| Eri-silkworm(Philosamia Cynthia ricini) is a kind of silk spinning insect of Lepidoptera Saturniidae. It is not only an important economic insect but also an excellent research object of molecular genetics. The cloning of Philosamia Cynthia ricini acetyl cholinesterase gene(Pcrace1), selection of reference genes for transcription expression analysis in Philosamia Cynthia ricini and comparison of phoxim resistance of Philosamia Cynthia ricini are all very important, which can help to elucidate the metabolic pathway of Philosamia Cynthia ricini at the molecular level. The research will also lay an important foundation for the study of economical traits of the Eri-silkworm, regulation and important economic traits, disease resistance genes of growth and development of Philosamia Cynthia ricini. At the same time, the study on the pesticide resistance of Philosamia Cynthia ricini has certain practical significance of reducing the pesticide poisoning during the pesticide production process. 1、Selection of Reference Genes for Transcription Expression Analysis in Philosamia cynthia riciniReal-time quantitative PCR was carried out in four tissues and nine different developmental stages respectively. First of all, the standard curve was made. Then the relative quantitative approach was used to handle Ct values. At last, the geNorm and NormFinder softwares were combined to analyze the transcriptional expression stability of four genes including β-actin、GAPDH、18S rRNA and 16 S rRNA in different tissues and developmental stages. The results showed that the transcription of β-actin in blood, fat body, midgut, silkgland and larvae, pupae, adult and egg of Philosamia Cynthia ricini is most stable compared with the other there genes, which is suitable tobe choosed as the reference gene for quantitative analysis. 2、Cloning and expressing of Pcrace1 from Philosamia cynthia riciniAcetylcholinesterase gene(Pcrace1) was cloned from Philosamia Cynthia ricini larvae head tissue using RT-PCR and RACE Technology. The length of the cloned cDNA of Pcrace1 was 2374 bp. Its open reading frame(ORF) was 2067 bp, with untranlated regions of 207 bp and 100 bp in 5’ and 3’ ends respectively, which encodes 688 amino acid residues. The encoded protein has a molecular weight of 77.7kD and an isoelectric point of 6.275. A systematic analysis based on the acetyl cholinesterase homologous amino acid sequences showed the closest relationship between Philosamia Cynthia ricini and Cydia pomonella. Pcrace1 gene of Philosamia Cynthia ricini was expressed in different tissues and periods of time, with the highest expression in the head. In addition, the 5th instar larva and the 1st instar larva showed the highest and lowest expression level respectively in the different development periods. 3、Comparison of Phoxim Resistance of Philosamia cynthia riciniPhoxim solution was diluted to eight concentration gradients from 35mg/L to 70 mg/L. Leaf dipping method was used to determine the LC50 of different concentrations of phoxim in 3-day old, 5th instar Philosamia Cynthia ricini within 24 h with a LC50 of 51.81mg/L. Quantitative Real-time PCR was carried out to detect the expression changes of Pcrace1 and Pcrace2 in different tissues(blood, fat body, silk gland, midgut, head) of Philosamia cynthia ricini after intake of 50mg/L phoxim. The result showed that the expression of Pcrace1 and pcrace2 in different tissues are changing after the sddition of phoxim.For example, the expression of Pcrace1 has raised 10.66 times and 26.50 times in head and fat body compared with the controls. Besides, the expression of Pcrace2 has also raised 24.73 times and 14.56 times respectively. However, the expression of Pcrace1 and Pcrace2 has little change in midgut and silk gland and no change in blood. These results above showed that Pcrace1 and Pcrace2 play an important role in metabolic detoxification in the bead and fatty body, a little in the midgut and silk gland and little in the blood. |
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