不同氮处理茶树实时定量PCR内参基因筛选和验证

为筛选不同氮处理下茶树(Camellia sinensis)实时荧光定量PCR(q RT-PCR)试验体系中最佳内参基因,以茶树幼叶、成熟叶和幼根为材料,应用qRT-PCR分析GAPDH、18S rRNA、β-actin、RPL13、a-tubulin、Ru BP等6个内参基因在不同氮浓度和氮源处理下的表达变化,借助GeNorm和NormFinder程序对候选内参基因稳定性进行评价。结果表明,在不同氮浓度和氮源处理下,GAPDH、b-actin和RPL13表达稳定性较好,GAPDH+β-actin组合稳定性最佳,可用作茶树基因表达研究的内参基因;而a-tubulin和RuBP表达稳定性较差,不适合做内参基因。为进一步证实上述结果,分别以GAPDH、a-tubulin、GAPDH+β-actin组合为内参基因,分析茶树幼叶中硝酸根转运蛋白基因(CsNRT1.2)和铵根转运蛋白基因(Cs AMT1.1)的表达水平,发现以GAPDH和GAPDH+β-actin组合为内参基因时,目的基因的相对表达量随处理时间延长变化规律基本一致;而以a-tubulin为内参基因时,目的基因的变化规律与前两者存在明显差别。因此,根据特定试验体系选择合适的内参基因对于qRT-PCR定量结果的准确性和可靠性具有重要意义。

Screening and Validation of Reference Genes for Quantitative Real-time PCR Analysis in Tea Plant (Camellia sinensis) under Different Nitrogen Nutrition

The objective of this study was to select the most reliable reference genes for qRT-PCR analysis of target tea plant genes under varying nitrogen source and availability. We chose 6 housekeeping genes which included five commonly used and one new candidates to systematically assess their expression levels at three different tissues (young leaves, mature leaves and roots) under different nitrogen regimes by qRT-PCR. GeNorm and NormFinder software were used to analyze and evaluate the data for reference genes. The results indicated that GAPDH, β-actin and RPL13 are the best reference genes for normalizing target gene expression in tea plant under different nitrogen nutrition, whereas a-tubulin and RuBP are not suitable in many experimental conditions and the best combination (GAPDH+β-actin) was recommended. Meanwhile, the expression levels of CsNRT1.2 and CsAMT1.1 in young leaves of tea plants were analyzed. The results showed that the variation tendency of CsNRT1.2 and CsAMT1.1 are exactly consistent when using GAPDH and GAPDH+β-actin as reference genes. However, the expression levels of these genes are showed significant differences when a-tubulin was used as a reference gene. Thus, validation of suitable reference genes for specific condition can guarantee the accurate quantification of the target genes in qRT-PCR analysis.