Functional Analysis Of Heterologous Transfor Mation Of GsMML Gene On Enhancing Acid Aluminum Resistance In Arabidopsis Thaliana

Aluminum toxicity is one of the major factors which limits crop growth on acidic soils.Plant transcription factors were found to respond to aluminum stress.In this study,GsMML gene was cloned from the Glycine soja BW69 line,and the GsMML transgenic lines were obtained by heterologous transformation of Arabidopsis thaliana.The results from molecular identification and acid-resistant phenotypic identification showed that GsMML enhanced the tolerance of transgenic Arabidopsis to acid aluminum stress.The main results obtained are shown as follows:GsMML gene?No.100805092?was derived from the acid-resistant gene expression profile of Glycine soja with a full length sequence of 1213 bp and open reading frame length of 726 bp which encodes 241 amino acid residues.Analysis of the protein domain revealed that GsMML protein contained a MADS-box domain from 2^ndto 77^thamino acids and a K-box domain from 81^thto 169^thamino acids.It is speculated that Gs MML protein is a member of the MADS-box transcription factor family.The expression pattern of GsMML gene was investigated by Real-time RT-PCR.The results showed that GsMML gene expresses in root,stem,leaf,flower and pod with the highest level in root of Glycine soja BW69 line.Under the condition of acidic aluminum stress,GsMML was up-regulated with the aluminum concentrations showing the expression pattern of increasing gradually first and then decreasing slowly.The expression of GsMML gene was up to the highest level at the 50?M of Al Cl₃ with 3 times'expression level of the control treatment.The expression pattern of GsMML gene was also analyzed by time course.The results showed that there was no difference in the expression of GsMML gene at 6 h and 12 h between the aluminum treatment?pH4.3?and the control treatment?pH5.8?with the relative expression close to 1.At the condition of acidic aluminum stress?pH4.3?,the GsMML expression was first increased up to the highest level at 6 h with about 3.5times'level of the control treatment.While at the points of other treatment time,there was no significant expression difference of GsMML with the expression level close to 1-1.5times of the control treatment.The fusion vector pYL322-d1-eGFP-GsMML was constructed by recombinant DNA technique to insert the coding sequence of GsMML into the downstream of GFP sequence.The subcellular localization analysis was carried out by PEG-mediated method using the recombinant vector and the empty vector to transform Arabidopsis protoplasts.The results indicated that GsMML protein was found to be transiently expressed in the nucleus.The plant expression vector pTF101.1-GsMML was constructed by recombinant DNA technique after the full-length sequence of GsMML gene was cloned from Glycine soja BW69.The transformant plants were obtained by heterologous transformation of Arabidopsis thaliana and screened with glufosinate for T₁ generation seedlings.The results of molecular identifications showed GsMML gene had been integrated into the Arabidopsis thaliana genome by PCR and was overexpressed by Real-time RT-PCR in different transgenic Arabidopsis lines.Treatment of T₃ transgenic Arabidopsis lines were carried out with acid aluminum stress,the results showed that the relative root lengths of GsMML transgenic Arabidopsis lines were more than those of wild type at a certain aluminum concentration.Under the condition of 50?M AlCl₃?pH4.5?,the dissociative proline content of GsMML transgenic Arabidopsis lines were about twice level higher than those of wild type.The results suggested that GsMML overexpression enhanced the tolerance of transgenic Arabidopsis to acid aluminum stress.