紫云英实时荧光定量PCR分析中内参基因的选择

发布时间：2018-04-30 13:41

本文选题：紫云英 + 实时荧光定量PCR　；参考：《农业生物技术学报》2017年08期

【摘要】：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。
[Abstract]:In real-time fluorescence quantitative PCRQ RT-PCRanalysis, the selection of appropriate internal reference genes is a prerequisite to ensure the accuracy of the detection results. The genomic information of Astragalus sinicus is scarce, so the selection of internal reference gene is particularly important. In this study, seven housekeeping genes (GAPDHHN Cons7ELF1B tubulin Ubiquitin E2Actin A and Actin 20) were screened. The expression of homemaker gene was studied by qRT-PCR technique. The results were analyzed by using GeNorm Norm Finder and Best Keeper software in different tissues (root, stem, leaf). Stability of expression in leaves under flower and pod stress and abiotic stress (drought, waterlogging, salt stress, abscisic acididae ABA) stress. The results showed that seven internal reference genes were analyzed in different tissues and organs and under different stress treatments. The stability of Cons7 was the most stable under salt stress and waterlogging stress, and the expression of tubulin tin-20 and GAPDH were most stable in different tissues, respectively. Therefore, when qRT-PCR was used to analyze the difference of gene expression in different organs and tissues, Tubulin could be selected as the corrected reference gene, while Cons7 could be selected as the corrected reference gene when compared with abscisic acid treatment, salt stress and waterlogging stress. In comparison with drought stress, GAPDH was selected as the corrected reference gene. The results of this study provide a stable and reliable internal reference gene for studying the expression of target gene in different physiological processes.
【作者单位】：浙江省农业科学院环境资源与土壤肥料研究所;江西省农业科学院土壤肥料与资源环境研究所;中国农业科学院农业资源与农业区划研究所;
【基金】：浙江省自然科学基金(No.LY14D010004) 公益性行业(农业)科研专项(No.201103005)
【分类号】：Q943.2;S551.9

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