不同氮处理茶树实时定量PCR内参基因筛选和验证

发布时间：2018-06-18 23:01

本文选题：茶树 + 内参基因　；参考：《茶叶科学》2016年01期

【摘要】：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。
[Abstract]:In order to screen the best internal reference genes of tea Camellia sinensis (Camellia sinensis) real-time fluorescence quantitative PCR system, the young leaves, mature leaves and young roots of tea plants were used as materials. QRT-PCR was used to analyze the expression changes of six internal reference genes GAPDHN 18s rRNAand 尾 -actinus RPL13a tubulin Ru BP under different nitrogen concentration and nitrogen source treatment. The stability of candidate internal reference genes was evaluated by GeNorm and Norm Finder program. The results showed that the combination of GAPDH 尾 -actin and GAPDH 尾 -actin had the best stability under different nitrogen concentration and nitrogen source treatment, but the expression stability of a-tubulin and RuBP was poor, so it was not suitable to be used as internal reference gene. In order to confirm the above results, GAPDH 尾 -actin combinations were used as internal reference genes, respectively. The expression levels of nitrate transporter gene (CsNRT1.2) and ammonium transporter gene (Cs AMT1.1) in young tea leaves were analyzed. It was found that when GAPDH and GAPDH 尾 -actin combinations were used as internal reference genes, GAPDH and GAPDH 尾 -actin combinations were used as internal reference genes. The relative expression of the target gene was basically consistent with the treatment time, but when a-tubulin was used as the internal reference gene, the change rule of the target gene was obviously different from the former two. Therefore, it is important for the accuracy and reliability of the quantitative results of qRT-PCR to select the appropriate internal reference gene according to the specific test system.
【作者单位】：中国农业科学院茶叶研究所国家茶树改良中心;
【基金】：浙江省茶树农业新品种选育重大科技专项(2012C12905) 国家茶叶产业技术体系(nycytx-23)
【分类号】：S571.1

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