| Nilaparvata lugens is one of the most important worldwide economic pests of rice.Nilaparvata lugens often suck the host plant juice,destroy their nutrition transmission,damage the host plant,and seriously affect the yield and quality of rice.At present,the control of Nilaparvata lugens mainly depends on chemical pesticides,but the abuse of pesticides leads to the increased resistance of them to pesticides,which makes the control of them more difficult.Therefore,it has become a goal for scientist to seek a new method of preventing Nilaparvata lugens with high efficiency and environmental protection.MIF proteins belong to the secretory protein family and have been shown to be highly conserved among the major species and are an important gene affecting the feeding behavior of insects,but so far there are few studies which have been made to discover the MIF genes of Nilaparvata lugens.A MIF gene of N.lugens was cloned by bioinformatics analysis,and its expression and function were studied.The main findings are as follows:1. Cloning and sequence analysis of BphMIF1 geneBphMIF1 gene was cloned from Nilaparvata lugens,which consists of 360 bases and encodes a 119 amino acid protein with a molecular weight of 12.95 kda,isoelectric point of 7.239.the amino acid sequence alignment revealed that BphMIF1 was highly conserved with other stinging mouthparts insects and evolved close to the MIF genes from B.tabaci.2. Analysis of spatiotemporal expression of BphMIF1 geneAccording to the analysis of the expression of BphMIF1 gene of b Nilaparvata lugens at different age and adult stage,the expression of BphMIF1 gene was significantly different in all ages,and there was no significant difference in the two wing-type in adult stage,but the expression was higher than that in other ages at the end of 4th nymphoid stage.Guess the reason may be that the stinger mouthpiece insect develops to mature nymph stage,need a lot of nutrition,the process of increasing intake,stimulate its secretion of more saliva,resulting in up-regulation of BphMIF1 gene expression..Besides,to elucidate the expression of BphMIF1 in different parts of the body,the expression of head,thorax and abdomen was analyzed.The results showed that the expression of BphMIF1 gene in the abdomen of Nilaparvata lugens was higher than that in other parts,but there was no significant difference between head and thorax.3. Prokaryotic expression of BphMIF1 geneThe BphMIF1 was constructed and pET-28a-BphMIF1 with pET-28a prokaryotic expression vector.The pET-28a-BphMIF1 plasmid was transformed into escherichia coli receptive state and SDS-PAGE analysis was carried out after small scale induced expression.BphMIF1 protein was expressed in supernatant and precipitate.after adjusting the induction conditions,it can be expressed in large quantities after 28℃,0.5mmol/l IPTG induction for 4 h,and there is no obvious effect on the expression when the concentration is adjusted to 1.0 mmol/l.The IPTG of 0.5 mmol/l was chosen as the final concentration,and the induction time was 4 h4. Preliminary identification of BphMIF1 gene’s functionRNAi experiment on brown planthopper with ds RNA,mixed artificial feed of synthetic BphMIF1 gene.The q PCR results showed that the expression of BphMIF1 gene was down-regulated in 48 h after silencing,and reached very significant level after 72 h.The survival and weight of Nilaparvata lugens in experimental group and control group decreased significantly. |
