Researches Of The Thymidylate Kinase And The TRNA-isopentenyltransferase Genes From Paulownia Witches’-broom Phytoplasma

Paulownia witches’-broom (PaWB) is one of important and most representative forestdisease caused by PaWB phytoplasma in China. The disease distributes in vast areas from theShanhaiguan to the Yangtze River basin, and it reduced paulownia production. The thymidylatekinase (TMK), catalysing the transfer of a terminal phosphoryl group from ATP to dTMP, iscrucial to both the de novo synthetic and the salvage pathways for pyrimidinedeoxyribonucleotides. TMK exists in various organisms and is an important rate-limitingenzyme in the process of DNA replication and is crucial for the survival of organisms. Manyresearches, such as PCR amplication, sequencing and polymorphism analysis of the PaWB tmkgenes, prokaryotic expression, enzyme activity of the PaWB TMK proteins, effect of thePaWB TMK proteins on growth and propagation of the transformed E. coli, the expression ofthe PaWB tmk genes in the enriched phytoplasmas as well as in the infected host plants, wereconducted in this thesis. These researches laid the theoretical foundations for exploration ofbreeding and pathogenic mechanism of phytoplasma and drug design and screening forphytoplasma disease chemothraphy and disease integrated control.The thymidylate kinase gene (tmk) diversity, variation, predicted protein function domainsand evolution from paulownia witches’-broom phytoplasmas Pishan strain (PaWBPs) in Hebeiprovince and Ji’an strain (PaWBJan) in Jiangxi province were comparatively analyzed usingdirect and cloned DNA sequencing of PCR products followed by DNAMAN and MEGAsoftware.52and24different tmk-ahomologues respectively from cloned93sequences ofPaWBPs and41of PaWBJan in total were identified, whereas the tmk-b gene sequence wasrelatively conserved, with99.8%similarity and only one amino acid variation of the predictedproteins between both strains. All tmk-a ORFs were classified into tmk-a-1(639bp) andtmk-a-2(627bp). The ratio tmk-a-1/tmk-a-2respectively in PaWBPs and PaWBJan was2.5and3.3, while five of cloned tmk-a-1sequences of PaWBPs were the same as one in PaWBJan as well as tmk-Y of jujube witches’-broom phytoplasma. In addition, up to48.1%and41.7%of predicted tmk-a pesudogenes in total tmk-a were found respectively in PaWBPs andPaWBJan due to the multiple locus mutation. Rich sequence variability was found in tmk-ahomologues of PaWBPs and PaWBJan and there exist three functional domains in animo acidsequences of both tmk-a and b which are necessary for thymidylate kinase activity. Thephylogenetic analysis showed that all the tmk-a homologues of both strains with tmk-X and Ywere clustered into clade I, while tmk-b with wheat blue dwarf tmk-2into clade III, suggestingthat tmk-b nucleotide sequences consistant with highly conesrved16S rDNA can be used forthe classification of phytoplasmas on16Sr group levels whereas diverse tmk-a might be helpfulfor analyzing the genetic variation and diversity of different phytoplasma strains.The alignment and similarity analysis of amino acid sequence were conducted amongPaWBPS TMK-a-1, PaWBPS TMK-a-2, PaWBPS TMK-b, onion yellow wild-type line(OY-W) TMK-a, OY-W TMK-b, white blue dwarf (WBD) TMK-a and WBD TMK-b. Theamino acid sequence similarity value was96.65%between PaWBPS TMK-b and WBDTMK-2,99.03%between PaWBPS TMK-b and OY-W TMK-b,90.57%between PaWBPSTMK-a-1and PaWBPS TMK-a-2, ranged from87.32%to99.26%among PaWBPS TMK-a-1,PaWBPS TMK-a-2, WBD TMK-1and OY-W TMK-a, and from22.22%to25.95%amongTMK-a and TMK-b of PaWBPS, OY-W and WBD phytoplasma. The pET28a system was usedto generate a polyhistidine (polyHis)-tagged TMK fusion protein. The polyHis-tagged TMKswere expressed in Escherichia coli BL21(DE3) cell induced by IPTG and purified undernative conditons by Ni-NTA chelating colum. Then, the activity of three TMKs was measuredusing enzyme-coupled assay respectively. Results showed that the TMK-b had thymidylatekinase activity (85.960.74U·mg-1), whereas the specific activity of TMK-a-1and TMK-a-2proteins was hardly detected.To study the effect of the PaWB TMK-a-1, TMK-a-2and TMK-b proteins on the growthand propagation of transformed E. coli BL21(DE3) in which the three proteins were expressed,the growth curves were determinated by measuring optical density of broth culure at600nm (OD600). The result indicated that the overexpression of PaWB TMK-a-1, TMK-a-2andTMK-b proteins could significantly promote E. coli BL21(DE3) growth and propagationduring late logarithmic growth. The TMK-b had a stronger effect than the TMK-a-1andTMK-a-2, and there was no significantly different effect between the TMK-a-1and TMK-a-2.Therefore, we assumed that the TMK-a-1and TMK-a-2which had no specific thymidylatekinase activity phosphating the dTMP substate in vitro may still played an important role inphytoplasma cells in the different way from the TMK-b.To detect the genes expression at the transcriptional level, the DNA probes of the PaWBtmk-a-1, tmk-b genes as well as16S rDNA of PaWB as control gene were prepared. Northernblot analysis revealed that there were a large number of transcribed16SrRNA in PaWBphytoplasmas, but the clear hybridaztion signal was also detected from the healthy paulownia.Using the DNA probes specified to the PaWB tmk-a-1, tmk-a-2genes, however, the mRNAtranscription of PaWB tmk-a-1and tmk-b genes were not detected. The reason may be the lowlevel of transcribed mRNA of the PaWB tmk-a-1and tmk-b genes in the tested materials ortheir special temporal and spatial expression pattern yet not revealed so far. To explore thereason why there existed a definite16S rRNA band when the healthy paulownia total RNA wasused to analyze by Northern blot, the similarity analysis was conducted. The result showed thatthe sequence similarity value between PaWB16SrDNA and chloroplast16SrDNA fromgenomes of Arabidopsis thaliana, Nicotiana tabacum L., Populus trichocarpa Torr.&Grayand Chlamydomonas reinhardtii were all above70%. Thus, we deduced that the stronghybridaztion band in the Northern blot analysis of healthy paulownia total RNA was originatedfrom16S rDNA of paulownia chloroplast.To study the proteins expressed in PaWB phytoplasma, preparation of the polyclonalantisera using the PaWB TMK-a-1and TMK-b proteins as immunogens was completed byimmuizing the German rabbits. The results from Western blot, FITC fluorescece microscopyand immunoelectron microscopy analysis showed that the PaWB TMK-a-1and TMK-bproteins were expressed in a relativly low level in enriched phytoplasmas and in vitro cultured infected plants with typical witches’-broom symptoms. However, a similar size band withPaWB TMK protein was also detected in the total proteins extracted from the healthypaulownia by the polyclonal antisera to the PaWB TMK-a-1and TMK-b proteins; and FITCfluorescence was also observed in the parenchymal cells. Animo acid multiple sequencealignment among PaWB TMK-a-1, PaWB TMK-a-2, AtTMPK and Paulownia host TMKderived from the available transcriptomic data of Paulownia was conducted and the antigenicpeptide sequences were predicted. The results revealed that there existed many highlyhomologous antigenic peptide sequences among these four proteins, which may lead to theemergence of a similar size band, cross serological reaction with PaWB TMK protein in totalproteins extracted from healthy paulownia. In addition, the observation of immunoelectronmicroscopy using the PaWB TMK-a-1and TMK-b antibodies showed that there were a largenumber of immune colloidal gold particles located in the nucleus and chloroplast, which mayalso reveal the reason at the subcellular level why there existed the unexpected bands whendetected by Western blot. These results may be able to provide strong suggestion as well asexperimental evidence for the endosymbiont theory to explain the phytoplasma origination andevolution.The isopentenyltransferases (IPTs), which transfer an isopentenyl group fromdimethylallyldiphosphate (DMAPP) to the N6atom of free or bound adenosine nucleotides,while tRNA-IPTs catalyse the isopentenylation of tRNA to play important roles in the genetranscription as well as cytokinin synthesis through the tRNA degradation. The growth curvesof transformed E. coli BL21(DE3) in which the PaWB tRNA-IPT protein was overexpressedshowed that the tRNA-IPT could remarkably promote the bacterial propagation. In addition,as compared with the tRNA-IPT mutant arising from frameshift base change to stop codon,the growth and propagation of tRNA-IPT and blank control strains were repressed at0.2to1.0mM IPTG addition, whereas the propagation of tRNA-IPT mutant were not inhibited,which suggests that this mutant was able to overcome the inhibitory effect of IPTG on thegrowth of E. coli. To study the PaWB tRNA-IPTs function further, a plant expression vector, Cam35S-GFP tRNA-IPT with GFP reporter gene, was constructed successfully. TheAgrobacterium-mediated transformation of Arabidopsis thaliana was performed by thepollen tube pathway through foral dip, however, no target gene transformed seed was notobtained so far.