The Function Analysis Of Petunia Hybrida PMADS9 Gene

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.It 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 introduced PMADS9 into petunia(Petunia hybrida)via Agrobacterium tumefaciens mediated method,to research the gene function.The main results are as follows:1.Establishment of petunia regeneration systemPetunia line A01 was choose to be the transformation plant material.The sterile young leaves of petunia were cultured on MS containing 2.0 BA and 1.0 mg/L NAA to induce bud initiation directly. Regenerated buds were rooted on 1/2 strongth MS medium.2.Optimization of transformation systemYoung sterile leaves,which were cut into 5×5mm²,dipped into Agrobaterium(OD₆₀₀0.5-0.8) for 30s was choose for petunia transformation.The leaves were cultured on different MSI(MS+BA2.0 mg/L+NAA0.1mg/L+sucrose 3%+gelose 0.7%,pH5.8)respectively,which including different concentration of Km.The result showed that the callus could not be induced when the concentration of Km was 50mg/L.The seedlings were cultured on different 1/2 MS respectively,including different concentration of Km.The result showed that the seedlings could not be root when the concentration of Km was 30mg/L.3.Introduction of PMADS9 into petuniaPMADS9 gene was introduced into petunia(Petunia hybrida)via Agrobacterium tumefaciens mediated method.7 PMADS9(+)independent and 8 PMADS9(-)kanamycin-resistant petunia plants were obtained.PCR detection showed that the gene PMADS9 was integrated into they genome.4.Phynotypes of transformed petunia and function analysis of PMADS9 geneThe PMADS9(+)transformed petunia flower opening later than wide type,suggest that overexpression of PMADS9 gene defer opening of petunia flower.The number of PMADS9(+) transformed petunia's leaves in a flower branch was less than wide type's when the first flower opening,,and the flower life-span was longer than wide type,suggest that PMADS9 overexpress delay the flower senescence.