Bioinformitics Analysis Of Genes Of Phosphatidic Acid Biosynthetic Pathway And Expression Of DGK Gene In Apple

Plants often suffer various stresses during their whole life, among these stresses, abiotic stresses like heat, cold, drought, salt are major factors that impact and limit the growth and development of plants. The abiotic stress will effect the agriculture production, and even lead to death of the crop, thereby influence agriculture production efficiency seriously. As a consequence, the study of abiotic stress related genes has significant theoretical and practical meaning.Phosphatidic acid(PA) is an important signaling molecule in both plants and animals, it already promises to rival the importance of the classic second messengers Ca2+ and cAMP. In plants, the production of PA can be triggered by a lot of biotic and abiotic stresses, including pathogen infection, drought, salt, wounding and cold. Two signaling pathways are predominantly held responsible for PA formation responses to abiotic stresses, one is formed directly by phospholipase D(PLD), which hydrolyzes structural phosphlipids to produce PA; the other one is via the sequential action of phospholipase C(PLC) and diacylglycerol kinase(DGK). It is observed that PLD, PLC and DGK all play key roles in the pathway of formation of PA signal. The gene identification and function study have made considerable progress and development in model plant Arabidopsis and rice(Oryza sativa). However, the research on these three genes family mostly concentrate upon herb, there is far less information about these genes family for woody plant species.In this study, the bioinformatic analysis of PLC, DGK and PLD gene family and the expression analysis of DGK genes were performed. The main results are as follows:1.Bioinformatic analysis of PLC gene family in Malus domesticaThrough bioinformatic analysis, we got 13 PLC protein sequences from apple protein database. Apple PLC gene family can be divided into two subfamily based on their gene architectures, protein sequence comparison and phylogenetic relationship with Arabidopsis PLCs, PI-PLC subfamily has 7 genes and NPC subfamily has 6 genes. The protein molecular weight of PI-PLCs is between 64 KDa and 79 KDa, and the isoelectric point in the range of 5.90 to 8.31. The protein molecular weight of NPCs is between 25 KDa and 52 KDa, and the isoelectric point in the range of 5.22 to 9.28. The number of exons in PI-PLC subfamily is all 9 except MdPLC2, this is identical with Arabidopsis. All the NPC members contain 2-4 exons and this intron-exon pattern is similar to Arabidopsis and rice.The predication of sub-cellular localization of PLC genes suggested that mostly PI-PLCs concentrated in cytoplasm and mitochondrial matrix, 3 NPC genes located in nucleus and the other 3 located in cytoplasm, endoplasmic reticulum membrane and outside, respectively.2.Bioinformatic analysis of PLD gene family in Malus domesticaIn this study, we have made a genome-wide search of PLD gene and identified 15 genes encoding PLDs in apple. These numbers where similar to the number of PLD genes present in the Arabidopsis(12), rice(17) and grape(11) genomes. Based on the presence of C2, PX and PH, signal peptide within their N-terminal regions, all the PLD family members were assigned to three main subgroups: C2-PLD, PXPH-PLD and SP-PLD, containing 11, 3 and 1 members respectively. In addition, based on the protein sequence comparisons, the conserved domain composition and the phylogenetic relationship with Arabidopsis PLDs, all the apple PLD genes were classified into 6 subgroups, α,β,δ,ε,ζ and φ, the gene number in each subgroup is 4, 2, 3, 2, 3 and 1. All apple PLDs contained two conserved HKD domains, meanwhile, the C2-PLDs contained one C2 domain in their N-terminal regions, the PXPH-PLDs contained both PX and PH domains and an N-terminal signal peptide was identified in the SP-PLD. The predication of sub-cellular localization of PLD genes revealed that most of MdPLDs located in nucleus and peroxysome.3.Bioinformatic analysis of DGK gene family in Malus domesticaWe have identified 8 DGK genes from apple protein database using bioinformatic ways. The results of evolutionary relationships and gene structure analysis indicate that MdDGKs fall into three phylogenetic clusters: ClusterⅠ, ClusterⅡ and Cluster Ⅲ, as also described for the AtDGKs. The genes in ClusterⅠ all contain 7 exons, which is completely same with Arabidopsis; in Cluster Ⅱ, there is only one gene that contain 12 exons while the corresponding genes in Arabidopsis have 9 to 12 exons. The genes in Cluster Ⅲ all contain 12 exons except MdDGK4, this is highly conserved with Arabidopsis. Motif analysis results showed that the number, size and pattern of motifs of MdDGKs in the same cluster are highly conserved. All of the MdDGKs were found to contain a DGK catalytic domain and a DGK accessory domain. In addition, genes in ClusterⅠ harbor two C1 domains and one trans-membrane domain on the N-terminal region. The protein molecular weight of DGKs is between 54 KDa and 82 KDa, and the isoelectric point in the range of 6.08 to 8.74.4. Expression of DGK genes in different tissue and under three abiotic stressesSix of DGK genes were chosen to analysis their expression pattern in different tissue and under three environment stimuluses. The results showed that all genes are expressed in leaf, shoot, root, flower and fruit of Malus prunifolia. Except MdDGK7, all genes had high expression levels in the shoot. In addition, they were induced significantly when response to drought, salt and ABA. All the results may have revealed that DGK genes play a role in signal transduction in response to these three environment stimulus. Four genes that response to abiotic stresses have been cloned from Malus prunifolia and the sequences have been submitted to NCBI GenBank, the accession number are KM099880, KM099881, KM099882 and KM099883, respectively.