Functional Identification Of GmPME2 And GmFDH And Effects To Aluminum Resistance Of Tobacco(Nicotiana Tabacum)

Aluminum,as the third most abundant element behind oxygen and silicon,is the most abundant mental element in soil which exists in form of aluminosilicate and its oxide and do no harm to plant.But acid soil can turn aluminum into soluble ion with the form of Al³⁺which was released to soil resulting in inhibition of root growth,depressing the absorption of root to water and mineral nutrient in soil and finally crop failure.Our previous study found that Gm PME2 was significantly down-regulated under aluminum treatment but the Gm FDH was significantly up-regulated which indicated the two genes were associated with aluminum tolerance.By investigating the aluminum resistance mechanism,this research may provide gene resources for breeding of aluminum tolerance plant.Tamba black soybean（Glycine max cv.Tamba）,an aluminum-resistant leguminous plant,as the model plant to investigate aluminum resistance,is used to research the function of Gm PME2 and Gm FDH exerting in defensing Al toxicity.We cloned Gm PME2 and Gm FDH from Tamba black soybean via reverse transcription PCR（RT-PCR）.The aluminum functional identification and aluminum tolerance analysis was carried out and the main results were as follow:（1）The coding regions of GmPME2 were 900 bp in length and coded for 299amino acids with an isoelectric point of 8.85.The instability index of Gm PME2 was30.11 indicating a stability protein.The expression analysis in Tamba black soybean by q RT-PCR showed that the expression level of Gm PME2 under Al stress（p H 4.5,0.5mmol/L Ca Cl₂,50μmol/L Al Cl₃）decreased after 18 h.The highest expression was in root（especially in 0–2 cm of root）,and the expression of Gm PME2 was decreased by the increase of aluminum concentration.Transient expression in Nicotiana benthamiana indicated Gm PME2 mainly located in cell wall.The expression vector,p BI121-Gm PME2-e GFP,was constructed by recombinant clone and transformed into Nicotiana tabacum by agrobacterium mediated transformation.9 positive transgenic tobaccos in DNA and RNA level was obtained.3 positive tobaccos with higher RNA expression level were identified by Western blot.The aluminum resistance analysis of the 3 transgenic tobacco lines showed that the activity of pectin methylesterase was1.17-1.33 times to wild type.The relative root elongation in transgenic tobaccos was42.85%-85.71%of the wild type.Hematoxylin,Evans blue and morin staining revealed a deeper stain in transgenic tobaccos than that in wild type.The content of MDA in the root of transgenic tobaccos was 2.28-2.57 times of wild type which meant that over-expression of Gm PME2 enhanced aluminum sensitivity in transgenic tobaccos.Though the activity of POD and SOD,the secretion of citric acid and the expression of Nt ALS3 and Nt MATE（aluminum sensitive marker genes）in the root of transgenic tobaccos were increased in compared with wild type,but it not enough to make up the toxicity to root by over-expression of Gm PME2.The results all above indicated that Gm PME2 was an aluminum sensitive gene and overexpression of Gm PME2 could decrease the aluminum tolerance of plant.（2）The coding regions of GmFDH were 1128 bp in length and coded for 375amino acids with an isoelectric point of 6.87.The instability index of Gm FDH was26.83 indicating a stability protein.The expression analysis in tamba black soybean by q RT-PCR showed that the expression level of Gm FDH under Al stress（p H 4.5,0.5mmol/L Ca Cl₂,50μmol/L Al Cl₃）was significantly increased.The highest expression was in root（especially in 2-4 cm of root）,and the expression of Gm FDH showed no remarkable difference in treatment with different Al concentration.Therefore,the concentration of 50μmol/L was selected for further experiment.The highest m RNA level in treating with different metal ions was Al.The FDH activity of Tamba black soybean was increased by the decrease of p H,increase of Al concentration,increase of Al-stimulated time and the increase of external fomate concentration.The formate content of tamba black soybean root tips was increased by the decrease of p H,increase of Al concentration,increase of Al-stimulated time and the increase of external fomate concentration.The aluminum content of tamba black soybean root tips was decreaed by the decrease of p H,increase of Al concentration,increase of Al-stimulated time and the increase of external fomate concentration.The expression vector,p BI121-Gm FDH-e GFP,was constructed by recombinant clone and transformed into Nicotiana tabacum by agrobacterium mediated transformation.11 positive transgenic tobaccos in DNA and RNA level was obtained.3 positive tobaccos with higher RNA expression level were identified by Western blot.The aluminum resistance analysis of the 3 transgenic tobacco lines showed that the relative root elongation in transgenic tobaccos was 2.20-2.60 times of the wild type.Hematoxylin,chromazurine,Evans blue and morin staining revealed a lighter stain in transgenic tobaccos than that in wild type which meant the Al resistance in transgenic tobaccos was remarkably enhanced in compared with wild type.The content of MDA in the root of transgenic tobaccos was83.33%-58.33%of wild type and 70.83%-76.67%of H₂O₂ content in compared with wild type.The POD and SOD activity were 1.08-1.33 times and 1.10-1.24 times respectively compared to wild type.The citric acid content in transgenic tobacco root tips was 1.84-2.10 times to wild type which demonstrated that transgenic tobaccos had higher tolerance to Al compared with wild type.The relative expression of Al sensitive marker gene Nt ALS3 and Nt MATE showed no obvious difference between transgenic tobaccos and wild type.The results all above revealed that over-expression of Gm FDH enhanced the aluminum tolerance to transgenic tobaccos indicated that Gm FDH was an Al tolerance gene which provided the gene resource to improve Al tolerant leguminous varieties.