Cloning And Expression Analysis Of GmDREPP Gene And Its Effect To Aluminum Resistance

Aluminum（Al） toxicity is a key factor in limiting the growth and yield of forages and crops in acid soils,and Al accumulates mainly in the meristem and elongation region of the plant root tip with ionic form in acid soils.Free Al³⁺can prevent cell division by inducing lipids to produce free radicals and cause ROS damages,and inhibiting mitosis as well as DNA synthesis.Besides,Al³⁺can change the structure of cell wall to reduce the cell wall malleability.Then the growth of plant root system is finally inhibited.Developmentally-regulated plasma membrane polypeptide（DREPP）,acting as one kind of plant-specific protein,is involved in the response and regulation of plants to biologic and abiotic stresses.Tamba black soybean（Glycine max cv.Tamba）is an aluminum-resistance cultivar originated from Japan.In previous experiments of our laboratory,the GmDREPP gene expression was significantly up-regulated in the apical chipset data under acid soil conditions and in the transcriptome data under aluminum stress in Tamba black soybean.Therefore,it is speculated that the gene is related to aluminum tolerance of plants,but its aluminum tolerance function is not clear.In this study,GmDREPP gene was cloned and its function was identified so as to provide theoretical basis for aluminum tolerance breeding of herbage.The CDS sequence encoding the GmDREPP protein was cloned from Tamba black soybean,and the expression of GmDREPP in Tamba Black Soybean was analyzed by q RT-PCR to research its tissue expression and response characteristics to abiotic stress.In addition,the overexpression vector named p CXSN-GmDREPP was constructed and transformed into wild type tobaccos mediated by agrobacterium to obtain the transgenic tobaccos.To investigate the aluminum resistance,the transgenic tobacco lines（GmDREPP-2,GmDREPP-4,GmDREPP-5）selected by RT-PCR were challenged by Al（p H4.5,0.5 mmol/L Ca Cl₂,50μmol/L Al Cl₃）compared with wild type（WT）.The results were as following:A.Bioinformatics and q RT-PCR analysis of GmDREPP in Tamba black soybeanIn this study,GmDREPP gene was obtained from Tamba black soybean by molecular cloning.The length of the whole sequence is 624 bp and encodes 207 amino acids.GmDREPP is located on the surface of cell membrane and has no predicted cross membrane region and signal peptide.The molecular formula is C₁₀₂₈H₁₆₄₆N₂₅₄O₃₄₆S₁,and the theoretical isoelectric point is 4.95.The highest amino acid composition is glutamate（21.3%）,followed by lysine（17.4%）and alanine（10.1%）,and the lowest are arginine and tryptophan（both 0.5%）.Instability index is more than 40,which means GmDREPP is an unstable protein.Its amino acids from 1～177 sites are its functional domain,and that is the unique conserved domain of DREPP family.The result of constructing evolutionary tree shows that GmDREPP has high homology in the same family.GmDREPP expressed in roots,stems,leaves and cotyledons,while the expression level in root tips was the highest in root system.Besides,the m RNA level of GmDREPP increased with the increase of the Al³⁺concentration,but had no significant changes when Al³⁺concentration was higher than 50μmol/L.B.Construction of the overexpression vector with GmDREPP gene and genetic transformation of tobaccoThe CDS sequence was cloned,and the overexpression vector,p CXSN-GmDREPP,was constructed and transformed into wild type tobaccos to obtain the 42 transgenic tobaccos.DNA was extracted for PCR detection and 7 positive plants were obtained,and then RNA was extracted for RT-PCR detection to obtained 5 plants with expression activity.The three expressed lines（GmDREPP-2,GmDREPP-4,GmDREPP-5）were selected from the 5 transgenic tobaccos to measure the relative expressions and analyze the aluminum resistance.C.Identification of aluminum resistance of transgenic tobaccosThree transgenic lines（GmDREPP-2,GmDREPP-4,GmDREPP-5）and WT were treated with 50μmol/L Al³⁺for 2 weeks,and results showed that the relative elongation of transgenic tobacco roots was up to 2.6 times of the wild type（P<0.01）.Also,indicating a reduction in aluminum toxicity,root tips stains of the transgenic tobaccos using Evans blue and hematoxylin manifested transgenic tobaccos were lighter than WT after treated with Al³⁺.These results exposed that the overexpression of GmDREPP gene enhanced the aluminum resistance of tobaccos.D.Analysis of aluminum resistance of transgenic tobaccoNt ALS3 and Nt MATE in tobacco were regarded as symbolictic genes of resistance to aluminum,and the expression of Nt ALS3 and Nt MATE in the transgenic tobaccos roots remarkably increased by 1.9 times（P<0.01）and 1.7 times（P<0.01）after treated with 50μmol/L Al³⁺.In addition,compared with WT,the expression levels of Nt ALS3and Nt MATE in transgenic plants were increased by 1.4～1.6 times（P<0.01）and 1.8～2.0times（P<0.01）,respectively.And the secretion of citric acid increased more than 2.9times（P<0.01）in order to facilitate the complexation of Al³⁺to improve aluminum resistance.Also,the activities of POD and SOD increased significantly（P<0.01）,more than 1.8 and 1.9 times of WT,and MDA content in root system was decreased by 56%（P<0.01）after aluminum treatment.It could be seen that the overexpression of GmDREPP can enhance the aluminum resistance of transgenic tobacco by up-regulating the expression of Nt ALS3 and Nt MATE which were proved to be the key genes to alleviate aluminum stress,increasing the activity of root citric acid secretion and antioxidant enzyme SOD&POD as well as reduceing the activity of MDA prominently in the root system.Conclusion:In this study,GmDREPP gene was cloned from the aluminum-resistant plant Tambo black soybean.GmDREPP gene is a member of DREPP family,with a full length sequence of 624 bp,encoding 207 amino acids.GmDREPP protein,located on the cell membrane surface,has the unique conserved structural domain of DREPP family.Through the test of aluminum resistance of tobacco with GmDREPP gene,this study proved that GmDREPP is aluminum resistant and can be used in providing aluminum-resistant genetic resources for aluminum resistance varieties of alfalfa and other forages in breeding.