Gao Xue-Ke, Zhang Shuai, Luo Jun-Yu, Wang Chun-Yi, Lü Li-Min, Zhang Li-Juan, Zhu Xiang-Zhen, Wang Li, Lu Hui, Cui Jin-Jie
Institute of Cotton Research, Chinese Academy of Agricultural Sciences, State Key Laboratory of Cotton Biology, Anyang, Henan 455000, China.
Institute of Cotton Research, Chinese Academy of Agricultural Sciences, State Key Laboratory of Cotton Biology, Anyang, Henan 455000, China.
Gene. 2017 Dec 30;637:211-218. doi: 10.1016/j.gene.2017.09.057. Epub 2017 Sep 28.
Lysiphlebia japonica (Ashmead) is a predominant parasitoid of cotton-melon aphids in the fields of northern China with a proven ability to effectively control cotton aphid populations in early summer. For accurate normalization of gene expression in L. japonica using quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), reference genes with stable gene expression patterns are essential. However, no appropriate reference genes is L. japonica have been investigated to date. In the present study, 12 selected housekeeping genes from L. japonica were cloned. We evaluated the stability of these genes under various experimental treatments by RT-qPCR using four independent (geNorm, NormFinder, BestKeeper and Delta Ct) and one comparative (RefFinder) algorithm. We identified genes showing the most stable levels of expression: DIMT, 18S rRNA, and RPL13 during different stages; AK, RPL13, and TBP among sexes; EF1A, PPI, and RPL27 in different tissues, and EF1A, RPL13, and PPI in adults fed on different diets. Moreover, the expression profile of a target gene (odorant receptor 1, OR1) studied during the developmental stages confirms the reliability of the chosen selected reference genes. This study provides for the first time a comprehensive list of suitable reference genes for gene expression studies in L. japonica and will benefit subsequent genomics and functional genomics research on this natural enemy.
日本柄瘤蚜茧蜂（Lysiphlebia japonica (Ashmead)）是中国北方棉田棉瓜蚜的主要寄生蜂，具有在初夏有效控制棉蚜种群的能力。为了使用定量逆转录聚合酶链反应（RT-qPCR）对日本柄瘤蚜茧蜂的基因表达进行准确归一化，具有稳定基因表达模式的内参基因至关重要。然而，迄今为止尚未对日本柄瘤蚜茧蜂的合适内参基因进行研究。在本研究中，从日本柄瘤蚜茧蜂中克隆了12个选定的看家基因。我们使用四种独立算法（geNorm、NormFinder、BestKeeper和Delta Ct）和一种比较算法（RefFinder），通过RT-qPCR评估了这些基因在各种实验处理下的稳定性。我们鉴定出在不同阶段表达最稳定的基因：DIMT、18S rRNA和RPL13；在不同性别中为AK、RPL13和TBP；在不同组织中为EF1A、PPI和RPL27，以及在取食不同食物的成虫中为EF1A、RPL13和PPI。此外，在发育阶段研究的一个靶基因（气味受体1，OR1）的表达谱证实了所选内参基因的可靠性。本研究首次提供了一份适用于日本柄瘤蚜茧蜂基因表达研究的合适内参基因综合列表，将有利于对这种天敌后续的基因组学和功能基因组学研究。
