【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,營養(yǎng)學,遺傳學和蛋白組學等的相應變化,不僅藻種產(chǎn)量大幅度下降,而且為藻種的鑒定帶來了困難。因此為實現(xiàn)對螺旋藻形態(tài)變化的改良和調(diào)控,有必要解析螺旋藻形態(tài)建成的分子機理。本研究以來源于單藻絲培養(yǎng)物中發(fā)生形態(tài)分化的直線形和螺旋形個體為對象,利用iTRAQ蛋白組學和質(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)合生物信息學技術(shù)對篩選到的差異蛋白從參與的生物過程、細胞定位、分子功能三方面進行分析,通過實時熒光定量PCR技術(shù),對所得到的蛋白組學數(shù)據(jù)從轉(zhuǎn)錄組水平進行驗證,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)建成的分子機理及代謝調(diào)控機制。試驗研究結(jié)果如下:1.分別對直線形和螺旋形螺旋藻的藻絲體長度、螺徑、螺距、螺旋數(shù)及生長曲線、藻藍蛋白、別藻藍蛋白、葉綠素a、類胡蘿卜素等形態(tài)指標和生理指標進行測定,掌握不同形態(tài)螺旋藻之間的形態(tài)及生理指標差異,為后續(xù)研究對象的選擇提供充分的依據(jù)。2.通過iTRAQ蛋白組學及質(zhì)譜技術(shù),對與螺旋藻形態(tài)建成相關(guān)蛋白進行篩選鑒定。以FDR≤1%,去除反庫數(shù)據(jù)和比值為空白的無效值,作為可信蛋白篩選標準;蛋白豐度倍數(shù)變化大于2(上調(diào)表達)或小于0.5(下調(diào)表達),作為差異蛋白篩選標準。iTRAQ-LC-MS/MS分析共匹配到185123個圖譜,譜圖利用率為37.6%,去除可信度較低的肽段,共鑒定到30508個特定肽段,根據(jù)可信蛋白篩選標準,去掉冗余結(jié)果,最終鑒定到2156個蛋白。TJSD2/TJSD3組共篩選出165個差異蛋白,TJBC4-1/TJBC4-2組中共篩選出167個差異蛋白,兩組共有的差異表達蛋白35個。3.對篩選的差異蛋白進行生物信息學分析,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。通過對代謝途徑的分析,構(gòu)建了螺旋藻形態(tài)建成預測模型,用于初步解析螺旋藻形態(tài)建成機理。并通過實時熒光定量PCR對模型中的蛋白質(zhì)數(shù)據(jù)進行驗證。以蛋白組學和轉(zhuǎn)錄組學分析具有相同變化趨勢的蛋白質(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)絡圖,解析了蛋白質(zhì)的互作網(wǎng)絡。4.通過對兩組螺旋藻中的共性差異蛋白進行分析,對螺旋藻形態(tài)建成起關(guān)鍵作用的pgm基因,進行原核表達驗證。構(gòu)建pgm基因的融合表達載體,并成功轉(zhuǎn)入大腸桿菌中進行表達。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)進行功能驗證提供理論依據(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：2369378