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Cloning And Functional Analysis Of DNA Methyltransferase MtMET1 Gene In Medicago Truncatula

Posted on:2024-03-01Degree:MasterType:Thesis
Country:ChinaCandidate:Z Y LiuFull Text:PDF
GTID:2543307079495764Subject:Grass science
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DNA methylation is widely found in eukaryotes,and is involved in many biological activities,such as embryonic development,cell differentiation,gene silencing,transposon element inhibition and stress response.It is an important epigenetic modification pathway that was found earliest and studied most deeply.DNA Methyltransferase 1(MET1)is an important DNA methylase,which plays a key role in the process of DNA methylation and is mainly responsible for the methylation of symmetric Cp G sites.MET1 protein is also extremely conserved in a variety of organisms.Medicago truncatula is a self-pollinating alfalfa plant in the leguminous family.It has the characteristics of small ploidy,small genome,short growth cycle,and easy transformation.It has become a model species for leguminous research.In view of the lack of research on DNA methylation in legumes,the DNA methyltransferase gene of Medicago truncatula(R108)was cloned in this study,and using bioinformatics tools to analyze its structure characteristics.Through the transformation of Arabidopsis thaliana,we preliminarily analyzed its gene function.Meanwhile the genetic system of Medicago truncatula was optimized.We successfully obtained Medicago truncatula plants by overexpressing Mt MET1 gene.It is expected to provide a reference for improving leguminous plants from the perspective of DNA methylation.The main research results are as follows.1.The CDS sequence of Mt MET1 gene of Medicago truncatula is 4665 bp in length,encoding 1554 amino acids,with a theoretical isoelectric point of 5.51,fat coefficient of 74.58 and instability coefficient of 45.48,which is an unstable protein.Mt MET1 is a hydrophilic protein without signal peptide and transmembrane region,and its subcellular localization predicts its localization in the nucleus.There are a lot of random coils andα-helical structures in the secondary and tertiary structures,meanwhile there are five key functional domains.Protein homology analysis showed that the Mt MET1 protein of Medicago truncatula was closely related to Pisum sativum,Trifolium pretense and Cicer arietinum of leguminous plants,but was far away from Triticum aestivum and Aegilops tauschii of gramineous plants.2.The plant over-expression vector p CAMBIA3301-Mt MET1 was successfully constructed by homologous recombination method.The Agrobacterium tumefaciens strain GV3101 was used as the transformation engineering bacterium.Arabidopsis thaliana was successfully transformed through the flower dipping method.The T3generation homozygous transgenic Arabidopsis thaliana seeds were obtained under the screening of Glufosinate-ammonium.The drought and salt tolerance of transgenic Arabidopsis thaliana was decreased under salt stress and drought stress.3.The genetic transformation system of Medicago truncatula was optimized.The sterile leaves of Medicago truncatula were selected as explants.Agrobacterium GV3101 was the infected strain.200 mg/L Cefotaxime Sodium(Cef)and Timentin(Tim)were antibacterial antibiotics.The optimal callus induction medium was Murashige and Skoog medium(MS medium)+2,4-Dichlorophenoxyacetic acid(2,4-D)(2.0 mg/L)+6-Benzylaminopurine(6-BA)(0.2 mg/L).Glufosinate-ammonium(PPT)(2.0 mg/L)was the optimal screening concentration.At present,a total of 33 PPT resistant transgenic plants were obtained,and 16 positive transgenic plants were obtained by Polymerase chain reaction(PCR),with a positive rate of 48.48%.
Keywords/Search Tags:DNA methylation, Medicago truncatula, Gene cloning, MtMET1, Genetic transformation, Functional analysis
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