Molecular Cloning And Functional Analysis Of Leaf Development Gene BrLOM2 In Non-heading Chinese Cabbage

Non-heading Chinese cabbage originated in China,and it was one of the most popular crop in the Yangtze River Basin and south area of China.Non-heading Chinese cabbage had three varieties which were ordinary non-heading Chinese cabbage,savoy and caitai.These led to the phenomenon that non-heading Chinese cabbage had variety of leaf shapes.Leaf shape had influence on the adaptability to environment and commodity value.So it was important to chose the best non-heading Chinese cabbage to meet market demands.We used RNA-Seq to analysis the DEGs in leaf-smoothed and leaf-lobed non-heading Chinese cabbages,and expored the candidate genes of leaf development.Cloned BrLOM2 gene,which participated in leaf development,and its expression and response to exogenous hormone were studied,then uncovered its function by Agrobacterium tumefaciens mediated transformation of Arabidopsis thaliana.The main results are described as followings.1.RNA-Seq technology was applied to analysis the differern expressions between leaf-smoothed and leaf-lobed non-heading Chinese cabbages.The results indicated that after filtration of low-quality reads,a total of 54223970 and 54840708 high-quality reads were acquired,respectively.Among these high-quality reads,70.42%?smoothed-leaf?and 70.19%?lobed-leaf?could be accurately mapped to the reference genome,thus validated the RNA-Seq data and the reference sequence.A total of 564 DEGs were identified,among which 302 and262 were detected to be up-and down-regulated,respectively.GO enrichment analysis showed these DEGs were involved in pathways like cellular process,metabolic process,cell,cell part,binding and catalytic activity.KEGG pathway analysis showed that these DEGs were involved in biosynthesis of secondary metabolites and plant hormone signal transduction.Expression analysis of DEGs with RT-PCR was in consistent with those of RNA-Seq data,thus furtherly confirmed the reliability of RNA-Seq results.With RT-PCR,we found 7candidate genes participating in leaf development of non-heading Chinese cabbage,which were TEOSINTE BRANCHED1,CYCLOIDEA,and PCF13?TCP13??Bra001032?,DEVELOPMENT-RELATED PcG TARGET IN THE APEX4?DPA 4??Bra009179?,LOST MERISTEM2?LOM2??Bra014479?,GROWTH REGUL ATING FACTOR6?GRF6??Bra015184?,CYP79B2?Bra017871?,Arabidopsis thaliana V-H+-PPase?Avp1??Bra018135?and LateMeristem Identity1?LMI1??Bra009510?.Using near-isogenic lines of leaf-lobed and leaf-smoothed non-heading Chinese cabbages as experimental materials,BrLOM2 gene was cloned by homology cloning techniques and its expression and response to exogenous hormone were studied.Sequence analysis indicated that ORF of BrLOM2 consisted of 549 bp,encoding 182 amino acids and locating on nuclear.The deduced amino acids possessed GRAS conserved domain.Phylogenetic analysis showed that BrLOM2 had the highest evolutionary relationship with Chinese Cabbage?XP₀09138823.1?and Brassica napus?XP₀13707740.1?.The results at transcriptional level indicated that expression of BrLOM2 was main in leaf and shoot apice,and the expression in root was higher than that in flower.During leaf development process,the expression of Br LOM2 in leaf-smoothed non-heading Chinese cabbage was higher than that of leaf-lobed type at the same developping period.Along with plant growth,its expression in leaf-smoothed non-heading Chinese cabbage was decreasing gradually and that in leaf-lobed non-heading Chinese cabbage was increasing,respectively.When treated with GA3,BrLOM2 showed up-regulationat first,then down-regulation.While treated with 6-BA,BrLOM2 showed down-regulation firstly,then up-regulation.In conclusion,the expressions of BrLOM2 in two margins of non-heading Chinese cabbage were different,had time-specific feature and responded to hormone.All results indicated that the gene may plays a role in leaf development.The open reading frame?ORF?of BrLOM2 was cloned into the pCAMBIA2301-35S-Nos vector,and a RNA interference?RNAi?expression vector,pRNAi-BrLOM2,was contructed.The constructs was transformed into Col-0 using Agrobacterium-mediated transformation method.Transgenic lines were obtained by antibiotic resistance screening ?PCR detection and phenotypic observation.