Molecular Characteristics And Differential Expression Analysis Of Cu/Zn-SOD Gene In Peanut (Arachis Hypogaea L.)

Peanut (Arachis hypogaea L.) is a widely cultivated commercial plant in the world，which is also one of the most important oilseed and cash crop in China. Oxidative damagegenerated by drought is the primary limitation factor for the yield and quality of peanut. Forthis reason, reaserch on the molecular mechanism of response to drought stress in differentcultivars is particularly essential and valuable for accelerating the process of breedingdrought-resistant varieties. As the key enzymes of antioxidant protection system, superoxidedismutase (SOD, EC1.15.1.1) plays important role in resisting oxidative stress induced bydrought. Based on PCR, we obtained cytoplasm Cu/Zn-SOD gene (AhCSD1) andchloroplastic Cu/Zn-SOD gene (AhCSD2) from peanut cultivars and dopild wild speices. Bysequences alignment, we explored the molecular origins of AhCSD1AhCSD2in cultivatedpeanut. In addition, further study on expression pattern of AhCSD2and the differences amongvarieties were performed upon stress induced by PEG and drought. The major results areabstracted as follows:(1) Cloning and sequence analysis of AhCSD1in peanutAnalysis showed the two cDNA sequences of AhCSD1in Shanhua9, labeled asrShCSD1-1and rShCSD1-2, were98.18％identical with10SNP sites, which were alllocated in the open reading frame. The corresponding genomic DNA sequences, named asgShCSD1-1and gShCSD1-2, were97.08％identical with64differences in total,27of whichwere SNP sites. Three different recognition sites of restrictive enzyme were found, in addition.Sequences analysis showed AhCSD1contained8exons and7introns. The rCSD1-2sequences in the4cultivars were completely identical, while99.68%homologous as torCSD1-1with6SNP sites in the coding region, causing the number53,75, and147differences of the corresponding amino acid sequences. Molecular evolutionary analysisdisplayed gShCSD1-1and gShCSD1-2came respectively from A-and B-genome ofcultivated peanut, and A. ipaensis was the donor of B-genome of cultivars. Compared with A.kuhlmannii, A. duranensis from the same A-genome had a closer relationship with peanutcultivars.(2) Cloning and sequence analysis of AhCSD2in peanut The two cDNA sequences from Shanhua11, rCSD2-1and rCSD2-2shared a homologyof98.56%with11SNPs in total, were764bp in full-length with624bp of ORF. Thecorresponding DNA sequences, gSh11CSD2-1and gSh11CSD2-2, were2395bp and2428bpin length, with the homology of96.54%with84different sites,46of which were SNP. Thealignment showed8exons and7introns were contained within AhCSD2with5differentspecific recognition sites of endonuclease. The derived amino acid sequences, labeled asAhCSD2-1and AhCSD2-2,207aa in length, were both predicted to contain a chloroplastictransit peptide from1to53aa with6differences, the2differences in mature peptide regionmight resulted in three dimensional structure changes. For different cultivars, gCSD2-2were100％identical, but99.98%with3SNP sites in the intronic region as to gCSD2-1among the11cultivars. Molecular evolutionary analysis of AhCSD2displayed gCSD2-1and gCSD2-2came respectively from A-and B-genome of tetraploid cultivated peanut, and A. ipaensis wasthe donor of B-genome. A. cardenasii resembled sequences from cultivars most, followed byA. duranensis.(3) Expression analysis of AhCSD2in different peanut cultivars upon PEG stressThe results indicated the dynamic changes of SOD activity and transcriptional level ofAhCSD2among different drought-tolerant varieties were highly significant (p<0.01) uponPEG stress, of which in drought-resistant Shanhua11and Jihua4were substantially high thanthat of drought-sensitive Baisha1016and Krapt.st.16. SOD activity and expression level ofAhCSD2maximized separately at24h and12h under PEG stress, which were also in highlysignificant positive correlation (r>0.95, p<0.01). The results proved SOD activity wasregulated by AhCSD2at transcriptional level in peanut under PEG stress.(4) Expression analysis of AhCSD2at transcriptional level under drought stress indifferent cutivarsThe correlation between SOD activity and expression of AhCSD2was highly significantpositive. The expression of AhCSD2at transcriptional level under drought stress was in highlysignificant positive correlation with drought resistance of cutivars. Therefore, thetranscriptional expression of AhCSD2can be used as assessment indicators for mechanism ofdrought resistance in different peanut cultivars.