| The brown planthopper(Nilaparvata lugens St?l)is a hemipteran insect pest of rice.Nilaparvata lugens has strong reproductive ability,rapid population development,and strong adaptability to traditional chemical pesticides or resistant rice varieties.Therefore,it is a difficult problem in rice breeding to control this pest.Autophagy plays an important role in maintaining normal physiological functions and coping with stress for cells and organisms.It is of great importance to study the effects of autophagy-related genes on brown planthoppers and to analyze their potential as targets for pest control.In this study,two autophagy-related genes NlATG1 and NlATG13 of the ATG1 kinase complex were screened based on the comparative transcriptome information,analyzing the interaction between brown planthoppers from different rice cultivars.The full-length c DNAs of these two genes were cloned,and the gene expression at different developmental stages and tissues of brown planthopper was analyzedby real-time quantitative PCR(RT-q PCR).The potential of these two genes in the control of brown planthopper was studied by RNAi(RNA interference).The results in detail are as follows:1.Cloning and functional study of the autophagy related gene NlATG1The full-length 2502 bp c DNA of NlATG1 gene was cloned by RACE method.It was predicted that NlATG1 contains a 2040 bp open reading frame that encodes a protein containing 679 amino acids,with a protein kinase domain.Evolutionary analysis found that ATG1 of different species has homology and is most closely related to Recilia dorsalis.RT-q PCR results showed that the NlATG1 gene was expressed at different developmental stages of the brown planthopper,and the expression level was higher in the nymphs,but lower in female and male adults,and the expression level was independent of the wing type.NlATG1 gene was also expressed in different tissues,among which,the head,midgut,ovary and fat body have higher expression levels,and the expression in the thorax was the lowest.N1ATG1 was subjected to RNA interference by microinjection.The results showed that the interference effect was good.The gene expression in the interference group was significantly lower than that in the control group within 8 days.The interference efficiency was highest on the fourth day,and the expression level decreased to 14.6%.Different tissues and organs of Nilaparvata lugens treated for 4days were dissected,and the expression of NlATG1 in each tissue of the interference group was significantly reduced by RT-q PCR analysis.RNAi treatment resulted in a decrease of autophagic vesicles in the midgut epithelial cells,a significant accumulation of glycogen particles,and a decrease in the width and area of mitochondria.At the same time,RNAi treatment caused a decrease in ATP content in brown planthoppers.Interfering with the NlATG1 gene significantly reduced oviposition amount and hatchability.The oviposition amount of ds NlATG1-treated females mateed with ds GFP-treated males,was significantly reduced,only 3% of the control group(ds GFP-treated females mated with ds GFP-treated males);there was no significant difference in oviposition amount between ds NlATG1-treated males mated with ds GFP-treated females compared to the control,,but the hatching rate was only 33.8% to the control group,indicating that RNAi of NlATG1 affects male fertility.Interference with the NlATG1 gene also significantly reduced the survival rate of brown planthoppers,especially females after eclosion.On the 8th day after eclosion,the survival rates of males and females in the control group were86.7% and 93.3%,while the survival rates of females and males treated with ds NlATG1 interference decreased to 6.7% and 58.3%,respectively.In addition,the molting of some dead brown planthoppers did not go well,suggesting that the gene may also be related to molting.2.Cloning and functional study of the autophagy related gene NlATG13The full-length 1325 bp c DNA of the NlATG13 gene was cloned by the RACE method.It was predicted that NlATG13 contains a 1203 bp open reading frame that encodes a protein containing 400 amino acids.A phylogenic tree of ATG13 was constructed based on the amino acid sequences from brown planthoppers and other species,analysis revealed that ATG13 had homology with different species.RT-q PCR results showed that the NlATG13 gene was expressed at different developmental stages of the brown planthopper.The expression level was higher in3-5 instar nymphs,lower in 1-2 instar nymphs and adults.NlATG13 gene is also expressed in different tissues,the relative expression in the head,midgut and fat body were higher,and the expression in the thorax was the lowest.RNA interference was performed on NlATG13 by microinjection.The results showed that the interference effect was well and the interference efficiency was the highest on the 4th day.Compared with the ds GFP control group,the expression level of the ds NlATG13 treatment group decreased to 16.9% on the 4th day.RNAi treatment of the target gene resulted in an accumulation of glycogen particles in the midgut epithelial cells and a decrease in ATP content,but did not result in a decrease in autophagy.Interference with NlATG13 gene significantly reduced the survival rate of brown planthoppers,and the survival rate at day 10 was 41.4% when the control group was 85.6%.In summary,this study cloned two autophagy-related genes Nl Atg1 and Nl Atg13,and reveaded the expression profiles of these two genes.The functions of 2 genes were explored through RNAi,and the effect of autophagy on brown planthopper was explored using epithelial cells of midgut as a model,which provided new ideas for later researches on autophagy of incomplete insect and provided new potencial targets for brown planthopper control. |
