Cloning And Functions Analysis Of Disordered Proteins MsZFP Gene In Alfalfa

Intrinsically disordered protein is a class of protein with normal biological activity which has more dynamic tautomeric structure. Intrinsically disordered protein can caused complex interactions diversity through a combination with a variety of molecules in molecular regulatory networks, thereby they play an important role in plant adaptation to variety stress. Transcription factor MsZFP having a finger-like domain which was predicted as an intrinsically disordered protein. Ms ZFP take part in cell differentiation, regulation of gene expression, embryonic development, and enhance plant resistance. In this study, alfalfa(Medicago sativa L) MsZFP was cloned, and transformed into tobacco. Results obtained from this study are expected to lay the foundation of MsZFP gene function research in resilience mechanism. The main findings are as follows:1. Cloning and sequence analysis of MsZFP geneMsZFP was cloned by RT-PCR. The gene contains 762 bp open reading frame which encode 253 amino acids with two single conserved C2H2-type domain. According to the disorder analysis of amino acid, MsZFP was found to be intrinsically disordered proteins. By building evolutionary trees with homologous genes from other species, we found that MsZFP gene's source of genetic kinship closer with the relatives gene from truncatula, soybean and peanut.2. Construction of plant expression vector pCBM-MsZFPPlant expression vectors pCBM-35S-MsZFP containing MsZFP gene which have glufosinate selection marker gene and constitutive promoter 35 S was constructed.3. MsZFP gene transformation into tobaccoMsZFP gene transfered into tobacco was using Agrobacterium-mediated method. 43 PPT resistant lines were obtained. 25 positive transgenic plants were screened by qRT-PCR verification from resistant plants, and showed high expression level in transgenic 3, 15 and 23 lines.4. Analysis of resistance of MsZFP transgenic tobaccoProcessing wild-type(WT) and transgenic tobacco 2 h under-4 ?, observed that the WT leaves wilting significantly, its relative conductivity significantly higher than transgenic tobacco. After low temperature stress(0 ?) treatment, we found that transgenic tobacco MDA content was significantly lower than the WT, but proline, soluble protein content was significantly higher than that of WT. Description MsZFP gene can improve cold tolerance of plants.The same part of WT and transgenic tobacco leaf discs were immersed in 300 mM NaCl solution for 7 days, transgenic tobacco plants remain green while the WT obvious chlorosis. Chlorophyll content of transgenic tobacco was significantly higher than that of WT, indicating that MsZFP gene play an important role in improving salt stress tolerance of plant.