| Harmonia axyridis Pallas is an important predator for a variety of pests.The application of H.axyridis in the field not only has a strong control effect on aphids and other pests,but also can protect the ecological environment,which is one of the very important biological control methods.One of the keys of insect reproduction is vitellogenesis.Vitellogenin is mainly synthesized in fat body,then secreted into hemolymph,and transported to ovary through vitellogenin receptor-mediated endocytosis,so as to form vitellin which provides nutriments and energy for oocyte maturation and embryo development.Previous studies in our laboratory showed that vitellogenesis of H.axyridis was mainly regulated by juvenile hormone(JH).The sequence and function of vitellogenin gene(Vg)were also analyzed,but the molecular characteristics of vitellogenin receptor(VgR)and the JH regulation mechanism of vitellogenesis have not been clarified.Therefore,in this study,on the basis of feeding on artificial diet,the female adults were dripped exogenous hormone JHIII on the relationship between JH and ovarian development dynamics was explored,and then carried out molecular identification and expression analysis on the reproductive related gene VgR,JH receptor Met and downstream transcription factor Kr-hl.Meanwhile,the effect of JH on the transcription level of target genes was revealed.Finally,the target genes were silenced by RNA interference technology to clarify the role of each target gene in the reproduction of female adults of H.axyridis,which to provide an important theoretical basis for clarifying the regulation of reproductive pathway of H.axyridis by JH.The main results are as follows:1.On the basis of feeding H.axyridis with artificial diet,the female adults on the 2nd day after emergence were dripped with different doses of JHⅢ(40,80,120 and 160 ng,with acetone as the control).The effects of exogenous hormones on the ovarian development of female adults were observed.The results showed that the ovarian development of females was accelerated under 80 and 120 ng JHⅢ,and the promotion effect of 120 ng was better.On the 7th day after the treatment,a large amount of yolk was deposited and the eggs were full,but the vitellogenesis was inhibited at low dose of 40 ng and high dose of 160 ng.At the dose of 120 ng,the length and width of the ovary and the first oocyte of female adults were larger than those of the control group as a whole.On the 7th day after treatment,the length(2.57 mm)and width(2.51 mm)of the ovary were significantly higher than those of the control group(2.00 mm and 1.99 mm),and the length(1.16 mm)and width(0.57 mm)of the first oocyte were significantly higher than those of the control group(0.82 mm and 0.40 mm).2.HmVgR gene(GenBank No.MG787232)was successfully cloned by PCR combined with the transcriptome database of H.axyridis.Its open reading frame(ORF)was 5,447 bases encoding a protein of 1,779 amino acids.It contains signal peptides and conserved domain motifs of low-density lipoprotein receptor family,such as ligand binding domain,epidermal growth factor precursor homology domain,O-linked sugar domain,transmembrane domain and cytoplasmic domain.The HmVgR protein also contains 9 N-glycosylation sites,50 O-glycosylation sites and 90 phosphorylation sites.The predicted protein molecular weight and theoretical isoelectric point were 200.56 kDa and 5.10,respectively.Phylogenetic analysis showed that the HmVgR was clustered within the Coleoptera,and the similarity with Coleoptera insect Colaphellus bowringi VgR was the highest(52.91%).The results of expression profile showed that HmVgR gene was specifically expressed in female adults and their ovaries,which was positively correlated with the ovarian development dynamics of H.axyridis.It was expressed from day 3 of female adult after eclosion,the expression was stable from day 5 to day 13,peaked on day 19 day,and then decreased.3.HmMet gene(GenBank No.MH619532)was successfully cloned.Its ORF was 1,524 bases encoding a protein of 507 amino acids with a predicted molecular weight of 58.14 kDa and a isoelectric point of 8.65,including 4 N-glycosylation sites and 66 phosphorylation sites.HmMet protein belonged to bHLH-PAS family,contained bHLH,PAS-A,PAS-B and PAC conserved domains,and no signal peptides.Phylogenetic tree analysis and multi sequence comparison showed that HmMet was clustered within Coleoptera and had the highest similarity with Coleoptera Tribolium castaneum Met(49.71%),and domain comparison showed that PAS-B was the most conservative,while PAS-A was relatively low.The expression profiles at different developmental stages showed that the HmMet mRNA expression increased from day 1 to day 3 of female adult after eclosion,the first expression peak appeared on day 5,followed by a slight fluctuation(7～19 days),and then decreased after the expression peaked on day 21;the results of tissue expression profile showed that the expression of HmMet gene was the highest in ovary of female adults.The ORF of HmKr-hl gene(GenBank No.MK629763)was 1,453 bases encoding a protein of 482 amino acids,with 42 phosphorylation sites but no N-glycosylation sites and signal peptides.The HmKr-h1 protein contained eight typical C2H2 conserved zinc finger domains with a molecular weight of 54.16 kDa and isoelectric point of 8.87.The phylogenetic results showed that HmKr-h1 had the closest genetic relationship with Coleoptera,and the highest similarity with Coleoptera insect T.Castaneum(70.49%),in which Zn1 domain was the least conservative.The results of expression profiles at different developmental stages showed that the expression was the highest in female adults,which with a small expression from day 1 to 3,followed by an upward trend,the first expression peak appeared on day 7,followed by a downward trend(9～17 days),and then a downward trend after the expression peak appeared on day 19,which was consistent with the dynamic change of endogenous JH titer of H.axyridis;The results of tissue expression profile showed that the expression level was higher in fat body of female adult.4.JHⅢ experiment showed that the gene expressions of HmMet,Hm Vg1,Hm Vg2 and HmVgR were significantly up-regulated on day 5 after 120 ng dose treatment,which was 2.78,13.14,21.48 and 8.28 times higher than that of the control group,respectively,while the gene expression of HmKr-hl was not different from that of the control group.It is speculated that the gene transcription level is highly expressed before yolk deposition,and JH participates in the transcriptional expression of Vg and VgR genes by inducing the expression of Met gene.5.Using RNA interference technology,it was confirmed that HmMet,HmKr-hl and HmVgR genes were very important in the reproductive process of H.axyridis.Silencing HmMet gene significantly reduced the relative expression of HmKr-h1,HmVg1,HmVg2 and HmVgR genes;silencing HmKr-hl gene resulted in a significant decrease in the transcription levels of HmVgl,HmVg2 and HmVgR genes,and inhibited the HmMet mRNA level;injection of dsHmVgR effectively silenced HmVgR gene and significantly inhibited the transcription level of Hm Vgl and Hm Vg2 genes.The silencing of target genes can delay ovarian development and reduce the fertility of female adults.Compared with dsGFP control(6.33 d),the pre-oviposition period was significantly prolonged by 1.67 and 2.00 d under dsMet and dsVgR treatment,respectively,but there was no significant difference under dsKr-h1 treatment;the total number of eggs laid within 10 days after injection of dsMet,dsKr-h1 and dsVgR were 92.67,207.33 and 258.83 respectively,which were significantly lower than those in dsGFP control group(684.83);the hatching rates of eggs treated with dsKr-hl and dsVgR were 81.03%and 49.05%respectively,which were significantly lower than that of dsGFP control(94.88%),but there was no significant difference between dsMet treatment and control.In conclusion,through the study on the transcription level and function of HmMet,HmKr-h1 and HmVgR genes,and the effect of JH on the transcription and expression of target genes,this paper preliminarily reveals the molecular mechanism of JH regulating the reproductive development of female adults of H.axyridis by its receptor Met gene and downstream transcription factor Kr-h1. |
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