向日葵BADH基因表达的定量PCR检测方法建立与初步应用

Development and Application of a Real-time RT-PCR Approach for Quantification of BADH gene Expression in Helianthus annuus

向日葵不是盐生作物,但具有较强的耐盐和耐旱能力。甜菜碱醛脱氢酶基因(BADH)是植物重要的抗逆基因之一。检测BADH基因表达有助于研究向日葵对逆境胁迫的响应机制。因此本文以真核延伸因子1A(eEF1A)为内参,采用SYBR Green I荧光染料法,建立向日葵BADH基因表达的定量PCR检测方法。BADH和eEF1A的扩增曲线基线平整、指数区明显,熔解曲线上仅显示单一特异峰,扩增效率分别为1.90和1.96,Cq值平均变异系数分别为0.51%和0.65%。这些数据表明检测方法特异性强、重复性好、结果可靠。应用建立的检测方法初步检测了盐和干旱胁迫对向日葵BADH基因表达的影响,结果显示BADH受盐和干旱胁迫的诱导表达。

Sunflower （Helianthus annuus） possesses the ability to tolerate salt and drought stress, although it is not a halophyte. BADH （betaine aldehyde dehydrogenase） gene is one of the most important stress-resistant genes in plant. Detecting BADH gene expression is useful for researchers to characterize the stress-resistant mechanism of sunflower. Real-time quantitative PCR assay conditions should be properly evaluated before detecting BADH gene expression. So the aim of this study was to develop a real-time PCR approach to detect BADH gene expression in sunflower. A real-time PCR approach for quantification of BADH gene expression was developed with eEFIA （ eu- karyotic translation elongation factor 1A）as reference gene, using SYBR Green I. All amplification curves were S- shaped with three phases：flat baseline, exponential amplification phase and plateau. All melting curves showed only a single peak. The amplification efficiencies of BADH and eEF1A were 1.90 and 1.96 respectively. The average variation coefficients of Cq value were 0.51% ~ 0.65% respectively. The developed approach had advantage of specific, accurate and reproducible. The mRNA expression levels of BADH gene in sunflower were preliminarily detected using the approach. The result showed salt and drought stress induced the expression of BADH gene in leaf.