Molecular Cloning And Functional Analysis Of Three Genes In Chitin Synthesis Pathway In Bactrocera Dorsalis(Hendel)

The oriental fruit fly, Bactrocera dorsalis Hendel, belonging to Bactrocera macpuart in Tetrphitidae of Diptera, infecting a variety of fruits and vegetables, is a worldwide pest. Once intruded into our country, it quickly spread to Guangdong, Guangxi, Taiwan, and other coastal regions. Up to now, it has continued to spread to the north of China, including Jiangsu, Zhejiang, Hubei and other provinces or cities. It causes huge economic losses because of the larvae feeding in the internal of the fruits and vegetables. It is difficult to control the pest using traditional chemical due to that it is hard to find it in the fruits. As friendly to environment and harmless to human, insect growth regulator will become to a main direction researches in the future. The targets of the insect growth regulator are different, including the one-insect chitin. Chitin, widely exists in arthropods, nematodes and yeast, has aroused the researchers’ extensive interest due to particularity existence. It has a lot of advantages of researching and designing new type environmental protection pesticides to achieve the goal of pest prevention and control in the field. Chitin biosynthesis is a complex process which needs participation of eight different enzymes at least. Insects can’t survive from lacking of chitin if chitin production was blocked by any step of chitin synthesis. In this paper, we studied three genes in the chitin synthesis pathway (chitin synthase2, CHS2; glucose-6-phosphate isomerase, G6PI; UDP-N-acetylglucosamine pyrophosphorylase, UAP) using molecular cloning, quantitative expression, food and no food stimulation, as well as chitin content and inhibitor bioassay. Here, we did a preliminary study on these genes in oriental fruit fly. The mainly results are as follows:1. Full-length cDNA obtains and sequence analysis of three chitin synthesis gene BdCHS2, BdG6PI and BdUAP in B. dorsalis.A large fragment of cDNA sequences from several contigs were taken from the known transcriptome dataset. The missing part of the cDNA was obtained by rapid amplification cDNA end (rapid amplification of cDNA ends, RACE) technology. Totally, three full-length cDNA sequences of genes in the chitin biosynthesis were isolated from B. dorsalis. The GenBank numbers of these genes were as follows:BdCHS2, KC354694, BdG6PI, JQ925874and BdUAP, JX434756. The open reading frame of these genes encoded amino acid sequences were derived by sequence analysis software. Furthermore, protein physicochemical properties, conservative motif, transmembrane helical structure and potential physiological function were predicted by the corresponding software. To understanding the genetic distance between these three genes and counterparts in other species, we constructed the phylogenetic tree by downloading the cDNA sequence and with Mega5.04using Neighbor Joining method. The results enriched and developed the genetic information of chitin synthesis pathway genes, and provided favorable conditions in-depth exploration of the mechanism in chitin synthesis.2. Expression profile of these three chitin synthesis pathway gene BdCHS2, BdG6PI and BdUAP at spatio-temporal distribution analysis in B. dorsalis.Five different tissues (fat body, midgut, Malpighian tubule, integument and trachea) were dissected under anatomical lens. Then extracted total RNA, and synthesis of the first chain of cDNA, expression studies on three chitin synthesis pathway gene BdCHS2, BdG6PI and BdUAP was detected by qPCR. The results were as follows:BdCHS2expressed the highest level in the midgut than in any other tissues; BdG6PI was mainly distribute in the tissues of fat body and Malpighian tubule; BdUAP was mainly distribute in the tissues of midgut, Malpighian tubule and integument; all of them expressed the lowest level in the tissues of trachea. Quantitative expression in different tissues enriches the genetic information about these genes.Different developmental stages (egg,1-8day old larvae,1-d,4-d,7-d,10-d old pupa and fresh emerge adult) were obtained. Then extracted total RNA, and synthesis of the first chain of cDNA, different developmental stage express level on three chitin synthesis pathway genes BdCHS2, BdG6PI and BdUAP was detected by qPCR. The results were as follows:The gene of BdCHS2, BdG6PI and BdUAP expressed in the whole developmental stages, especially expressed higher level in feeding stage while lower level in other stage. Theses genes had different level in their corresponding stages.3. Feeding-mediated changes in transcript levels of BdCHS2, BdG6PI and BdUAP in the midgut in B. dorsalisIn order to test and verify the microscopic changes under the feeding-mediated in the midgut of the larvae stage in B. dorsalis, we examined the changes in transcript levels of BdCHS2, BdG6PI and BdUAP in the larvae of B. dorsalis by feeding on the artificial diet or not. The results were as follows:The condition of feeding food or not did significant influence on the genes BdCHS2and BdG6PI in the midgut,. However, it did not have significant influence on the gene BdUAP in the midgut.4. The influence of diflubenzuron on the larvae of B. dorsalisIn order to explore whether the diflubenzuron did significantly influence on the gene BdCHS2expression level, we did a biological assay of diflubenzuron on the first day larvae by using impregnation method in B. dorsalis. In this study, the first day larvae were treated by different concentrations of diflubenzuron for10s, then checked the result after48h. we got that the lethal concentration of50%(LC50) of the diflubenzuron was42.05mg/L. We also found that the larvae died due to it can not molting in the treatment. qPCR analyses were indicated that diflubenzuron had no significant difference between the control and the treatment of the expression level of gene BdCHS2.5. Chitin content assayTo know the chitin content in the midgut by the treatment of feeding-mediated, we detected the chitin content of the midgut. The result showed that:No matter first feeding for24h then starvation for24h or first starvation for24h then feeding for24h, the chitin content all had significant difference. The change tendency had dramatic the same as the expression level of the gene BdCHS2. Furthermore, we detected the chitin content in the different developmental stage (egg,1-d,2-d,3-d,4-d,5-d,6-d,7-d old larvae,1-d,4-d,7-d old pupa and fresh emerge adult). We found that it was increased by a geometry power shape curve from egg to the last day larvae. Also, the chitin content of the whole developmental stage had a linear correlation with the expression level of the gene BdCHS2.In summary, three completely(BdCHS2, BdG6PI and BdUAP) cDNA sequences of genes from the chitin synthesis pathway were sequenced and study like this enriches the genetic information of B. dorsalis. It will provide the conditions to do further research in chitin anabolism. Based on these genes, spatial and temporal expression profile of five different tissues and fourteen different developmental stages were analyzed by qPCR. In addition, these three genes’ function is discussed in this paper by feeding-mediated treatment. Further more, in order to know the chitin content in each of the developmental stage. At last, we tested the effect of inhibitor diflubenzuron on the first day larvae and the expression level of the gene chitin synthase2in B. dorsalis, which provided a foundation for further studies in inhibitors. In one word, the study of three genes on chitin synthesis pathway and related functions is useful for looking for the target sites which are valuable for pest prevention and control. The studies here have important theoretical and realistic significance due to providing the basic molecular information for development of new pesticides and achieve the purpose of pest prevention and control in the field.