Clone And Preliminary Functional Analysis Of A Lobed-leaf Gene BrcLL1 In Chinese Cabbage (Brassica Rapa Ssp.Chinensis)

Chinese cabbage(Brassic rapa ssp.chinensis)derived from China is an important leafy vegetable.The variation of leaf margin is the main reason for the diversity of the leaf shape,in which the lobed-leaf is a very important economic or marker character,and studying the mechanism of gene regulation has a certain application and theoretical significance.On the basis of previous study,we mapped the fine interval which controlled this trait and screened the candidate gene that is temporarily named as BrcLL1(B.rapa.ssp.chinensis lobed-leaf 1).After we have cloned the gene and analyzed the temporal and spatial expression,and carried on the preliminary functional verification by transgenic technique.The results are as follows:1.Map-based cloning BrcLL1By mapping of the target gene,our group mapped the gene in the range of two markers:Bra9511 and Bra9512,but no encoding genes were found in this range.Therefore,we used the invisible individuals of F2 large population to rescreen the neighbor markers 3KBra9510 and Bra9510-4H,that respectively derived from the promoter and gene inside of encoding gene of BrcLL1,and finally we got the results that the two markers were co-segregating with the candidate gene BrcLL1.Based on the transcriptome profiles,it was found that the expression of Bra009510 was down regulated in the lobed leaf mutant.we could determine Bra009510 to be the candidate gene.2.Moleculer clone of BrcLL1Respectively,we used three pairs of NILs as research materials to clone the BrcLL1 of the round-leaf and lobed-leaf materials.We predicted the gene had been duplication in lobed-leaf through the gDNA that of the round-leaf material were 1635 bp and just had one copy,however,of the lobed-leaf material,had two copies and they were 1635 bp and 1390 bp respectively;but the two materials were consistently having only one cDNA sequence(693bp).By comparing the sequences using the software DANMAN,we found that the BrcLL1 gene had three exons and two introns.3.Expression analysis of BrcLL1We used one pairs of NIL as research materials to detect qRT-PCR expression levels of BrcLL1.The results showed that in the cotyledon stage,expression of BrcLL1 gene inlobed-leaf were significantly higher than that in round-leaf materials.Its expression trend was first increased and then decreased in two materials with Ga3 treatment,while the results produced a opposite effect with Cytokines treatment.4.Vector construction and genetic transformationTo verify the function of the BcLL1 gene,we have built plant over expression vector named pCAMBIA2301-BrcLL1-a and pCAMBIA2301-BrcLL1-b of the lobed-leaf material.Then we used the floral dip method to convert the over expression vector pCAMBIA2301-BrcLL1 to the wild type Arabidopsis thaliana,and preliminary got their resistance transgenic T1 generation plants that had not seen the anticipated phenotype.