The Physiological And Biochemical Response And Differentially Expressed Proteins Under Low Temperature Stress In Citrus Reticulata Blanco

Citrus reticulata Blanco belongs to the subfamily of Rutaceae,which is small evergreen tree or shrup.The south of Shaanxi Province is the main place of Citrus reticulata Blanco planting in Shaanxi,and also is the northernmost region of Citrus reticulata Blanco cultivation in China,which is more easily to be limited by periodicity freezing injury.Three varieties of Citrus reticulata Blanco seedlings（Miju,Shiranui,Kumquat）were used to investigate the influence of low temperature（2℃,72 h）on leaf morphology,accumulation of O₂-.and related physiological indexes.Meanwhile,analysis of principal component was used to measure the cold resistance.Two-dimensional electrophoresis（2-DE）and MALDI-TOF/TOF-MS were used to study protein expression of Citrus reticulata Blanco seedlings under low temperature.In this study the corresponding cold preventive measures were put forward after the investigation and regularity research of periodicity freezing injury of Citrus reticulata Blanco in southern of Shaanxi Province since 1991.The results were showed as follows:1.Different degree of leaf wilting or curling was caused by 2℃low temperature stress in 3 varieties of seedlings,and Miju was heavily wilted,Shiranui was second.Histochemical localization of O₂^-.indicated that,Miju accumulated the most content of O₂^-.,but there was no significant changes in Shiranui and Kumquat.During low temperature stress,the relative electric conductivity（REC）and malondialdehyde（MDA）content about cell membrane system were increased,and Miju increased more than others,which indicated that the cell membrane system of Miju was damaged more serious.The osmotic adjustment substance,Proline（Pro）and soluble sugar were all increased,while Kumquat changed more prominent than Miju and Shiranui.The analysis of principal component showed that the correlation of MDA and the cold resistance was the highest,thus it could be used as an index to evaluate the cold resistance of Citrus reticulata Blanco seedlings.The order of cold resistance of 3varieties of Citrus reticulate Blanco was Kumquat>Shiranui>Miju.2.Tris-HCl buffer extraction method and three chloroacetic acid acetone（TCA-acetone）precipitation method were compared,and it was founded that amount of protein was extracted from TCA-acetone precipitation method was more,thus TCA-acetone precipitation method was more suitable for the preparation of two-dimensional electrophoresis.Two-dimensional gel electrophoresis technology was used to separate the proteins of Shiranui and Kumquat.194 proteins were identified from the protein pattern of Shiranui,94 protein spots could be matched,and 35 different protein spots were detected,5 proteins were up-regulated,30proteins were down-regulated.168 proteins could be detected from the protein pattern of Kumquat,77 proteins could be matched,14 proteins were changed significantly,and 7 spots were up regulated,7 were down regulated.Through proteomic analysis by 2-DE coupled with MADLI-TOF/TOF-MS,8 proteins were identified.Molecular chaperones,photosynthesis,nitrogen metabolism and signal transduction were involved in the response of Citrus reticulata Blanco seedlings to low temperature.3.Investigation and research on periodical freezing injury in southern Shaanxi showed that,six times of freezing injury occurred from 1991 to 2016.Recently,Citrus reticulata Blanco freezing injury cycle was shortening.Under cold stress,the freezing injury in Citrus reticulata Blanco planted on the slop was less severe than that planted on the flat and gully bottom,and the old trees were less severe than the young.The investigation also showed that,good cultivation and management and high grafting could effectively reduce the impact of freezing injury.