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柠檬色百合实时荧光定量PCR内参基因筛选
梁蕤1,3,徐雷锋3,毕蒙蒙3,王静3,唐玉超3,郝泽慧3,4,刘一洁3,5,杨盼盼3,明军3*,杜方2*
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(1.山西农业大学 园艺学院, 山西 太谷 030801;2.山西农业大学 城乡建设学院, 山西 太谷 030801;3.中国农业科学院 蔬菜花卉研究所, 北京 100081;4.北京农学院 园林学院, 北京 102206;5.青岛农业大学 园林与林学院, 山东 青岛 266109)
摘要:
为筛选柠檬色百合(Lilium leichtlinii)实时荧光定量PCR稳定的内参基因,以柠檬色百合不同发育时期花被片、根、茎、叶和鳞片为试验材料,测定比较总类胡萝卜素含量;使用RT-PCR和荧光定量PCR检测9个候选内参基因(AP4、Actinβ-TUBCYPeIFGAPDHRH2、UBC和18S)特异性及表达水平,利用geNorm、NormFinder、Bestkeeper、ΔCT程序和RefFinder网站综合评估候选内参基因表达稳定性;选用八氢番茄红素酶基因PSY对内参基因稳定性进行验证,最终确定合适且稳定的内参基因。结果表明:1)柠檬色百合花被片发育过程中ActinAP4为9个候选内参基因中最稳定的内参基因,UBC为最不稳定的内参基因。2)柠檬色百合不同器官间eIFAP4为候选内参基因中最稳定的内参基因,18S为最不稳定的内参基因。3)在柠檬色百合花被片发育过程和不同器官间,以筛选出最稳定的内参基因为参照计算所得PSY基因表达情况与实际测得总类胡萝卜素含量变化趋势一致;以筛选出最不稳定的内参基因作为参照计算所得PSY基因表达情况与总类胡萝卜素含量变化趋势不同甚至相反。研究表明在试验中不能随意使用内参基因,需要在使用前对其进行筛选、评价和验证,同时研究结果为柠檬色百合花被片发育过程和不同器官间基因表达分析提供了稳定的内参基因。
关键词:  柠檬色百合  实时荧光定量PCR  内参基因  类胡萝卜素
DOI:10.11841/j.issn.1007-4333.2023.02.06
投稿时间:2022-08-22
基金项目:山西省基础研究计划(自由探索类)自然科学研究面上项目(20210302123416);国家自然科学基金项目(32172624,31801899);山西农业大学横向科技合作项目(2021 NYGG-1-11)
Validation of reference genes for quantitative real-time PCR in Lilium leichtlinii
LIANG Rui1,3,XU Leifeng3,BI Mengmeng3,WANG Jing3,TANG Yuchao3,HAO Zehui3,4,LIU Yijie3,5,YANG Panpan3,MING Jun3*,DU Fang2*
(1.College of Horticulture, Shanxi Agricultural University, Taigu 030801, China;2.College of Urban and Rural Construction, Shanxi Agricultural University, Taigu 030801, China;3.Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China;4.College of Landscape Architecture, Beijing University of Agriculture, Beijing 102206, China;5.College of Landscape Architecture and Forestry, Qingdao Agricultural University, Qingdao 266109, China)
Abstract:
In order to select and validate the real-time PCR reference genes stably expressed in Lilium leichtlinii, tepals in different stages, root, stem, leaf and scale were taken as study objects. This study measured and compared the total carotenoids contents, analyzed the specificity and the expression levels of AP4, Actin, β-TUB, CYP, eIF, GAPDH, RH2, UBC and 18S in L. leichtlinii by RT-PCR and qRT-PCR. GeNorm, NormFinder, Bestkeeper, ΔCT and RefFinder were used to evaluate expression stability of these candidate reference genes. PSY gene was used to verify the stability of reference genes. The results showed that: In tepals during development of L. leichtlinii, AP4 and Actin were the most stable genes and UBC was the least stable gene among 9 candidate reference genes. In different tissues, eIF and AP4 were the most stable, 18S was the most unstable. In addition, the expression levels of PSY calculated using the most stable reference genes were consistent with the trend of total carotenoids contents during tepal development process and at different tissues. The expression level of PSY calculated using unstable reference genes was different and even opposite to the trend of total carotenoids contents. In conclusion, reference genes should not be used randomly in test, and they need to be evaluated and verified before use. This study provided the stable reference genes for the analysis of gene expression during tepal development and different tissues in L. leichtlinii.
Key words:  Lilium leichtlinii  reverse transcription real-time polymerase chain reaction  reference gene  carotenoid