Cloning and Expression Analysis of miR156-targeted SPL9 Gene of Strawberry
|School||Shenyang Agricultural University|
|Keywords||Fragariaxananassa Gene cloning SPL miR156 Real-time quantitative PCR|
SPL gene family that encodes the proteins considered to be a transcription factor is a specific gene family of plants, and is also the target gene of miR156. It is reported that SPL transcription factors were involved in a variety of physiological and biochemical processes, such as development of flowers and fruits, plant morphogenesis, sporogenesis, stress responses, hormone signal transduction and plant phase change. In recent decades, SPL gene is isolated from Arabidopsis, maize, rice, grape, pepper, trifoliate orange and so on. However, the research of its function has been limited to Arabidopsis and other model plants. In order to lay the foundation for plants phase change and strawberry culture plants "juvenility return" research, we cloned full-length SPL gene of strawberry, and studied the expression profiles of FaSPL9, miR156 and miR172 in different periods of strawberry seedling plants and micropropagated-plants by real-time quantitative PCR. The main results were as follows.1. Five SPL gene fragments were isolated from strawberry using degenerate primer with homology cloning method. The lengths of these fragments are 120 bp,141 bp,342 bp,544 bp and 773 bp, and two of them have the miR156 target sequence. The nucleotide identity compared with Arabidopsis thaliana or apple was higher than 78%.2. The 5’and 3’RACE methodologies were used to isolate full-length sequence of strawberry SPL gene, which shares the highest nucleotide identity with Arabidopsis thaliana AtSPL9, so we named it FaSPL9. The cDNA full length of FaSPL9 is 1337 bp, and coding sequence is 1143 bp which encoded the predicted protein of 381 amino acid residues including a SBP domain composed by 79 amino acid residues.3. FaSPL9 gene fragments is isolated from strawberry cultivars Toyonoka, Tudla, Albion, Tochiotome,Benihoppe and Line 08-4-1 using primers designed according to the full-length gene sequences obtained from Allstar. These fragments shared 99.18% nucleotide and 98.73% amino acid identity.4. With the real-time quantitative PCR based on Taqman probe, expressions of miR156 and FaSPL9 were compared among different periods of strawberry seedling plants and micropropagated plants, and the expression of miR172 among different periods of strawberry culture plants was detected. The results indicated that with the growth of seedling age a decrease in transcript levels of miR156 was found, the transcript levels of FaSPL9 is increased in seedling plants and the transcript levels of miR172 showsed a upward trend in micropropagated plants.5. FaSPL9 mutant that miR156 target sites eliminated without any amino acid change is obtained by Rapid site-directed mutagenesis methodologies.