Selection and Validation of Reference Genes for Gene Expression Analysis in Vigna angularis Using Quantitative Real-Time RT-PCR

Adzuki bean (Vigna angularis) is one of the most important legume crops in Asian countries like China, Japan and Korea due to its nutritious protein and starch contents. In spite of its economic importance, gene expression analysis system for gene function verification of adzuki bean is still absent. Therefore, reference genes for gene expression analysis based on the quantitative real time PCR (qRT-PCR) were screened in current study. A total of nine general housekeeping genes, including ACT, Fbox, ZMPP, GAPDH, EF, PP2A, UBC, UBN and PTB were evaluated for their expression stability by qRT-PCR in four adzuki bean cultivars, three different tissues, four abiotic stress and one biotic stress. The best group of candidates as reference genes were as follows: PTB and ACT for different cultivars; EF and UBN for different tissues; ACT and ZMPP for biotic stress and waterlogging stress; Fbox and UBC for salinity-alkalinity stress; Fbox and PTB for drought stress. Our results will provide a more accurate and reliable normalization of qRT-PCR data in adzuki bean.

红小豆（Vigna angularis）因其富含优质蛋白质与淀粉，是中国、日本、韩国等亚洲国家至关重要的豆科作物之一。尽管其经济价值举足轻重，但目前仍缺乏用于红小豆基因功能验证的基因表达分析体系。为此，本研究基于实时定量PCR（quantitative real-time PCR，qRT-PCR）技术，筛选适用于基因表达分析的内参基因。本研究共选取9种常规持家基因，包括ACT、Fbox、ZMPP、GAPDH、EF、PP2A、UBC、UBN及PTB，通过qRT-PCR在4个红小豆品种、3种不同组织、4种非生物胁迫条件及1种生物胁迫条件下，对其表达稳定性进行了评估。筛选得到的最优内参基因组合如下：不同品种条件下为PTB与ACT；不同组织条件下为EF与UBN；生物胁迫与涝渍胁迫条件下为ACT与ZMPP；盐碱胁迫条件下为Fbox与UBC；干旱胁迫条件下为Fbox与PTB。本研究结果可为红小豆qRT-PCR数据的归一化分析提供更为精准可靠的依据。