Cloning Of PLD CDNA From Agropyron Cristatum (L.) Gaertn And Expression And Function Analysis Of PLD Gene Under Drought Stress

In this study,the full cDNA sequence of phospholipase D from Agropyron cristatum(L.)Gaertn was cloned by RACE technigue.The sequence of PLD cDNA,composition and characteristics of amino acid of PLD were analyzed with bioinformatics software. The physical and chemical properties of PLD in different species was also compared. The protein secondary and tertiary structure of PLD of Agropyron cristatum(L.)Gaertn was predicted.The signal peptide, hydrophobicity, transmembrane domain, local structural disorder of the PLD were analyzed. A phylogenetic tree was drown among the PLD proteins from 15 different species. The expression of the PLD gene was analyzed by real-time fluorescent quantitative PCR under different drought stress time and level of PEG6000. Six physiological indices of leave of Agropyron cristatum(L.)Gaertn was measured under drought stress, including proline, POD, CAT, MDA, Ca2+and soluble sugar. Correlation was analyzed between the relative expression of PLD gene and the physiological indices by SPSS13.0 software.The results showed as follows:1 The full length cDNA was 2967 bp which consist of open reading frame encoded 812 amino acids, 128 bp 5'untranslated regions and 368 bp 3'untranslated regions. The start codon was ATG and the termination codon was TAG. The sequence identity was 89%～99% compared with Agropyron mongolicum, Lolium temulentum, Oryza sativa and Zea mays.2 The relative molecular weight of PLD of Agropyron cristatum (L.)Gaertn was 91.832KD and its isoelectric point was 5.335. It was dissolvable protein without signal peptide and transmembrane domain.The secondary structure of PLD was mainly composed of random coil. The catalytic activity site composed of HKD motif near C-terminal and in the middle of tertiary structure.3 The expression of PLD gene of Agropyron cristatum (L.)Gaertn went throught all up-down-up-down style with the increasing of stress time. The expression of PLD gene was up-down-up style with the increasing of stress level.4 The transcriptional level was significantly correlated with the physiological indices(proline, POD and solube sugar)under different stress time and level.The results showed PLD of Agropyron cristatum(L.)Gaertn played a crucial role in drought resisting procell.this study laid a foundation for undersranding the regulatory mechanism and function of PLD gene.