Study On Freezing Resistance And DNA Binding Proteins Associated With Freezing Tolerance Of Kumquat

DNA-binding proteins are proteins that have a specific or general affinity for either single or double stranded DNA. They mainly include histones, transcriptional factors, DNA polymerase, RNA polymerase, single-stranded DNA binding protein, helicase, ligase, restriction endonuclease, telomerase, and etc. The variation of the binding style between DNA-binding protein and DNA would triger a series of changes on chromosome. And this variation plays a critical role on DNA replication, recombination, transcription, chain degradation and other biological processes.Freezing injury is a kind of meteorological hazards of agriculture, which mainly occurs on overwintering crops, fruit trees and economic forests. Thus it is a huge threat to agricultural production. For example, citrus production in U.S. and citrus production in China suffered huge losses due to freezing injury. Fortunella margarita is an evergreen tree and has the capablity to withstand freezing temperature, some diseases and insects. Therefore, it is a great research object for the isolation of freezing-resistant genes and the investigation of anti-freezing mechanism in plants.In this project, Fortunella margarita was used as the primary material for establishing a freezing tolerance experiment model. Low temperature stress-responsive DNA-binding proteins in the cold acclimation of Fortunella margarita were analyzed by 2-D electrophoresis and mass spectrometry techniques. Furthermore, the transcription regulation mechanism of Fortunella margarita’s freezing resistance process was investigated.Two-year old, vigorous and similar-sized Fortunella margarita (Lour.) Swingle seedlings were transplanted in cultivate pots and grown under greenhouse conditions(25℃)for 3 weeks; then some seedlings were selected at random and transferred to grow in a growth champer(4℃)for cold acclimation for 2 weeks. During the establishment of freezing tolerance experiment model, growth pots and low parts of seedling stalks were coverd lightly by cotton materials and treated under -10℃for 60min; The cold stress-treated seedlings which displayed certain degrees of injury were regarded as the experimental group, while those untreated seedlings with no cold injury would be the control group; The sucessful establishment of freezing tolerance experiment model lays a solid foundation for subsequent study.In addition, the physiologic and biochemical index under the freezing tolerance experiment model were examined. It was found that cold acclimation can effectively improve the content of soluble protein, soluble sugar and proline. And this may be one of the reasons explained the capacity of cold resistance in Fortunella margarita. It was also found that SOD, CAT and POD were coordinated with each other to maintain the relative low level of free radicals. Thus the free radicals’damage to membrane was largely reduced and then the stress tolerance capability in plants was improved in this way. The mathematic model of the oxygen radicals elimination by anti-oxidization enzymes is as following, N=X—b×X×Y×Zi—c×X×Y×Z2 (N:oxygen radicals responsible for the produce of MDA in lipid membrane peroxidization; X:the content of oxygen radicals in plants; Y:SOD activity in plants Z1: CAT activity in plants Z2:POD activity in plants)Microcrystalline cellulose was used to immobilize DNA through a process that phosphate groups in single-stranded DNA fragment can bind with hydroxyl groups to form Phosphodiester bonds. And techniques based on DNA re-denaturalization characteristics were used to acquire double-stranded DNA chromatography ligand from the stabilized DNA.DNA-binding proteins in leaves of Fortunella margarita were prepared by dsDNA chromatography ligand, and then mixed with purified genome DNA for chromatin reconstitution. The result indicates that DNA-binding proteins prepared by DNA-cellulose chromatography technique can combine with genome DNA for chromatin reconstitution.SDS-PAGE was applied to isolate DNA-binding proteins; then LC-MS-MS was taken to analyze these DNA-binding proteins and 1614 peptide sequences were acquired. Bioinformatics analysis indicates that among these peptide sequences.28 kinds of protein were identified. And quite a name of unmatched peptides were found to have structural characteristics similar with some classic domain structures in transcription factors.Under the freezing tolerance experiment model, the comparision of DNA-binding proteins in leaves of Fortunella margarita with different treatments was also conducted. The result indicates that there are differences in the quality and quantity of expressed DNA-binding proteins during Fortunella margarita’s cold acclimation. Also there is a fact of methionine oxidization in DNA-binding proteins. Therefore. this phenomenon indicates that methionine oxidization may be one of the regulators in transcription regulation.