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1. Selection and evaluation of potential reference genes for gene expression analysis in greenbug (Schizaphis graminum Rondani)
ZHANG Bai-zhong, LIU Jun-jie, YUAN Guo-hui, CHEN Xi-ling, GAO Xi-wu
Journal of Integrative Agriculture    2018, 17 (09): 2054-2065.   DOI: 10.1016/S2095-3119(18)61903-3
摘要433)      PDF    收藏
In order to precisely assess gene expression level, a suitable internal reference gene must be chosen to quantify real-time reverse transcription polymerase chain reaction (RT-qPCR) data.  For greenbug, Schizaphis graminum, a suitable reference gene for assessing the level of transcriptional expression of target genes has yet to be explored.  In our study, eight reference genes, elongation fator 1 beta (Ef1β), TATA box binding protein (TBP), alpha-tubulin (α-TUB), 18S ribosomal (18S), 28S ribosomal (28S), glyceraldehyde-3-phosphate (GAPDH), actin (ACT), and ribosomal protein L18 (RPL18) were evaluated in S. graminum at different developmental stages, tissues, and insecticide treatments.  To further explore whether these genes are suitable to serve as internal control, three software-based approaches (geNorm, BestKeeper, and NormFinder), ?Ct method, and one web-based comprehensive tool (RefFinder) were employed to analyze and rank the tested genes.  The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated according to normalized gene expression data of three target genes, heat shock protein gene (HSP70), cytocrome P450 gene (SgraCYP18A1), and glutathione S-transferase (GST).  We found that the most suitable reference genes varied considerably under different experimental conditions.  For developmental stages, α-TUB and 28S were the optimal reference genes; for different tissues, 18S and ACT were suitable reference genes; for insecticide treatments, 28S and α-TUB were suitable for normalizations of expression data.  In addition, 28S and α-TUB were the suitable reference gene as they had the most stable expression among different developmental stages, tissues and insecticide treatments.  This should be useful for the selection of the suitable reference genes to obtain reliable RT-qPCR data in the gene expression of S. graminum.
 
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2. Insecticide induction of O-demethylase activity and expression of cytochrome P450 genes in the red imported fire ant (Solenopsis invicta Buren)
ZHANG Bai-zhong, KONG Fan-chao, WANG Hua-tang, GAO Xi-wu, ZENG Xin-nian, SHI Xue-yan
Journal of Integrative Agriculture    2016, 15 (1): 135-144.   DOI: 10.1016/S2095-3119(15)61072-3
摘要2119)      PDF    收藏
The red imported fire ant (Solenopsis invicta) is a global major invasive pest, and has caused significant economic, social and environmental impacts since its invasion to mainland of China in 2004. To date, chemical control has been the most effective measure. However, the long-term use of chemicals would lead to an unexpected rebound. To understand the risks and explore the mechanisms of detoxification or induction to insecticides in S. invicta, the O-demethylase activity and expression of cytochrome P450 genes of workers and queens, and the effects of chlorpyrifos and fipronil exposure in workers were investigated. Biochemical assays showed the O-demethylase activity of cytochrome P450 was significantly higher in workers than in queens (1.66-fold), and was significantly induced in workers exposed to chlorpyrifos and fipronil, reaching a maximum (3.00- and 1.95-fold) at 48 h and then decreasing dramatically compared to controls (exposed to acetone counterpart). The relative expression levels of 12 cytochrome P450 expressed sequence tags (ESTs) in workers were significantly higher than in queens (from 2.3- to 36.4-fold). Multiple cytochrome P450 genes (except 9E4) were co-up-regulated (from 1.5- to 2.86-fold) in workers exposed to fipronil. These results indicated that the increased O-demethylase activity may result from the increased transcription levels of cytochrome P450 related to detoxification of insecticides in S. invicta. It appears that cytochrome P450 plays an important role in enhanced metabolic detoxification of insecticides. At the same time, it also provides the theoretical basis for resistance management and rational usage of insecticides to control S. invicta.
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