Molecular Cloning And Expression Analysis Of GAPDH And OPDR From Puccinellia Tenuiflora

Puccinellia tenuiflora as a pasture in the Northern, has strong salinity resistance. Studying stess resistance genes and the related mechanism at molecular level is more significant for improving salt tolerance and soil utilization. Previously we have screened some highly expressed genes on saline-alkaline stress by microarray technology, including the key enzymes of metabolism GAPDH and OPDR. This experiment is to clone full-length sequences of GAPDH and OPDR by some known sequences from Puccinellia tenuiflora-suppression subtractive hybridization library (suppression subtractive hybridization library, SSH). And to use bioinformatics tools analyzing the sequences of genes and proteins, constructing the phylogenetic trees. Meanwhile studying the expression of the two genes under different Na2CO3 treatment. It can lay a foundation for studying the salt resistance mechanisim of Puccinellia tenuiflora. The results of this study are as follows:(1) Using Puccinellia tenuiflora as material, construst Puccinellia tenuiflora RACE cDNA library. Then use RACE technology to clone full-length sequences of GAPDH and OPDR genes from Puccinellia tenuiflora.(2) The cloned sequences of GAPDH and OPDR are respectively 1030 bp and 978 bp. Known from the bioinformatics analysis of genes:Their ORFs can encode 342 and 378 amino acid polypeptide chains respectively. GAPDH protein contains two conserved regions, they belong to the Gp_dh_C superfamily and the Gp_dh_N superfamily. The Blast analysis shows that:GAPDH gene in Puccinellia tenuiflora has the same gene homology as the ones in Festuca rrundinacea and barley, with the high homologous rate of 97%, OPDR gene and some genes related to the synthesis of OPDR in Oryza sativa Japonica Group have the same gene homology, such as OPDAR1 gene, with the high homologous rate of 84%.(3) Treat the grass with different concentration of Na2CO3. Northern blotting shows:the expression of GAPDH and OPDR genes in leaves have increased compared with the control; Meanwhile treating Puccinellia tenuiflora with Na2CO3 for different periods, the expression of the two genes in leaves and roots have increased significantly compared with the control. The results above all reveal that GAPDH and OPDR genes are related to the salt tolerance of Puccinellia tenuiflora.