摘要
【目的】分析藤茶高通量转录组序列,从中挖掘出黄酮类化合物合成相关基因,为进一步揭示藤茶黄酮类化合物生物合成调控机制提供理论参考。【方法】分别采集藤茶的幼叶和成熟叶,提取其总RNA构建c DNA文库,采用Illumina HiSeqTM4000高通量测序平台对藤茶叶片进行转录组测序,经过滤处理后运用Trinity组装,将获得的Unigene与Nr、Nt、Pfam、Swiss-Prot、GO、KO和KOG 7个数据库进行比对注释,并预测Unigenes的编码区序列(CDS);基于KEGG信号通路富集分析,发掘藤茶黄酮类化合物合成相关基因。【结果】藤茶叶片转录组测序获得82126236条原始测序序列(Raw reads),过滤处理后得到80156972条高质量序列(Clean reads),进一步组装拼接得到92472条Unigenes,平均长度为1208 bp,N50长度为1780 bp,其中,至少在1个数据库注释的Unigenes有84217条,占Unigenes总数的91.07%,有8944条Unigenes在7个数据库均被注释,占Unigenes总数的9.67%。在GO数据库成功注释的41116条Unigenes可分为生物学过程、细胞组分和分子功能三大类,共56个小类;在KOG数据库注释的14553条Unigenes可分成25类,其中,一般功能预测注释成功的Unigenes最多(1946条);其次是翻译后修饰、蛋白质翻转、分子伴侣(1776条),参与次生代谢物质的生物合成、转运和降解的Unigenes较少,仅有319条;KEGG信号通路富集分析发现,共有15262条Unigenes注释到128条KEGG信号通路,以注释为代谢的Unigenes最多,为8694条,其中筛选获得有98个黄酮类化合物合成相关基因,分别编码苯丙烷代谢通路的3种关键酶和类黄酮代谢通路的14种关键酶。藤茶叶片转录组Unigenes与Swiss-Prot和Nr数据库比对,获得52582条CDS序列,ESTScan 3.0.3预测获得35535条CDS序列。【结论】藤茶在细胞过程、代谢过程、单有机体过程、细胞和细胞部分、结合和催化活性能力分布的基因较丰富,在一般功能、翻译、翻译后修饰、蛋白质翻转及分子伴侣的基因表达量较高,具有较强的碳水化合物代谢能力。多种关键酶基因参与藤茶黄酮类化合物的生物合成,推测其生物合成途径存在多条分支,调控机制也较复杂。
【Objective】The study aimed to obtain the high throughput transcriptome sequence information and explore the genes related to the flavonoid biosynthesis in Ampelopsis grossedentata(Hand.Mazz.)W.T.Wang.It provided the theoretical basis for further revealing the biosynthetic pathway and regulation mechanism of flavonoids in A.grossedentata.【Method】The young and mature leaves of A.grossedentata were collected respectively for the extraction of total RNA which was used to construct cDNA library.The transcriptome of A.grossedentata leaves was sequenced using an Illumina HiSeqTM 4000 high throughput platform.The sequences were assembled with Trinity after data filtering.The obtained Unigenes were compared and annotated with seven databases,including Nr,Nt,Pfam,Swiss-Prot,GO,KO and KOG,and the coding region sequences(CDS)were predicted by Blast and ESTScan software.KEGG signal pathway enrichment analysis was used to identify genes related to flavonoid synthesis in A.grossedentata.【Result】A total of 82126236 raw reads were obtained with the transcriptome sequencing.After reads filtering,a total of 80156972 clean reads were obtained,which were used to assemble and merge into 92472 Unigenes with an average length of 1208 bp and a N50 length of 1780 bp.Of these unigenes,84217(91.07%)were successfully annotated in at least one of the Nr,Nt,Swiss-Prot,KEGG,COG and GO databases,and 8944 unigenes(9.67%)were annotated in all databases.Among them,41116 Unigenes annotated in GO database were matched to 56 sub-classes of biological function,cell component and molecular function,and 14553 unigenes annotated in KOG database were divided into 25 sub-classes.The number of the unigenes annotated in the general function was the greatest(1946);the post translational modification,protein turn over and chaperones was the second(1776);the unigenes involved in the biosynthesis of secondary metabolites,transportation and degradation were less(only 319).The KEGG signal pathway enrichment analysis results showed that a total of 15262 unigenes were participated in 128 KEGG pathways.The unigenes annotated with metabolism were the greatest in number(8694),98 of which participated in the flavonoid synthesis pathway of A.grossedentata.They encoded three key enzymes of phenylpropane metabolic pathway and 14 key enzymes of flavonoid metabolic pathway.By comparing with Swiss-prot and Nr database,52582 CDS were obtained.A total of 35535 CDS sequences were obtained via ESTScan 3.0.3 prediction.【Conclusion】A.grossedentata has abundant genes related to cell process,metabolic process,single organism process,cell and cell part,binding and catalytic activities.It shows a high level of gene expression in general function,translation,post translational modification,protein turn over,chaperones.And it has a strong carbohydrate metabolic ability as well.Many key enzymes are involved in the biosynthesis of flavonoids in A.grossedentata,suggesting that there might be multiple branches in the biosynthetic pathway,and the regulation mechanisms might also be complex.
作者
许明
杨志坚
黄学敏
郑金贵
XU Ming;YANG Zhi-jian;HUANG Xue-min;ZHENG Jin-gui(Key Laboratory of Crop Biotechnology,Fujian Province University,Fuzhou 350002,China;College of Agriculture,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Forestry Bureau of Yongchun County,Fujian Province,Yongchun,Fujian 362600)
出处
《南方农业学报》
CAS
CSCD
北大核心
2020年第8期1797-1805,共9页
Journal of Southern Agriculture
基金
国家科技支撑计划项目(2013BAD01B05)
福建农林大学科技创新专项(KFA17424A)。
关键词
藤茶
转录组
黄酮类化合物
基因挖掘
高通量测序
Ampelopsis grossedentata(Hand.Mazz.)W.T.Wang
transcriptome
flavonoids
gene mining
highthroughput sequencing
