Gene Function Analysis Of SABP2 And SAMT In Poplarinteractions With Botryosphaeria Dothidea

When plants of annual herb are being invaded by a pathogen,salicylic acid(SA)producted at the inoculation site can be converted into methyl salicylate(MeSA)under the action of the gene of salicylic acid methyltransferase(SAMT).Part MeSA be evaporated into the air and are converted into SA again at uninoculated site in the plant through the gene of Salicylic acid-binding protein 2(SABP2).These processes play an important role in plant systemic acquired resistance(SAR)and signal transduction of SA.However,the function of SAMT and SABP2 in perennial woody plants needs to be further verified.Our research mainly contains two parts,one part is the cloning of the SABP2 and SAMT genes in 84 K poplar(Populus alba × Populus glandulosa)and genetic transformation,the other part is validation of SABP2 and SAMT genes function about transgenic seedlings after inoculation with Botryosphaeria dothidea.Cloning of the two genes: In this study,two pairs of primers,which were designed according to the complete cds of Populus trichocarpa SAMT and populus tomentosa SABP2,were used to amplify the SAMT gene and SABP2 gene of 84 K popular by polymerase chain reaction.The product of PCR was ligated to pGM-T cloning vector and transformed into TOP10 competent cells chemically.Then,clones were obtained and sequenced.Bioinformatics analysis was also conducted by various online software.The results are as follows.SABP2 gene ORF full-length cDNA sequence is 789 bp,encoding 263 amino acids.Sequence analysis and comparison show that 84 k poplar SABP2 gene's sequence homology with other plant is as high as 76% ~ 98%.To some extent,the comparison results also reflected the SABP2 conservative in structure.Using bioinformatics software to predict the protein function,shows its may have intermediary metabolism function,belongs to the alpha/beta folding hydrolase esterase families.This feature is consistent with the partial function of the redox enzyme and hydrolytic enzymes predicted by previous researchers.At the same time,this result also consistent with the previous research in the tobacco.In tobacco,the function of SABP2 was predicted that its belong to the esterase of alpha/beta folding hydrolase families members,and through the restructuring experiment proves its display esterase activity,and by combining the SA to stimulate the activity of esterase then signal may be produced.SAMT gene's ORF,coding of 364 amino acids,was 1094 bp.Through the predictions of the physical and chemical properties,hydrophobicity,domain,function,secondary structure and subcellular localization using bioinformatics tools,we got some results which showed that the gene was a hydrophilic protein,which belonged to the methyltransferase 7 family and located in the cytoplasm.The results were consistent with the previous research of the gene in herbs.Vector construction and genetic transformation: Using Gateway cloning technology,SABP2 and SAMT over-expression and RNAi carrier were constructed successfully,then by agrobacterium mediated " leaf dish transformation" genetically modified plants were got.SABP2 and SAMT over-expression each strain named OE-SABP2 and OE-SAMT.SABP2 and SAMT silence each strain named R-SABP2 and R-SAMT.Then choose suitable strain and inoculation experiment was carried out.The results are as follows:SAMT is closely related to SA and MeSA in poplar.SA can be transformed to MeSA in infected and uninfected part after inoculation with B.dothidea.In theinoculation period,the changing trends of SAMT in organization is consistent with SA of OE-SAMT plant and the Me SA content and gasification MeSA in transgenic plants after inoculation compared with wild type were significantly or extremely significantly rise,SA content decreased significantly at the same time;While MeSA contentwas reduced in R-SAMT plant tissues after inoculationsignificantly,and the evaporated MeSA into the air was reduced at the same time.SABP2 is closely related to SA and MeSA in poplar too.MeSA can be converted to SA in infected and uninfected sites.During the inoculation,in OE-SABP2 plant,SA content was increased in the inoculation site,and the MeSA content and gasification MeSA were decreased significantly or extremely significantlycompared with wild type.These results is consistent with predecessors' studying which SABP2 can catalyze MeSA to SA.The decrease of MeSA in tissues and the volatilization content were thoughta part of MeSA were converted SA by SABP2.Unvaccinated parts in OE-SABP2 poplar,the contents of MeSA and SA all significantly reduced compared with wild typeis due to less volatile MeSA in the vaccination site then MeSA converted fewer SA.Compared with the wild type SA content also significantly reduced in R-SABP2 plants in the late inoculation.PR-1 cannot serve as a sign of SAR in poplar.Whether the plant disease resistance are more closely relationship with the content of SA.At the end of the inoculation,all the strain compared to the wild type,the OE-SABP2 plants showed disease resistance than the wild type were due to the highest content of SA in the plant.After inoculation OE-SAMT plantswere lower than the wild type in the plant disease resistance.The reason is the high expression of SAMT led to the high expression of PR-1,and the high expression of PR-1 in plants generally are more susceptible to disease,and whether the PR-1 were resistant todisease related to the kinds of pathogenic bacteria.R-SAMT and R-SABP2 plants compared with wild-type plants are also more susceptible to disease,also related to PR-1 large increase expression after inoculation.In the process of genetic transformation,over-expression or interference a particular gene can cause other genes expression change.This were more obvious in the plant trials inOE-SAMT.Over-expression of SAMT in plants,The expression of PR-1 were increased before inoculation and the SABP2 expression was rise after inoculation.In our trials,the changes of the related gene causes the OE-SAMT plants become more susceptible to disease than wild type at the inoculation end may be associated with the change of its other genes.There was no balance and constraint relationship between SAMT and SABP2.The result was inconsistent with previous speculation,which higer expression of SAMT was because increasing SA in tissues was toxic to plant and needed convert MeSA into air through SAMT.Our studies have shown that SAMT and SABP2 genes in poplar is closely related to biosynthesis of MeSA and SA.After inoculation withB dothidea,SAMT over-expression plants can significantly improve the MeSA of evaporate into the air and tissues,the content of the SA levels was low in the organization at the same time.Silence of the SAMT can decrease the contents of MeSA.The synthesis of MeSA in OE-SABP2 plants also significantly reduced and the content of SA was increaseed in tissues.So OE-SABP2 plant showed disease resistance significantly.