谷子萌发期响应干旱胁迫的基因表达谱分析

Gene Expression Profiling of Foxtail Millet(Setaria italica L.) Under Drought Stress During Germination

【目的】谷子是一种耐旱作物,通过二代测序技术获得大量的谷子萌发期响应干旱胁迫的差异基因,进而挖掘谷子萌发期抵御干旱的关键基因及其相关的分子机制。【方法】以晋谷45为材料,谷子萌发期分别用18%PEG-6000干旱胁迫(处理组)和蒸馏水(对照组)处理种子并测定1、10和18 h种子的SOD、POD和CAT活性。SOD活性用氮蓝四唑(NBT)法测定,POD活性用愈创木酚法测定,CAT活性用比色法测定;对萌发10和18 h种子的对照组和处理组构建c DNA文库并进行差异基因表达谱分析;利用Bowtie将reads比对到参考基因组,采用RSEM对bowtie的比对结果进行表达量估计;使用DESeq进行差异表达基因分析;利用NR、Swiss-Prot、KEGG、COG和GO在线数据库对差异基因进行功能注释,挖掘调控谷子萌发的关键基因;利用q RT-PCR验证测序结果的可靠性。【结果】处理组的SOD活性整体比对照组高,而POD活性和CAT活性与之相反;随着萌发时间的变化,SOD活性在不断地增加,但CAT和POD活性逐渐减小。基因表达谱序列与所选参考基因组序列高度一致,基因表达呈现出高度不均一性。通过高通量测序最后获得35 470个基因,以RPKM≥0.01为筛选标准,对照样本中分别筛选出24 030和24 486个表达基因,PEG干旱胁迫处理10和18 h的样本分别筛选出24 019和23 877个表达基因;差异表达基因分析表明,谷子萌发10和18 h分别筛选出456和545个差异基因,其中87和267个上调表达基因,369和278个下调表达基因;GO功能显著性富集分析表明,差异基因主要涉及代谢过程,细胞进程和响应刺激;KEGG富集分析表明,差异基因参与到苯丙烷代谢和植物激素信号转导过程;通过q RT-PCR对5个差异基因在干旱胁迫下种子萌发时的表达分析表明,其表达趋势与表达谱分析结果基本一致。【结论】差异表达基因广泛涉及到糖、蛋白质、核酸等生物大分子代谢、次生代谢和能量代谢等过程;Sn RK2和PAL可能在干旱胁迫下调节种子的萌发。

【Objective】Foxtail millet（Setaria italica L.）is a drought tolerant crop.The objective of this research is to get a lot of differently expressed genes during germination in response to drought stress by high-throughput sequencing,then to obtain the key gene and the related molecular mechanism at seed germination stage in foxtail millet under drought stress.【Method】Seed of Jin Gu45was treated with 18%PEG-6000（PEG-stress）and distilled water germination（control sample）at 1 h,10 h and 18 h as a test material,and the activities of SOD,POD and CAT were measured,respectively.SOD activity was assayed by nitro blue tetrazolium（NBT）method.POD activity was determined by guaiacol method,and the activity of CAT was measured by colorimetric method.Sample of control and PEG-stress that germinated for 10h and 18h were used to construct c DNA library by gene expression profiling technology.We compared reads to the reference genome by using Bowtie and analysed the result by using RSEM.Differential expression analysis used DESeq.The functional annotation of differently expresse genes were obtained by using NR,Swiss-Prot,KEGG,COG and GO online databases.The key genes that regulate germination in foxtail millet was obtained through analyse DEGs.The reliability of sequencing results was comfirmed by q RT-PCR.【Result】The SOD activity of PEG-stress sample was higher than that in the control sample,but the activity of POD and CAT were lower than that in the control group.With the time of germination changes,the activity of SOD was increased,but the activity of CAT and POD was gradually decreased.The sequences of gene expression profile was highly consistent with the selected reference genome sequence,and the gene expression was highly heterogeneous.Expression analysis showed a total of 35 470 genes,and with the selection criteria of RPKM≥0.01,there were 24 030and 24 486 genes in the control samples and 24 019 and 23 877 genes in the samples under PEG drought stress,respectively.456 and545 DEGs were screened out during millet germination at 10h and 18h under drought stress,in which 87 and 267 DEGs were up-regulated and 369 and 278 DEGs were down-regulated.GO enrichment analysis showed that these DEGs were mainly relating to metabolism process,cell stimulation and response process.The KEGG enrichment analysis showed that these DEGs were associated with phenylpropanoid metabolism and plant hormone signal transduction.The results obtained from five genes tested by RT-PCR agreed with the trend of regulation identified by gene expression profile.【Conclusion】DEGs were widely involved in the metabolism of biomacromolecule such as sugar,protein,nucleic acid,secondary metabolism and energy metabolism.Sn RK2 and PAL genes may regulate seed germination in foxtail millet under drought stress.