Overexpression of OsTZF8, a CCCH-Tandem Zinc finger, increases drought tolerance in rice by regulating stress related genes

Purpose: Next-generation sequencing (NGS) has been utilized for systems-based analysis of rice plants. The goals of this study were to compare the transcriptome between non-transgenic (NT) control and OsTZF8 overexpressing transgenic plants. Methods: Total RNAs were extracted from the whole plants of OsTZF8 overexpressing plants (T4 generation, line number #20) and non-transgenic (NT) plant using RNeasy plant mini kit (Qiagen, Germany) according to the manufacturer’s instruction. cDNA libraries were prepared from total RNAs using TruSeq RNA sample Prep kit (v2) (Macrogen, Korea). Two biological replicates were analyzed by RNA-sequencing analysis. Single-end sequences were obtained using IRGSP (v 1.0) and raw sequence reads were trimmed to remove adaptor sequence, and those with a quality lower than Q20 were removed using the Trimmomatic 0.32 software (Bolger et al., 2014). To map the reads to reference genome, all reads were assembled with annotated genes from the Rap-DB database [http://rapdb.dna.affrc.go.jp; IRGSP (v 1.0)] using TopHat software (https://ccb.jhu.edu/software/tophat/index.shtml). Conclusions: Our study has identified downstream candidate genes regulated by overexpression of OsTZF8. Non-transgenic (NT) control and OsTZF8 overexpressing trangenic plants were grown in soil for three weeks. Three-week-old plants were harvested and used for RNA-sequencing analysis to identify differentially expressed genes between NT and OsTZF8 overexpressing transgenic rice plants.

研究目的：下一代测序（Next-generation sequencing, NGS）技术已被应用于水稻植株的系统级分析。本研究旨在比较非转基因（non-transgenic, NT）对照与OsTZF8过表达转基因水稻植株的转录组（transcriptome）。实验方法：分别提取OsTZF8过表达T4代#20株系转基因植株与非转基因对照植株的全株总RNA，操作参照德国Qiagen公司的RNeasy plant mini kit试剂盒说明书进行。使用韩国Macrogen公司的TruSeq RNA sample Prep kit（v2版），以总RNA构建cDNA文库。本研究设置两个生物学重复，开展RNA测序分析。采用IRGSP（v1.0）参考基因组获取单端测序序列，使用Trimmomatic 0.32软件（Bolger等，2014）对原始测序读段进行质控修剪：去除接头序列，同时过滤掉质量值低于Q20的读段。为将读段比对至参考基因组，使用TopHat软件（https://ccb.jhu.edu/software/tophat/index.shtml），将读段与Rap-DB数据库[http://rapdb.dna.affrc.go.jp; IRGSP (v 1.0)]注释的基因进行组装。研究结论：本研究鉴定出受OsTZF8过表达调控的下游候选基因。非转基因对照与OsTZF8过表达转基因植株种植于土壤中培养三周，收获三周龄植株用于RNA测序分析，以鉴定非转基因对照与OsTZF8过表达转基因水稻植株间的差异表达基因。