Cloning And Expression Analysis Of Salt-resistant Gene Of Peanut

Arachis hypogaea L.is the most important oil plant in our courtry, but salt stress is the major factor that limits and decreases the output of peanuts with the water crisis all around the world and overly high soil salinity. So finding the key factor of salt and understanding the mechanism salt tolerance, and improving peanuts salt-tolerant characters become imperative task. With the development of the molecular biological technology, many plant salt-responsive genes were cloned. In this study, NHX1 and SAMDC are cloned successfully from peanut and their structures and functions are analyzed by bioinformatic. AhNHX1 and AhSAMDC gene expression under different salt stress conditions in peanut are also analyzed by real-time quantitative PCR technology. The results were as follows:1. Degenerate Primers were designed based on the conserved sequences of the vacuolar Na⁺/H⁺ antiporter genes from other plants. A novel vacuolar Na⁺/H⁺ antiporter gene was isolated from Arachis hypogaea by RT-PCR and RACE approach, and named as AhNHX1, The cDNA 2331bp in length including an open reading frame (ORF) of 1620bp, a 5'untranslated region (5'UTR) of 450bp and a 3'untranslated region (3'UTR) of 261bp with a ploy (A) tail encodes a protein of 539 amino acids with a deduced molecular mass of 58.8 Kda and a pI of 7.23. The AhNHX1 protein shares 84.07%, 83.73%, 82.99%, 82.92%, 82.69%, 82.53% and 81.89% homology with the GmNHX1, CkNHX1, Tr NHX1, GoNHX1, LtNHX1, MsNHX1 and RpNHX1 respectively. AhNHX1 has twelve putative transmembrane domains, the N-terminal of which contains the conserved amiloride-binding sites (⁸⁴LFFIYLLPPI⁹³). Inaddition,two potential N-glycosylation sites were found within the AhNHX1 protein, at Asn50 and Asn293, respectively. These results suggested that the AhNHX1 protein is glycosylated. The gene has been submitted to GenBank database,the accession number: HM590627.2. Degenerate Primers were designed based on the conserved sequences of the SAMDC genes from other plants. A novel SAMDC gene was isolated from Arachis hypogaea using RT-PCR and RACE approach,and named as AhSAMDC,The cDNA 1984bp in length including an open reading frame(ORF) of 1077bp, a 5'untranslated region (5'UTR) of 741bp and a 3'- untranslated region (3'UTR) of 166bp with a ploy(A) tail. The AhSAMDC cDNA encodes a protein of 358 amino acids with a deduced molecular mass of 39.1Kda and a pI of 4.99. There are two uORF in 5'UTR and a polya with the signal of AATAAA in 3'UTR; The AhSAMDC protein shares 84.92%, 77.72%, 81.01%, 81.84% and 83.52% homology with the chickpea, lima bean, pea ,soybean and sickle medic respectively. There are two structural domains of SAM-decarbox and the PEST, which are ⁷⁰LSESSLF⁷⁶ and ²⁴⁸TIHVTPEDGFSYASFE²⁶³ respectively. The gene has been submitted to GenBank database, the aeeession number: HM070023.3. 18S rRNA gene were used as the control for AhNHX1 and AhSAMDC gene expression analysis by real-time quantitative PCR technology under different salt stress conditions in peanuts. The results showed that there is an expression of AhNHX1 and AhSAMDC gene under whether or not salt stress, belongs to constitutive expression; Moreover the expression of two genes has also tissue specificity and time differences: Under the low concentration of salt stress(50, 100 mM NaCl), the expressions of AhNHX1 and AhSAMDC genes in root augment with salt concentration increases, increase after the first reduce under high concentration of salt stress (150, 200, 250 mM NaCl), but in leaf and stem reduce with salt concentration increase. In different times, The expressions of AhNHX1 and AHSAMDC genes in root and leaf were highest quantity after 24 h salt stress, and was in stem after 48 h and 72 h salt stress respectively.