Time course response of Synechocystis PCC 6803 to UV irradiation. Synechocystis sp. PCC 6803

The responses of the transcriptome of Synechocystis PCC 6803 to UV-irradiation were measured at time points over 36 h. Irradiation was provided by Sylvania soft white DuluzR compact fluorescent 23W bulbs (Osram Sylvania Ltd, Mississauga, Canada), a 20W RS UV-B medical light with a spectral maximum at 310 nm (model ‘TL’, Philips, Holland), and 15W black lights each with a spectral maximum at 368 nm (model F15T8-BL, General Electric, USA). Total quantum scalar irradiance was measured with a model QSL-100 meter (Biospherical Instruments Inc., San Diego, CA). The flux densities of the UV-A and UV-B components of the spectrum were measured with DIX series UV-B and 365A sensors, respectively, with a Spectroline DRC-100X digital radiometer (Spectronics Corporation, Westbury, NY). In these experiments full illumination represented a continuous photon flux density in the visible range of 330 μmol photons m-2 s-1, with UV-A and UV-B maxima of 3.8 x 10^6 and 0.8 x 10^6 mW m-2, respectively. All values reported were the incident fluxes within culture vessels at the immediate surface of the cell suspensions. Aliquot cultures (in duplicate) were harvested after 0, 15 min, 1 h, 3 h, 6 h, 12 h, 24 h and 36 h of UV-irradiation. For each time point, total RNA were isolated from stressed and unstressed cells, reverse-transcribed, differentially labelled (dye swapped), hybridized together (stressed versus unstressed samples) and analyzed with DNA glass microarrays (two slides per each time point) (Custom-commercial array : CyanoCHIP version 2.0, TAKARA). To identify differentially expressed genes, the median of the normalized ratio of Cy5/Cy3 intensity was calculated for each spot of the replicated dye-swap. The results of the analysis were carefully examined to exclude the dye effect between the 2 Cy-swapped arrays. Keywords: UV-irradiation, desiccation, Synechocystis PCC 6803, cyanobacteria, time course, transcription Overall design: Goal - Compare the responses of cyanobacterium Synechocystis sp. PCC 6803 to UV-irradiation and dehydration/desiccation stress. Origin of biological sample - The axenic strain Synechocystis sp. PCC 6803 was obtained from the American Type Culture Collection (ATCC 27184). Special attention was given to maintain all the Synechocystis cultures under the same conditions to minimize environmental variation. Brief description - Ultraviolet (UV) light and desiccation stresses often co-occur in natural environments, making their combined effects important selective factors within microbial populations. The effects of these stresses on the cyanobacterium Synechocystis sp. PCC 6803 was assessed through transcriptional analyses using differential display and microarray assays. Experimental factors and design- Axenic Synechocystis cultures were grown at 25°C in BG-11 medium, on a rotary shaker at 70 rpm, at a photon flux density of 200 µmol photons m-2 s-1. At the midpoint of the exponential phase of growth (~ 10^6 to 10^7 cells ml-1), 25-ml aliquots of the cell suspension were subjected to UV-irradiation (in 50-ml flasks) with a continuous photon flux density in the visible range of 330 µmol photons m-2 s-1. UV-A and UV-B maxima were 3.8 x 10^6 and 0.8 x 10^6 mW m-2, respectively. Cells were harvested after 0, 15 min, 1 h, 3 h, 6 h, 12 h, 24 h and 36 h of irradiation for disruption and rapid recovery of RNA. As controls, RNA was obtained from Synechocystis cells grown without UV-irradiation. Cells were disrupted with glass beads and RNA was extracted with a buffer of acidified phenol (65°C), sodium acetate pH 5.1, EDTA and sodium dodecyl sulfate), followed by ethanol precipitation. The RNA (10 µg) was annealed with 20 µg Random RT primer and reverse transcription into cDNA was achieved with Superscript II reverse transcriptase enzyme for 2h, at 42°C. The assay contained dGTP, dCTP, dATP and dTTP, each a final concentration of 0.5 mM. After hydrolysis of RNA with 0.1M NaOH and 10 mM EDTA, then neutralization, cDNAs were purified using a Quiagen QIAquick PCR purification kit as modified in the Genisphere 3DNA Array 350RP protocol. Two experimental replicates of RNAs from time points 12 ml, and two experimental replicates from 0.5 ml, of dehydration, were used for hybridization to microarrays. Three experimental replicates of RNAs from time point 3h of UV-irradiation, and three experimental replicates from time point 36h of UV-irradiation, were used for hybridization to microarrays. Quality contro-l DNAse treatment (DNAse free, Ambion) was for 3 h at 37 °C. This treatment was sometimes repeated for 2 h until the absence of any detectable DNA was checked by the absence of product after 40 cycles of PCR amplification. The quality and purity of RNAs was judged by A254/280 (1.9 to 2.1) and by the integrity of 23S and 16S rRNA resolved through gel electrophoresis. The ribosomal subunits gave sharp and bright bands that were comparable among all samples. The quality of the random primed cDNA samples was checked by SDS-PAGE after ligation of Cy3/Cy5 cap sequences. Test arrays were performed to determine the optimum hybridization and washing conditions of the TAKARA’s microarray chips.

本研究测定了集胞藻PCC 6803（Synechocystis PCC 6803）的转录组对紫外线照射的响应，采样时间覆盖36小时。
本次实验采用三种光源进行照射：分别为Sylvania软白光DuluzR紧凑型23W荧光灯泡（欧司朗喜万年有限公司，加拿大密西沙加）、峰值波长310nm的20W RS型UV-B医用灯（型号‘TL’，飞利浦，荷兰），以及峰值波长368nm的15W黑光灯（型号F15T8-BL，通用电气，美国）。
总量子标量辐照度采用QSL-100型辐照计（美国加州圣地亚哥Biospherical仪器公司）进行测定；光谱中UV-A与UV-B组分的通量密度则分别采用DIX系列UV-B传感器、365A传感器，结合Spectroline DRC-100X型数字辐射计（美国纽约州韦斯特伯里Spectronics公司）完成测定。
本实验中全光照条件下可见光范围的连续光子通量密度为330 μmol·m⁻²·s⁻¹，UV-A与UV-B的最大通量密度分别为3.8×10⁶和0.8×10⁶ mW·m⁻²。所有报道的数值均为细胞悬浮液表层处培养容器内的入射通量。
每份等分培养物设置生物学重复，分别在紫外线照射0、15分钟、1小时、3小时、6小时、12小时、24小时及36小时后收集样本。对于每个时间点，分别从受胁迫与未受胁迫的细胞中提取总RNA，经反转录、差异标记（染料互换）后，将受胁迫样本与未受胁迫样本进行杂交，随后采用DNA玻璃微阵列（每个时间点两张芯片，定制商业化芯片：CyanoCHIP 2.0版，TAKARA）进行分析。
为鉴定差异表达基因，针对染料互换重复实验的每个探针点，计算Cy5/Cy3信号强度归一化比值的中位数。对分析结果进行严格校验，以排除两张染料互换芯片间的染料效应偏差。

关键词：紫外线照射、脱水胁迫、集胞藻PCC 6803（Synechocystis PCC 6803）、蓝细菌、时间序列、转录

实验设计概述：
研究目标——比较蓝细菌集胞藻PCC 6803对紫外线照射与脱水胁迫的响应。
生物样本来源——无菌集胞藻PCC 6803菌株购自美国典型培养物保藏中心（ATCC 27184）。为最小化环境差异，所有集胞藻培养过程均严格保持一致的培养条件。
研究概况——紫外线（UV）与脱水胁迫在自然环境中常同时存在，二者的联合效应是微生物种群中重要的选择压力。本研究通过差异显示与微阵列分析技术，探究上述两种胁迫对蓝细菌集胞藻PCC 6803的影响。

实验因素与设计：
无菌集胞藻培养于BG-11培养基中，培养温度25℃，摇床转速70 rpm，光子通量密度为200 μmol·m⁻²·s⁻¹。当培养至指数生长期中期（细胞密度约为10⁶~10⁷ cells·ml⁻¹）时，取25 ml细胞悬浮液置于50 ml培养瓶中进行紫外线照射处理，其可见光范围的连续光子通量密度为330 μmol·m⁻²·s⁻¹，UV-A与UV-B的最大通量密度分别为3.8×10⁶和0.8×10⁶ mW·m⁻²。
分别在照射0、15分钟、1小时、3小时、6小时、12小时、24小时及36小时后收集细胞，用于裂解并快速提取RNA。设置对照组：采集未经过紫外线照射的集胞藻细胞以提取RNA。
细胞通过玻璃珠法裂解，采用酸化苯酚（65℃）、pH 5.1的乙酸钠、EDTA及十二烷基硫酸钠（SDS）缓冲液提取RNA，随后经乙醇沉淀纯化。取10 μg RNA与20 μg随机反转录引物退火，于42℃下利用SuperScript II反转录酶进行2小时反转录反应以合成cDNA。反应体系中dGTP、dCTP、dATP及dTTP的终浓度均为0.5 mM。
采用0.1 M NaOH与10 mM EDTA水解RNA后中和体系，随后按照Genisphere 3DNA Array 350RP实验方案优化后的步骤，使用Qiagen QIAquick PCR纯化试剂盒纯化cDNA。
针对12小时脱水处理的样本设置两份生物学重复RNA样本，针对0.5小时脱水处理的样本同样设置两份生物学重复RNA样本，均用于微阵列杂交；针对3小时与36小时紫外线照射的样本，分别设置三份生物学重复RNA样本用于微阵列杂交。
质量控制：采用无DNA酶的Ambion试剂盒于37℃下处理3小时以去除基因组DNA，该步骤有时需重复2小时，直至经40轮PCR扩增无特异性产物生成，确认无任何可检测到的DNA残留。RNA的质量与纯度通过A254/A280比值（1.9~2.1）以及经凝胶电泳分离的23S和16S rRNA完整性进行评估，所有样本的核糖体亚基均呈现清晰明亮的条带且组间可比性良好。
随机引物合成的cDNA样本质量通过连接Cy3/Cy5接头序列后的SDS-PAGE电泳进行检测。通过预实验杂交确定TAKARA微阵列芯片的最优杂交与洗涤条件。