转录组和蛋白组.
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食品微生物专题
利用转录组学和蛋白组学技术研究乳酸菌 胁迫条件下自身应答机制
郝彦玲副教授
食品科学与营养工程学院 2013年9月30日
1、转录组学测序技术介绍
转录组（transcriptome）：广义上指某一生理条件下，细
胞内所有转录产物的集合，包括信使RNA、核糖体RNA、 转运RNA及非编码RNA；狭义上指所有mRNA的集合。
利用2-D gel 结合实时定量进行研究，创新点是推测出丙酮酸的 代谢流向饱和脂肪酸，从而增强了膜的rigidity and impermeability.
2011：
转录组：基因芯片技术 蛋白质组：2D-DIGE技术 在0.2%的 oxgal中培养，316基因，42个蛋白质发生显著变化。
蛋白质双向电泳技术：是等电聚焦电泳和SDS-PAGE的组合，即 先进行等电聚焦电泳（按照pI分离），然后在垂直的方向再进行 SDS-PAGE（按照分子大小），经染色得到的电泳图是个二维分 布的蛋白质图。 优点： 双向电泳技术具有很好 的分辨率和灵敏度，可以同时 显示组织或细胞内各种蛋白， 高分辨率确保蛋白质最大程度 的分离。 缺点：重复性是很不理想。高 重复性有利于凝胶间的对比。
Results and Discussion
（1）GG Alter cell surface properties and expression multiple ABC-type multidrug transporter in response to bile. （2）Two-component regulatory systems and bile salt hydrolase modulate cellular response to bile （3） Bile induces common stress response in GG . （4） Bile affects central metabolic processes
（2）Several enzymes involved in carbohydrate catabolism, including the key enzyme of the so-called bifidobacterial shunt, are overexpressed in cells grown in the presence of bile salts
荧光差异凝胶电泳技术（2D-DIGE）
采用专有的荧光染料与多重样本和图象分析的方法，在 同一块胶上可同时分离多个由不同荧光标记的样品。
iTRAQ技术
• Exposure to bile is a serious challenge to the viability of probiotics because the concentration of bile acids in the human small intestine typically varies between 0.2 and 2% • When challenged with bile, bacteria are known to modify their cell envelope properties such as cell membrane fatty acid composition, peptidoglycan composition and membrane charge . • Bile stress can also cause deleterious effects, including protein misfolding and denaturation, DNA damage, secondary structure formation in RNA, and intracellular acidification .
基因组（genome）：指的是细胞或生物体中所有 的DNA，包括所有的基因和基因间隔区。
转录组具有时空特异性
From Sanger to Next Generation Sequencing
2、蛋白组学技术的介绍
蛋白质组(Proteome)：指由一个基因组或一个细胞、 组织表达的所有蛋白质。 蛋白质双向电泳技术(2D -Gel) 荧光差异凝胶电泳技术（2D-DIGE） iTRAQ技术
典型文章
实验手段：DNA 芯片和实时定量PCR。 推测结果：通过转录组发现Genes encoding three transport systems belonging to the major facilitator superfamily (MFS), Bbr_0838, Bbr_0832, and Bbr_1756, and three ABC-type transporters, Bbr_0406-0407, Bbr_1804-1805, and Bbr_18261827在胆盐胁迫下表达水平发生显著变化，推测可能参与胆盐胁迫。 创新点：利用胆盐缺陷型的L. lactis NZ9000 lmrCD 作为宿主，通过异源表 达证明其参与胆盐抗性。
14 and 22 spots were significantly overexpressed at the 0.6g/L and 1.2g/L bile salts. Only 4 spots were significantly down-regulated at the both concentrations. 3 spots were only detected only when bile salts were added to the medium.
(1) Ox bile extract promotes induction of general stress response proteins the maturation of newly synthesized proteins, refolding and degradation of denatured proteins and DNA repairs, such as GroEL and DnaK
（3）生理指标的检测： End metabolic products （lactate, acetate and formate）and F6PPK were detected.
源自文库
（4）Regulation by bile salts of proteins involved in transcription, translation, and metabolism of amino acids and nucleotides.
利用转录组学和蛋白组学技术研究乳酸菌 胁迫条件下自身应答机制
郝彦玲副教授
食品科学与营养工程学院 2013年9月30日
1、转录组学测序技术介绍
转录组（transcriptome）：广义上指某一生理条件下，细
胞内所有转录产物的集合，包括信使RNA、核糖体RNA、 转运RNA及非编码RNA；狭义上指所有mRNA的集合。
利用2-D gel 结合实时定量进行研究，创新点是推测出丙酮酸的 代谢流向饱和脂肪酸，从而增强了膜的rigidity and impermeability.
2011：
转录组：基因芯片技术 蛋白质组：2D-DIGE技术 在0.2%的 oxgal中培养，316基因，42个蛋白质发生显著变化。
蛋白质双向电泳技术：是等电聚焦电泳和SDS-PAGE的组合，即 先进行等电聚焦电泳（按照pI分离），然后在垂直的方向再进行 SDS-PAGE（按照分子大小），经染色得到的电泳图是个二维分 布的蛋白质图。 优点： 双向电泳技术具有很好 的分辨率和灵敏度，可以同时 显示组织或细胞内各种蛋白， 高分辨率确保蛋白质最大程度 的分离。 缺点：重复性是很不理想。高 重复性有利于凝胶间的对比。
Results and Discussion
（1）GG Alter cell surface properties and expression multiple ABC-type multidrug transporter in response to bile. （2）Two-component regulatory systems and bile salt hydrolase modulate cellular response to bile （3） Bile induces common stress response in GG . （4） Bile affects central metabolic processes
（2）Several enzymes involved in carbohydrate catabolism, including the key enzyme of the so-called bifidobacterial shunt, are overexpressed in cells grown in the presence of bile salts
荧光差异凝胶电泳技术（2D-DIGE）
采用专有的荧光染料与多重样本和图象分析的方法，在 同一块胶上可同时分离多个由不同荧光标记的样品。
iTRAQ技术
• Exposure to bile is a serious challenge to the viability of probiotics because the concentration of bile acids in the human small intestine typically varies between 0.2 and 2% • When challenged with bile, bacteria are known to modify their cell envelope properties such as cell membrane fatty acid composition, peptidoglycan composition and membrane charge . • Bile stress can also cause deleterious effects, including protein misfolding and denaturation, DNA damage, secondary structure formation in RNA, and intracellular acidification .
基因组（genome）：指的是细胞或生物体中所有 的DNA，包括所有的基因和基因间隔区。
转录组具有时空特异性
From Sanger to Next Generation Sequencing
2、蛋白组学技术的介绍
蛋白质组(Proteome)：指由一个基因组或一个细胞、 组织表达的所有蛋白质。 蛋白质双向电泳技术(2D -Gel) 荧光差异凝胶电泳技术（2D-DIGE） iTRAQ技术
典型文章
实验手段：DNA 芯片和实时定量PCR。 推测结果：通过转录组发现Genes encoding three transport systems belonging to the major facilitator superfamily (MFS), Bbr_0838, Bbr_0832, and Bbr_1756, and three ABC-type transporters, Bbr_0406-0407, Bbr_1804-1805, and Bbr_18261827在胆盐胁迫下表达水平发生显著变化，推测可能参与胆盐胁迫。 创新点：利用胆盐缺陷型的L. lactis NZ9000 lmrCD 作为宿主，通过异源表 达证明其参与胆盐抗性。
14 and 22 spots were significantly overexpressed at the 0.6g/L and 1.2g/L bile salts. Only 4 spots were significantly down-regulated at the both concentrations. 3 spots were only detected only when bile salts were added to the medium.
(1) Ox bile extract promotes induction of general stress response proteins the maturation of newly synthesized proteins, refolding and degradation of denatured proteins and DNA repairs, such as GroEL and DnaK
（3）生理指标的检测： End metabolic products （lactate, acetate and formate）and F6PPK were detected.
源自文库
（4）Regulation by bile salts of proteins involved in transcription, translation, and metabolism of amino acids and nucleotides.