Isolation And Construction Of Expression Vector Of BnAGL15 Gene From Brassica Napus,PMADS9 Gene From Petunia Hybrida And Introduction Of BnAGL15 Gene Into Petunia Hybrida Mediated By Agrobacterium Tumefaciens

MADS-box genes have been isolated from numerous eukaryotic organisms and encode transcription factors involved in growth and development. The MADS-box genes were named after four of the originally cloned members-MCM1, AG, DEFA and SRF-which all possess a highly conserved 56 amino acid motif within their DNA-binding domains. The MADS region linked specifically to the coalescent site of target gene and formed a homogenous or heterogenous dimer to regulate it, which can make different kinds of floral organs for many flowering plants. So they are important factors to control the development of floral organs and to make homeotic transformations. It is very useful to analyze the floral organ identity genes in order to understand the molecular model of plant floral organ development.AGL15 (AGAMOUS-like 15) is a mermber of MADS-box family.lt was found that overexpression of AGL15 affects floral organ senescence.The effects of AGL15 could be distinguished from the effects of the ethylene to senescence. PMADS9, a mermber of AGL15 subfamily, was isolated from petunia and the function was not conformed. This experiment obtained BnAGL15 gene from rape (Brassica napus) genome DNA and PMADS9 gene from Petunia (Petunia hybrida) young floral bud cDN A by PCR amplification, and construction of them expression vector respectively.We also introduced BnAGL15 into petunia (Petunia hybrida) via Agrobacterium tumefaciens mediated method.The main results are as follows:1.Cloning of BnAGL15 and construction of the plant expression vectorThe BnAGL15 gene fragment was amplified by meas of PCR using rape (Brassica napus) genomic DNA as template and a pair of specific oligonucleotides at 5' and 3'-end as primer. Sequence analysis showed that the fragment consisted of 1695bp and encoded 264 amino acids residues deduced from DNA sequence.which had 7 introns. Comparison with previously published sequence showed 98.47% homologies in nucleotide sequence and 99.25% in amino acid sequence respectively.The plant expression vector pBIN-BnAGL15 was constructed.2.Cloning of PMADS9 and construction of the plant expression vectorsThe PMADS9 gene fragment was amplified by meas of PCR using petunia (Petunia hybrida) young floral bud cDNA as template and a pair of specific oligonucleotides at 5'-and 3'-end as primer. Sequence analysis showed that the fragment consisted of 811bp,which contains an open reading frame of 810bp, and encoded a polypeptide of 269 amino acids. Comparison with previously published sequence showed 99.75% homologies in nucleotide sequence and 99.26% in amino acid sequence respectively.The plant expression vectors pBL-PMADS9 (+) and pBIN-PMADS9 (-) were constructed.3.Introduction of BnAGL15 into petuniaBnAGL15 gene was introduced into petunia (Petunia hybrida) via Agrobacterium tumefaciens mediated method.6 independent kanamycin-resistant petunia plants were obtained and 4 plants were alive. PCR detection showed that the BnAGL15 gene was integrated into they genome.