非生物胁迫下青花菜内参基因的筛选  

Screening of internal reference genes under abiotic stress in broccoli

作　　者：焦鹏 刘院梅 裴徐梨 冯鹏宇 唐征[2] 荆赞革 李金林 徐谦[2] JIAO Peng;LIU Yuan-mei;PEI Xu-li;FENG Peng-yu;TANG Zheng;JING Zan-ge;LI Jin-lin;XU Qian(College of Agronomy and Life Sciences,Kunming University,Kunming 650214,China;School of Agriculture and Biotechnology,Wenzhou Vocational College of Science and Technology,Wenzhou,Zhejiang 325006,China)

机构地区：[1]昆明学院农学与生命科学学院,昆明650214 [2]温州科技职业学院农业与生物技术学院,浙江温州325006

出　　处：《西南农业学报》2024年第3期513-523,共11页Southwest China Journal of Agricultural Sciences

基　　金：云南省科技厅基础研究专项青年项目(2019-1-C-25318000002236);云南省地方本科高校(部分)基础研究联合专项(2018FH001-044,202301BA070001-21);浙江省自然科学基金项目(LY18C150006);浙江省十四五农业新品种选育重大科技专项(2021C02065-4-2);温州市科技项目(2019ZX007-2)。

摘　　要：【目的】检测非生物胁迫下常见内参基因的表达稳定性,筛选出青花菜的适宜内参基因。【方法】以青花菜高代自交系“KU19-3”为试验材料,利用已有的转录组数据和文献,筛选出31个候选内参基因,对其在不同非生物胁迫下(盐、干旱、渍水、弱光、高温和低温)的表达水平进行荧光定量PCR检测,采用geNorm、Normfinder和BestKeeper软件分析其表达稳定性,从而筛选出适宜的内参基因。【结果】31个候选内参基因的扩增特异性良好,且表达水平差异明显。其中BOL029171的Ct值最小,表达量最大;而BOL001470的Ct值最大,表达量最小。其他基因的Ct值介于20~40。3种软件综合分析结果表明:BOL034565和BOL008820在高温胁迫下最稳定,BOL043724和BOL005937在弱光胁迫下表达稳定性最好,BOL043724和BOL001470、BOL005937和BOL006136分别在低温和盐胁迫下表达最稳定,BOL024326和BOL025875、BOL008820和BOL001788分别在渍水和干旱胁迫处理下表达最稳定。综合上述得出BOL006136和BOL008820可作为非生物胁迫下青花菜的通用内参基因。【结论】本研究筛选出青花菜在非生物胁迫下通用内参基因是BOL006136和BOL008820,结果可为以后开展青花菜逆境胁迫下相关基因的表达特征研究和青花菜抗逆种质创建提供一定的科学基础。【Objective】In order to detect the stability of common reference genes under abiotic stress,the suitable reference genes were identified in broccoli.【Method】The broccoli inbred line‘KU19-3’was used as the material.Using existing transcriptome data and literatures,31 candidate reference genes were selected and their expression levels were quantified by fluorescent quantitative RT-PCR under different abiotic stresses(salt,drought,waterlogging,weak light,high temperature and low temperature).The expression stability of these candidate reference genes was analyzed using geNorm,Normfinder,and BestKeeper software to identify suitable reference genes.【Result】The results of amplification showed that 31 candidate reference genes had good specificity,and their expression levels were obvious different.The Ct value of BOL029171 was the smallest and its expression level was the largest.Meantime,the Ct value of BOL001470 was the largest and its expression level was the smallest.The Ct values of other genes were between 20 and 40.A comprehensive analysis of three software showed that BOL034565 and BOL008820 had the highest stability under high temperature stress,BOL043724 and BOL005937 exhibited the best stability under weak light stress,BOL043724 and BOL001470,BOL005937 and BOL006136 had the most stable expression under low temperature and salt stress,respectively,and BOL024326 and BOL025875,BOL008820 and BOL001788 demonstrated the most stable expression under waterlogging and drought stress,respectively.Based on the above,BOL006136 and BOL008820 genes could be selected as universal reference genes under abiotic stress in broccoli.【Conclusion】In the study,BOL006136 and BOL008820 were identified as the universal suitable reference genes under different abiotic stresses in broccoli.The result could provide a scientific basis for the future research on the expression characteristics of related genes under different abiotic stresses and establishment of anti-stress germplasm of broccoli.

关 键 词：青花菜 内参基因 实时荧光定量PCR 非生物胁迫 

分 类 号：S635.3[农业科学—蔬菜学]