Functional Research Of APPR-1-P Enzyme Family And ABA Acceptor Function In Salt Acclimation

ADP-ribose-1’’-monophosphatase (APPR-1-P) is an important processingenzyme in cells, participating in splicing the tRNA procedures and catalyzingADP-ribose-1’’-monophosphate into ADP-ribose. We found ADP ribosylation playedan important role in complex biological processes, such as DNA damage and repair,transcriptional activation and repression, mitosis, cell proliferation and differentiation,cell apoptosis and programmed cell death.There are two members of APPR-1-P processing enzyme family, AT1G69340and AT2G40600. Also, our results showed that many difference between them in thelevels of gene expression, the expression tissues, the mutant phenotype and the rolesin plant development and in the response to circumstance stress and so on. Maybe,AT1G69340participates in embryo development and AT2G40600plays an importantrole in the MeJA-mediated signalling.Soil salinity is one of the major environmental factors limiting plant growth,future food productivity and the area of cultivated land through the worldwide,therefore the studies on the regulatory molecular mechanisms of plant adaptation tosalt stress and the searches for strategies to increase plant salt tolerance will havegreat theoretical and practical significance. Plants can acquire the improvement oftheir salt resistance through gradual salt acclimation, while the molecular mechanismsare unclear.In this work we pretreated the mutants of pyl, pyl8and pyl12with low salt andthen treated these mutant with high salt to know if the three genes of ABA receptorsplay a key role in salt acclimation by observing these mutant phenotypes under theexperimental conditions.In our work, there were some aspects as below:1. Through the analysis of bioinformatics, we identified two genes, AT1G69340and AT2G40600in Arabidopsis genome. We analyzed the amino acid sequences andphysico-chemical properties of both genes encoding proteins in Col-0ecotype. By theanalysis of MACRO domain, we found, although there exist large variations at thelevel of amino acids, the spatial structures were almost the same. In our work, we used the genome data of19Arabidopsis ecotypes to evaluate the differences in theregions of promoters in both genes; moreover, we got the expressions of both genes indifferent tissues in Col-0with the data of AtGenExpress and their expressions inseedlings in19ecotypes based on RNA-Seq data. The results showed, there weredifferent expression patterns for the same gene among ecotypes. Based on theexpression data in Col-0, the expression pattern of both genes were very similarexcept in flowers and seeds. These results implied that the regulatory mechanisms ofthese genes’ expressions had changed in these ecotypes for the diversities oftranscription factors and transcription factor binding sites.2. We analyzed the expression of AT1G69340and AT2G40600in roots, stems,leaves, flowers, siliques in Col-0ecotype with semi-quantitative RT-PCR andqRT-PCR. The results showed the expression of AT1G69340in roots, stems andflowers were low. The expressions of AT1G69340in leaves and siliques was higherthan in roots, stems and flowers and the expression in leaves was the most. The resultwas associated with the analysis in bioinformatics.3. By the means of PCR, we evaluated the genotypes of T-DNA insertionmutants of AT1G69340(stock numbers: CS808255、CS839860) and AT2G40600(stock numbers: CS830160) ordered from ABRC. We did not get the homozygousmutants of AT1G69340because homozygous mutants were lethal, while we got thehomozygous mutants of AT2G40600. We analyzed the expression of AT2G40600inhomozygous mutants with semi-quantitavie RT-PCR and qRT-PCR and found theexpression of AT2G40600was up-regulated. The mutant of AT2G40600was a gain offunction mutant.4. The analysis of mutant phenotypes: we found there were undeveloped embryosacs in young siliques and shriveled seeds in mature siliques of T-DNA insertionmutants of AT1G69340. These abnormal seeds did not germination to establishseedlings. All the results could explain well the reason that we could not gain thehomozygous mutants of AT1G69340--this gene homozygous mutant is lethal. Further,we observed the phenotype of AT2G40600mutant under different stresses and foundthat there were phenotype differencees between T-DNA mutant of AT2G40600andwild type of Arabidopsis thaliana which grew in medium containing methyljasmonate (MeJA), and the growth of mutant seedlings showed insensitive to MeJA. 5. To analyze the functions of both genes, we cloned the full length cDNA ofAT1G69340and generated the over-expression construct of pCAMBIA3301NH-AT1G69340. To gain the functional complementation individual, the construct wereintroduced into the mutant plants of CS808255and CS839860byAgrobacterium-mediated transformation, respectively. The overexpression constructof pCAMBIA3301H-AT2G40600was transformed into wildtype Arabidopsis thalianaand got the AT2G40600overexpressing plants. Futher, we will compare thedifferences in responses to MeJA between over-expression individuals and T-DNAinsert mutants.6. We transformed the expression vector of pCAMBIA3301-PAtAPPR1Pases::GUS towild Arabidopsis thaliana using Agrobacterium-mediated flower dipping and gotthe basta resistance plants. We analyzed the tissue specific expression of genes usingGUS-staining.7. We transformed the over-expression vector of pCAMBIA3301-GFP-AtAPPR1Pases into wild Arabidopsis thaliana using Agrobacterium-mediated flowerdipping. We got the basta resistance plants and analyzed the subcellular localizationof both AtAPPR1Pases through the obsearvation GFP.fluorescence signal.8. Because the T-DNA insertion mutant of At2G40600was function-gainedmutant, to dissect the gene function, we got the silencing construct ofpCAMBIA3301-AT2G40600-00604G2TA and transformed the vector into wildArabidopsis thaliana to get the down-regulated expressing plants which were used toanalyze their response to MeJA.9. We analyzed the interaction of AT1G69340and AT2G40600with otherproteins using yeast-two hybrid experiments.10. To get the functions of ABA receptors of Arabidopsis thaliana in saltacclimation, in our work, we used the T-DNA insertion mutants ordered from ABRCas material and got the homozygous T-DNA mutant individuals evaluated by PCR.Then, we pretreated the mutant seedlings with low salt firstly and then with high saltto gain some information of ABA receptor responses to salt acclimation. But, inpresent, we had not got the ideal results.