The Biological Characteristics of Spiroplasma Isolated from Gadfly and Fly and Searching for Pathogenic Related Factors of Spiroplasma Melliferum

Spiroplasmas are helical prokaryotes without cell wall. The main hosts of spiroplasma are insects and plants. The relationship between most spiroplasmas and hosts are mutualism, but some spiroplasmas are pathogenic on plants and animals. Spiroplasmas in insects of Tanbanidae and Tyrphidae have been reported and a number of spiroplasma strains have been isolated, indicating that there are abundant spiroplasma resources in insects of Tanbanidae and Tyrphidae. Report on the spiroplasma in insects of Tanbanidae and Tyrphidae in China is rare. This research focused on biodiversity and geographic diversity investigation of spiroplasma in insects of Tanbanidae and Tyrphidae. Another purpose of this research was seeking of the pathogenicity-related factors of Spiroplasma melliferum CH-1involved in the pathogenesis. On the basis of S. melliferum CH-1genomic DNA sequencing, potential pathogenicity-related factors were analyzed by bioinformatics methods. Then pathogenicity-related gene was expressed in vitro. This thesis will be helpful for further research on the pathogenic mechanism of S. melliferum CH-1. The main results are as listed as followed:(1) A number of spiroplasmas were isolated from110gadflies and40flies samples which were collected from Nanjing, Yongkang, Guilin. Seventeen isolates were purified and used to examine basic biological characteristics. These17spiroplasma isolates were almost similar in morphological, physiological and biochemical characteristics. These isolates were helical in the logarithmic growth phase. All the isolates could pass through a pore size of0.22μm and0.45μm microporous membrane. Their colonies exhibited granular or irregular shape in solid medium. Growth pH of spiroplasma iaolates varied from5.2to10.2and pHopt was about7.3. They could metabolize glucose, D-fructose and mannitol, while NM1108-5, NM1208-5, NM1208-6could not use mannitol. None of the isolates could metabolize arginine or urea. They grew fast and reached the logarithmic growth phase in18h. Unidirectional serological tests showed that the spiroplasma isolated from insects of gadfly and fly were distantly related to S. melliferum CH-1(honeybee spiroplasmosis), S. eriocheiris (Chinese mitten crab tremor disease) and S. mirum (suckling mouse cataract).(2) Genomic DNA was extracted by modified method of CTAB/NaCl.16S rDNA sequences were then amplified. Phylogenetic tree suggested that all isolates were divided into two branches, Apis and Citri-Chrysopicola-Mirum respectively. Most spiroplasma isolaes were clustered with S. turonicum with a bootstrap value of100%, while NM1108-5, NM1208-6was clustered with S. gladiatoris, NM1208-5was clustered with S. syrphidicola. In order to further verify the results of16S rDNA sequences, interval transcribed spacer sequences (ITS) and the gene sequences encoding the RNA polymerase beta subunit (rpoB) were also conducted as the markers for phylogenetic analysis. It distinguished different strains effectively. According to the biological characteristics, serological characteristics and phylogenetic analysis of the isolates, the isolates from gadfly NM1108-5, NM1208-6were preliminarily identified S. gladiatoris, and NM1208-5was S. syrphidicola; DCY1207-1from fly and other13spiroplasma isolates such as NM1108-1etc. from gadfly were identified S. turonicum.(3) Basing on the results of the genome sequencing of Spiroplasma mellifeum CH-1, we searched and analyzed chitinase gene which was unique in S. melliferum through bioinformatics methods,(a) Length, physical and chemical properties of the chitinase amino acid sequences from different bacteria were different. But all the chitinase amino acid sequences contained five conserved sequences:LGGAD, DIDLEQ, MAPEPYL, DAAA, MTWS.(b) There was no trans-membrane domains in chitinase from S. mellifeum CH-1. But there was a match with Glycosyl hydrolases family18which had functional domains with degradation of carbohydrates. Curl and a-helix were major structural element in secondary structure. The β-folded and β-turn distributed on the entire structure. The target protein with the model sequence could be considered to be similar on three-dimensional structure with index of46.72%.(c) Chitinase from S. melliferum CH-1was distant with chitinase from other bacteria. The evolutionary relationships were divided into four branches.(4) According to bioinformatics analysis, the chitinase gene, chitin deacetylase gene and P54gene from S. mellifeum CH-1could be associated with the pathogenicity. The full length of three genes were successfully cloned. Complete chitin deacetylase gene and P54gene were expressed while part of the chitinase gene was expressed in E. coli. We confirmed that TGA in S. melliferum CH-1encoded tryptophan instead of the termination codon. These results provide further research on function of related factors in pathogenesis of S. melliferum CH-1.