药用大黄幼苗转录组高通量测序及蒽醌类生物合成基因筛选

High-throughput Transcriptomic Sequencing of Rheum officinale Baill. Seedlings and Screening of Genes in Anthraquinone Biosynthesis

目的获得药用大黄(Rheum officinale Baill.)转录组信息特征及蒽醌类生物合成通路基因。方法采用高通量测序平台Illumina Hi Seq^(TM)2000 150PE进行大黄幼苗转录组测序并进行系统的生物信息学分析,结合文献报道筛选蒽醌类生物合成通路基因。结果转录组测序共得到9. 34 Gb有效数据(clean data),62 240 114条高质量读长(reads)(SRA数据库注册号SRP160410)。Trinity组装成82 229个单基因(unigene),平均长度1 082 nt。BLAST分析显示64 361条unigene(78. 23%)在NR、NT、Swiss-port、PFAM、KOG数据库得到注释信息;基因本体论(gene ontology,GO)分类的生物过程、细胞组分和分子功能进一步分为57分支; KEGG分析发现1 253条unigenes参与17个次生代谢标准通路。195条基因编码蒽醌类生物合成相关的MVA、MEP、莽草酸和聚酮途径关键酶,166条CYP450基因、66条糖基转移酶基因可能参与次生代谢物的修饰。MISA分析发现转录组包含16 378个简单重复序列(simple sequence repeats,SSRs)。结论利用高通量测序技术和生物信息分析获得药用大黄转录组信息特征,为后期功能基因鉴定、次生代谢途径解析及其调控机制研究奠定基础。

OBJECTIVE To obtain the transcriptomic characteristics of Rheum officinale Baill.and screen anthraquinone biosynthesis genes.METHODS R.officinale Baill.seedlings were subjected to high-throughput transcriptomic sequencing via Illumina Hi SeqTM2000 150 PE platform,followed by systemic bioinformatics analyses.Then the anthraquinone biosynthesis genes were uncovered based on previously published studies.RESULTS The transcriptome sequencing analyses produced a total of 9.34 Gb clean data,resulting in 62 240 114 clean reads(SRA acc.SRP160030).Using trinity assembling,a total of 82 229 unigenes were finally obtained,with an average length of 1 082 nt.BLAST analyses showed that 64 361 unigenes(78.23%)were successfully annotated against the NR,NT,Swiss-port,PFAM,and KOG databases.GO classification of biological process,cellular component,and molecular function further contained 57 subgroups.KEGG analysis revealed that 1 253 unigenes were involved in 17 secondary metabolic standard pathways.Totally 195 unigenes were found to encode key enzymes of MVA,MEP,shikimic acid,and polyketide pathways that were mostly correlated to the anthraquinone biosynthesis.It was found that 166 and 66 unigenes,encoding cytochrome P450 s and UDP-glycosyltransferases respectively,were related with the modification of secondary metabolites in R.officinale Baill.Meantime,MISA prediction produced a number of 16 378 simple sequence repeats(SSRs).CONCLUSION The transcriptomic characteristics of R.officinale Baill.seedlings are revealed by high-throughput sequencing technology along with bioinformatics analyses,better for further functional gene characterization,secondary metabolism pathway dissections,and their regulatory mechanisms in this species.