Cloning And Characteristic Analysis Of The Bre Family Genes In Helicoverpa Armigera(Lepidotera:Noctuidae)

Glycosphingolipids（GSLs） play important roles in cellular biology of vertebrate and invertebrate organisms, such as cell differentiation, tumor metastasis and cell co-ordination. GSLs also serve as receptors for different bacterial toxins, such as vibrio cholera and shigella dysenteriae. Glycosyltransferases are very important in the synthesis of glycosphingolipids（GSLs）, and the variations of glycosyltransferases have been proved to be related with Bt resistance. In insects, glycosyltransferases have many large groups, the occurrence of related variations have a lot of uncertainty. It is not only the important catalytic enzyme in the synthesis of GSLs, but also may be important in the modification of the toxin receptors, such as cadherin（CAD）, aminopeptidase N（APN）, alkaline phosphatase（ALP） and glycoproteins, so the mutations of glycosyltransferases may influence several important receptors at the same time. In this study, we preliminary studied Harmbre2-4 genes of H. armigera with the methods of molecular biology and bioinformatics technology, and the main research contents and results are as follows:1. We firstly cloned the full length sequences of ORF of Harmbre2 genes in H. armigera and submitted them to the GenBank. The full length ORF of Harmbre2（GenBank KR362875）; Harmbre3（GenBank KR362876） and Harmbre4（GenBank KR362877） are 1011 bp, 1377 bp and 1275 bp; which encoded predicted proteins of 336, 458 and 424 amino acids with a predicted molecular masses of 38, 52 and 48 kda respectively, and their isoelectric points were 8.75, 8.62 and 8.90. Protein sequence analysis revealed that only HarmBre4 codify for a signal peptide. We predicted there were five transmembrane helices in HarmBre3, only one transmembrane helix in HarmBre4, and none in HarmBre2, all of the three glycosyltransferases have the conservative sequences of homologous species. The amino acid sequence of HarmBre3 shared 86% identity with that of glycosyltransferase precursor from B. mori, 40 BmorBre3（GenBank NP₀01243979.1）, and 83% with beta-1,4 mannosyltransferase from P. xylostella, PxylBre3（GenBank ADB79796.1）. Similarly, the amino acid sequence of HarmBre2 and HarmBre4 also shared high identity with the homologous Bre2 and Bre4 proteins of other lepidopteran species.2. We use Real time PCR to analyze the transcript levels of bre genes in different tissues and in different developmental stages of Harmbre2-5 in H. armigera. The results in the different developmental stages showed that transcripts for Harmbre2, Harmbre3, and Harmbre5 were highly expressed in the egg stage compared with other developmental stages. Harmbre3 and Harmbre4 also showed high expression in the adult stage. Expression levels of the four genes in the larval and pupal stages are relatively low as compared with other stages, and there were no significant differences among the different larval stages and pupal stages of the four genes. it showed that all of the four genes expressed at very low levels in the gut tissues, especially in the midgut. The highest expression level of Harmbre2 was detected in the Malpighian tubule, while expression levels in other tissues were relatively low and showed no differences among all of them. Transcripts of Harmbre3 were maximally expressed in the head and peritrophic membrane（PM）, while Harmbre4 was highly expressed in the Malpighian tubule and Harmbre5 has high expression in the PM and Malpighian tubule.3. Three of the four Harmbre genes, excluding Harmbre4, were successfully expressed in Hi5 cells. We investigated the subcellular localization of HarmBre proteins in these insect cells. Fluorescence microscopic observations revealed that the proteins were dispersed in cytoplasm and were not localized in the cell nucleus.4. We down-regulated the Harmbre2-5 gene by RNAi, and used Real- time PCR to detect the silence efficiency. Results showed that after injection the siRNA of target genes, the expression level of Harmbre2 was reduced about 30%, while the expression levels of Harmbre3-5 were reduced about 50%.5. We analyzed the different gene structures and expression levels of Harmbre2-5 between the susceptible strain and resistant strains by RT-PCR and qPCR. Results show the gene structures and expression levels of Harmbre3-5 have no difference between susceptible and resistant strains in LF strains, but Harmbre2 expressed significantly higher in LF low resistant strains compared to susceptible strain, and its 5’UTR region missed a few bases in resistant strains compared to susceptible strain. 5’-UTR region has relationship with the gene expression level, so we speculated that the differences may be related to Bt resistance of H. armigera.In conclusion, Harmbre showed highly expression in the egg and the adult stage, and in the larval and pupal stages were relatively low. It also showed that all of the four genes expressed highly in the Malpighian tubule, PM and the head, and expressed at very low levels in the gut tissues, especially in the midgut. Cell level experiment showed that glycosyltransferases were mainly dispersed in cytoplasm. The variation analysis of susceptible and resistance strains found that Harmbre2 expressed higher in H. armigera of low level resistance, and its 5 ’UTR region had mutation of several bases. We speculated that the up-regulated and mutation of Harmbre2 may give rise to Bt resistance of H. armigera. Our study made a preliminary research for the function of glycosyltransferase genes in H. armigera, and laid foundation for further research of its specific functions and its relationship with Bt resistance of H. armigera. This may explore new Bt resistance mechanisms in H. armigera.