Study On The Changes Of Granulation And The Activity Of Pectin Methylesterase In Later Ripening Navel Orange(Citrus Sinensis Osbeck)

Citrus,ranking first among fresh fruit in the world,is the third largest trade agricultural product,and it is also one of the important cash crops.Juice sac granulation is a physiological disorder,which leads to lignification,dehydration of juice sac cell and poor quality of commodity value of fruit.Previous studies had shown that low temperature was the main cause of later ripening navel orange granulation before harvest.The anatomic structure showed that granulation juice sac was often associated with the formation of secondary wall and the thickening of juice sac cell wall.Pectin methylesterase catalyzes the removal of the methyl group from homogalacturonan,and pectin methylesterase inhibitor regulates its activity by forming a 1:1 reversible complex with PME,both of which take a significant part in cell wall modification during fruit ripening and plant development.In this study,lanelate navel orange(Citrus sinensis Osbeck ‘Lanelate’ Navel orange)selected from Three Gorges Reservoir Area in Hubei Province was used as experimental material and PME/PMEI family genes were identified from Citrus genome.To gain insight into the roles of pectin methylesterase in juice sac granulation progress in Citrus and its application prospect in the prevention and control of granulation,we performed a genome-wide characterization of the PME/PMEI family,including gene family bioinformatics analysis,expression patterns,subcellular localization,transcription factor interaction analysis and functional characterization.The main results of this study are as follows:1.53 PME genes and 45 PMEI genes were identified,which were respectively divided into four branches and three branches by phylogenetic analysis.Analysis of gene structure and conserved domain showed that the gene structure and conserved sequence of the same clade of CsPME/CsPMEI protein were similar.Chromosome localization,gene replication and collinearity analysis showed that,53 CsPMEs and 45 CsPMEIs were unevenly distributed on other Citrus chromosomes except Chr8.6 pairs of CsPMEs and 9 pairs of CsPMEIs were tandemly duplicated,17 pairs of CsPMEs and 6 pairs of CsPMEIs were segmentally repeated.A total of 27 homologous PME gene pairs and 20 homologous PMEI gene pairs were identified between Citrus and Arabidopsis,and 16 homologous PME gene pairs were identified between Citrus and Oryza sativa.The above results provided an important basis for further study on CsPME/CsPMEI gene function.2.Transcriptome data analysis of four different granulation degrees of ‘Lanelate’navel orange fruits showed that,11 CsPMEs and 6 CsPMEIs were differently expressed between granulated fruit and normal fruit.The expression pattern was further verified by q RT-PCR,and the expression of most CsPMEs were consistent with the transcriptome data except CsPME22,and the expression of all differentially expressed CsPMEIs were consistent with the transcriptome data.q RT-PCR results showed that all the differentially expressed genes were induced by low temperature.Therefore,the CsPME/CsPMEI gene families were involved in the process of juice sac granulation induced by low temperature.Subcellular localization analysis showed that CsPME35,CsPME40,CsPME52 and CsPMEI19 were located in the cytoplasm,CsPME6 and CsPME34 were located in the vacuole membrane,CsPME3 and CsPME21 were located in the cell wall,and CsPMEI32 was located in the plasma membrane.And these genes might be synthesized in cells and transported to the cell wall to play a role.3.Yeast one-hybrid assay showed that the transcription factor Cs RVE1 directly interacted with the CsPME3 promoter.Dual luciferase transcriptional activity assay(LUC)was further measured in tobacco leaves,and it was found that Cs REV1 significantly induced pro:CsPME3 activity.q RT-PCR results showed that the expression level of Cs RVE1 was also induced by low temperature treatment at 3h and 48 h.Therefore,Cs REV1 might play a regulatory role in the process of juice sac granulation induced by low temperature,through activating CsPME3.Stable transformation and latter analysis in navel orange callus showed that over-expression of CsPMEI19 significantly increased the total pectin content,which was consistent with previous results of pectin content change in granulated juice sac.And the expression of CsPMEI19 was significantly upregulated at 48 h and 72 h in response to low temperature induction.In conclusion,this study not only comprehensively classified the two gene families of PME/PMEI in Citrus,but also provided new insights into the biological functions and regulatory patterns of pectin methylesterase during the process of juice sac granulation in navel orange fruit.