Gene Expression Analysis And CDNA Cloning Of Chitinase5and10of White Backed Planthopper,Sogatella Furcifera (Horvath)

The important function of chitinase as a chitin digestive enzyme has attracted increasing research focus as one of the potential targets for controlling many insect pests. The white backed planthopper, Sogatella furcifera (Hemiptera:Delphacidae), destroys plants by extracting fluid as well as ovipositing in tissues and has been considered as an increasingly significant pest of rice in Asia. The damage caused by this insect has greatly affected sustainable rice production and food security in various countries when the world is facing serious challenges such as increasing human population and climate change. Pest management remains a vital strategy in agriculture since most insects continue adapting and evolving to defeat the new methods of pest control. One of the novel strategies used in the control of insect pests is inhibiting their growth and developmental processes through the interference of chitinase synthesis. The study validated two chitinase genes (Cht5and10), determined their expression profile and analyzed the evolution of two chitinase genes.Two chitinase genes, cht5and cht10, were selected for further study. The gene sequences were obtained from transcriptome data. RT-PCR was used to validate the contigs revealing that gene sequences were accurate. Blastx analysis indicated that the two chitinase genes belonged to GH18Chitinase-like family. Two chitinase genes, cht5and cht10, were used to determine their expression pattern in this study. The insects used for analysis were from1st to5th larval stages and adults. At all stages both male and female were used. In general, the expression patterns of both chitinase genes had similar results. Compared with the expression levels at the2nd and3rd stages, Cht10significantly decreased at the4th stage, and then dramatically increased at the5th instar larval stage. The decrease was both in male and female adults. Cht5exhibited the highest mRNA expression during the5th larval stage. Compared with the expression of the2nd and3rd stages, cht5expression level was normal but significantly decreased at the4th stage and increased at the5th stage. After the5th stage, cht5expression level decreased severely at the adult stages. The cht5revealed the highest expression in the5th larval stage than cht10suggesting that cht5might have more important biological function in S. furcifera as compared to cht10. Furthermore, identifying the expression profile of the chitinase at a particular stage of development could help in manipulating the genes for the benefit of insect control.The study obtained21cht5and cht10protein sequences from different species. ORFinder translated nucleotide sequences into protein sequences which were verified using BlastP. A phylogenetic tree was constructed using Mega5.10program. The selected chitinase genes were clustered into two parts consisting of SfuCht5and SfuCht10. Cht5was clustered with Nasonia vitripennis designating a small group and a big group with eleven other species which were Anopheles gambiae, Chilo suppressalis, Ostrinia furnacalis, Choristoneura fumiferana, Papilio xuthus, Agrotis ipsilon, Phyllonorycter ringoniella, Mamestra brassicae, Mythimna separata, Spodoptera exigua, and Spodoptera frugiperda. Cht10was clustered with seven species comprising Plutella xylostella, Camponotus floridanus, Tenebrio molitor, Tribolium castaneum, Culex quinquefasciatus, Monochamus alter natu, and Aedes aegypti. The phylogenetic tree provided evidence of the evolutionary relationships and similarities among the five different insect orders (Lepidoptera, Coleoptera, Diptera, Hemiptera and Hymenoptera).