低磷胁迫下盾叶薯蓣中甾体皂苷生物合成关键酶基因的筛选及鉴定  

Screening and identification of key enzyme genes for steroidal saponin biosynthesis in Dioscorea zingiberensis under low phosphorus stress

作　　者：谢彩侠[1] 李伟峰[1] 龚海燕[1] 李雅静[1] 张娟[1] 刘庆普 雷敬卫[1] 王丰青[2] XIE Cai-xia;LI Wei-feng;GONG Hai-yan;LI Ya-jing;ZHANG Juan;LIU Qing-pu;LEI Jing-wei;WANG Feng-qing(Henan Provincial Engineering Technology Research Center for Quality Control and Evaluation of Traditional Chinese Medicine,Henan University of Chinese Medicine,Zhengzhou 450046,China;Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[1]河南中医药大学河南省中药质量控制与评价工程技术研究中心,河南郑州450046 [2]河南农业大学,河南郑州450046

出　　处：《中国中药杂志》2022年第10期2623-2633,共11页China Journal of Chinese Materia Medica

基　　金：国家重点研发计划项目(2017YFC1700705);河南省高等学校重点科研项目(20A360016)。

摘　　要：为了探讨低磷胁迫下盾叶薯蓣甾体皂苷生物合成关键酶基因的响应特征,该研究设置重度磷胁迫组、中度磷胁迫组与正常磷组3种处理,利用盆栽方式模拟盾叶薯蓣的低磷胁迫实验。分别在胁迫初期、胁迫中期、胁迫后期采取盾叶薯蓣植株,利用分光光度法测定盾叶薯蓣不同组织部位的甾体总皂苷含量,确定对低磷胁迫响应的关键时期,并利用BGI 500测序平台获得响应低磷胁迫关键时期盾叶薯蓣样本的转录本信息,构建转录组数据库,分析盾叶薯蓣甾体皂苷生物合成途径催化酶基因的表达量与甾体总皂苷含量的相关性,筛选甾体皂苷生物合成途径中响应低磷胁迫的催化酶基因,并利用实时荧光定量PCR(qRT-PCR)分析其表达模式。结果发现,低磷胁迫后盾叶薯蓣各组织部位甾体总皂苷含量具有明显的组织特异性,胁迫初期为盾叶薯蓣响应低磷胁迫的关键时期;转录组测序共获得了101593个unigene,有77.35%在NT、NR、SwissProt、KOG、GO、KEGG等数据库中得到注释;根据甾体皂苷的生物合成途径,鉴定了其已知甾体皂苷生物合成途径关键酶基因的256个转录本,将其表达量FKRM与总甾体皂苷含量做相关性分析,发现编码18个催化酶的69个转录本的表达量与甾体总皂苷的含量呈显著相关性;qRT-PCR分析表明,低磷胁迫下几个关键的催化酶基因在4个部位中呈现不同的表达模式。因此,低磷胁迫下盾叶薯蓣中甾体总皂苷含量及调控甾体皂苷生物合成关键酶基因的表达均发生变化,该研究为阐明盾叶薯蓣甾体皂苷成分生物合成响应低磷胁迫的分子机制提供一定的生物学信息。To investigate the responses of key enzymes involved in steroidal saponin biosynthesis of Dioscorea zingiberensis to low phosphorus stress,we designed three treatments of severe phosphorus stress,moderate phosphorus stress,and normal phosphorus level.The D.zingiberensis plants were collected at the early,middle,and late stages of treatment.The content of total steroidal saponins in different tissues of D.zingiberensis was determined by spectrophotometry for the identification of the critical stage in response to low phosphorus stress.BGI 500 sequencing platform was employed to obtain the transcript information of D.zingiberensis samples at the critical stage of low phosphorus stress,and then a transcriptome library was constructed.The correlation between the expression of genes involved in steroidal saponin biosynthesis and the content of total steroidal saponins was analyzed for the screening of the key enzyme genes in response to low phosphorus stress.Further,the expression patterns of these genes were analyzed by real-time fluorescence PCR(qRT-PCR).The content of total steroidal saponins in D.zingiberensis had obvious tissue specificity under low phosphorus stress,and the early stage of stress was particularly important for D.zingiberensis to respond to low phosphorus stress.A total of 101593 unigenes were obtained by transcriptome sequencing,of which 77.35%were annotated in NT,NR,SwissProt,KOG,GO,and KEGG.A total of 256 transcripts of known key enzyme genes in the biosynthetic pathway of steroidal saponins were identified.The expression levels of 69 transcripts encoding 18 catalytic enzymes were significantly correlated with the content of total steroidal saponins.The qRT-PCR results showed that several key enzyme genes presented different expression patterns in four tissues under low phosphorus stress.The results indicated that the content of total steroidal saponins and the expression of key enzyme genes regulating steroidal saponin biosynthesis in D.zingensis changed under low phosphorus stress.This study

关 键 词：盾叶薯蓣 低磷胁迫 甾体总皂苷 关键酶 实时荧光定量PCR 

分 类 号：S567.239[农业科学—中草药栽培]