紫菜精子囊细胞发育机制初探和胁迫下内参基因的筛选

发布时间：2018-05-03 06:12

本文选题：精子囊细胞 + 细胞分化　；参考：《中国科学院大学(中国科学院海洋研究所)》2017年硕士论文

【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(18S,GAPDH,EF3,RPS,TubA,ACT,TubB)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,EF3与18S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,18S与TubA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。
[Abstract]:As the main large cultivated red algae in China, porphyra has not only higher economic value, but also higher theoretical research value because of its unique biological characteristics. In this paper, two kinds of common porphyra were studied as follows: 1. During sexual reproduction, the pale yellow spermatozoa in the striatum of porphyra yezoensis was striped with the crimson brown fruit cell. Observed under microscope, the leaf striatum materials of different developmental stages were obtained, including vegetative cell stage, sperm sac development apparent 2 cell phase, 4 cell phase and 8 cell phase, and their soluble proteins were extracted. The differential expression of protein was analyzed by iTRAQ labeling method, and the regulation mechanism of spermatozoa differentiation and development and the metabolic pathway involved in the differentiation and development of spermatozoa were preliminarily explored. The results showed that the up-regulated proteins were mainly involved in the replication and assembly of chromosomes, including histone, tubulin, molecular chaperone, etc. Some proteins may be involved in the formation of chromatin remodeling complexes to regulate the transcriptional process. In addition, the related subunits of proteasome mediated protein degradation system were up-regulated in varying degrees. During the formation of sperm sac cells, the cell cycle was accelerated by the regulation of ubiquitin proteasome degradation pathway. The active recycling of proteins provides a material basis for cell transformation from vegetative growth to reproductive growth, and up-regulates the expression of phosphokinase, phosphatase system, calmodulin and other regulatory factors at different stages of sperm sac development. It also indicated that the original metabolic network in the cell was regulated during the differentiation of spermatozoa, and the energy metabolism in which the phosphate group participated was active, which laid the energy foundation for the formation and release of spermatozoa. At the same time, the protein subunits of phycobiliary protein, oxygen releasing complex, electron transporter and so on, which were involved in photosynthesis, were down-regulated in varying degrees, which proved that the process of cell nutrition accumulation was slowed down. The energy consumption assimilation process of photosynthesis coupling was also decreased. The phosphatase gene up-regulated during the development of spermatozoa cells was screened, its open reading frame was cloned, and the recombinant expression vector pPICZ 伪 A-pp2c was constructed, and the recombinant vector was transformed into Pichia pastoris. A gene expression system based on methanol was established. The expression conditions of exogenous protein were optimized. However, the purification of the final target protein was not successful. The annual yield of porphyra chinensis is much higher than that of porphyra yezoensis, and the excellent stress response mechanism of porphyra yezoensis has been a hot and important issue. Using the method of absolute quantitative analysis and software analysis, we studied the expression of the common internal reference gene of porphyra sinensis under different salinity and different light exposure, the expression of GAPDHN EF3 RPS tubula ACTACTACTACTACTACTB) was studied, and the high salt was screened out respectively. The stable genes were expressed under high light stress. It was concluded that Ef3 and 18s were relative quantitative and reliable internal reference genes for RT-qPCR under salinity stress, while 18s and TubA were ideal reference materials for RT-qPCR under light stress. The results of this study laid a foundation for the further study on the stress resistance mechanism of porphyra chinensis.
【学位授予单位】：中国科学院大学(中国科学院海洋研究所)
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;Q945.4

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