| MADS-box, a very important regulatory factor in the process of plant growth and development, is a kind of gene family which widly exists in plants. It participates in vegetative growth, seed coat development, ovary development, embryogenesis, root formation and symbiotic induction. At present, the research of MADS-box genes regulation mechanism has become a hotspot in the field of plant molecular biology. AGL6 family genes belong to AP1/AGL9 group in MIKC subfamily in MADS-box genes, which functions are redundancy. Studies have shown that AGL6 gene is associated with floral organ development.Chongqing Engineering Research Center for Floriculture had cloned CpAGL6 (GenBank accession number:FJ807387) from Wintersweet(Chimonanthus praecox (L.)Link), it was 948 bp. RT-PCR of different tissues and different stages of Wintersweet flower showed that CpAGL6 specifically expressed in Wintersweet flower organs, and participated in the formation of floral primordium. Promoter is an important regulatory factor on the transcriptional level. It determines the temporal and spatial expression patterns and intensity of gene through combining a variety of transcription factors. Researchers have done many study of AGL6, but few about the promoter of AGL6. In order to clarify the expression characteristics of CpAGL6 gene promoter in plants, we cloned the upstream regulatory sequence of the gene from Wintersweet, and preliminary studies the function of the promoter. The results are as follows:1. Three nested primers have been designed near the 5’side of CpAGL6, the regulative sequence (1266 bp) of the flower development-related gene CpAGL6 promoter was cloned from genomic DNA of Chimonanthus praecox by hiTAIL-PCR. Bioinformatics analysis revealed that the promoter sequence contained basic cis-elements, such as TATA-box and CAAT-box and many elements involved in the plant abiotic stress.2. In order to study the function of CpAGL6 promoter, a promoter-reporter vector was constructed, and introduced into tobacco by Agrobacterium-mediated method. The result of transient expression indicated that the sequence had the function to drive reporter gene GUS in tobacco.3. The transgenic plants were obtained after Kanamycin resistance screening, GUS histochemical staining and PCR analysis.4.Quantitative detection of GUS enzyme activity showed that:CpAGL6 promoter could drive the GUS gene exclusively express in leaves, stems and flowers of transgenic tobacco, and there were significant differences of GUS enzyme activity among the flowers in different stages, almost no expression in roots. The results indicated that CpAGL6 promoter mainly driven GUS reporter gene expression in floral organs and green organs or tissues of tobacco.5. GUS enzyme activity of transgenic plants increased under different treatments, including darkness, gibberellin (GA3) and low temperature. The results indicated that CpAGL6 promoter play a very important role in abiotic stress resistance.6. The T2 generation of transgenic tobacco seedling GUS expression pattern time testing results showed that only 7 d tobacco seedlings coloring is very shallow, the rest can clearly see the blue spots in each period, and spot color depth along with the growth process of gradually deepening. It suggested that the regulation role of promoter may increase with the growth of tobacco. |
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