary studies were carried out on the molecular mechanism of gonad development in triploid shrimp, and the results have established a firm basis for further dissection on the mechanism of gonad development and sex determination in crustacean. Some important progresses are as follows: Comparative studies on meiosis behavior of spermatocytes between diploid and triploid males were carried out by synaptonemal complex (SC) analysis. Spermatocytes of diploid shrimp showed typical morphological characteristics of eukaryote SC, with complete synapsis of bivalents. No recognizable bivalent associated with sex chromosomes was observed in spermatocytes of diploid shrimp. However, differences in morphology of SC, including unsynapsed univalents, bivalents, totally paired trivalents with non-homologous synapsis, partner switches and triple synapsis were identified at early pachytene stage of triploid spermatocytes. Triple synapsis was especially common at late pachytene stage in spermatocytes of triploid shrimp. The abnormal synapsis behavior in triploid shrimp may be responsible for the production of triploidy sperms. To identify key molecular changes involved in ovary development of triploid shrimp, two subtracted cDNA libraries between triploid and diploid shrimp ovaries were constructed using suppression subtractive hybridization (SSH) technique. For the forward library (triploid ovary as tester), 54 genes were identified. For the reverse library (diploid ovary as tester), 16 genes were identified. Using semi-quantitative RT-PCR, the differential expression profiles of eleven selected genes were confirmed in the ovaries of triploid and diploid. These genes fell in gene categories with a wide range of functions. The results indicated that triploidy affects the dynamic gene regulatory network in triploid ovary. This study established a firm basis for future investigation on characterization of crucial molecular events for normal ovarian development in shrimp. To further dissect exact gene functions for gonad development of shrimp, three differentially expressed genes between diploid and triploid ovary, PCNA (proliferating cell nuclear antigen), CAS/CSE1 (cellular apoptosis susceptibility protein/chromosome segregation 1) and SSRF (spermatogonial stem-cell renewal factor) were characterized on certain aspects. These results have set firm basis for further functional analysis of these genes in ovary development and gene regulatory mechanism for triploids. FcPCNA expressed especially high in proliferating tissues of shrimp such as gonad and haematopoietic tissue (HPT). It showed significant expression level in diploid ovary compared to the triploid counterparts. Different expression profiles of FcPCNA in HPT were obtained after challenged by two pathogens, indicating different defense mechanisms of shrimp against these two pathogens. FcPCNA showed high identity and conservation with PCNAs reported in other species, which suggests that it may have conserved functions in shrimp. It has been reported that PCNA can be served as a marker for cell proliferation activity, which can be very helpful for studies in shrimp development, and also it is helpful for comparative development studies between diploid and triploid. CAS/CSE1 expressed especially high in diploid ovary, and its mRNA can be detected in cytoplasm and around nucleus in the oocytes. It can be served as the maternal factor which is necessary for early embryo development. The conserved domain for importin-β family proteins can be found at N terminal of its amino acid sequence, which suggests that the role in nucleocytoplasmic transport is important for this gene in development regulation. In addition, CAS/CSE1 was successfully expressed in E. coli, which will be utilized for further functional studies. SSRF expressed especially high in triploid ovary. For the diploid normal tissues, it showed the highest expression level in nerve, indicating the potential role of this gene for neuron development. The amino acid sequence of this gene shows high identity with TP (thymine phosphorylase) of other organisms. In addition, SSRF was successfully expressed in E. coli, which will be utilized for further functional studies; researches on the enzyme activity and function dissection are required to be carried out.
页数	152
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1479
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	谢玉素. 中国明对虾三倍体性腺发育机理的初步研究[D]. 海洋研究所. 中国科学院海洋研究所,2009.

条目包含的文件
文件名称/大小	文献类型	版本类型	开放类型	使用许可
10001_20071800681201（5936KB）			限制开放	--	浏览