Chitinase,Chtin Deacetylase Genes Of Spiroplasma Melliferum And Influence Of Spiroplasma On Anti-ros Relative Enzymes Within Apis Mellifera

Spiroplasmas(Mollicutes)are kind of spiral wall-less prokaryotic microorganisms which infect arthropods and plants.Spiroplasma is phylogenetical close to Bacilli and other free-living Firmicutes.The "climb disease" of honeybee is common in China,while Spiroplasma melliferum is among the main pathogens.Its basic biological information was detail studied,but pathogenic mechanism remains unknown.Basing on results from comparative genomics and proteomics,chitinase and chitin deacetylase gene were detected in S.melliferum while absence in other non-pathogenic spiroplasmas to insects(such as S.citri and S.kunkelli).This result suggested that these two genes may be responsible for invasion of spiroplasmas to host.Chitinase and chitin deacetylase gene were cloned and expressed in E.coli.Then their enzyme activity and enzymatic properties of chitin deacetylase were conducted.On the other hand,honeybee exhibited a series of oxidation-reduction reaction after infected by spiroplasmas.Changes of anti-ROS system related metabolic enzymes had been observed in Apis mellifera.Firstly,though application of bioinformatics and online forecasting tools,we predicted the physicochemical properties,functional domains and Spiroplasma melliferum CH-1 chitin deacetylase to reveal the phylogenetic relationship of spiroplasmas with other bacteria.(1)The molecular weight of S.melliferum CH-1 chitin deacetylase was 26 kD;(2)Acidic/basic amino acid ratio was smaller than other bacteria,mainly expressed hydrophilic,no transmembrane region and signal peptide,the entire peptide chain was located outside the cell membrane and the protein may be exocrine;(3)Alignment with other bacteria showed that chitin deacetylase protein contain three conserved motifs which could be essential functional domains.Bioinformatic analysis revealed the basic assumptions and spatial structure of the protein,which was helpful to understand the function and target protein expression.Secondly,since UGA was read as a tryptophan codon and not as a termination signal in most mollicute species,expression of mollicute genes by cloning in E.coli has been limited.In order to explore pathogenesis of the chitinase and chitin deacetylase(CDA)in Spiroplasma melliferum CH-1,the chiA/chid gene was cloned and expressed by expression vector pET-28a(+)s followed by NTA-Ni2+ column,Western blot,UV spectrophotometry to analyze its enzymatic properties.(1)ChiA and chid were successfully cloned.Active chitin deacetylase protein was expressed while chitinase protein was expressed as inclusion body in E.colit ChiA/chid contained a 768/672 bp coding sequence,encoding a protein with 32 kD/28 kD composed of 225/223 amino acids;(2)We used two methods to made chitinase protein inclusion body refolding,but failed to obtain active protein.Eukaryotic expression system can be selected in the future.Thirdly,for the active chitin deacetylase protein,UV spectrophotometry was used to detect the enzyme activity and enzymatic properties.(1)Enzyme activity of chitin deacetylase protein was 10.14 U·mL-1;(2)The optimum temperature and optimum pH was 50? and 7.0-7.5 respectively.The enzyme was insensitive to temperature.After 30 minutes treatment in different temperature gradient between 35?-60?,CDA enzyme can still maintain more than 60%viability.The pH stability experimental study showed that the enzyme was relatively alkali resistance;(3)Monovalent metal ions Na+ and K+ showed a promote tendency to CDA activity.There were three kinds of tendency on divalent metal ions,namely Ca2+,Mg2+ mainly for inhibition,Zn2+ had no significant effect on the CDA enzyme activity,Fe2+ could promote the activity.Trivalent metal ions Fe3+ and Al3+ could affect the CDA activity in low concentration,mainly expressed promotion.Fourthly,after feeding log phase Spiroplasma melliferum CH-lto Apis mellifera,variation of anti-ROS system related enzyme activity was studied during time periods.(1)On the third day,the Apis mellifera exhibited "climb disease" symptoms.On the fifth day,the mortality rate reached the maximum.(2)On the third day,the catalase(CAT)first decreased then increased with the extension time of infection on 5th,7th day,but still hadn't obvious difference compared with the control group.(3)The activity of SOD and GSH-Px increased obviously in the initial infection time and reached the maximum on fifth day,then slightly declined.All results showed that the Apis mellifera could induce free radical scavenging system to resist pathogen infection.All the understandings contribute to the interactions between spiroplasma and honeybee.