基于转录组测序的枇杷五环三萜合成途径差异基因的分析  

Differential Genes Analysis of Pentacyclic Triterpenoid Synthesis Pathway Based on Loquat(Eriobotrya japonica) Transcriptome Sequencing

作　　者：李惠华[1] 常强[1] 王伟[1] 曾碧玉[1] 徐剑[1] 苏明华[1] 

机构地区：[1]福建省亚热带植物研究所/福建省亚热带植物生理生化重点实验室,厦门361006

出　　处：《农业生物技术学报》2018年第2期222-233,共12页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.31501735);药用植物资源的应用研究(No.20150318-1)

摘　　要：枇杷(Eriobotrya japonica)叶是中国传统药用植物资源,五环三萜类化合物(pentacyclic triterpenoids)作为枇杷的主要活性成份具有广泛的药理作用和重要的生物活性,同时也是枇杷三萜类物质的主要组成。经细胞悬浮培养产生的五环三萜类化合物含量远高于原植株。本研究以原植株枇杷叶片、愈伤组织(悬浮培养的必经阶段)、2个富含五环三萜类化合物的调控,共4个样品进行了转录组测序,用生物信息学方法进行Unigene的从头测序拼接,蛋白质功能注释,代谢通路分析。结果共获得123 685个Unigene,平均长度为804.44 bp。三萜合成相关途径中,叶片(样本Ⅰ)分别与培养细胞(样本Ⅱ,样本Ⅲ,样本Ⅳ)的基因表达差异较大,愈伤组织阶段(样本Ⅱ)与富含五环三萜类目标产物的不同调控(样本Ⅲ,样本Ⅳ)之间的基因表达模式相近。在三萜骨架形成和修饰代谢途径中,以样本之间差异基因的交集结合已报道的重要基因进行筛选,从整体上分析了枇杷五环三萜类化合物代谢途径中的关键性酶为乙酰辅酶A酰基转移酶(acetyl-CoA acetyltransferase,AACT),鲨烯合成酶(squalene synthase,SQS),鲨烯环氧酶(squalene monooxygenase,SQE),香树素合酶(amyrin synthase,AS),细胞色素P450(cytochrome P450,CYP450),糖基化转移酶(UDP-glycosyltransferase,UGT),这些酶相关基因或不同家族基因成员的表达水平与目标产物的含量有一定相关性,为进一步深入研究此物种三萜合成途径提供理论依据。同时,枇杷愈伤组织阶段五环三萜类化合物合成途径已经大量开启,是枇杷细胞悬浮培养的重要中间阶段。本研究通过Illumina HiSeq 4000平台,首次利用转录组测序技术对枇杷细胞悬浮培养后五环三萜类活性成分富集的遗传基础进行分析,为进一步完善植物体五环三萜类化合物的合成机制提供理论依据,同时也为枇杷细胞悬浮培养生产萜类物质的人工调控提供基础资Loquat（Eriobotrya japonica） is an important plant species resources of traditional Chinese medicine. It has been reported that the main activity components in E. japonica were pentacyclic triterpenoids,which have been extensively known for its important and wide biological activity. Comparing with original plant plants, the content of tpentacyclic triterpenoids in E. japonica obtained by suspension culture is much higher. In this study, the genetic basis of these active components enrichment after suspension culture wasanalyzed to improve the synthesis mechanism of tricyclic triterpenoids in plant and provide reference for the artificial regulation of terpenoids in large-scale loquat cell suspension culture. The loquat leaves（sampleⅠ）,the loquat callus on solid media（the necessary stage of suspension culture, sampleⅡ）, the loquat suspension cell lines（two regulations rich in pentacyclic triterpenoids, sample Ⅲ and sample Ⅳ） were sequenced by Illumina Hiseq4000. The results are as follows： De novo assembly, protein function annotations, Gene Ontology（GO） annotations and Metabolic pathway analysis（the Kyoto Encyclopedia of Genes and Genomes）of Unigenes were obtained using bioinformatics. A total of 123 685 Unigenes were obtained with an average length of 804.44 bp. The genes related to triterpene synthesis expression profiles of samples Ⅰ and three other cultured cells（samples Ⅱ, Ⅲ, Ⅳ） were significantly different, but the difference was not significant among the three cultured cells. From expression profile of the differentially expressed genes in the skeleton formation and modification metabolic pathways of triterpenoid, 10 potential-selection genes were selected by the intersection of the different expressed genes between the samples and the importance of the genes reported.Those genes are two acetyl-CoA acetyltransferase genes（AACTs）, squalene synthase gene（SQS）, two squalene monooxygenase-like genes（SQEs）, two amyrin synthase genes（ASs）, cytochrome

关 键 词：枇杷 细胞悬浮培养 五环三萜 转录组 

分 类 号：S667.3[农业科学—果树学]