Cloning And Salt Beet M14 Lines BvM14-SAMS2 Gene Response Under Oxidative Stress

Sugar Beet M14line is rich in gene resources for high quality traits, and alsopossess strong tolerance to abiotic stresses, including salt, cold stress and droughtstress. The Sugar Beet M14line is an uncommon material that can grow and developwell under the biotic and abiotic stresses. Using the Sugar Beet M14line as theexperiment material, a total of75differentially expressed proteins points under500mM NaCl stress have been obtained by using proteomics technology. In addition,58unigenes sequences have been obtained from the level of transcription screeningusing a SSH technology. A protein spot containing BvM14-SAMS2was found,which is the only matching protein spot between the proteomics and transcriptomicsexperiments.In this study, the1538bp full length of BvM14-SAMS2gene cDNA was clonedby using RACE, which contains the largest open reading frame of1182bp andcoding393amino acids. Subcellular localization studies showed that theBvM14-SAMS2protein may be in the cytoplasmic matrix. Homologous sequenceanalysis found that a98%similarity exists between the SAMS protein in Sugar BeetM14and that in Arabidopsis.The expression of BvM14-SAMS2gene was analyzed in Sugar Beet M14lineusing Real-Time PCR. BvM14-SAMS2gene in Sugar Beet M14line showed thehighest level of expression in roots, followed by stems, leaves and flowers. Forexample, its expression in the roots is4.26fold higher than in the leaves. Underdifferent concentrations of NaCl treatment, with the roots and the leaves under0mMNaCl as the control. The expression of BvM14-SAMS2was compared between theleaves and roots. Along with the increase of salt concentration, the expression ofBvM14-SAMS2gene is increased to149percent under the500mM NaCl treatment.The expression of BvM14-SAMS2gene in the roots were stable, while in the leavesincreased gradually. It is clear that salt stress induced the expression ofBvM14-SAMS2gene in Sugar Beet M14line.We constructed a prokaryotic expression vector pET28a-BvM14-SAMS2, and transformed into BL21(DE3). We focused on the prokaryotic expression system, andmeasured the concentration of the purified protein to be73.57μg/ml. The activity ofBvM14-SAMS2protein is1.24U/mg by using Western blot.In the meantime, we constructed an eukaryotic expression vector pCAMBIA1305.1-BvM14-SAMS2, and transformed into Agrabacterium tumefaciens EHA105.In addition, we obtained the wild type(WT) and SAMS gene knockout(KO)Arabidopsis thalianas using an inflorescence infection method. We also gained thepositive transgenic plants of T1generation. With the wild type(WT) and the SAMSgene overexpression(OX) as the control, the SAMS gene recovery type(CO) andSAMS gene knockout(KO) type Arabidopsis thalianas were determined under saltand oxidative stress respectively. Fresh weight, root length, chlorophyll, proline,MDA, the activity of SOD, POD, CAT and SAMS were measured. The resultsshowed that shifting the BvM14-SAMS2gene into OX and CO plants led to highertollerance to salt and oxidative stresses. It is cleaely demonstrated that theBvM14-SAMS2gene could enhanc the tolerance ability of plants under salt andoxidative stress.