两品种当归转录组分析及抗旱关键基因挖掘

Transcriptome Analysis of Two Cultivars of Angelica sinensis and Excavation of Key Gene for Drought Resistance

目的对当归[Angelica sinensis(Oliv.)Diels]主栽品种“岷归1号”和“岷归2号”进行转录组测序差异比较分析,挖掘当归响应干旱胁迫关键基因。方法以两品种当归的新鲜叶片和根组织为研究材料构建cDNA文库,利用二代高通量测序平台Illumina HiSeq^(TM)4000进行测序分析,从差异表达基因中筛选响应干旱胁迫的关键基因。结果转录组测序共获得584423236条高质量序列,Q20(碱基量≥20%)与Q30(碱基量≥30%)占比分别在97.47%、92.64%以上,鸟嘌呤和胞嘧啶所占比例(GC)含量是42.78%~43.15%。两品种当归叶片和根中共筛选得到1894个差异表达基因(DEGs),数目分别为674和1220个,共有差异表达基因为338个。基因本体(GO)富集结果表明,两品种当归在同一组织部位中的DEGs注释分类主要包括细胞过程、代谢过程和催化活性等功能。京都数据库与基因组百科全书(KEGG)分析发现差异表达基因均在植物-病原互作、植物-MAPK信号通路、苯丙烷生物合成和植物激素信号转导等通路中显著富集,精细分类注释结果与GO及KEGG分析结果趋势相符合。基于功能注释结果,挖掘到抗旱相关基因60个,选取HVA22C、KRP1、PUB23、DREB1B、Bp10通过实时荧光定量聚合酶链反应(qRT-PCR)验证其表达量,结果表明,其基因表达水平与转录组测序基因表达趋势一致。结论两品种当归在脱落酸调节、渗透调节、活性氧清除和其他功能蛋白质调节等抗旱途径方面存在一定差异性,筛选出的抗旱基因可为进一步研究当归响应干旱胁迫的分子机制提供数据参考。

OBJECTIVE To excavate the key genes of drought stress response of A.sinensis,and carry out the comparative analysis with the transcriptome data of the main cultivars‘Mingui 1’and‘Mingui 2’of Angelica sinensis(Oliv.)Diels.METHODS With the fresh leaf and root tissues of two cultivars of A.sinensis as materials,a cDNA library was constructed.The Illumina HiSeq^(TM)4000,a second-generation high-throughput sequencing platform,was used for sequencing analysis,and key enzyme genes in response to drought stress were screened from differentially expressed genes(DEGs).RESULTS A total of 584423236 clean reads were obtained from transcriptome sequencing,in which the percentage of Q20(base amount≥20%)and Q30(base amount≥30%)were above 97.47%and 92.64%,and the GC content ranged from 42.78%to 43.15%.A total of 1894 DEGs were screened from the leaves and roots of two cultivars of A.sinensis,the numbers of which were 674 and 1220,respectively,and they had 338 shared DEGs.The GO enrichment results showed that the annotation classification of DEGs of two cultivars of A.sinensis in the same tissue part mainly included cellular process,metabolic process and catalytic activity.KEGG analysis found that the DEGs were significantly enriched in plant-pathogen interaction,MAPK signaling pathway-plant,phenylpropanoid biosynthesis and plant hormone signal transduction pathways.The detailed classification annotation results were consistent with the trends of GO and KEGG analysis.Based on the functional annotation results,60 drought resistance genes were excavated.HVA22C,KRP1,PUB23,DREB1B and Bp10 were selected to verify their expression levels by qRT-PCR.and the results showed their genes expression level were consistent with the transcriptome sequencing gene expression trends.CONCLUSION The two cultivars of A.sinensis have some differences in drought resistance pathways such as abscisic acid regulation,osmoregulation,scavenging of reactive oxygen species and regulation of other functional proteins,and the screened drought resistance genes can provide data references for further research on the molecular mechanisms of A.sinensis in response to drought stress.