Identifcation And Expression Analysis Of Glucose-6-phosphate Dehydrogenase Genes In Strawberry (Fragaria×ananassa)

Strawberry (Fragaria x ananassa), a horticultural berries plants, belongs to Rosaceae Fragaria perennial herbaceous plants. Strawberry is a fruit of economic and nutritional importance and also a model species for fleshy fruits and genomics in Rosaceae. This topic studied the cryogenic of glucose-6-phosphate dehydrogenase (G6PDH, EC1.1.1.49) and antioxidant enzymes in the process of low temperature, obtained G6PDHs and other13key genes involved in cold response by cloning and4key genes from Genbank, and analyzed these gene expression changes under low temperature stress. The main research contents and results were as follows:1Determined the activity of G6PDH and reactive oxygen species (ROS) including super oxygen ions (O2·-) and hydrogen peroxide (H2O2), antioxidant enzyme system including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX), and malondialdehyde (MDA), soluble sugar content of strawberry during low temperature. The results show that the significant increase in G6PDH activity was found to be closely correlated to the levels of SOD, POD and MDA, and O2·-during cold acclimation. It is suggested that the enhancement of cold resistance of strawberry induced by cold acclimation is related to the distinct increase in G6PDH activity. On one hand, G6PDH activates NADPH oxidase to generate ROS; on the other hand, it may be involved in the activation of SOD, POD, CAT and APX, and promote many other important enzymatic reactions. Then, these factors further resulte in the elevation of membrane stability and cold resistance of strawberry. Thus we proposed a model of the physiological role of G6PDH in the enhancement of cold resistance.2Strawberry, riching in pigment, protein, polysaccharide, have the compound such as phenol which prone to browning effect. In order to obtain high quality of tissue total RNA in strawberry for the subsequent cold response gene cloning and expression analysis,7kinds of methods of total RNA extraction were summarized. Proved by this experiment, we found that3%CTAB3was appropriate for RNA extracting from all tissues of strawberry and total RNA obtained were of sufficient quality to be used for downstream experiments. 3Obtained13key genes involved in cold response by means of similar electronic cloning form strawberry under low temperature, including3G6PDHs (FaG6PDH-Cy, Accession No. KC433888; FaG6PDH-P1, Accession No. KC433889; FaG6PDH-P2, Accession No. JQ260862.1),4SODs (FaCu/Zn SOD, Accession No. KC433884; FaFe SOD1, Accession No. KC433886; FaFe SOD2, Accession No. KC433887; FaMn SOD, Accession No. KC433893),2APXs (FamAPX, Accession No. KC433892; FapAPX, Accession No. KC433895),1CAT (FaCAT, Accession No. KC433883),2glutathione peroxidase gene (FaGPX2, Accession No. KC433890; Fa GPX6, Accession No. KC433891),1NADPH oxidase gene (FaNADPH, Accession No. KC433894).4The chemical and physical properties, amino acid sequence, phylogenetic tree and protein3D structure of three types of G6PDH protein were analyzed by bioinformatics methods. Results show that the G6PDH of strawberry share the homology of over99%with wild strawberries, and share the homology above80%with Arabidopsis thaliana, potato, wheat and rice. The three proteins are soluble and strong hydrophilicity, and the N-terminal of FaG6PDH-P1, FaG6PDH-P2are found a period of about60amino acid sequence of signal peptide. Phylogenetic tree analysis found that FaG6PDH-Cy, FaG6PDH-P1, FaG6PDH-P2respectively gather together with each type of G6PDH family, then respectively together with dicotyledonous plants such as Vitis vinifera, Arabidopsis thaliana, Ricinus communis, Glycine max and Populus trichocarpa. The species status in the phylogenetic tree is consistent with its biological classification status. Three types of protein all contain two conservative functional domains, NADP combination domain and G6P combination domain.5The expression of13key genes involved in cold response obtained by cloning and4genes from Genbank (FacAPX Accession No. AF159630.1; dehydroascorbate reductase gene FaDHAR2Accession No. HM045477; monodchydroascorbate reductase gene FaMDAR Accession No. JQ320104.1; glutathione reductase gene FaGR Accession No. JQ339738) were analyzed by technique of real-time quantitative PCR during low temperature adversity. The results show that although G6PDHs, NADPH and all kinds of resistant enzyme genes adapted differently to the situation of low temperature, plants in cold acclimation can be roughly divided into two stages according to the expression trend, namely the early stage of the reaction, ability of tolerance in the late stage. The stage of early emergency response stage, gene expression quantity is gradually increasing to increased tolerance ability. Then, because of the oxidative burst, membrane structure damaged and been poisoned, some cold response genes expression quantity has decreased. But after the plants adapted to low temperature gene expression quantity increased again. Some genes such as FaG6PDH-P1, FaMDAR and FaGPX6are so strong that express continue to increase in the whole process of low temperature adversity. Interestingly, FaCAT highly expressed in the normal growth of plants, but decreased expression in the low temperature. High levels of genes obtained through the low temperature adversity could be a main reason for enhancing plant cold resistance during low temperature adversity.6Combining physiological response of various enzymes and cold response related genes expression in low temperature stress, both of them were increased significantly in the process of cold acclimation or de-acclimation, and then prompt the stability of cell membrane and increase cold resistance of strawberry. Most of cold response genes in de-acclimation are still maintaining high levels of gene expression and enzyme activity, so it will be a simple and effective method for plants to acquire chilling resistance through cold acclimation in seedling stage.