Transcriptomics Analyses of tobacco leaves and roots sample during water-deficit. Nicotiana tabacum

Microarray experiments were performed on the roots and leaves samples seperately using custom based Nimblegen platform (12plex). This is a time-course study. The plants were grown in controlled growth chamber condiditons. The seeds were sown in sterile petri plates with modified half-strength MS agar medium. The seedlings were then aseptically transferred into sterile polycarbonate container with half-strength MS liquid medium. Seedlings were grown for 2 weeks in an incubator set at 25 oC with constant light and subjected to dehydration for 20min, 40min, 1hr, 2hrs, and 4hrs. Overall design: We used a hydrophonics system for the trancriptome profiling of tobacco (cv Burley 21) root and leaves samples to study plant reponses during water- deficit or drought. Plants were grown in controlled growth chamber conditions and subjected to water deficit condition after 2 weeks.The plants were transferred to empty containers without touching to nullify any possibility of wounding and harvested subsequently. For 0mins, plants were taken out of the well-watered conditions and transfered to empty boxes and immediately harvested for both tissues and hence called 0-min dehydrated tissue. Similarly other time-points were processed( for instance, 20 min of dehydration, plants were dehydrated for 20 min after being taken out from the container and then harvested). Tissues for three biological replicates and 20 plants per biological replicates were collected for each time-point. RNA was isolated from both tissues using RNeasy Kit from Qiagen and quality was accessed with Nano-drop as well as with Agilent-Bioanalyzer using RNA-600 kit. 20ug of sample was processed at Micro Array Facility.

本研究基于定制化尼姆布金（Nimblegen）12plex芯片平台，分别对根、叶样本开展微阵列实验。本研究属于时间进程类实验。实验植物在可控环境生长箱条件下培养：将种子播种于添加改良型1/2浓度MS琼脂培养基的无菌培养皿中，随后将无菌幼苗转接至装有1/2浓度MS液体培养基的无菌聚碳酸酯容器内。将幼苗置于温度设定为25℃、持续光照的培养箱中培养两周，随后分别进行20分钟、40分钟、1小时、2小时及4小时的脱水处理。

实验整体设计如下：本研究采用水培系统，对烟草（栽培品种白肋21，Burley 21）的根、叶样本进行转录组表达谱分析，以探究植物在水分胁迫或干旱条件下的响应机制。实验植物在可控生长箱中培养，于培养两周后施加水分胁迫处理：将植株转移至空容器中且避免触碰植株，以消除机械损伤的潜在影响，随后进行样本收获。对于0分钟处理组，将植株从充分供水的培养环境中取出并转移至空盒后立即收获根、叶组织，因此该组样本被称为0分钟脱水组织。其余时间点样本的处理流程与此类似：例如20分钟脱水组，植株从培养容器中取出后先进行20分钟脱水处理，随后收获组织。每个时间点均设置三份生物学重复，每份生物学重复取材自20株植株的组织。采用凯杰（Qiagen）RNeasy试剂盒从两类组织中提取RNA，并通过Nano-drop分光光度计及搭载RNA-600试剂盒的安捷伦生物分析仪（Agilent-Bioanalyzer）检测RNA质量。在微阵列实验中心对总量为20 μg的样本完成芯片处理。