本文選題：精子囊細(xì)胞 + 細(xì)胞分化　； 參考：《中國(guó)科學(xué)院大學(xué)(中國(guó)科學(xué)院海洋研究所)》2017年碩士論文

【摘要】：作為我國(guó)主要的大型栽培紅藻,紫菜不僅具有較高的經(jīng)濟(jì)價(jià)值,也因其獨(dú)特的生物學(xué)特征,同時(shí)具有較高的理論研究?jī)r(jià)值。本論文以兩種常見紫菜為研究對(duì)象開展了如下兩方面研究:1.在有性生殖階段,條斑紫菜葉狀體中淡黃色的精子囊器與深紅褐色的果胞呈條紋狀相間分布。在顯微鏡下觀察、切割獲得了不同發(fā)育時(shí)期的葉狀體材料,包括營(yíng)養(yǎng)細(xì)胞期,精子囊發(fā)育表觀2細(xì)胞期、4細(xì)胞期、8細(xì)胞期,提取其可溶性蛋白,并采用iTRAQ標(biāo)記法進(jìn)行了蛋白差異表達(dá)分析,初步探索了精子囊分化發(fā)育調(diào)控機(jī)制及參與的代謝路徑。結(jié)果顯示,明顯上調(diào)的蛋白主要涉及染色體的復(fù)制、組裝,包括組蛋白、微管蛋白、分子伴侶蛋白等,一些蛋白可能通過參與形成染色質(zhì)重塑復(fù)合體而對(duì)轉(zhuǎn)錄過程進(jìn)行表觀調(diào)控。此外,26 S蛋白酶體介導(dǎo)的蛋白降解系統(tǒng)相關(guān)亞基出現(xiàn)不同程度的上調(diào),在精子囊細(xì)胞形成過程中,通過泛素—蛋白酶體降解途徑的調(diào)控,細(xì)胞周期運(yùn)轉(zhuǎn)加快,因此,蛋白質(zhì)的循環(huán)利用活躍,為細(xì)胞由營(yíng)養(yǎng)生長(zhǎng)向生殖生長(zhǎng)轉(zhuǎn)化提供了物質(zhì)基礎(chǔ);精子囊發(fā)育不同階段,磷酸激酶及磷酸酶系統(tǒng)、鈣調(diào)蛋白等調(diào)控因子表達(dá)上調(diào),也說明細(xì)胞內(nèi)原有的代謝網(wǎng)絡(luò)在精子囊分化過程中發(fā)生了調(diào)整,磷酸基團(tuán)參與的能量代謝活躍,為精子的形成及釋放奠定了能量基礎(chǔ)。與此同時(shí),參與光合作用過程的藻膽蛋白、放氧復(fù)合體、電子傳遞體等的蛋白組成亞基均出現(xiàn)不同程度的下調(diào),證明細(xì)胞營(yíng)養(yǎng)積累過程減緩,與光合作用偶聯(lián)的耗能同化過程也有所下降。2.篩選了在精子囊細(xì)胞發(fā)育過程中上調(diào)的磷酸酯酶基因,克隆其開放閱讀框,并構(gòu)建了重組表達(dá)載體pPICZαA-pp2c,通過將該重組載體轉(zhuǎn)化入畢赤酵母,建立了以甲醇為營(yíng)養(yǎng)的異源基因表達(dá)系統(tǒng)。并對(duì)外源蛋白表達(dá)條件進(jìn)行了優(yōu)化。但最終目的蛋白的純化未成功。3.壇紫菜為我國(guó)特有的紫菜栽培品種,年產(chǎn)量遠(yuǎn)超條斑紫菜,紫菜優(yōu)異的抗逆響應(yīng)機(jī)制一直是研究的熱點(diǎn)與重點(diǎn)問題。我們采用絕對(duì)定量與軟件分析相結(jié)合的方法,對(duì)不同鹽度、不同光照處理下的壇紫菜常見內(nèi)參基因(18S,GAPDH,EF3,RPS,TubA,ACT,TubB)的表達(dá)進(jìn)行了研究,分別篩選出了高鹽、高光脅迫條件下表達(dá)相對(duì)穩(wěn)定的基因,認(rèn)定壇紫菜在鹽度脅迫條件下,EF3與18S是進(jìn)行RT-qPCR相對(duì)定量可信賴的內(nèi)參基因,而在光脅迫條件下,18S與TubA則是理想內(nèi)參。本研究結(jié)果為后續(xù)紫菜抗逆機(jī)制研究的開展奠定了基礎(chǔ)。
[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 偽 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.
【學(xué)位授予單位】：中國(guó)科學(xué)院大學(xué)(中國(guó)科學(xué)院海洋研究所)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2;Q945.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王旭雷;馬穎超;魯曉萍;姜波;沈宗根;陸勤勤;沈頌東;盧山;何林文;牛建峰;汪文俊;夏邦美;茅云翔;陳昌生;謝潮添;駱其君;邵魁雙;陳偉洲;王廣策;;法紫菜生物多樣性及其栽培生物學(xué)基礎(chǔ)[J];海洋科學(xué);2017年02期

2 陸勤勤;周偉;鄧銀銀;;條斑紫菜栽培的經(jīng)濟(jì)效益與生態(tài)效益概述[J];中國(guó)水產(chǎn);2016年01期

3 潘光華;楊睿靈;王廣策;;條斑紫菜精子囊細(xì)胞色素含量及光合活性的研究[J];天津科技大學(xué)學(xué)報(bào);2012年02期

4 胡世界;羅素蘭;張吉貞;長(zhǎng)孫東亭;;巴斯德畢赤酵母表達(dá)系統(tǒng)及其高水平表達(dá)策略[J];生物技術(shù);2007年06期

5 宋素勝;謝道昕;;泛素蛋白酶體途徑及其對(duì)植物生長(zhǎng)發(fā)育的調(diào)控[J];植物學(xué)通報(bào);2006年05期

6 周文君;李峗;戴繼勛;;條斑紫菜自由絲狀體的形態(tài)結(jié)構(gòu)觀察[J];中國(guó)水產(chǎn)科學(xué);2006年02期

7 陳建明;俞曉平;程家安;;葉綠素?zé)晒鈩?dòng)力學(xué)及其在植物抗逆生理研究中的應(yīng)用[J];浙江農(nóng)業(yè)學(xué)報(bào);2006年01期

8 ;Isolation and Characterization of Photosystem Ⅱ of Porphyra yezoensis Ueda[J];Acta Biochimica et Biophysica Sinica;2004年11期

9 莫良玉,吳良?xì)g,陶勤南;高等植物GS/GOGAT循環(huán)研究進(jìn)展[J];植物營(yíng)養(yǎng)與肥料學(xué)報(bào);2001年02期

10 王素娟,馬凌波;壇紫菜幼苗無性繁殖的再研究[J];上海水產(chǎn)大學(xué)學(xué)報(bào);1994年Z1期

相關(guān)博士學(xué)位論文 前1條

1 周偉;條斑紫菜葉狀體不同區(qū)域光合作用比較分析及相關(guān)蛋白質(zhì)組學(xué)研究[D];中國(guó)科學(xué)院研究生院（海洋研究所）;2013年

相關(guān)碩士學(xué)位論文 前1條

1 王麗;條斑紫菜性別差異基因的篩選和初步分析[D];蘇州大學(xué);2008年

，

本文編號(hào)：1837320