Chen Canbin, Wu Jingyu, Hua Qingzhu, Tel-Zur Noemi, Xie Fangfang, Zhang Zhike, Chen Jianye, Zhang Rong, Hu Guibing, Zhao Jietang, Qin Yonghua
1State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables/Key Laboratory of South China Horticultural Crop Biology and Germplasm Enhancement, Ministry of Agriculture, College of Horticulture, South China Agricultural University, Guangzhou, 510642 China.
2French Associates Institute for Agriculture and Biotechnology of Drylands, The J. Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, 84990 Sede Boqer, Israel.
Plant Methods. 2019 Jul 8;15:70. doi: 10.1186/s13007-019-0455-3. eCollection 2019.
A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in quantitative real-time PCR (qRT-PCR) analyses. However, there is no absolute universality in reference genes among different species. It's hard to find an ideal reference gene to fit for different tissues and growth periods. Pitaya () is commercially produced as a new fruit crop at a large scale in tropical and subtropical regions. To date, there is no report on the identification of the most reliable reference genes for qRT-PCR normalization in pitaya.
In this study, six candidate reference genes i.e. , , , - and were selected from thirty-nine typical candidate reference genes to determine the most stable reference genes for qRT-PCR normalization in different tissues, temperature stresses and fruit developmental stages of pitaya. Among the six candidate reference genes, and - were the most stable gene according to calculations of three statistical methods (GeNorm, NormFinder and BestKeeper) while and showed the lowest expression stability. The six candidate reference genes were further validated by comparing expression profiles of key genes related to betalain biosynthesis at flesh coloration stages of Guanhuahong () and Guanhuabai () pitayas. was recommended the best reference gene for accurate normalization of qRT-PCR data.
In this study, the stability of the selected reference genes for normalizing the qRT-PCR data were identified from pitaya. was the most stably expressed genes in different tissues and fruit developmental stages in pitaya. The present work provides the first data of reference gene identification for pitaya and will facilitate further studies in molecular biology and gene function on and other closely related species.
合适的内参基因是保证定量实时荧光定量PCR（qRT-PCR）分析结果准确可靠的重要前提。然而，不同物种间内参基因不存在绝对的通用性。很难找到一个适用于不同组织和生长时期的理想内参基因。火龙果（）作为一种新型水果作物在热带和亚热带地区大规模商业化种植。迄今为止，尚无关于火龙果qRT-PCR标准化最可靠内参基因鉴定的报道。
本研究从39个典型候选内参基因中筛选出6个候选内参基因，即、、、-和，以确定火龙果不同组织、温度胁迫和果实发育阶段qRT-PCR标准化最稳定的内参基因。根据三种统计方法（GeNorm、NormFinder和BestKeeper）的计算，6个候选内参基因中，和-是最稳定的基因，而和的表达稳定性最低。通过比较“关花红”（）和“关花白”（）火龙果果肉着色阶段与甜菜碱生物合成相关关键基因的表达谱，进一步验证了这6个候选内参基因。推荐作为qRT-PCR数据准确标准化的最佳内参基因。
本研究鉴定了火龙果中用于qRT-PCR数据标准化的选定内参基因的稳定性。是火龙果不同组织和果实发育阶段表达最稳定的基因。本研究首次提供了火龙果内参基因鉴定的数据，将有助于对火龙果及其他近缘物种进行进一步的分子生物学和基因功能研究。
