Expression Patterns And Functional Analysis Of Chitinase Gene Am Cht11 In Honey Bee Apis Mellifera Ligustica

Chitin is an important component of the insect epidermis and the midgut peritrophic membranes.It plays an important role in supporting the insect body and protecting insects from external damage.In the life activities of insects,chitin is continuously synthesized and degraded to meet the needs of insect growth and development.Insect chitinase acts on chitin and decomposes it into small-molecule oligosaccharides or oligosaccharides.It plays a very important role in regulating the balance of the chitinous renewal membrane and insect molting.Apis mellifera have been widely studied by researchers as a significant economic and social insect for flower pollination.A lot of essays report that insect chitinase in the degradation of chitin in molting and epidermal regeneration while few reports on the function of insect chitinase in the intestine of Apis mellifera.In this study,the Am Cht11(Bee Base accession number GB47539)of the Apis mellifera chitinase GH18 family member was selected as the research object.The c DNA sequence of the Am Cht11 was cloned and identified.The Am Cht11 amino acid sequence characteristics were analyzed by bioinformatics and followed by real-time fluorescence quantitative PCR.The Am Cht11 was expressed in various developmental stages of the Apis mellifera larvae and in the major body tissues of the adults.Finally,the Apis mellifera was fed with the ds RNA to silence the expression of the Am Cht11 in the intestine,and analyzed with the tissue section together that the basic characteristics of intestinal histology,body weight,and survival of Italian honey bees.Then the function of Am Cht11 in Italian honeybees was initially discussed.The main findings are as follows:1.Bioinformatics characteristics of chitinase Am Cht11 from Apis melliferaIn this study,bioinformatics analysis was used to analyze the physicochemical properties,conserved motifs,functional domains,and phylogenetic classification characteristics of the amino acid sequence of the Apis mellifera chitinase Am Cht11,and predicted the advanced structure of protein.The results showed that the Am Cht11 encodes 468 amino acids with a protein size of 53.4 k D and an isoelectric point p I of7.08.The amino acid sequence contains a transmembrane domain and a functional catalytic domain unique to the chitinase GH18 family,and its catalytic domain including four amino acid motifs: "Rxxxxx GG","GAMx Lxx Nx D","Mx YDxx F","FPGx DLDWEP" are highly conserved.The similarity of the protein structure with the typical chitinase structural model reached 37.25%,mainly consisting of α-helix(41.03%),β-sheet(11.75%),random coil(27.99%)and extended chain(19.23%).Phylogenetically,it is closely related to the chitinase chitinase Tc Cht11 and classified in the VIII subfamily of the chitinase GH18 family.2.Cloning and spatio-temporal expression characteristics of the chitinase gene Am Cht11 from Apis mellifera.We extracted the total RNA of Apis mellifera adults and first-strand c DNA was synthesized by reverse transcription.The full-length c DNA of the chitinase gene Am Cht11 from Apis mellifera was 1404 bp by PCR using c DNA as a template.Real-time quantitative PCR analysis revealed that the Am Cht11 was expressed in the head,foot,chest,epidermis,fat body and intestine tissues of the adult,obviously,it had a relatively higher expression level in the intestinal tissue.During the development of larvae,Am Cht11 gene was expressed at the 2-6th day of age,and began to increase significantly from the 5th day of age.We presumed that this change for the larvae,may be to preparate to enter a different developmental stage.3.The function of the Am Cht11 in intestinal tissues of Apis mellifera.The Am Cht11 gene ds RNA was expressed in Escherichia coli with food at a concentration of 40 μg/ml then to feed the aldults Apis mellifera for 20 days continuously.The results showed that the expression of Am Cht11 in the intestine of Italian honeybee adults was significantly down-regulated after 10 days and 20 days after adding ds RNA,indicating that the addition of ds RNA could effectively silence the expression of the intestinal Am Cht11 gene.In adult appearance and macroscopic morphology of the intestine,there was no significant difference between the test group and the control,but the observation of the intestinal microscopic section revealed that the midgut peritrophic membranes was significantly thickened after silencing the Am Cht11 gene.In addition,body weight and cumulative survival of Italian honeybees in the ds RNA-treated group were significantly reduced.In summary,This study hypothesized that the addition of Am Cht11 ds RNA in vitro significantly interfered with the silence of the expression of that in the intestinal tract of Apis mellifera.This leads to a thickening of the midgut peritrophic membranes due to the difficult degradation of chitin,which in turn affects the bee’s absorption of food nutrients,which ultimately results in a decrease in the weight and an increased mortality rate of the Apis mellifera.