Cloning And Expression Analysis Of Superoxide Dismutase Genes(SOD) From Stylosanthes Guianensis

Stylosanthes guianensis is an excellent forage grass widely cultivated in tropical regions.However,cold damage,Stylo anthracnose and other stress factors seriously affect the production of Stylosanthes guianensis.Superoxide dismutase(SOD)is a kind of Metalloenzyme that can remove the reactive oxygen species(ROS).It is widespread in animals,plants and microorganisms,which is closely related to the stress resistance of plant.In this study,2 superoxide dismutase(SOD)genes were cloned at first time from leaves of the S.guianensis cv.Reyan No.2,CIAT184,and their expressions in the different tissues and in the leaves treated with chemical inducer were detected.The main results were as follows:1 One C u/Zn-containing Superoxide dismutase gene and O ne Mn-containing Superoxide dismutase gene was cloned by RT-PCR and RAC E techniques,and was named as SgCu/Zn-SOD and SgMn-SOD,respectively.The full-length cDNA of SgCu/Zn-SOD(GenBank number: KP100654)was 913 bp and had a 624 bp open reading frame(ORF)coding 207 amino acids,which had a predicted molecular mass of 22.1k Da.The DNA sequence of the SgCu/Zn-SOD(GenBank number: K R758758)gene was 1908 bp,which contained 8 exons and 7 introns.The full-length c DNA of SgMn-SOD(GenBank number: KR701610)was 965 bp and had a 714 bp open reading frame(ORF)coding 237 amino acids,which had a predicted molecular mass of 26.2k Da.The DNA sequence of the Sg Mn-SOD(GenBank number: KR758757)gene was 2414 bp,which contained 6 exons and 5 introns.2 Two promoter sequences of SgCu/Zn-SOD and SgMn-SOD were obtained by Tail-PCR.They were 766 bp and 568 bp long,respectively.The two of them contain TATA-box?CAAT-box and light responsive element.Besides,the promoter sequences of SgCu/Zn-SOD contains cis-regulatory element invo lved in endosperm expression and salicylic acid responsiveness and the promoter sequences of SgMn-SOD contains cis-acting regulatory element involved in circadian control.3 The q-PCR results showed that the expression of SgCu/Zn-SOD and SgMn-SOD had significant difference(P<0.05)in tissues including root,stem and leaf.The expression of SgCu/Zn-SOD was highest in leaf,while SgMn-SOD was in root.4 SgCu/Zn-SOD and SgMn-SOD were differently expressed in the leaves treated with chemical inducer(SA,MeJA and INA).After treating with SA,the expression of SgCu/Zn-SOD was higher than CK at 24 h,36h,48 h and 96 h,while SgMn-SOD was higher than CK at 24 h,36hand 96 h.In MeJA treatment,the expression of SgCu/Zn-SOD was higher than CK at 24 h and 48 h,while SgMn-SOD was higher than CK at 24 h.In INA treatment,both of them were was higher than CK at 12 h,24h and 48 h.5 SOD activity increased in the leaves treated with chemical inducer(SA,MeJA and INA).In this three treatments,the SOD activities were all higher than CK at 24 h,36h and 48 h.SgCu/Zn-SOD and SgMn-SOD gene were inserted into plasmid vector PBI121-GFP to construct plant express vector PBI121-SgCu/Zn-SOD-GFP and PBI121-SgMn-SOD-GFP,which is prepared for subcellular localization.