Expression And Function Analysis Of Four Chitinase Genes In Striped Rice Borer, Chilo Suppressalis

Rice striped stem borer, Chilo suppressalis, is one of the most important rice pests worldwide. The larvae of the rice stem borer feed within plant stems, causing severe rice crop losses annually. In addition, it can damage water bamboo, broad bean, sugarcane, broomcorn and many other economic important crops. In recent years, Chilo suppressalis severely damaged rice in China. Chitin is the endocuticle and exocuticle of insects. It almost occupied 40% of insect cuticle. During insect’s developmental stage, old cuticle is digested while a new one is deposited. Chitinases participate in chitin degradation during the molting process. The rice striped stem borer, Chilo suppressalis, was chosen as the target of our study.1) We obtained four chitinase genes in Chilo suppressalis by bioinformatics methods. We validated the genome sequences by cloning. It demonstrated high identity between the validated sequences and genome sequences. The results revealed that the genome sequence is reliable. The amplified chitinase sequences were used to BLASTX against the GenBank database of NCBI, showing that the similarity was 50-90% with other species. We classified the four genes to three groups according to domain analysis and phylogenetic analysis, indicating that Cs-Chtl belongs to the group III, Cs-Cht3 belongs to the group II, Cs-Cht2 and Cs-Cht4 belongs to the group IV.2) We collected samples at different developmental stages to study the expression profile of four chitinase genes. RT-PCR results revealed that, Cs-Chtl and Cs-Cht3 were highly expressed at pupa stage. Cs-Cht2 and Cs-Cht4 were highly expressed at larval stage. These results showed that Cs-Chtl and Cs-Cht3 is related with pupation and eclosion, and Cs-Cht2 and Cs-Cht4 is related with larval ecdysis.3) We knocked known four chitinase genes in Chilo suppressalis by RNA interference, indicating that Cs-Chtl, Cs-Cht2, Cs-Cht3 and Cs-Cht4 play important roles at different developmental stages. After injection of dsCs-Chtl, the treated group delayed eclosion compared to the GFP group, with abnormal phenotypes. Altogether, we conclude that Cs-Chtl plays important role at eclosion. Injection of dsCs-Cht2 and Cs-Cht4 led to mortality as compared to control group animals, demonstrating that Cs-Cht2 and Cs-Cht4 involved in the larval ecdysis.4) We predicted the miRNAs that targeted on the genes in the pathway of chitin degradation and synthesis. We also predicted the second structure and analyzed the characteristic values for the four miRNAs targeted the chitinase genes. It appears high similarity with other species.The chitinase genes existed mainly in the insect, as there is no chitin in the high animals and plants. Understanding the functional characterization of chitinase and chitinase-like proteins has implications for eco-friendly pest management. With the strengthening of people’s awareness of environmental pollution, there is more urgent demand for eco-friendly pest management. Recent research showed that, insect chitinase and other insecticidal factor has synergistic insecticidal effect, for example, the Baculovirus and Bacillus thuringiensis spores. Studying the RNA interference of chitinase genes, may lead to novel practical applications in managing this insect pest.