Cloning Of Glutathione Peroxidase And Selenium-binding Protein Gene In Aril Of Pomegranate And Their Expression Analysis

Pomegranate (Punica granatum L.) was adapt to the arid environment and adverse condition, therefore, it could be cultivated widely, the fruit of which was a sort of nutrient-rich ones people were willing to consume in daily life. Glutathione peroxidase (GPX), a kind of peroxiredoxins, was relative to lots of biotic stress and abiotic stress. Selenium-binding protein (SBP) was a type of selenoprotein which took part in antioxidant response. Both GPX and SBP gene had been linked to selenium and plaied important roles in antioxidant response. To study on the expression of the two genes in pomegranate plant, RACE methods were used to clone GPX and SBP gene with pomegranate seeds material, and Realtime PCR was used to anysis the expression of GPX and SBP in different tissues under different concentrations of selenium treatment, determine of gene expression and the glutathione peroxidase enzyme activity under salt stress. We also measured selenium content of different tissues and cultivars of pomegranates.Progresses of this research are following:1. Pomegranate seeds cDNA as template, RACE was used to get the sequence of GPX. The full length of GPX is 872bp, CDS is 504bp,168 amino acids were coded.5’ untranslated region length is 123bp,3’untranslated region length is 245bp. The gene we cloned is named as PgGPX, genebank accession is KJ143522. The full length of SBP is 1638bp, CDS is 1440bp,478 amino acids were coded.5’untranslated region length is 44bp,3’untranslated region length is 154bp. The gene we cloned is named as PgSBP, genebank accession is JX472277.2 The expression of PgGPX gene increase in leaf deal with the NaCl solution of 150mmol/L,300mmol/L and 450mmol/L.Under the 150 mmol/L Salt Stress,the expression reached the highest value in 3h and 4.80 times water treatment.lt dropped to 1.36 times in 12 h but up to 3.88 times in 48h.Under the 300 mmol/L Salt Stress,the expression reached the highest value in 3h and 8.17 times water treatment.It dropped to 4.06 times in 12 h but up to 18.77times in 48h.Under the 450 mmol/L Salt Stress,the expression reached the highest value in 3h and 15.32 times water treatment.lt dropped to 4.59 times in 12 h but up to 16.51 times in 48 h.3 It make clear that the glutathione peroxidase activity rise in the same cuttings leves by Oh,3h,6h and 12h deal with 150mmol/L NaCl.In the initial state of glutathione peroxidase activity was 47.43U. the enzyme activity is lower than the initial value in 3h and the value is 30.53U.Then the enzyme activity gradually increased and more than the initial value and the values are 53.37U and 67.77U in 6h and 12h.4 The expression of PgGPXand PgSBP genes has difference in the same cuttings deal with different concentration of sodium selenite solution.In the treatment of sodium selenite, the expression of PgGPX was higher than that of CK and reach a maximum value at 10mmol/L concentration of sodium selenite treatment which is 3.86 times higher than CK.Then the expression decreased and 1.76 times in 20mmol/L and 1.67 times in 30mmol/L.The expression of PgGPX deal with 40mmol/L rise 3.08 times than CK.The expression of PgSBP in the control 10mmol/L treatment was 1.98 times higher than CK(Expression of PgSBP in the water is 0.5 times of PgGPX).The expression level of PgSBP was 0.45 times of CK when the concentration reached 20mmol/L.The expression of PgSBP increased and more than the CK value when the concentration reached 30mmol/L,but the expression value is always less than 10mmol/L.It indicate that a small amount of exogenous selenium intake promotes the expression of PgSBP and excessive intake inhibiting the expression of PgSBP but always plays a promoting role in PgGPX.5.We measured the selenium purity of 8 cultivars of pomegranate and their four different tissues leaf, juice, peel, seed we selected before. The results show that the content of selenium in the same tissue has no obvious difference between different cultivars; the content of selenium between different tissues has significant difference in the same cultivar, and content order from high to low is Leaf, Seed, Peel and Juice.