Studies On Heat Shock Proteins And Molecular Typing Of Wolbachia In Pteromalus Puparum (Hymenoptera: Pteromalidae)

Heat shock proteins (Hsps) are a group of conserved proteins in evolution, whichexist in all eukaryotic and prokaryotic organisms. Hsps are very important fororganisms to survive in the stress. Wolbachia are maternally inherited bacteria thatspread widely in arthropods and can induce cytoplasmic incompatibility (CI),parthenogneesis, male killing and feminization. In this study, 6 heat shock genes inthe Pteromalus puparum were cloned and characterized, and their functions were alsostudied. Moreover, an investigation to determine the diversity of Wolbachia in P.puparum from 13 geographically distributed native populations in both southern andnorthern China were undertaken by comparing the sequences of Wolbachia 16S rRNA,ftsZ and wap genes. The results are summarized as follows:1 Cloning and characterization of the hsps in the P. puparumSix full-length ORFs coding Hsps involved five Hsp families were cloned by themethods of Blast search, PCR/RT-PCR, RACE and cDNA library screening.The full-length ORF of the Pphsp90 gene was 2148 bp which was predicted toencode a 716 amino acid peptide with a deduced molecule weight of 82.2 kDa, and nointron was found. The full-length ORF of the Pphsc70-1 gene was 1968 bp which waspredicted to encode a 656 amino acid peptide with a deduced molecule weight of 71.3kDa, and three introns were found. The full-length ORF of the Pphsc70-2 gene was1923 bp which was predicted to encode a 641 amino acid peptide with a deducedmolecule weight of 70.9 kDa, and one intron was found. The full-length ORF of thePphsp60 gene was 1719 bp which was predicted to encode a 573 amino acid peptidewith a deduced molecule weight of 60.6 kDa, and three introns were found. Thefull-length ORF of the Pphsp40 gene was 1095 bp which was predicted to encode a365 amino acid peptide with a deduced molecule weight of 39.6 kDa. The full-lengthORF of the Pphsp20 gene was 573 bp which was predicted to encode a 191 aminoacid peptide with a deduced molecule weight of 21.7 kDa.2 The Pphsps expression in relation to different stressorsTaqMan-MGB probes and the corresponding primers were designed to constructa system for real-time fluorescence quantitative RT-PCR to detect the changes inexpression levels of the six Pphsps in the adult wasps under the stressors, such as hightemperature (30～42℃for 1h), low temperature (3～15℃for 1h), starvation (0～24h) and heavy metals (0.5～5 mM Cd²⁺/Cu²⁺ treated 24h or 60h). Under the stress of high temperature, the expression of six Pphsps could beinduced. The genes, Pphsp20, Pphsp60, Pphsc70-2 and Pphsp90, had highestexpression levels after heat shock at 36℃for 1 h, while the expressional peakscorresponding to the genes, Pphsp40 and Pphsc70-1 were observed at 39℃.Under the stress of low temperature, with the exclusion of the Pphsp60 gene, theexpression of the other five Pphsps could be induced. The highest expressional levelsof the five genes were observed at -3℃.Under the stress of starvation, the expressional levels of the Pphsp40 andPphsc70-2 had no significant changes during starvation. The expression levels ofPphsp20 increased significantly at the beginning 6 h of starvation, and then decreasedgradually to the levels that were significantly lower than those of the control. Pphsp60,Pphsc70-1 and Pphsp90 mRNA levels increased significantly after 24 h of starvation.Under the stress of Cd²⁺, after 24 h treatment, the Pphsp40 mRNA levels weregradually elevated with the increase of the concentrations. However, the other fivePphsps mRNA levels reached the highest peak at the concentration of 0.5 mM, andthen reduced with the increase of the concentrations. After 60 h treatment, thePphsp40 mRNA levels were also gradually elevated with the increase of theconcentrations. Pphsp60 mRNA levels increased significantly at the concentration of0.5 mM. Pphsc70-1 expression levels increased significantly at the concentration of50 mM. The other three genes mRNA levels had no significant difference compare tothe controls or lower than those of controls.Under the stress of Cu²⁺, after 24 h treatment, the Pphsp40 and Pphsc70-2mRNA levels were gradually elevated with the increase of the concentrations.However, the other four genes mRNA levels reached a maximum at the concentrationof 50 mM. After 60 h treatment, only Pphsp20 and Pphsp90 mRNA levels had1.32-fold and 1.30-fold, respectively, greater than that of the control at theconcentration of 0.5 mM. However, the other treatments of Pphsps wereapproximately identical with or lower than those of the controls.In addition, the expression of the Pphsc70-1 gene in the pupae during thermalstress (40℃for 0～8 h) and recovery was also quantified using real-time quantitativePCR (SYBR Green dye). A significantly elevation of Pphsc70-1 expression wasobserved following heat treatment, however, continued exposure to heat shock orrecovery caused the expression of induced mRNA to gradually decline to levels that were significantly lower than those of control pupae.3 Expression of Pphsps in insect cells and Escherichia coliThe six Pphsps were successfully expressed in the insect cell line Tn-5B1-4 byusing Bac-to-Bac Baculovirus Expression System. By subcellular localizationanalysis, the expressional products of the six Pphsps located in the cytoplasm of theinsect cells.In addition, the six Pphsps were also expressed in the E. coli strain Bl21 (DE3)for the next functional study on the PpHsps. However, only five genes (except ofPphsp40) were successfully expressed. Further analysis showed that the expressionalproducts of the five Pphsps were dissolved.4 Purification of PpHsps and their functions under the thermal stressThe effects of overexpressed fusion PpHsps on the survival of E. coli and thestability of E. coli proteins during high-temperature stress were studied. The resultsshowed that the five expressed fusion proteins (PpHsp90, PpHsc70-1, PpHsc70-2,PpHsp60 and PpHsp20) could partly maintain the stability of E. coli proteins up to80℃. However, in functional bioassays (1 h at 50℃) for recombinant PpHsps, only E.coli cells overexpressing PpHsp20 and PpHsp90 proteins had the significantlyincreased survival rate comparing to those lacking PpHsp. Recombinant PpHsp20 andPpHsp90 were purified by Ni-NTA resin. Measuring the light scattering of luciferaserevealed that only PpHsp20 prevented the aggregation of luciferase.5 Cloning and sequence analysis of wsp, ftsZ and 16S rRNA genes fromWolbachia in the 13 populations of P. puparum in ChinaThe results showed that all the 13 populations were infected with B-Wolbachia.In the 13 populations, we found four distinct sequences for wsp (wPup1, wPup2,wPup3 and wPup4), two distinct sequences for both ftsZ(fPup1 and fPup2) and 16S(16SPup1 and 16SPup2). Moreover, recombination of wsp gene within distinctsequences was found.In addition, only one sequence was found for wsp, ftsZ and 16S rRNA in the allthe southern populations with the exclusion of Shanghai population. However, two orthree distinct sequences for the three genes were found in the all northern populations.The results indicated that two Wolbachia strains present in the northern populations,whereas only one Wolbachia strain present in the southern populations (exceptShanghai population).