| Locusta migratoria is a worldwide agricultural pest with a wide area of disaster relief and serious disasters.In addition to its clustering and migratory nature,the locust has a strong reproductive capacity,which is responsible for the large population and serious damage.Intersegmental membrane plays an important role in insect movement,mating and spawning.The abdomen stretching into the soil depends on the ductility of the intersegmental membrane.The length of the female locust in adult is about 2 cm-3 cm,and when it is laying,the abdomen can extend to 8 cm-10 cm.This large extension is related to the highly stretched membrane(intersegmental membrane)between the body segments.In this paper,we studied the transcriptome sequencing of male and female intersegmental membrane in adult,and screened two differentially expressed cuticle protein genes.The molecular characteristics and expression characteristics of these two cuticle protein genes were analyzed and based on RNA interference(RNAi)technology combined with transmission electron microscope(TEM),scanning electron microscope(SEM)and animal behavioral intelligence real-time monitoring technology to explore its membrane structure formation and oviposition behavior in the interfacial biological functions.The main results are as follows:(1)Screening and identification of differentially expressed genes in intersegmental membrane of male and female in L.migratoriaThe results of intersegmental membrane ductility measurement showed that there were significant differences in the interfacial ductility of male and female.The intersegmental membrane samples of male and female were taken,and the total RNA was extracted for transcriptome sequencing.A total of 428 differentially expressed genes in intersegmental membrane of male and female locust were obtained.The cuticle protein genes Lm Abd-1 and Lm Abd-6 were found and their different expression quantity reached 40% and66.5%,respectively in intersegmental membrane.RT-q PCR results showed that the expression levels of Lm Abd-1 and Lm Abd-6 in the female intersegmental membrane were 1.5 and 125.6 times that of the male,respectively.(2)Molecular properties and expression analysis of LmAbd-1 and LmAbd-6Bioinformatics analysis showed that both LmAbd-1 and LmAbd-6 gene sequences contain two exons,and their amino acid sequences contain one signal peptide and one chitin binding domain 4;among them,Lm Abd-1 is a glycosylation-modified cuticle glycoprotein,which belongs to the CPR family RR-1 subclass cuticle protein.Different tissue expression analysis showed that Lm Abd-1 and Lm Abd-6 were highly expressed in the cuticle(especially intersegmental membrane)and had differences in male and female expression(female was significantly higher than male).Else,Lm Abd-1 and Lm Abd-6 were highly expressed at the 4-5,5-6,6-7 abdominal segments;Expression analysis at different developmental stages indicated that Lm Abd-1 and Lm Abd-6 were highly expressed in the early stage of the nymphal stage,and the expression level in the adult stage was significantly higher than that in the nymphal stage.The results of juvenile hormone analogue(JHA)induced showed that the expression levels of Lm Abd-1 and Lm Abd-6 were up-regulated,respectively,and interfered with the receptor gene Met and its corresponding gene Kr-h1,Lm Abd-1 and Lm Abd-6 were down-regulated respectively,which indicated that JH signaling can regulate the expression of LmAbd-1 and LmAbd-6.(3)Polyclonal antibodies preparation and localization of LmAbd-1 and Lm Abd-6The prokaryotic expression vector was constructed,transformed into E.coli,and the recombinant proteins of Lm Abd-1 and Lm Abd-6 were expressed and purified.The recombinant protein was immunized to New Zealand white rabbits and mice respectively to prepare Lm Abd-1 rabbit polyclonal antibody and Lm Abd-6 mouse source.Lm Abd-1 and Lm Abd-6 were tissue-localized by immunohistochemistry using the prepared polyclonal antibodies.The results showed that both Lm Abd-1 and Lm Abd-6 were present in the endocuticular layer and overlapped with chitin,but not in the abdomen cuticle.(4)Effects of LmAbd-1 and LmAbd-6 on the development and intersegmental membrane structure in L.migratoriaRNA interference specific primers of LmAbd-1 and LmAbd-6 were designed,ds RNA was synthesized in vitro,and ds GFP was used as a control.10 μg ds RNA was injected into the body from the junction of the 2nd and 3rd abdominal segments in 5th instar female nymph with 3 parallel experiments were setted up.Lm Abd-1 RNA interference,Lm Abd-6 RNA interference,Lm Abd-1 and Lm Abd-6 RNA interference,observed the development of the molting and intersegmental membrane in L.migratoria.The results showed that after silencing Lm Abd-1 and Lm Abd-6,the abnormal phenotype of internode film exposure and intersegmental membrane and cuticle could not be properly nested in the abdomen of females.Compared with the control group,the scanning electron microscope showed that the cuticle of the intersegmental membrane of Lm Abd-1 and Lm Abd-6 was irregularly arranged,the interlamellar gap was reduced,and distortion and overlap appeared.Transmission electron microscopy results it was shown that,compared with the control group,the intersegmental membrane endocuticle layer of the treatment group was reduced,so that the entire cuticle layer structure was thinned.(5)Effects of Lm Abd-1 and Lm Abd-6 on the oviposition behavior in L.migratoriaUsing the above RNA interference method,after injecting ds RNA to interfere with the target gene(3 sets of parallel experiments: Lm Abd-1 RNA interference,Lm Abd-6 RNA interference,Lm Abd-1 and Lm Abd-6 RNA interference),the female locusts from the molting to the adult Put it into the incubator with video monitoring system to continue normal feeding,and put the same number of normal male adult locusts,set ds GFP control experiment,observe and record the interfacial film extension,mating,deep underground times and spawning time and other behavioral data,the number of female adults in the soil and the time of spawning in the soil during the unit time;after the end of spawning,the number of oocysts and the number of eggs in the soil of three parallel experimental groups and the control group were counted,and females were dissected and the number of residual eggs in the abdomen was counted.The results showed that compared with the control group,after silencing Lm Abd-1 and Lm Abd-6 respectively,the number of times the abdomen protruded into the soil significantly increased,and the time per time into the soil was significantly shortened,and the eggs produced by locusts were significantly shortened.The number of capsules and the number of eggs were significantly reduced,and the oocysts that did not normally produce remained in the ovary of the locust.In this paper,studied the molecular characteristics and expression characteristics of Lm Abd-1 and Lm Abd-6,learned the effects of intersegmental membrane structure and the the oviposition behavior in male and female locusts by ds Lm Abd-1 and ds Lm Abd-6,which aimed to search the new molecular targets for locust control and provides new ideas and methods for effective management of locust disasters. |
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