| Nilaparvata lugens St(?)l is an insect pest of rice with strong reproductive ability and great harm.Reducing the survival and reproduction ability of N.lugens is of great significance for controling the N.lugens and ensuring the rice production.Cuticle plays an important role in the maintenance of insect normal physiological function and insecticide resistance.We previously found that inhibiting of autophagy using RNAi affects the molting process of N.lugens,but the exact mechanism is currently unknown.In this study,based on the previous transcriptome results of the interaction between N.lugens and rice varieties,an autophagy-related gene NlATG5was screened and the temporal and spatial expression pattern of this gene in the N.lugens was analyzed by RT-q PCR.RNA interference(RNAi)was used to study the effect of NlATG5 gene on the fecundity of N.lugens.RNAi combined with transcriptome analysis was employed to investigate the differential expressed genes associated with NlATG5,and preliminarily explore the molecular mechanism of autophagy affecting the molting of N.lugens.According to the transcriptome results,Cpr21L,a cuticular protein gene affected by RNAi of NlATG5 was selected and its core fragment of Cpr21L gene was cloned,and the temporal and spatial expression pattern of this gene in N.lugens was analyzed by RT-q PCR.The effect of this gene on the fertility and cuticular structure of N.lugens was investigated by RNAi.This study aims to evaluate the potential of the Cpr21L gene as targets for the control of N.lugens.The results are as follows:1.Cloning and functional analysis of autophagy related gene NlATG5 in N.lugensFirstly,according to the sequence of NlATG5 gene previously obtained from the transcriptome in the early stage of our research group,primers were designed to clone the full-length c DNA.The results showed that the full-length c DNA sequence of NlATG5 gene in N.lugens was 1334 bp.RT-q PCR analysis showed that the expression of NlATG5 in nymphs and males was relatively stable,and the expression of NlATG5 in females was significantly higher than that in nymphs and males.The expression of NlATG5 in N.lugens head was significantly higher than that in other tissues,but the expression in thorax was the lowest.The NlATG5 gene was interfered by RNAi.The results of RT-q PCR showed that the interference effect was good.Four days after injection of ds NlATG5,the expression of NlATG5 decreased significantly to 41.4%of the ds GFP control group.At this time,the survival rate of the 4th instar nymphs decreased to about50%,all N.lugens died within 8 days and most of the death of N.lugens occured during molting.The decrease of NlATG5 gene expression would also affect the fecundity of N.lugens.Injection of ds NlATG5 decreased the female experimental group(ds NlATG5♀×ds GFP♂),the hatching rate was only 54.1%of the control group(ds GFP♀×ds GFP♂),which was significantly different from the control group.By observing the morphology of eggs under a microscope,we found that ds NlATG5 affected the development of eggs laid by females in the experimental group.Eggs in the experimental group may have stopped developing before 3 days of age compared to eggs in the control group.2.Analysis of participating transcriptome after RNAi of NlATG5 geneTranscriptome analysis showed that RNAi of NlATG5 significantly changed the expression of 24 genes,including 6 up-regulated genes and 18 down-regulated genes.Eight differentially expressed genes(DEGs)were selected from the transcriptome data,and the expression of these eight DEGs was analyzed by RT-q PCR.The correlation coefficient between RT-q PCR and FPKM results was high(R~2=0.86),which showed that the transcriptome data for RNAi of NlATG5 gene was reliable,and the information in the transcriptome could be used for subsequent experimental analysis.Transcriptome analysis showed that the above 24 DEGs were involved in the biological processes of autophagy,DNA replication,transcription,translation,and transportation,etc.In particular,the cuticular protein gene Cpr21L(111051450)and collagenα-1 gene(l11063651)was down regulated in responding to NlATG5RNAi.3.Cloning and functional study of cuticular protein gene Cpr21L in N.lugensFirstly,the core sequence of Cpr21L gene was obtained from the participating transcriptome,and the primers were designed in the software according to the obtained sequence.The results showed that there was a 728 bp core sequence of Cpr21L gene in the N.lugens.RT-q PCR showed that Cpr21L was expressed in both nymphs and adults of the N.lugens.With the development of the N.lugens,the expression of Cpr21L increased in nymphs.The expression of Cpr21L in female adults was generally low,except at the first day of emergence.By contrast to the female adults,the expression of Cpr21L was relatively high in male adults,although it showed a decreasing trend.Spatially,the expression of Cpr21L in the head of the N.lugens was significantly higher than that in other tissues.The expression of Cpr21L in the ovary was the lowest,just 3%of the head,and the expression of Cpr21L in testis and epidermis was higher,but there were no significant differences between testis/epidermis and thorax/fat body/gut.To verify Cpr21L was really a CP gene,RNAi of Cpr21L was performed on the fourth-instar nymphs.RT-q PCR results showed that after 2 days of treatment with ds Cpr21L,the expression of Cpr21L significantly decreased to16.0%compared to the GFP control.RNAi of Cpr21L significantly decreased the survival of the N.lugens after 2 days of treatment with ds Cpr21L,and almost all N.lugens died at day 8.In addition,RNAi of Cpr21L also affected the growth and development of the N.lugens,for example,when the N.lugens of the GFP control group reached the fifth instar,those of ds Cpr21L-treated group remained at the fourth instar.To study the effects of Cpr21L on the structure of cuticle of the N.lugens,the fourth-instar nymphs were injected with ds Cpr21L,using ds GFP as control.Samples were taken 48 hours later for transmission electron microscopy(TEM).TEM results showed many lamellar body-like structures in the cuticular cells of N.lugens injected with ds GFP;in contrast,there were fewer lamellar body-like structures in N.lugens injected with ds Cpr21L.In addition,the volume of lamellar body-like structures in the treated group ds Cpr21L were smaller than those in the GFP control,which indicated that inclusion in the lamellar body-like structure,probably the Cpr21L protein,was reduced.It should be noted that the cuticle of the ds Cpr21L treated group was thinner than that of the control group.In particular,the endocuticle of the N.lugens in the control group showed about 21 endocuticular lamellae,while the endocuticle of the N.lugens injected with ds Cpr21L was only about nine endocuticular lamellae,which may have been caused by the decreased synthesis and secretion of Cpr21L protein.In addition,the decrease of Cpr21L gene expression also affected the fecundity of N.lugens.ds Cpr21L had a significant effect on the male of N.lugens,and it could seriously change the body shape and the morphology of reproductive organs in N.lugens.In terms of appearance,the males injected with ds Cpr21L were shorter and smaller than those in the ds GFP control group,especially in the abdomen.In addition,in males injected with ds GFP,there were six sharp pepper shaped testicular tubes,while in males injected with ds Cpr21L,the testicular tubes were small spherical,and their vas deferens became thinner and had nodules.Taken together,NlATG5 gene mediated autophagy is essential for N.lugens.Autophagy affects the formation of the cuticle probably by regulating the expression,transportation and secretion of cuticle proteins(such as Cpr21L).RNAi of NlATG5and Cpr21L inhibited the fecundity of N.lugens,indicating that NlATG5 and Cpr21L gene have some potential in the controlling of N.lugens. |
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