發(fā)布時(shí)間：2018-12-09 12:40

【摘要】：螺旋藻形態(tài)是衡量螺旋藻品質(zhì)的重要因素,但螺旋藻在培養(yǎng)過(guò)程中易受到外界環(huán)境的影響,形態(tài)發(fā)生改變,并伴隨一系列生理學(xué),營(yíng)養(yǎng)學(xué),遺傳學(xué)和蛋白組學(xué)等的相應(yīng)變化,不僅藻種產(chǎn)量大幅度下降,而且為藻種的鑒定帶來(lái)了困難。因此為實(shí)現(xiàn)對(duì)螺旋藻形態(tài)變化的改良和調(diào)控,有必要解析螺旋藻形態(tài)建成的分子機(jī)理。本研究以來(lái)源于單藻絲培養(yǎng)物中發(fā)生形態(tài)分化的直線(xiàn)形和螺旋形個(gè)體為對(duì)象,利用iTRAQ蛋白組學(xué)和質(zhì)譜分析技術(shù),探討其蛋白質(zhì)組的差異變化,獲得與螺旋藻形態(tài)建成相關(guān)的差異蛋白,并結(jié)合生物信息學(xué)技術(shù)對(duì)篩選到的差異蛋白從參與的生物過(guò)程、細(xì)胞定位、分子功能三方面進(jìn)行分析,通過(guò)實(shí)時(shí)熒光定量PCR技術(shù),對(duì)所得到的蛋白組學(xué)數(shù)據(jù)從轉(zhuǎn)錄組水平進(jìn)行驗(yàn)證,旨在找到調(diào)控螺旋藻形態(tài)建成的關(guān)鍵基因,揭示螺旋藻形態(tài)建成的分子機(jī)理及代謝調(diào)控機(jī)制。試驗(yàn)研究結(jié)果如下:1.分別對(duì)直線(xiàn)形和螺旋形螺旋藻的藻絲體長(zhǎng)度、螺徑、螺距、螺旋數(shù)及生長(zhǎng)曲線(xiàn)、藻藍(lán)蛋白、別藻藍(lán)蛋白、葉綠素a、類(lèi)胡蘿卜素等形態(tài)指標(biāo)和生理指標(biāo)進(jìn)行測(cè)定,掌握不同形態(tài)螺旋藻之間的形態(tài)及生理指標(biāo)差異,為后續(xù)研究對(duì)象的選擇提供充分的依據(jù)。2.通過(guò)iTRAQ蛋白組學(xué)及質(zhì)譜技術(shù),對(duì)與螺旋藻形態(tài)建成相關(guān)蛋白進(jìn)行篩選鑒定。以FDR≤1%,去除反庫(kù)數(shù)據(jù)和比值為空白的無(wú)效值,作為可信蛋白篩選標(biāo)準(zhǔn);蛋白豐度倍數(shù)變化大于2(上調(diào)表達(dá))或小于0.5(下調(diào)表達(dá)),作為差異蛋白篩選標(biāo)準(zhǔn)。iTRAQ-LC-MS/MS分析共匹配到185123個(gè)圖譜,譜圖利用率為37.6%,去除可信度較低的肽段,共鑒定到30508個(gè)特定肽段,根據(jù)可信蛋白篩選標(biāo)準(zhǔn),去掉冗余結(jié)果,最終鑒定到2156個(gè)蛋白。TJSD2/TJSD3組共篩選出165個(gè)差異蛋白,TJBC4-1/TJBC4-2組中共篩選出167個(gè)差異蛋白,兩組共有的差異表達(dá)蛋白35個(gè)。3.對(duì)篩選的差異蛋白進(jìn)行生物信息學(xué)分析,鑒定的差異蛋白參與糖酵解TCA循環(huán),光合作用,光合作用-天線(xiàn)蛋白,淀粉與蔗糖代謝,脂多糖生物合成,光合生物固碳,卟啉,葉綠素代謝等多種代謝調(diào)控。通過(guò)對(duì)代謝途徑的分析,構(gòu)建了螺旋藻形態(tài)建成預(yù)測(cè)模型,用于初步解析螺旋藻形態(tài)建成機(jī)理。并通過(guò)實(shí)時(shí)熒光定量PCR對(duì)模型中的蛋白質(zhì)數(shù)據(jù)進(jìn)行驗(yàn)證。以蛋白組學(xué)和轉(zhuǎn)錄組學(xué)分析具有相同變化趨勢(shì)的蛋白質(zhì)為依據(jù),構(gòu)建了螺旋藻形態(tài)建成中心能量代謝網(wǎng)絡(luò)圖,解析了蛋白質(zhì)的互作網(wǎng)絡(luò)。4.通過(guò)對(duì)兩組螺旋藻中的共性差異蛋白進(jìn)行分析,對(duì)螺旋藻形態(tài)建成起關(guān)鍵作用的pgm基因,進(jìn)行原核表達(dá)驗(yàn)證。構(gòu)建pgm基因的融合表達(dá)載體,并成功轉(zhuǎn)入大腸桿菌中進(jìn)行表達(dá)。轉(zhuǎn)入螺旋藻pgm基因的大腸桿菌形態(tài)發(fā)生顯著變化,說(shuō)明螺旋藻pgm基因可調(diào)控大腸桿菌形態(tài)建成,為pgm基因在螺旋藻體內(nèi)進(jìn)行功能驗(yàn)證提供理論依據(jù)。
[Abstract]:The morphology of Spirulina is an important factor to measure the quality of spirulina, but it is easy to be affected by the external environment during the culture process, and the morphology changes with a series of corresponding changes in physiology, nutrition, genetics and proteomics. Not only the production of algae decreased significantly, but also brought difficulties for the identification of algae. Therefore, in order to improve and regulate the morphological changes of Spirulina, it is necessary to analyze the molecular mechanism of the morphogenesis of Spirulina. In this study, iTRAQ proteomics and mass spectrometric analysis were used to investigate the proteome variation of linear and spiral individuals derived from monophyta filamentum culture. The differential proteins related to the morphogenesis of Spirulina platensis were obtained, and the differential proteins were analyzed by bioinformatics from three aspects: biological process, cellular location and molecular function, and real-time fluorescence quantitative PCR technique was used to analyze the differential proteins. In order to find the key genes to regulate the morphogenesis of spirulina and reveal the molecular mechanism and metabolic regulation mechanism of the morphogenesis of Spirulina platensis, the proteomic data were verified from the transcriptional level. The experimental results are as follows: 1. The length, diameter, pitch, helix number, growth curve, phycocyanin, allophycocyanin, chlorophyll a, carotenoid and other morphological and physiological indexes of Spirulina spirulina were measured. To grasp the differences of morphological and physiological indexes among different forms of Spirulina platensis and to provide sufficient basis for the selection of objects for further study. 2. The proteins associated with Spirulina morphogenesis were screened and identified by iTRAQ proteomics and mass spectrometry. Using FDR 鈮，

本文編號(hào)：2369378