摘要
【目的】筛选适用于东方泽泻实时荧光定量PCR(qRT-PCR)的内参基因,并分析其鲨烯合酶基因(AoSS)的表达特性,为后续研究东方泽泻原萜烷型三萜生物合成调控及诱导机制打下基础。【方法】从东方泽泻转录组中选取内参基因18S rRNA、EF1α、GAPDH、ACT、UBQ和UBC,应用qRT-PCR检测其在不同组织和激素处理下的表达情况,并借助Delta CT、GeNorm、NormFinder、BestKeeper和RefFinder对其表达稳定性进行分析评价。以筛选到的内参基因分析AoSS基因在不同组织中的表达情况。【结果】18S r RNA、GAPDH和EF1α基因在东方泽泻不同组织的表达稳定性较高,但18S rRNA、UBC和EF1α基因在不同激素处理下的表达稳定性均较高。以18S rRNA、GAPDH和EF1α基因为内参分析AoSS基因在东方泽泻不同组织中的表达量情况,结果显示,AoSS基因在东方泽泻不同组织中均有表达,其表达情况基本一致,均在东方泽泻块茎中表达量最高,其次是叶柄和叶,而在根中表达量最低。【结论】18S rRNA、GAPDH和EF1α基因是研究东方泽泻不同组织中基因表达分析的首选内参基因;18S rRNA、EF1α和UBC基因是研究激素处理下基因表达的首选内参基因。AoSS基因的表达具有组织特异性,其可能在东方泽泻的生长发育中发挥重要的调控功能。
【Objective】The present study was to screen reference genes for real-time fluorescence quantitative PCR(qRT-PCR)research in Alisma orientale(Sam.)Juzep,research expression characters of its squalene synthase gene(AoSS)and lay foundation for future studies on regulation mechanism about protostane triterpenes biosynthesis in A.orientale.【Method】18S rRNA,EF1α,GAPDH,ACT,UBQ and UBC were selected as candidate reference genes from the transcriptome databases of A.orientale.The expressions of the six candidate reference genes in different tissues and different hormone treatments were compared by qRT-PCR,then their stability was evaluated by using five statistical softwares:Delta CT,GeNorm,NormFinder,BestKeeper and RefFinder.On this basis,the expression of AoSS in different tissues was analyzed by reference genes which had been screened.【Result】The expression stability of 18S rRNA,GAPDH and EF1a genes were high in different tissues of A.orientale and the expression stability of 18S rRNA,UBC and EF1a genes were high under hormone treatments.GAPDH,EF1αand 18S rRNA as reference genes were used to analyze the relative expression of AoSS in different tissues,the results showed that AoSS was expressed in all tissues,and the trend of expression was basically the same,and it was the highest in tubers,followed by the petiole and leaf,and that in root was the lowest.【Conclusion】In this study,18S rRNA,GAPDH and EF1αgenes are the preferred internal reference genes for gene expression analysis in different tissues of A.orientale.18S rRNA,UBC and EF1αgenes are the preferred internal reference genes for studying gene expression under hormone treatment.The expression of AoSS has a tissue-specificity in various tissues,which may play an important regulatory role in the growth and development of A.orientale.
作者
刘青芝
谷巍
孙云飞
吴启南
巢建国
桑晓华
王小浩
刘琪
LIU Qing-zhi;GU Wei;SUN Yun-fei;WU Qi-nan;CHAO Jian-guo;SANG Xiao-hua;WANG Xiao-hao;LIU Qi(College of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China;Jiangsu Hospital of Chinese Medicine,Nanjing 210029,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2018年第8期1467-1475,共9页
Journal of Southern Agriculture
基金
国家自然科学基金项目(81673534)
江苏省自然科学基金项目(BK20161576)
江苏省中药优势学科Ⅱ期建设项目(PAPD-2014)
江苏省中药资源产业化过程协同创新中心项目(ZDXM-3-24)
