Regulation of uptake hydrogenase and effects of hydrogen utilization on gene expression in Rhodopseudomonas palustris. Rhodopseudomonas palustris

We used transcriptome analysis to identify genes that were differentially expressed between wild-type (CGA010) and hupV mutant cells (CGA009) grown photoheterotrophically with malate under nitrogen-fixing conditions. We also compared the transcriptome of a hupS mutant with that of the wild type. Keywords: Genetic modification Overall design: RNA was isolated from various Rhodopseudomonas palustris strains that were grown to the mid-logarithmic phase of growth. Fluorescently labeled cDNA was prepared by direct incorporation of either Cy3-dCTP or Cy5-dCTP during a first-strand reverse transcription reaction. The hybridization mixtures containing the two labeled cDNA samples to be compared were applied to microarray slides that had been covered with Lifterslips (Erie Scientific Company, Portsmouth, NH). The slides were assembled with hybridization chambers (Corning, Corning, NY) and submerged in a 65ºC water bath. After 14-16 h of hybridization, the slides were washed and scanned with a ScanArray 4000XL scanner (PerkinElmer, Boston, MA). Images (Cy3 and Cy5) were captured as TIFF files and were analyzed with the image processing software ImaGene version 5.6 (BioDiscovery, Inc., El Segundo, CA). The software package lcDNA was used for data normalization and assessment of the statistical confidence intervals of gene expression. Duplicate calibration experiments and three comparative experiments using RNA from three separately grown cultures (three biological replicates) with duplicate slides for each (10 slides in total) were used to generate each data set.

本研究采用转录组分析（transcriptome analysis），旨在鉴定在固氮条件下以苹果酸为碳源进行光异养生长的野生型（wild-type）菌株CGA010与hupV突变体CGA009之间的差异表达基因。我们还对比了hupS突变体与野生型的转录组。关键词：基因修饰（Genetic modification）实验设计概述：从培养至对数生长中期的不同沼泽红假单胞菌（Rhodopseudomonas palustris）菌株中提取总RNA。通过在第一链反转录反应中直接掺入Cy3-dCTP或Cy5-dCTP，制备荧光标记的互补脱氧核糖核酸（cDNA）。将待比较的两种标记cDNA样品混合形成杂交液，滴加至覆盖有Lifterlips玻片（Erie Scientific公司，美国新罕布什尔州朴茨茅斯市）的微阵列芯片上。将芯片与杂交腔（Corning公司，美国纽约州康宁市）组装后，浸没于65℃水浴中进行杂交。杂交14~16小时后，对芯片进行洗涤，随后使用ScanArray 4000XL扫描仪（PerkinElmer公司，美国马萨诸塞州波士顿市）进行扫描。将Cy3与Cy5通道的图像以标记图像文件格式（TIFF）文件存储，并使用ImaGene 5.6版图像处理软件（BioDiscovery公司，美国加利福尼亚州埃尔塞贡多市）进行图像分析。使用lcDNA软件包完成数据标准化处理，并计算基因表达的统计置信区间。本数据集的生成采用如下实验方案：设置2次重复校准实验，以及3次基于3份独立培养物提取的RNA的比较实验（3次生物学重复），每次比较实验均设置2张重复芯片，总计使用10张芯片玻片。