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 gr...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 （a-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）, ACt 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, a-TUB and 28S were the optimal reference genes; for different tissues, 18S and ACTwere suitable reference genes; for insecticide treatments, 28S and a-TUB were suitable for normalizations of expression data. In addition, 28S and a-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.展开更多
The full-length c DNA of functionally-unknown salivary protein C002 in Schizaphis graminum was cloned using rapid amplification of c DNA ends（RACE） and designated as Sg C002（Gen Bank accession no. KC977563）. It is...The full-length c DNA of functionally-unknown salivary protein C002 in Schizaphis graminum was cloned using rapid amplification of c DNA ends（RACE） and designated as Sg C002（Gen Bank accession no. KC977563）. It is 767 bp long and encodes a protein of 190 amino acid residues with a predicted mass of 21.5 k Da and a predicted cleavage site of N-terminal signal peptide between the 24 th and the 25 th residues. Sg C002 is specifically expressed in salivary gland with the highest level at the 2nd instar. Introducing Sg C002-specific 476-si RNA, but not 546-si RNA to aphids through artificial diet significantly suppressed Sg C002 expression. Silencing Sg C002 gene led to lethality of the aphid on wheat plants, but not on pure artificial diet. Our study demonstrated that artificial diet-mediated RNAi can be a useful tool for research on the roles of genes in aphid salivary gland, and also provided new insights into the characteristics of C002 in wheat aphids.展开更多
基金the National Key Research and Development Program of China (2017YFD0201700)the Key Science and Technology Program (Agriculture) of Henan,China (182102110053)+2 种基金the Major Projects of Henan Institute of Science and Technology,China (14QN024)the Project of High-Level Talent Introduction of Henan Institute of Science and Technology,China (208010616003)the Scientific and Technological Innovation of Henan Institute of Science and Technology,China (208010616005) for the financial support given to the present research work
文摘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 （a-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）, ACt 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, a-TUB and 28S were the optimal reference genes; for different tissues, 18S and ACTwere suitable reference genes; for insecticide treatments, 28S and a-TUB were suitable for normalizations of expression data. In addition, 28S and a-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.
基金supported by the National Natural Science Foundation of China (30971920,31371946)the International Cooperation Project between China and Belgium (2010DFA32810)the Earmarked Fund for Modern Agro-Industry Technology Research System,China (CARS3)
文摘The full-length c DNA of functionally-unknown salivary protein C002 in Schizaphis graminum was cloned using rapid amplification of c DNA ends（RACE） and designated as Sg C002（Gen Bank accession no. KC977563）. It is 767 bp long and encodes a protein of 190 amino acid residues with a predicted mass of 21.5 k Da and a predicted cleavage site of N-terminal signal peptide between the 24 th and the 25 th residues. Sg C002 is specifically expressed in salivary gland with the highest level at the 2nd instar. Introducing Sg C002-specific 476-si RNA, but not 546-si RNA to aphids through artificial diet significantly suppressed Sg C002 expression. Silencing Sg C002 gene led to lethality of the aphid on wheat plants, but not on pure artificial diet. Our study demonstrated that artificial diet-mediated RNAi can be a useful tool for research on the roles of genes in aphid salivary gland, and also provided new insights into the characteristics of C002 in wheat aphids.
