Ectopic Expression Of SbMATE Gene To Improve The Aluminum Tolerance In Alfalfa

Aluminum is mainly composed of soil inorganic mineral elements, but Al3+or Al(OH)4+the presence of the soluble form to be toxic to all living cells. At low pH values, the toxic species of aluminum will be released from the solid phase into the soil solution or exchangeable aluminum adsorbed on the surface of the soil cation exchange sites. Aluminum toxicity in acidic soils is harmful to plant growth and soil microbial activities, which will lead to the plasma membrane hardens, increased lipid peroxidation, destruction of the plasma membrane charge balance via blocking the Ca2+and K+channel. Therefore, the study on plant aluminum tolerance is becoming increasingly important.Aluminum toxicity in acidic soils is the main factor that causes the inhibition of crop growth and reduction of crop yield, even if it is alfalfa, which is high-yield, nutrient-rich, strong regeneration and enjoy the "king of grass" in the world of crops. Alfalfa, which is adapted to neutral or slightly alkaline soil, is mainly distributed in the north of China. While due to the hot, rainy and humid climate characteristics, the southern region of China mainly covered with acidic soil, which severely limits the growth of alfalfa. Aluminum toxicity in acidic soils is one of the main factors causing difficulties in the growth of alfalfa. We hope to get a new line of alfalfa which has high resistance to aluminum toxicity through the plant genetic engineering techniques. Thus we could increase the alfalfa cultivation in the acidic soil of southern China, at the same time help to improve soil fertility and slow soil down acidification.To plants or other organisms, one of the most important mechanism against the toxic substances is the exclusion of toxic compounds in the organism. Multidrug and toxic compound extrusion (MATE) family is critical for the ability of crops to resist aluminum toxicity. The MATE family, which is common constituents of living organisms, is a new type of the secondary transporter gene family encoding transporter proteins. The typical characteristic of this family is encoding transporter proteins, which have12transmembrane domains. The MATE family members discharged multidrug and noxious substances in plants are mostly dependent on the transmembrane electrochemical gradient.SbMATE gene, which comes from sorghum, is the first well-investigated aluminum-tolerance gene in MATE family. The length of ORF sequence of the SbMATE gene is1803bp, and encodes a protein, which includes a12transmembrane domains by predicting, containing600amino acid residues. The SbMATE protein is the mainly membrane citrate transporter, and which is activated by aluminum. The SbMATE gene, which is closely related to the aluminum tolerance for sorghum, mainly expressed in the apical root under the condition of aluminum, and responsible for the secretion of citrate and aluminum citrate chelate from the apical root of sorghum.We selected the CaMV35S constitutive promoter and the AtPTl phosphorus deficiency-induced root specific promoter separately to drive the expression of SbMATE gene in alfalfa.To obtain the highly aluminum tolerance alfalfa plants, the method of gene expression analysis was used, meanwhile, the aluminum tolerance test was used to detect the aluminum content in the roots, stems, leaves, and the type and content of organic acid has also been detected. The primary results as bellow:1. Histochemical staining and PCR analysis of transgenic plants of alfalfa showed that the transgenic plants were obtained successfully.2. The content of Al in alfalfa plants was investigated. The results indicated that the content of Al in the roots of alfalfa plants was18.1%-30.7%lower in35S-SbMATE transgenic group, and8.8%-38.3%lower in AtPTl-SbMATE transgenic group compared with the control group. The reduction of the content of A1in AtPTl-SbMATE transgenic alfalfa plants is much more than that in35S-SbMATE transgenic group.3. The content of organic acid in alfalfa plants was detected. The results showed that the content of oxalic acid, citric acid and malic acid in the roots of alfalfa plants was down-regulated both in35S-SbMATE transgenic and AtPT1-SbMATE transgenic plants. The decrease of the content of organic acid in AtPTl-SbMATE transgenic alfalfa plants is about1.0fold more than that in35S-SbMATE transgenic group.4. The content of organic acid in stems and leaves of Alfalfa plant were detected. The results showed that, compared with control group, the content of citric acid in stems and leaves of AtPTl-SbMATE transgenic alfalfa plants were increased about3.41%to28.50%and4.27%to13.98%, respectively. And the citric acid and malic acid were down-regulated in stems of35S-SbMATE transgenic alfalfa plants, while the content of oxalic acid increased about10.68%-63.71%。These results implied that expression of SbMATE gene may improve the Al tolerance ability in alfalfa plants. And the aluminum tolerance ability of AtPTl-SbMATE transgenic alfalfa plants was obviously higher than that of35S-SbMATE transgenic alfalfa plants.The results obtained from this research could provide theoretical foundation for the further application of gene engineering to cultivate high Al tolerant varieties of alfalfa.