| Temperature is one of the main environmental factors that affect the growth and development of plants, and it is also one of the main adversities to reduce the yield and quality of forage. Alfalfa(Medicago falcata L.) is a kind of important legume forage, which has strong cold resistance and drought resistance. To dig out the cold resistance related genes from Alfalfa and study the function of these genes can help to reveal its molecular mechanism of cold resistance, and provide theoretical basis for the genetic improvement of cold resistance of other legume forage. Medicago truncatula(Medicago truncatula G.) is a legume model plant with weak cold resistance. By comparing the genes which have differences in response to low temperature in alfalfa and medicago truncatula, we can screen the genes related to cold resistance in alfalfa.In this paper, the genes which have differential response to low temperature in alfalfa and medicago truncatula were selected as the object of study, and they encode auxin response protein 15 and C2H2 type zinc finger protein. Using the sequences of the Mt IAA15 and Mt ZFP gene in NCBI database to design PCR primers. Mf IAA15 and Mf ZFP gene were amplified by RT-PCR from c DNA of the leaves of the cold treated alfalfa. After sequence analysis,the open reading frame(ORF) of the Mf IAA15 gene is 822 bp, predicted that the protein consisted of 273 amino acid residues. The ORF of the Mf ZFP gene is 1356 bp, predicted that the protein consisted of 451 amino acid residues. By bioinformatics analysis, the Mf IAA15 in this study is most similar to the At IAA18 in Arabidopsis and Mt IAA15 in Medicago truncatula in evolution. And it has four conserved domains which the typical auxin response protein has. Mf ZFP is a C2H2 type zinc finger protein, and it is most similar to the Mt IAA15 in Medicago truncatula in evolution. Mt ZFP and other zinc finger proteins of many species, such as soybean and Arabidopsis thaliana, are highlyconserved in the zinc finger domain. Online prediction indicated that Mf IAA and Mf ZFP are all hydrophilic proteins through the website(http://www.expasy.ch/tools/proscale.html),and indicated that they are all located in the nucleus through the website Psort(http://wolfpsort.org/).In order to study the function of Mf IAA15 and Mf ZFP, we constructed two over expression vectors: p CAMBIA3301-Mf IAA15 and p CAMBIA3301-Mf ZFP. and used 35 S promoter to drive their expressions. We transformed them into Medicago truncatula through agrobacterium's mediation. We obtained transgenic regenerated plants by Basta's screening. We extracted the genomic DNA of transgenic plants and their wild types by CTAB method and detected the selection marker gene bar by PCR. The result shows that,60 transgenic regenerated plants in which Mf IAA15 is expressed and 50 transgenic regenerated plants in which Mf ZFP is expressed are inserted with bar gene. In addition, we introduced the expression vector p CAMBIA3301-Mf IAA15 into Arabidopsis thaliana and harvested T0 generation seeds. We used 2 mg/L Basta to spray T1 generation plants and obtained 30 strains of anti-Basta plants. Through the identification by PCR, the result shows that the specific bands of bar gene can be amplified in these resistant plants. Because the amount of plant material is not much, so in this paper, we don't carriy out further DNA hybridization and RT-PCR identification. This study laid a foundation for the further study of the relationship between Mf ZFP and Mf IAA15 and the cold resistance of plants. |
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