Studies On Cloning, Expression And Characterization Of NtSR45in Tobacco

Global analyses of splicing of precursor messenger RNAs (pre-mRNAs) have revealed that alternative splicing (AS) is highly pervasive in plants. The high frequency of alternative splicing among the serine/arginine-rich (SR) family of proteins in plants has been linked to important roles in gene regulation during development and in response to environmental stress. SR45belongs to plant-specific SR protein family that is highly conserved in the plant kingdom. Studying the function of SR45protein is helpful to clarify those molecular mechanisms.For the study, we isolated the SR45gene from Nicotiana tabacum NC89by the method of bioinformatics and molecular biology. Then, NtSR45gene was analyzed by bioinformatics, expression patterns, subcellular localization and transcriptional activity methods. The main findings of our study were as follows:(1) The NtSR45gene has an ORF of1242bp and encodes a413amino acids protein. It belongs to the SR45subfamily and contains an N-terminal RNA recognition motif (RRM) domain and two C-terminal RS-rich domains, which might be involved in regulating its function. It was submitted to the NCBI database with accession number JX846906.(2) The transcript analysis revealed that the NtSR45transcript is higher in leaves than in roots and stems in tobacco seedlings while the expression quantity of flowers is in the above of roots, stems and leafs in mature plant.(3) The expression patterns of NtSR45in response to various stresses including cold, dehydration, salinity, hot, ABA, ethephon (ET), methyl jasmonate (MeJA), salicylic acid (SA) or gibberellins (GA), were studied via real-time PCR. Results showed that maximum expression is observed under cold, salinity, ABA, MeJA and GA, highlighting its role in enhancing biotic and abiotic stress tolerance. In addition, NtSR45transcript got down-regulated in response to cold, dehydration, salinity, ABA and ET. After MeJA、SA or GA treatment, NtSR45expression was up-regulated, their quantity of expression were both increased first and decreased afterwards. However, NtSR45expression was down-regulated first and up-regulated afterwards after hot treatment.(4) Fusion proteins consisting of NtSR45, RS1or RS2domain coupled to the GAL4DNA binding domain strongly activated transcription in yeast, while RRM domain did not activate the expression of the reporter gene. (5) The localization of SR45and five deletion mutants of SR45fused to GFP in onion epidermal cells shows that SR45protein localized both in nuclear and cytoplasmic, probably the nuclear localization was mediated by RS domain.