Molecular Colning And Expression Analysis Of C-Class And E-class MADS-box Genes And From Kale(Brassica Oleracea Var Acephala)

Brassica Oleracea var acephala is biennial herb,and it is a horticultural mutant of the Cruciferae Brassica species.Because of high nutritional value and the gorgeous color of the leaves,the kales are widely cultivated and used in landscape gardening.We found two mutants in breeding work.The wild type Brassica Oleracea has four petals arranged in cross,six stamens and one carpel.The two mutants are the petal-loss one,in which the number of petal arranges form zero to three,and the petal-increased one,in which the number of petal arranges from five to muti-wheel.This research mainly focuses on the analysis of function relationship and the expression pattern of temporal and spacial of MADS-box genes in kales,for example:BoaAG(C-class),BoaSEP1/2/3(E-class),BoaAGL6(the sisiter clade of E-class).so that we can lay a solid foundation for explaining the molecular mechanism of kale mutants and flower development.(1)The full-length cDNA sequences cloning of MADS-box genes in kalesUse RACE and other technologies to clone the full-length cDNA of MADS-box genes in Brassica Oleracea's.We achieved five genes which are BoaAG(accession number:KC984300)?BoaSEP1(accession number:KC967957)?BoaSEP2(accession number:KC967958)?BoaSEP3(accession number:KC967960)?BoaAGL6(accession number:KC984301).(2)The phylogenetic analysis of MADS-box genes of the kalesDetermine the affinity of BoaAG?BoaSEP1?BoaSEP2?BoaSEP3 and BoaAGL6?protein by builting a phylogenetic tree.Through the analysis of MICK structure,it suggests that BoaAG belongs to euAG lineage.BoaAGL6 belongs to the euAGL6 lineage.BoaSEPl belongs to the LOFSEP lineage?BoaSEP2 belongs to LOFSEP lineage?BoaSEP3 belongs to SEP3 lineage.By predicting the primary and secondary structures of BoaAG?BoaSEP1?BoaSEP2?BoaSEP3 and BoaAGL6,it found that all of them are hydrophobic basic proteins.(3)The expression patterns analysis of temporal and spacial of kales' MADS-box genes by using semi-quantitative and Realtime PCR methodBy analysising BoaAG?BoaSEP1?BoaSEP2?BoaSEP3?BoaAGL6 specific expression of temporal and spacial during the process of floral development,We found in the mutants,BoaAG hardly expressed in petals.But the expression of BoaSEP1/2/3?BoaAGL6 genes was found in petals in the three kales and it was the highest in the petal-increased mutant.The role of BoaAGL6 gene in Brassica Oleracea mutant's petal development is very important.(4)The constrution of the overexpression plasmids of the MADS-box genes of kalesRecombinant plasmids pUN1301-BoaAG?pUN1301-BoaAGL6 were successfully constructed and applied to Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral-dip method.The F2 seeds of the three transformant have been harvested.