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紫菜精子囊细胞发育机制初探和胁迫下内参基因的筛选
王霞
学位类型硕士
导师王广策
2017-05-31
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
关键词精子囊细胞 细胞分化 酵母表达系统 内参基因 胁迫响应
摘要作为我国主要的大型栽培红藻,紫菜不仅具有较高的经济价值,也因其独特的生物学特征,同时具有较高的理论研究价值。本论文以两种常见紫菜为研究对象开展了如下两方面研究:
1.在有性生殖阶段,条斑紫菜叶状体中淡黄色的精子囊器与深红褐色的果胞呈条纹状相间分布。在显微镜下观察、切割获得了不同发育时期的叶状体材料,包括营养细胞期,精子囊发育表观2细胞期、4细胞期、8细胞期,提取其可溶性蛋白,并采用iTRAQ标记法进行了蛋白差异表达分析,初步探索了精子囊分化发育调控机制及参与的代谢路径。结果显示,明显上调的蛋白主要涉及染色体的复制、组装,包括组蛋白、微管蛋白、分子伴侣蛋白等,一些蛋白可能通过参与形成染色质重塑复合体而对转录过程进行表观调控。此外,26 S蛋白酶体介导的蛋白降解系统相关亚基出现不同程度的上调,在精子囊细胞形成过程中,通过泛素—蛋白酶体降解途径的调控,细胞周期运转加快,因此,蛋白质的循环利用活跃,为细胞由营养生长向生殖生长转化提供了物质基础;精子囊发育不同阶段,磷酸激酶及磷酸酶系统、钙调蛋白等调控因子表达上调,也说明细胞内原有的代谢网络在精子囊分化过程中发生了调整,磷酸基团参与的能量代谢活跃,为精子的形成及释放奠定了能量基础。与此同时,参与光合作用过程的藻胆蛋白、放氧复合体、电子传递体等的蛋白组成亚基均出现不同程度的下调,证明细胞营养积累过程减缓,与光合作用偶联的耗能同化过程也有所下降。
2.筛选了在精子囊细胞发育过程中上调的磷酸酯酶基因,克隆其开放阅读框,并构建了重组表达载体pPICZαA-pp2c,通过将该重组载体转化入毕赤酵母,建立了以甲醇为营养的异源基因表达系统。并对外源蛋白表达条件进行了优化。但最终目的蛋白的纯化未成功。
3.坛紫菜为我国特有的紫菜栽培品种,年产量远超条斑紫菜,紫菜优异的抗逆响应机制一直是研究的热点与重点问题。我们采用绝对定量与软件分析相结合的方法,对不同盐度、不同光照处理下的坛紫菜常见内参基因(18SGAPDHEF3RPSTubAACTTubB)的表达进行了研究,分别筛选出了高盐、高光胁迫条件下表达相对稳定的基因,认定坛紫菜在盐度胁迫条件下,EF318S是进行RT-qPCR相对定量可信赖的内参基因,而在光胁迫条件下,18STubA则是理想内参。本研究结果为后续紫菜抗逆机制研究的开展奠定了基础。
其他摘要As the major cultivating red algae in China, Pyropia not only have high economic value, but also have high theoretical research value due to its unique biological characteristics. This thesis has focused on two species of Pyropia and carried on relevant research as follows:
1. During the sexual reproduction stage, thallus of Pyropia yezoensisis is striped with yellowish spermatangia and rufous carpogonia. Under microscopic observation, thalli of different growth periods were obtained and the total soluble proteins were prepared respectively. And then, proteomic analysis was performed using iTRAQ labeling method. The developmental phase from vegetative cell to the spermatangium tissues was emphasized to explore the potential development mechanism and the relevant metabolic regulation. Compared with the vegetative cells, the up-regulated proteins in spermatangium cells were chromosome assembly and replication related, including molecular chaperone, microtubulin and so on. Some proteins might participate in the epigenetic regulation of transcription process through forming chromatin remodeling complex. Some subunits of 26S proteasome also showed up-regulation during the development of spermatangium cells. The differentiation of the cells may speed up operation of the cell cycle via the ubiquitin-proteasome degradation pathway. Thus, protein recycling activities are active which provides the material basis for the transition from vegetative to reproductive growth. The up-regulation molecules such as kinase, phosphatase, calmodulin also indicated that the original metabolism network of cells has been adjusted during the development process. The energy metabolism that phosphate group participates provided the energy basis for the formation and release of germ cells. Meanwhile, the down-regulated proteins mainly participated in assimilation metabolism, including the subunits forming phycobiliprotein, oxygen evolving complex, electron carriers, which indicated that the energy-consuming assimilation process coupling with the photosynthesis declined at the same time.
2. Based on the screening of the up-regulated phosphatase gene during the spermatangium cell development, we cloned the open reading frame and then constructed the recombinant expression vector pPICZαA-pp2c. By transformation it into pichia pastoris, the methylotrophic heterologous gene expression system was constructed. We also optimized the expression conditions of the target gene, however, the protein was not purified successfully.
3. Pyropia haitanensis is one kind of cultivar exclusive to China, its annual production far exceeds the Pyropia yezoensis. And it is also considered to be the ideal material for study on the resistant mechanism of algae. Through absolute quantification and software analysis, we studied the expression level of seven genes, including 18SGAPDHEF3RPSTubAACT and TubB, under different salinity and light intensity conditions. And then we screened genes with relatively stable expression under these stress conditions respectively. We proposed that EF3 and 18S could be used as internal control genes to perform real-time quantitative pcr when the Py. haitanensis blades were subjected to salinity stress, and that TubA and 18S could be used as the internal control genes when the stress was from strong light. This result laid the foundation for the follow-up study on the resistance mechanism of the Pyropia haitanensis.
学科领域地球科学
语种英语
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136617
专题实验海洋生物学重点实验室
研究生部
作者单位中国科学院海洋研究所
推荐引用方式
GB/T 7714
王霞. 紫菜精子囊细胞发育机制初探和胁迫下内参基因的筛选[D]. 北京. 中国科学院大学,2017.
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