Gene Cloning And Functional Analysis Of Two Dsrna-Binding Proteins In Rice Planthoppers

The rice planthopper, Laodelphax striatellus Fallen (SBPH) and Nilaparvata lugens Stal (BPH), as important rice pests, can not only cause insect damage through host-feeding activities but also rice diseases by transmiting plant virus, which result in huge crop loss. Nowadays, efficient control technologies for rice planthoppers are needed to substitute traditional chemical control because of the gradually enhanced resistance caused by long-term use of insecticides. RNA interference (RNAi) has been demonstrated to interfere special life processes by silencing the target genes, which could be used not only to reveal the function of genes, but also provide a possible way for pest control. However, to realize the pest control with gene interference, the method efficiency and bio-safity must be confirmed first. Therefore, this study cloned from SBPH the homologous gene of TRBP and STAU, which were reported as key factors in RNAi in different organisms. And if these two genes founctioned in the rice planthopper RNAi were confirmed by feeding dsRNAs with chidinase gene as the report gene. This study would throw light on finding the key factor for gene interference and the way for safe and efficient use of RNAi technology in pest control. The main results are summarized as follows:1. Gene cloning of two dsRNA binding proteins in rice planthoppersAccording to the reported TRBP and STAU sequences, homologous genes from the transcriptional data of rice planthoppers were searched and PCR confirmed. Then, the special primers were designed, and the full length of the two genes was cloned with5’and3’RACE technology were used to clone, which were named as LsTRBP and LsSTAU and registed in GenBank by the number JN882650and JN882649, respectively. Sequence analysis showed that these two genes were2548bp and2650bp in length and both consist of an open reading frame. LsTRBP encoded a protein of339amino acids, with its theoretical isoelectric point PI=8.86, molecular mass of37.1kDa. LsSTAU encoded a protein of743amino acids, with PI=9.26, molecular mass of81.2kDa. Both the two genes had a characteristic dsRNA binding domain in their sequences. Phenogenical analysis showed that the similarity of these two genes with their corresponding homologous genes from other organisms was closely related to the phenogeny relationship of their hosts. Among the genes tested, the cloned genes shared highest similarity with those corresponding genes from Tribolium castaneum.2. Function analysis of TRBP in Laodelphgax striatellusThe founction of TRBP in the RNAi processes of SBPH was researched by feeding dsRNA. According to the results of previous research, Chitinase gene (Chi) the efficient interference gene resulting in death was selected as the report gene. The related conditions for feeding interference experiment were determined by the preparing tests. The results found that the ingestion of dsChi alone can result in significant mortality for test larvae (21.2%), which is4.1times that of the control (5.2%). Ingesting dsTRBP alone (5.6%) had no obvious effect. When dsChi was fed mixed with dsTRBP, its killing effect dispeared (2.3%). Thus, these results indicated that interference of TRBP gene can significantly reduce the gene interference effect of dsChi, which means that SBPH not only emplys TRBP as an important interference function protein, but also has the similar dsRNA metabolism and gene interference mechanism as other animals.3, Function analysis of STAU in Laodelphgax striatellusBy feeding dsRNA, the founction of STAU in the RNAi processes of SBPH was studied. Also the chitinase gene (Chi) was selected as report gene. The results found that ingestion of dsChi alone can resulted in significant mortality for the tested larvae (21.2%). But ingestion of dsSTAU alone (6.9%) did not cause any obvious mortality as compared with the control (5.2%). When dsChi was fed mixed with dsSTAU, the mortality of the tested larvae (16.7%) decreased, but was still high and not significantly different as compared to the treatment with dsChi alone. These results indicated that interference of STAU gene had little effect on the RNAi in SBPH.4, Function analysis of TRBP and STAU in Nilaparvata lugensBy feeding the brown planthopper with different dsRNAs the founction of TRBP and STAU genes in the RNAi processes was studied. The same report gene Chi was used and the experiments were carried out under the conditions desibned according to relevant documents of the brown planthopper RNAi. Similarly as in SBPH, the results found that ingestion of dsChi alone can cause a significant mortality for the test larvae (45.2%), which is6.4times that of the control ingesting dsEGFP(7.1%). Ingestion of dsTRBP alone (11.9%) or dsSTAU alone (12.7%) had no significant effect on mortality as compared with the control. Ingestion the mixture of dsChi and dsTRBP (18.6%) can significantly reduce the mortality caused by dsChi alone. But though the mixture of dsChi and dsSTAU (35.8%) could reduce the mortality, the reducing effect was not significant as compared with the control. Checking the expression level of the relevant target genes in the brown planthopper found that all dsRNAs could efficiently silence their own target gene. The silence of TRBP gene can affect Chi gene interference effect by dsChi, but not the silence of STAU gene.To sum up, this study proved that there were two kinds of dsRNA binding proteins, TRBP and STAU homologous, in rice planthoppers, and the whole length of the two genes were successfully cloned from SBPH. Through RNAi tests it was showed that TRBP was an important protein founctioning inRNAi process of rice planthoppers. It also revealed that planthoppers had similar dsRNA metabolism control mechanism as other animals. The function of STAU is different from TRBP, its action mechanism remains to be studied. The results founded the basis for further study on insect RNAi mechanism, and the development of safe and effective RNAi pest control technologies.