Screening and Validation of Housekeeping Genes of the Root and Cotyledon of Cunninghamia lanceolata under Abiotic Stresses by Using Quantitative Real-Time PCR.
Screening and Validation of Housekeeping Genes of the Root and Cotyledon of Cunninghamia lanceolata under Abiotic Stresses by Using Quantitative Real-Time PCR.
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非生物胁迫下杉木根和子叶看家基因的筛选及验证
DOI：
10.3390/ijms17081198
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发表时间：
2016-07-28
影响因子：
5.6
通讯作者：
Wan Y
中科院分区：
文献类型：
--
作者：
Bao W;Qu Y;Shan X;Wan Y
杉木（Cunninghamia lanceolata）是柏科（Cupressaceae）的一种速生且具有重要商业价值的针叶树。由于杉木全基因组序列的缺乏和遗传背景信息的相对贫乏，有必要鉴定和分析合适的管家基因（HKG）的表达水平，作为精确分析的内参。根据数据库分析和转录组测序结果，我们选择了5个候选HKG（Actin、GAPDH、EF 1a、18 S rRNA和UBQ）在杉木及其近缘种中具有保守性序列，并对其进行了定量分析。通过qRT-PCR测定了这些HKG在5种不同非生物胁迫下不同时间间隔的根和子叶中的表达水平。使用以下算法对数据进行统计学分析：Normalization、BestKeeper和geNorm。最后，应用RankAggreg合并从三个程序生成的序列，并根据共有序列对这些序列进行排序。这些HKG的表达水平在不同的非生物胁迫下表现出不同的稳定性。其中，Actin是根中最稳定的内参基因，GAPDH是子叶中最稳定的管家基因。我们还描述了一种基于其他非模式植物的从头测序数据库选择HKG的实验程序。
Cunninghamia lanceolata (Chinese fir) is a fast-growing and commercially important conifer of the Cupressaceae family. Due to the unavailability of complete genome sequences and relatively poor genetic background information of the Chinese fir, it is necessary to identify and analyze the expression levels of suitable housekeeping genes (HKGs) as internal reference for precise analysis. Based on the results of database analysis and transcriptome sequencing, we have chosen five candidate HKGs (Actin, GAPDH, EF1a, 18S rRNA, and UBQ) with conservative sequences in the Chinese fir and related species for quantitative analysis. The expression levels of these HKGs in roots and cotyledons under five different abiotic stresses in different time intervals were measured by qRT-PCR. The data were statistically analyzed using the following algorithms: NormFinder, BestKeeper, and geNorm. Finally, RankAggreg was applied to merge the sequences generated from three programs and rank these according to consensus sequences. The expression levels of these HKGs showed variable stabilities under different abiotic stresses. Among these, Actin was the most stable internal control in root, and GAPDH was the most stable housekeeping gene in cotyledon. We have also described an experimental procedure for selecting HKGs based on the de novo sequencing database of other non-model plants.
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