| In plants,the galactolipids monogalactosyldiacylglycerol(MGDG)and digalactosyldiacylglycerol(DGDG)are major constituents of photosynthetic membranes in chloroplasts.Synthesis of MGDG plays a vital role in plant growth and development.One of the key enzymes for the biosynthesis of galactolipid is MGDG synthase(MGD).Nowadays,a number of studies about MGD were addressed in dicotyledonous plants,while reports related to the type and function of MGD in monocotyledonous plants was limited.To investigate the type and function of the MGD in monocotyledonous plant,this study cloned three MGD homologous genes in rice(Oryza Sativa L.spp.japonica,Nipponbare),which is a model plant of the monocotyledonous plan;predicted the MGD types,subcellular localization and potential function in plant gowth and development by the bioinformatics softwares;studied the mechanism of MGDs regulating phosphorus deficiency by overexpressing OsMGD genes in tobacco.(1)Three MGD homologous genes in rice(Oryza Sativa L.spp.japonica,Nipponbare)were obtained by blasting the MGD gene sequences from Arabidopsis thaliana and rice(Indica subspecies)in NCBI database.OsMGD/02g?OsMGD/08 g and OsMGD/09 g were isolationed and cloned by RACE method.Then,we constructed the phylogenetic tree of MGD by aligning the sequences of these three MGD amino acid sequences with MGD amino acid sequences from other plants.Results showed that OsMGD/02 g possibly belonged to Type B MGD,the same evolutionary branch with AtMGD2 and AtMGD3.Whereas OsMGD/09 g was predicted to be Type A MGD which was in a same evolutionary branch with AtMGD1.OsMGD/08 g was predicted to be a new type and was specific in monocot.The transmembrane structures,subcellular localization and the cis-acting elements of these three OsMGDs were also predicted and analyzed through bioinformatics online software.It was showed that all OsMGDs were predicted to be hydrophilic protein,and no transmembrane region was observed.Subcellular localization prediction results showed that OsMGD/02 g and OsMGD/09 g were located in chloroplasts,while OsMGD/08 g in mitochondria.Stress related cis-elements were found in the promoter region of these three OsMGD genes.(2)The expression level of three OsMGD genes was detected in different tissue of rice using semi-quantitative RT-PCR.Results showed that OsMGD/02 g and OsMGD/09 g were highly expressed in leaf and stem and the expression levels in root and flower were relatively low.The expression of OsMGD/08 g in leaf,stem,root and flower,which was different with other two OsMGD genes,was quite low.The expression level of three Os MGD genes under phosphorus deficiency,drought stress and salt stress were also detected.All three treatments increased the expression of OsMGD/02 g in leaf first and decreased then,while the expression of this gene in root was keeping upregulated.However,OsMGD/08 g was strong upregulated only under phosphorus deficiency.The expression of OsMGD/09 g in leaf was changed differently under different stress,while unchanged in root under all stresses.(3)We constructed overexpression vectors of three OsMGD genes by Gateway system and obtained the transformed tobacco by the method of leaf disc cocultivation with positive Agrobacterium.After hygromycin in medium screening and qRT-PCR detecting,five transgenic lines overexpressing each OsMGD gene were obtained.According to the results of quantitative PCR,three transgenic lines were chosed to carry out the pot experiment.Compared with the wild-type plant,transgenic tobacco overexpressing different OsMGD gene presented increased chlorophyll content and net photosynthetic rate.Therefore,the growth rate and the leaf fresh weight of transgenic tobacco plants were increased.(4)To study the function and regulating mechanism of OsMGD homologous genes under phosphorus deficiency,wild type and transgenic tobacco plants were treated with low phosphorus stress by pot experiment.Compared with wild-type plant,transgenic tobacco plants showed significant fewer and lighter necrotic spots under phosphorus deficiency.Moreover,chlorophyll content and net photosynthetic rate in transgenic plants were significantly higher than that of wild type tobacco.Furthermore,Fv/Fm,? PS II and ETR in transgenic tobacco decreased slower than wild type.Therefore,transgenic tobacco plants overexpressing OsMGD homologous genes alleviated the damage caused by phosphorus deficiency and the tolerance to phosphorus deficiency of transgenic tobacco plants was higher than wild-type tobacco.(5)Lipid composition and components in tobacco old leaf were determined using gas chromatography.Compared with wild type tobacco,content of MGDG was higher in transgenic plants than wild type bocacco under phosphorus deficiency.Low phosphorus treatment increased the content of DGDG and the DGDG rised more in transgenic plants.These results showed that synthesis of MGDG and DGDG were enhanced in transgenic tobacco plants by overexpressing OsMGD homologous gene under phosphorus deficiency.Low phosphorus treatment also decreased the content of phospholipid and the phospholipid dropped less in transgenic plants.In addition,higher GL/PL and DGDG/MGDG were observed in transgenic plants under phosphorus deficiency,suggesting that more of the DGDG(double-membrane formation)transferred from plastid membrane to nonplastid membrane replaced the reduced of phospholipid,so as to maintain the integrity and stability of the membrane.Therefore,lipid remodeling,which was contributed to maintaining the integrity and stability of the membrane,was improved under phosphorus deficiency by regulating the synthesis of galactolipids.And then,the structure and function of the chloroplast and other organelles were maintained to alleviate the damage of low phosphorus stress on plant.Thus the tolerance of plant to phosphorus deficiency was improved.(6)Comparing the changes of physiological indexes and lipid composition of the transgenic tobacco plants,OsMGD/08 g overexpressing tobacco plant was more tolerant to phosphorus deficiency and followed the OsMGD/02 g overexpressing tobacco plant.OsMGD/08 g probably played the role in long-time phosphorus deficiency.DAG came from the degradation of the phospholipid might be used as the substrate for OsMGD/08 g to synthetize MGDG,which was predicted to be located on mitrochondria before.Then,possible DGD on mitochondria catalyzed MGDG to synthetize DGDG,which replaced the reduced phospholipid directly on mitochondria.After then,the integrity and stability of the membrane are maintained.While OsMGD/02 g probably played the role in short-time phosphorus deficiency. |
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