DNA Methylation And Chromatin Remodeling Regulates Leafy Head Formation In Chinese Cabbage

Chinese cabbage belongs to Cruciferae Brassica plants,and was originated from China,with high nutrition value,economic value,and research value.Chinese cabbage is widely cultivated in the middle and lower reaches of the Yangtze river and is the main cultivar of northern China.As its main nutrient storage organ of Chinese cabbage,the heads formation affects the its yield and commodity seriously.Research on the main factor which affects the heads formation and finding the key genes regulating heads formation will have a profound effect on the breeding of Chinese cabbage.Previous research indicates,ANGUSTIFOLIA3 binds to SWI/SNF chromatin remodeling complexes to regulate transcription during Arabidopsis leaf development.But its potential function in heads formation has been uncovered yet.Our research mainly explores the influences of DNA methylation and ANGUSTIFOLIA3(AN3)binding with SWI/SNF chromatin remodeling complexes on heads formation.In our study,we utilize the MeDIP sequencing and mining some key genes from the differential-methylated genes.qRT-PCR was conducted to validate the candidate genes,exploring the potential pathway which affects the heads formation.Moreover,Quantitative analysis was performed to detect the relative expression level of AN3 before and after heads formation.The main research results are as follows:1.We utilized the MeDIP sequencing technology and analyzed the methylated genes before and after heads formation.Totally 97,959,184 raw reads with length of 49bp was obtained,then 6,024,506(no-heading)and 77,462,058(heading)reads was mapped to the reference genome.The result indicated the whole methylation level was decreased in the process of heads formation.We find differential-methylated genes were abundant in metabolic pathways,biosynthesis of secondary metabolites pathways,plant hormone signal transduction and plant-pathogen interaction pathways.Finally,we selected some candidate genes from the plant hormone signal transduction pathway and performed real time qRT-PCR for validation.2.We detected the relative expression level of AN3 before and after heads formation.The result indicated the AN3 was down-regulated in the process of heads formation.We established VIGS system in Brassica rapa,and induced silenced in multiple organs of Brassica rapa.Then we completed functional verification by VIGS technology.We found that silencing of AN3 and its interacting gene BRM can contribute to the formation of heads earlier.It demonstrated that AN3 binds to SWI/SNF chromatin remodeling complexes to regulate transcription during heads formation in Chinese cabbage.3.We detected the expression level of ULTRAPETALA2(ULT2)in Chinese cabbage at the second leaf stage and fourth leaf stage,and found it was down-regulated in the leaf development.Then the endogenous gene ULT2 was silenced utilizing the plasmid of TYMV,finally the plants formed upward-curled leaf with disorderly growth.The result indicated that the ULT2 plays an important role in leaf shape.