基于iTRAQ定量蛋白质组学技术筛选螺旋藻形态建成相关蛋白
发布时间：2018-12-09 12:40
【摘要】：螺旋藻形态是衡量螺旋藻品质的重要因素,但螺旋藻在培养过程中易受到外界环境的影响,形态发生改变,并伴随一系列生理学,营养学,遗传学和蛋白组学等的相应变化,不仅藻种产量大幅度下降,而且为藻种的鉴定带来了困难。因此为实现对螺旋藻形态变化的改良和调控,有必要解析螺旋藻形态建成的分子机理。本研究以来源于单藻丝培养物中发生形态分化的直线形和螺旋形个体为对象,利用iTRAQ蛋白组学和质谱分析技术,探讨其蛋白质组的差异变化,获得与螺旋藻形态建成相关的差异蛋白,并结合生物信息学技术对筛选到的差异蛋白从参与的生物过程、细胞定位、分子功能三方面进行分析,通过实时荧光定量PCR技术,对所得到的蛋白组学数据从转录组水平进行验证,旨在找到调控螺旋藻形态建成的关键基因,揭示螺旋藻形态建成的分子机理及代谢调控机制。试验研究结果如下:1.分别对直线形和螺旋形螺旋藻的藻丝体长度、螺径、螺距、螺旋数及生长曲线、藻蓝蛋白、别藻蓝蛋白、叶绿素a、类胡萝卜素等形态指标和生理指标进行测定,掌握不同形态螺旋藻之间的形态及生理指标差异,为后续研究对象的选择提供充分的依据。2.通过iTRAQ蛋白组学及质谱技术,对与螺旋藻形态建成相关蛋白进行筛选鉴定。以FDR≤1%,去除反库数据和比值为空白的无效值,作为可信蛋白筛选标准;蛋白丰度倍数变化大于2(上调表达)或小于0.5(下调表达),作为差异蛋白筛选标准。iTRAQ-LC-MS/MS分析共匹配到185123个图谱,谱图利用率为37.6%,去除可信度较低的肽段,共鉴定到30508个特定肽段,根据可信蛋白筛选标准,去掉冗余结果,最终鉴定到2156个蛋白。TJSD2/TJSD3组共筛选出165个差异蛋白,TJBC4-1/TJBC4-2组中共筛选出167个差异蛋白,两组共有的差异表达蛋白35个。3.对筛选的差异蛋白进行生物信息学分析,鉴定的差异蛋白参与糖酵解TCA循环,光合作用,光合作用-天线蛋白,淀粉与蔗糖代谢,脂多糖生物合成,光合生物固碳,卟啉,叶绿素代谢等多种代谢调控。通过对代谢途径的分析,构建了螺旋藻形态建成预测模型,用于初步解析螺旋藻形态建成机理。并通过实时荧光定量PCR对模型中的蛋白质数据进行验证。以蛋白组学和转录组学分析具有相同变化趋势的蛋白质为依据,构建了螺旋藻形态建成中心能量代谢网络图,解析了蛋白质的互作网络。4.通过对两组螺旋藻中的共性差异蛋白进行分析,对螺旋藻形态建成起关键作用的pgm基因,进行原核表达验证。构建pgm基因的融合表达载体,并成功转入大肠杆菌中进行表达。转入螺旋藻pgm基因的大肠杆菌形态发生显著变化,说明螺旋藻pgm基因可调控大肠杆菌形态建成,为pgm基因在螺旋藻体内进行功能验证提供理论依据。
[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，
本文编号：2369378
[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，
本文编号：2369378
本文链接：https://www.wllwen.com/shoufeilunwen/benkebiyelunwen/2369378.html
