Cloning And Founctional Analysis Of Two Genes Involved In Dsrna Transport In Rice Planthopper

RNAi is defined that endogenous or exogenous double-stranded RNA (dsRNA) induced intracellular mRNA to specific degradation, resulted in silencing the target gene, and generated a corresponding functional phenotype deficiency.Small brown planthopper (Laodelphax striatellus, SBPH) and brown planthopper (Nilaparvata lugens, BPH) are both belong to Delphacidae, Fulgoroidea, Cicadomorpha, Hemiptera. And they are global important pests of grain crops, not only can damage crop by piercing, but also spread the virus. On the control of rice planthoppers, the application of chemical pesticides is the main method, which caused not only serious environmental pollution, destruction of ecological balance, but also the threat on health of human and other animal by increasing pesticide residues. Therefore, it is necessary to replace traditional pest control technique by more clean and safe control technology. In recent years, the control of rice planthoppers with RNAi technology has been probed.The intercellular transporting of dsRNA is the key step for inducing efficient RNAi. Based on the previous studies of the dsRNA transport, this study with planthoppers as test animal tried to confirm if sid-1-like and chc are involved in dsRNA transport in living insects, and found the base for further work to find practical ways to enhance the RNAi efficiency and safety. The main findings are summarized as follows:l.Clonning of cDNA full-length of sid-1-like and clathrin heavy chain gene from L. striatellusThe clathrin heavy chain(chc) and sid-1-like gene fragments were first searched from the transcriptome data of L. striatellus and verified by PCR experiments. Then, the specific primers were designed to clone the cDNA full length by RACE, and the sequences were analyzed with ready softwares in internet. The results showed that the cloned chc was5,347bp in length with an open reading frame (ORF) encoding a protein of961amino acids. The molecule of the encoded protein weighed110,1078kD showed PI of5.36, and had the characteristic clathrin7-fold repeat regions. The clustering analysis showed that it was homologous with the chc of other biological species, and gathered into one large class with those of other insects, which sheared92%or more similarity. The cloned sid-1-like was2,515bp in length, with a ORF encoding a protein of648amino acids. Its molecular weight was52,0588kD and PI was8.98. Topological analysis showed that this gene similar with the sid-1gene of nematode and cotton aphid had the charateristic11transmembrane regions. The clustering analysis showed that this gene was homologous with the sid-1-like genes of other biological species, with the similarity between22%-81%among those of insects. These results indicate that the cloned genes were chc and sid-1-like of L. striatellus, named Lschc and Lssid, respectively. This study laid the foundation for designing dsRNA and further reaserching on the role of these genes in L. striatellus RNAi.2.Effect of silencing sid-1-like or chc on RNAi in L. striatellusWith Lschi as a repoter gene, if silence of Lssid and Lschc has influence on the RNAi by dschi was checked. The results showed that the mortality of the larvae ingesting dssid or dschc (5.25%and3.98%) was not significantly different with that of the control ingesting dsEGFP (5.07%), and the relative expression level of Lschi was also similar (1.33,1.19and1.0). This indicated that ingestion of dssid or dschc separately did not affect Lschi expression level and did not change the mortality of the test larvae. However, ingesting dschi not only significantly inhibited the expression level of Lschi to40%that of the control, but also enhanced the mortality significantly to22.78%, which is4.6times that of the control ingesting dsGFP. When dschi was ingested together with dssid or/and dschc, its inhibition on the expression of Lschi and the lethal effect on test insect both dropped significantly. The corrected mortality of the larvae ingesting dssid+dschi (6.65%), dschc+dschi (6.61%) and dschi+dssid+dschi (7.43%) was significant lower than those ingesting dschi. Unanimously, the relative expression level of Lschi in these larvae (0.77,0.64and0.81) was significant higher than those ingesting dschi. None significant difference was found beween treatments with dssid+dschi, dschc+dschi and dschi+dssid+dschi. In addition, adjustment of the ratio of different dsRNA in mixture was found had no significant influence on the RNAi effect too. Thus, silencing Lssid or Lschc could depress significantly the RNAi in the L. striatellus, and to the similar degree, It obviously indicated that Lssid and Lschc are both the important function genes involved in RNAi of L. striatellus.3.Effect of silencing sid-1-like and chc on RNAi in N. lugens With Nlchi as a repoter gene, the effect of silencing Nlsid-1and Nlchc on RNAi of dschi was determined. The results showed that the larvae ingesting dssid (8.05%) and dschc (15.03%) showed obvious mortality as compared with those of control larvae ingesting dsEGFP (2.84%). But their Nlchi expression level (1.09and1.26) was similar to those treated with dsGFP(1.21). This indicated that ingestion of dssid or dschc separately did not affect the expression level of chi though it resulted in a little morality. However, ingestion of dschi separately could significantly inhibit the expression of Nlchi (33%of the control), and could increase the mortality significantly (28.29%), as much as14times that of the dsEGFP control. When dschi was mixed with dssid or/and dschc, its effect on depressing Nlchi expression and enhancing the mortality of the test larvae had both dropped significantly. The corrected mortality had no significant difference between the treatments with dssid+dschi(16.07%), dschc+dschi (19.63%) and dschi+dssid+dschi (19.65%), but was significant lower than ingesting dschi (28.29%), and higher than ingesting dsGFP (2.84%). It got the unanimous results to detect the Nlchi relative expression. There was no significant difference between the treatments of feeding three different mixtures (1.06,0.77and1.14), but were significant higher than dschi treatment and lower than the control. Thus, silence of Nisid and Niche could affect significantly the RNAi in N. lugens, and the inhibitory effect of silencing Nisid was a little better than silencing Niche. These results also suggested that Nisid and Niche are both the important function genes invoved in RNAi of N. lugens. In summary, this study had cloned the sid-1-like and clathrin heavy chain gene of L.striatellus, confirmed that the interference of these two genes can significantly reduce the RNAi effect of the two planthoppers. It reveals that sid and chc gene are both important functional genes involved in insect RNAi, and laid the foundation for further study on the mechanism for RNAI in insects and efficient use RNAi technology in entomology.