Glucose-6-phosphate Dehydrogenase Gene Cloning And Its Role In Lotus Response To Submerge

Nelumbo nucifera?lotus?is more famous traditional flower of Chinese.It has a profound cultural connotation and high ornamental value,economic value and ecological value.Market demand has expanded each year.Since the lotus can not grow in deep water,easy to be drowned,so limiting the scope of application of the lotus.Therefore,it is one of the goals is to cultivate the deep-water varieties of resistant to flooding.Submerged is abiotic stress that has significant harm on plant growth and development,and to limit the distribution and yield of crops.When flooding stress,the pentose phosphate pathway is a major pathway of glucose metabolism,part of the gene and enzyme systems in the active state of plant.Study confirmed that,as a key enzyme in the pentose phosphate pathway?PPP?,glucose-6-phosphate dehydrogenase?G6PDH?participation in the regulation response to flooding stress of plant.Early transcriptome data analysis shows that Glucose-6-phosphate dehydrogenase?G6PDH?are active in the lotus flooded,it may be involved in regulating waterlogging tolerance of plants.However,no study on this idea.In order to verify the role of glucose-6-phosphate dehydrogenase and its regulatory mechanisms involved in resistance to flooding of the lotus.The present study first from the genome-wide analysis identified three lotus G6PDH?NnG6PDH?gene,and they were identified in terms of Bioinformatics and Molecular Evolution,and in-depth study of the mechanisms involved in its regulation of flooding tolerance in lotus.Meanwhile,the effects of exogenous glucose,Na₃PO₄?G6PDH activity inhibitor?treatment and flooding stress-induced expression of NnG6PDH,and under flooding stress,exogenous glucose,Na₃PO₄?G6PDH activity inhibitor?Physiological response of lotus,and verified functionality of NnG6PDH.Provides important technical foundation for the next step to carry out anti-flooding transgenic breeding cultivate lotus varieties.The main results are as follows:1 Identification and analysis of glucose-6-phosphate dehydrogenase?G6PDH?gene family in the lotus.In this study,three lotus G6PDH genes?NnG6PDH 1 to 3?were identified and cloned using a genome-wide scanning approach.The full length cDNA of NnG6PDH genes were 1557 to 1779 bp and encoding a peptide of 518 to 600 amino acids.Analysis of the functional domain features indicated that NnG6PDHs protein contains typical conserved structures,including the C-terminal active site,N-terminal NAD?P?binding site.Phylogeny analysis and subcellular localization prediction show that NnG6PDH3 is a cytosolic isoform,while the other two genes are plastidic isoforms.Prediction and analysis of promoter region showed that the cis-acting elements,such as ERF 1,AG and HMG-I/Y were predicted in this promoter structure.Transcriptional analysis of gene suggested that they were expressed in different tissues.Among them,NnG6PDH3 could obviously be induced by submergence and cadmium stress,implicating their roles in maintaining redox balance and defending against oxidative stress.2 Function of G6PDH in the regulation of glucose tolerance of lotus floodingWith red lotus of Weishan Lake seedlings as test materials,treatment with the exogenous glucose at 0,1 ?M,10 ?M,100 ?M,1 mM,10 mM,and 100 mM,Submergence stress and 100 ?M Na₃PO₄.Study on NnG6PDH induced expression of and physiological response of lotus,The results show:With the extension of the time submerged,the content of sugar,especially glucose,in lotus petiole decreased significantly.Supplied with exogenous glucose could enhance tolerance of lotus petiole to submergence stress within 48 h.Thus also show that glucose can be used as a response signal affect glucose of expression or activity of genes to regulate the resistance of plants to flooding.Early transcriptome data analysis shows that glucose-6-phosphate dehydrogenase 3?NnG6PDH3?genes might act as downstream target of glucose.The transcripts of G6PDH3 and G6PDH activities were induced after exposure to either glucose or submergence.Further results showed that glucose-mediated protected effect could be reversed by Na₃PO₄?an inhibitor of G6PDH?.The activities of representative antioxidant enzymes were induced by submergence,which might contribute to lotus tolerance to submergence.However,the effect of submergence in inducing antioxidant enzymes were counteracted by the treatment of glucose,and enhanced by Na₃PO₄.This indicated that G6PDH has an inhibit effect on submergence-induced antioxidant enzymes.Further results showed that Glucose-induced non-enzymatic antioxidant,reduced glutathione,could be blocked by Na₃PO₄.Together,these results demonstrated suggested that the content of glucose might function as an important signal to regulate the response strategy of lotus in response to submergence,via G6PDH regulated GSH content in early stage of submergence or switch to activating antioxidant enzymes,which might make the response faster and economical.