| Spodoptera exigua(Hubner)is an important polyphagouspest with worldwide distribution in agriculture and forestry which often causes intermittent outbreaks.It has a wide range of hosts,including Salix integra,herbage and wild plants,as well as damaging vegetables and other field crops.At present,the prevention and control of beet armyworm is facing severe challenges in Agroforestry production.Due to its strong reproductive ability and high resistance to a variety of pesticides,it is urgent to develop new prevention and control methods.The synthesis and uptake of vitellogenin is the key process of vitellogenesis,which directly affects the reproductive potential of female adults.Meanwhile,down-regulated expression of the acetylcholinesterase gene will affect insect reproductive indexs.However,SeVg and SeVgR,as the key genes of vitellogenesis of beet armyworm,whether they are involved in acetylcholinesterase regulation of insect reproduction has not been analyzed.In this study,SeVg and SeVgR genes were cloned,expressed in prokaryotes and their expression pattern and functions were also studied.Meanwhile,the effects of SeAce on reproduction and SeVg/SeVgR gene were studied by RNAi method.It can help elucidate the reproductive regulation mechanism of beet armyworm at the molecular level and provides new idea for the prevention and control of beet armyworm.The main results were as follows:1.The full-length sequence of SeVg and SeVgR is cloned by RACE and RT-PCR.The open read frame(ORF)of SeVg was 5247 bp which encoded 1761 amino acids.The predicted molecular weight(MW)was about 200.48 KD and PI value was 8.84.Using NCBI conservative domain search,three relatively conservative functional domains were found in SeVg sequence,namely vitellogenin-N(29-725),DUF1943(779-1036)and VWD(1427-1595).Sequence analysis showed that the SeVgR open reading frame(ORF)was 5445bp,encoding 1814 amino acids,with theoretical MW of about 200.13 Kd and PI value of 4.45.SeVgR has two LBD,containing four and seven class A repeat domains,respectively.Based on phylogenetic tree of lepidopterian Vgs and Vg Rs,the beet armyworm is most closely related to Spodoptera litura and Helicoverpa armigera.2.The sequence of SeVg and SeVgR functional regions is cloned and prokaryotic expression vectors of pCzn1-Vg and Pet15B-Vg R were constructed.The specific and high purity SeVg and SeVgR polyclonal antibodies were prepared at a titer of 1:512000.Analysis of the temporal and spatial expression pattern of SeVg showed that SeVg m RNA was only expressed in the fat body of female insects but not in ovary,midgut and other tissues.QPCR results showed the expression of SeVg m RNA was mainly dectected in the end of the female pupa stage and female adult stage.SeVg m RNA expression level was weak at the end of the female pupa,first increased and then decreased after adult eclosion.It reached the expression peak at 48h after eclosion.Western blotting analysis showed SeVg protein began to synthesis at the end of the pupa and also reached the peak in 48h-adults after eclosion.RT-PCR and q PCR results showed that SeVgR m RNA was only expressed in the ovary of female adults,and it began to be expressed at the end of pupa stage,and reached the peak at 48-h-old adults.The expression pattern of SeVgR protein was basically consistent with that of SeVgR m RNA,which was synthesized at the end of pupa,but the peak of SeVgR expression level was delayed by 12h.3.After in vitro injection,the synthesis of dsRNA of the functional region of beet armyworm SeVgR can significantly reduce the expression level of SeVgR.Meanwhile,the ovary development of beet armyworm was also affected.For 0-h-old adults,24-h-old adults,48-h-old adults,the expression level of SeVgR was decreased by 79.35%,84.22%and 67.68%,respectively,in the Vg R-dsRNA group compared with the control group injected with GFP-dsRNA.By anatomical observation of the ovary of 0-h-old adults,24-h-old adults,48-h-old adults of S.exigua,it was found that the ovarian development progress was significantly delayed in the the Vg R-dsRNA group compared with the control group.Compared with the control group,the length of ovarian tube decreased by 23.92%and the number of mature eggs was relatively fewer in the Vg R-dsRNA group.The size of eggs was also smaller compared with the control group.The average number of eggs laid by a single female adult in the Vg R-dsRNA group was only 170,Which was significantly lower than that in the control group(blank control group and GFP-dsRNA injection,451 and 420).4.The function of SeVg was studied by injecting different concentrations of Vg-dsRNA at different target to Vg functional region sequence in the late pupa stage.The results showed that compared with the control group,only injection of high concentration Vg-dsRNA(10 ug·ul-1)could significantly reduce the expression of SeVg of 0-h-old adults and 24-h-old adults,delay the development of ovary,and decrease the spawning quantity.Similarly,when siRNA was injected at different concentrations,Vg-siRNA at high concentrations could successfully interfere with the expression of SeVg at 24-h-old adults.5.The mortality rate of the ds SeAce injection group was determined after 72h compared with the ds GFP group and blank control group.The mortality rate of the ds SeAce1 and ds SeAce2injection group was 56.7%and 24.6%,respectively,while only 14.7%of the mortality rate was found when injected with ds GFP,suggesting that SeAce1 may play a more important role in larval development by regulating the neurotransmission of choline compared with SeAce2.The expression levels of SeVg of the ds SeAce1 and ds SeAce2 injection group were decreased,and the spawning quantity was also significantly reduced(P<0.05),but there was no significant difference in the expression level of SeVgR(P>0.05).Besides,the SeVg expression level and spawning capacity of the ds SeAce2 injection group were significantly lower than that of ds SeAce1injection group.The results indicate that SeAces play an important role in reproductive and other atypical cholinergic functions by regulating the SeVg expression of S.exigua. |
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