药用大黄全长转录组测序分析及Ⅲ型聚酮合成酶家族基因鉴定  

Full-length Transcriptome Sequencing of Rheum officinale Baill.and Identification of Genes in PKSⅢFamily

作　　者：杜鹃 王雪茹 李依民 杜桥 高静 王楠 彭亮[1] 张岗 DU Juan;WANG Xue-ru;LI Yi-min;DU Qiao;GAO Jing;WANG Nan;PENG Liang;ZHANG Gang(College of Pharmacy,Shaanxi University of Chinese Medicine/Shaanxi Qinling Chinese Herbal Medicine Application Development Engineering Technology Research Center,Xi'an 712046,China;Key Laboratory for Research and Development of"Qin Medicine"of Shaanxi Administration of Traditional Chinese Medicine,Shaanxi University of Chinese Medicine,Xi'an 712046,China;State Key Laboratory of Research and Development of Characteristic Qin Medicine Resources(Cultivation),Shaanxi University of Chinese Medicine,Xianyang 712083,China;State Key Laboratory of Dao-di Herbs,Beijing 100700,China)

机构地区：[1]陕西中医药大学药学院/陕西省秦岭中草药应用开发工程技术研究中心,陕西西安712046 [2]陕西中医药大学陕西省中医药管理局“秦药”研发重点实验室,陕西西安712046 [3]陕西中医药大学省部共建特色秦药资源研究开发国家重点实验室(培育),陕西咸阳712083 [4]道地药材国家重点实验室,北京100700

出　　处：《中国现代中药》2023年第3期533-543,共11页Modern Chinese Medicine

基　　金：国家自然科学基金项目(81973430,82104334);陕西省科技计划项目(2021ZDLSF04-01);陕西省中医药管理局专项(2021-QYZL-02);咸阳市创新服务能力支撑计划(科技领军人才)项目(L2022CXNLRC009);中央本级重大增减支项目(2060302);陕西中医药大学“秦药”品质评价与资源开发学科创新团队项目(2019-QN01)。

摘　　要：目的:利用全长转录组测序技术挖掘药用大黄蒽醌类成分合成中的关键酶Ⅲ型聚酮合成酶(PKSⅢ)家族基因。方法:利用PacBio SequelⅡ平台进行药用大黄全长转录组测序,数据通过非冗余蛋白(NR)、Swiss-Prot、京都基因与基因组百科全书(KEGG)、真核生物相邻类的聚簇(KOG)、基因本体(GO)数据库进行功能注释。筛选PKSⅢ家族基因全长并进行编码蛋白生物信息特征分析,借助MEGA 6.0构建PKSⅢ家族基因系统发育进化树。结合RNA-Seq数据分析,运用TBtools计算PKSⅢ家族基因的相对表达量。结果:共获得原始数据55.50 GB,52960条高质量转录本,平均长度1712.56 bp;50007条Isoforms在NR、Swiss-Prot、KEGG和KOG数据库中得到注释。GO分类注释到生物过程、细胞组分和分子功能3个类别的65个小组。KEGG代谢通路共有17637条转录本被注释于137条通路中,其中标准次生代谢通路15条。筛选到16个含开放阅读框的Isoforms,编码8个PKSⅢ家族基因,包括RoALS1-3(芦荟松合酶)、RoCHS1-3(查耳酮合酶)、RoSTS(二苯乙烯合酶)和RoPKS(聚酮合成酶)等,编码蛋白长度为391~393 aa,无信号肽或跨膜结构域,均定位于细胞质中。编码蛋白序列保守,磷酸化及糖基化位点和数目各异。大多数PKSⅢ家族基因Isoforms在药用大黄根和根茎中表达量高。药用大黄PKSⅢ家族基因与掌叶大黄、虎杖、拟南芥基因汇于一支,亲缘关系近。结论:获得药用大黄全长转录组信息特征,鉴定了8个药用大黄PKSⅢ家族基因,明确了序列及表达特征,为药用大黄蒽醌类成分合成的分子机制研究提供参考。Objective:To identify the genes of the typeⅢpolyketide synthase(PKSⅢ)family critical for anthraquinone biosynthesis in Rheum officinale Baill.by using the full-length transcriptome sequencing technology.Methods:PacBio SequelⅡplatform was employed to sequence the full-length transcriptome of Rh.officinale,and the obtained data were used for annotation against the Non-Redundant proteins(NR),Swiss-Prot,Kyoto Encyclopedia of Genes and Genomes(KEGG),clusters of euKaryotic Orthologous Groups(KOG),and Gene Ontology(GO)databases.After the full-length genes of the PKSⅢfamily were identified,the bioinformatics characteristics of the deduced proteins were analyzed.MEGA 6.0 was used to construct a phylogenetic tree of the PKSⅢgenes.TBtools was employed to determine the relative expression levels of PKSⅢgenes in three different tissues with the RNA-Seq data.Results:A total of 55.50 GB of raw data was obtained,including 52960 high-quality transcripts with an average length of 1712.56 bp.A total of 50007 isoforms were annotated in the NR,SwissProt,KEGG,and KOG databases.Sixty-five GO terms in three categories including biological processes,cellular components,and molecular functions were annotated.A total of 17637 isoforms were annotated in 137 KEGG pathways,of which 15 pathways were standard secondary metabolic pathways.Sixteen isoforms containing open reading frames were screened out,encoding eight PKSⅢgenes:RoALS1-3(aloesone synthase),RoCHS1-3(chalcone synthase),RoSTS(stilbene synthase),and RoPKS(polyketide synthase).The encoded proteins had the lengths ranging from 391 to 393 aa,without signal peptides or transmembrane domains,and were all localized in the cytoplasm.The proteins had conserved sequences,with varied sites and number of phosphorylation and glycosylation.Most PKS gene isoforms had high expression levels in the roots and rhizomes of Rh.officinale.The PKSⅢproteins of Rh.officinale were closely related to the counterparts from Rh.palmatum,Polygonum cuspidatum,and Arabidopsis thaliana.Conclusion:The

关 键 词：药用大黄 全长转录组 Ⅲ型聚酮合成酶 蒽醌 生物信息学 

分 类 号：R282[医药卫生—中药学]