Changes in gene expression due to CgtA-depletion in Vibrio cholerae.

A cgtA-depletion strain in V. cholerae was constructed and used to identify changes in gene expression that occur due to loss of the gene product. This information was then used to characterize the role of the gene, which was found to be involved in stress response. Keywords: Depletion strain, stress response V. cholerae cells from a cgtA-depletion strain were used. This strain requires arabinose for growth. Cells were grown to mid-log, washed, and split into two cultures, one with arabinose, one without. These cells were grown for one doubling time, then RNA was harvested using a Trizol method. Both depleted and non-depleted cell RNA was converted to cDNA, with one set incorporating Cy5, the other Cy3, with fluorophores reversed in the multiple analyses. RNA from both conditions was applied to a whole genome PCR microarray from V. cholerae N16961, containing two sets of every ORF. Data was analyzed by Genepix and Rosetta Resolver.

本研究构建了霍乱弧菌（Vibrio cholerae, V. cholerae）的cgtA基因耗竭菌株，用于鉴定因该基因产物缺失所引发的基因表达变化。随后利用该转录组变化信息解析该基因的功能，最终发现其参与应激响应过程。
关键词：耗竭菌株，应激响应
本实验使用的霍乱弧菌样本来自上述cgtA基因耗竭菌株，该菌株的生长依赖阿拉伯糖。将菌株培养至对数中期，洗涤后分为两组培养液：一组添加阿拉伯糖，另一组不添加。两组菌株继续培养至完成一个倍增周期，随后采用TRIzol试剂（TRIzol）提取总RNA。将耗竭组与非耗竭组的细胞RNA反转录为cDNA，其中一组标记Cy5荧光染料，另一组标记Cy3荧光染料；在重复实验中交换两组的荧光标记方式。将两组RNA分别应用于霍乱弧菌N16961全基因组PCR微阵列芯片，该芯片包含每个开放阅读框（Open Reading Frame, ORF）的两个重复位点。芯片数据通过Genepix与Rosetta Resolver软件进行分析。