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在实时荧光定量PCR(qRT-PCR)分析中,选择合适的内参基因是保证检测结果准确性的先决条件。紫云英(Astragalus sinicus)基因组信息匮乏,内参基因的选择尤为重要。本研究筛选了7个持家基因GAPDH、Cons7、ELF1B、Tubulin、Ubiquitin E2、Actin A以及Actin 20,利用qRT-PCR技术研究持家基因的表达情况,并采用GeNorm、NormFinder和Best Keeper软件,分析其在紫云英不同组织(根、茎、叶、花和荚果)和不同非生物胁迫(干旱胁迫、涝胁迫、盐胁迫、脱落酸(abscisic acid,ABA)胁迫)条件下叶片中的表达稳定性。结果表明,7种内参基因在紫云英不同的组织器官和不同的胁迫处理分析中,稳定性表现不尽相同。Cons7在盐胁迫和涝胁迫处理下最稳定,Tubulin、Actin 20和GAPDH分别在不同组织、ABA处理和干旱胁迫下表达最稳定。因此,采用qRT-PCR分析紫云英不同器官组织中的基因表达差异时,可选择Tubulin作为校正内参基因;而比较脱落酸处理、盐胁迫和涝胁迫处理时,可选择Cons7作为校正内参基因;比较干旱胁迫处理时,选择GAPDH作为校正内参基因。本研究结果为研究紫云英不同生理过程中目的基因表达提供了稳定可靠的内参基因。
In real-time quantitative PCR (qRT-PCR) analysis, the selection of a suitable internal control gene is a prerequisite for the accuracy of the test results. Astragalus sinicus genomic information is scarce, the choice of reference genes is particularly important. In this study, seven housekeeping genes, GAPDH, Cons7, ELF1B, Tubulin, Ubiquitin E2, Actin A and Actin 20, were screened by qRT-PCR and analyzed by GeNorm, NormFinder and Best Keeper softwares, Expression stability of leaves in different tissues (roots, stems, leaves, flowers and pods) and different abiotic stresses (drought stress, waterlogging stress, salt stress and abscisic acid (ABA) stress). The results showed that the stability of seven kinds of internal control genes in different tissues and organs of Astragalus sinicus and different stress treatment analysis were different. Cons7 was the most stable under salt stress and waterlogging stress. Tubulin, Actin 20 and GAPDH were the most stable under the different tissues, ABA treatment and drought stress respectively. Therefore, when using qRT-PCR to analyze the difference of gene expression in different tissues of Astragalus sinicus, Tubulin could be chosen as the correct reference gene. When compared with abscisic acid treatment, salt stress and waterlogging stress, Cons7 could be selected as the correction reference gene. In comparison with drought stress treatment, GAPDH was selected as the correct reference gene. The results of this study provide a stable and reliable reference gene for studying the expression of target gene in different physiological processes of Astragalus.