Cloning And Stability Analysis Of Reference Gene From Phyllonorycter Ringoniella

Reference gene is often as the reference to collate the gene expression levels. And it hasbeen applied in the study of gene expression in the field of agricultural pests such assilkworm, bollworm, planthoppers, citrus red mite, etc. Phyllonorycter ringoniella is animportant fruit pest which serious influencing fruit quality. With the development oftechnology, the research has reached genes level. However, the relevant reference gene hasnot been studied; these will seriously affect the accuracy of the experiment. So, this researchcomposed of three experiments, the comparison of RNA extraction method, the referencegene sequence cloned, and the best reference gene type and quantity at different life stages, indifferent tissues and exposed pesticide treatment were selected.The quality of RNA can be a critical factor in gene amplification, cloning analysis andquantitative expression. Therefore, this study compared three RNA extraction methodsthrough the integrity testing and analysis of purity, content. And then find the best way toRNA extraction of P. ringoniella.Select seven reference genes (α-Tubulin, β-Tubulin, β-actin, GAPDH, EF-1-α,18S andPGK) which were commonly used in the field of insects. And then degenerate primers of theseven reference genes were respectively designed to perform gene amplification bytraditional PCR, using the method of homologous genes of the related species. Finally, sevenreference gene segments from P. ringoniella were obtained; then recycling, cloning,sequencing and bioinformatics analysis of the internal genes were carried out.This study detect the expression of seven candidate reference genes at different lifestages, in different adult tissues, and3rd larvae exposed to pesticides from P. ringoniella.After analyzed by GeNorm and NormFinder, we proved that β-tubulin and β-actin is the mostsuitable reference genes at different life stages; β-actin and18S RNA gene is the mostsuitable reference genes in different tissues in adults. Besides, β-actin and β-tubulin canrespond to content variation of genes treated by pesticides.In a word, we compared three RNA extraction methods of P. ringoniella, and clonedseven reference gene fragments as well as analyzed them. These results indicate that the mostsuitable reference genes selecting at different life stages, in different adult tissues and under pesticide treatment from P. ringoniella.