| The small brown planthopper (SBPH), Laodelphax striatellus Fallen, as an important rice pest, can not only causes insect damage through host-feeding activities but also transmits virus and then causes rice virus diseases, which result in huge crop loss. Nowadays, efficient control technologies of SBPH 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 that could be used to interfere special life processes by silencing the target genes, besides to reveal the function of genes. Therefore, a new countermeasure of RNAi pest control can be opened up through exploring effective specific target genes and RNAi techniques by further study. Trehalase is an important regulatory enzyme in glucose metabolism of insects, which includes two kinds:soluble trehalase and membrane-bound trehalase. In this paper, we cloned full-length cDNA of trehalase genes of SBPH, and silenced these genes through feeding synthesized double-stranded RNA (dsRNA) to study the toxicological effects of RNAi on them. The main results are summarized as follows:1. By analysing the transcriptome data and carrying out RACE strategy, the full-length cDNA encoding two forms of trehalase were obtained from SBPH. Further analysis found that both of them have the typical characteristics of trehalase genes, shear higher similarity with the trehalase genes from other insects, and showing some phylogenetic relationships among their host species. Of which, the full-length of soluble trehalase gene named LSTre-1was composed of2042nucleotides. Its ORF encodes602amino acids, contains a signal peptide of25amino acids, but no transmembrane region. The full-length of membrane-bound trehalase gene named LSTre-2contains2619nucleotides, encoding618amino acids. It contains a signal peptide of26amino acids and two transmenbrane regions. The full-length cDNA of trehalase genes cloned from SBPH laid the foundation for the further research of RNAi effect.2. Lethal effect of RNAi targeting on the two forms of trehalase genes was studied by feeding the second instar nymphs of SBPH with dsRNA. The results showed that the mortality of the nymphs fed with dsTre-1(38.89%) and dsTre-2(27.72%) increased by24.5and17.4times, respectively, as compared with the control fed with dsGFP (1.95%). These experiments demonstrated that RNAi targeting on trehalase genes could result in significant lethal effect on SBPH, and especially targeting on LSTre-1.3. To demonstrate that the lethal effect by feeding dsRNA was resulted from silencing of trehalase genes, the transcript level of LSTre and the enzyme activities were tested. The results showed that feeding dsGFP had no obvious effect on the gene transcription and enzyme activity of trehalase. However, the hoppers treated with dsTre-1were found decreased their transcript level of the target gene by49.06%of LSTre-1expression, the expression of LSTre-2decreased21.53%and their trehalase activity by25.27%as compared with control, and with dsTre-2the transcript level decreased by17.53%of LSTre-1expression, the expression of LSTre-2decreased41.53%and trehalase activity by25.06%. These results indicated that feeding dsRNA of LSTre-1and LSTre-2could depress target gene expression, decrease trehalase enzyme activities and result in death.4. After confirming the lethal effect of SBPH was caused by silencing of trehalase genes, we further studied the influence of trehalase RNAi to the normal growth and development. The results showed that feeding dsGFP had no significant influence to body weight of the test hoppers, while the body weight of those test nymphs feeding dsTre-1and dsTre-2decreased significantly as compared with control. These results directly proofed that the silence of trehalase genes can not only caused lethal effect by decreasing the mRNA expression and enzyme activity but also prevented the normal growth and development of SBPH. |
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