| As a functional organ for photosynthesis,the leaves are vital to the growth and development of plants and affect the yield and flavor quality of leafy vegetables.Chinese kale is a vegetable in the Brassica oleracea group that uses the fat,tender bolting stem,and young leaves as the edible part.However,the transcriptional mechanisms regulating leaf development and morphogenesis during the growth of Chinese kale are still poorly understood,so it is important to study the molecular mechanism of mustard leaf development and morphology.In this paper,using ’Xiang Gu’ Chinese kale(Brassica oleracea cv.‘Xiang Gu’)as the experimental material,we first analyzed the differentially expressed genes of metabolic pathways combined with leaf transcriptome,and then selected key genes for bioinformatics analysis.Secondly,functional verification experiments of promoter vectors,gene overexpression vectors and gene knockout vectors were conducted for key members,and then verification experiments of yeast one-hybrid and protein interaction were conducted on potential upstream and downstream regulatory pathways of key genes.Finally,the phenotypic changes and related gene expression of transgenic plants were observed,and the mechanism of key genes in leaf development were clarified from epigenetic and molecular biology.The main research findings include the following four aspects:1 Analysis of differentially expressed genes of metabolic pathways in Chinese kale leaf development based on transcriptome sequencingIn order to study the main factors affecting the leaf development and metamorphic leaves of ’Xiang Gu’ Chinese kale(XG),12 samples of XG were sequenced.Found that the KEGG of the four comparison group DE-m RNAs was mainly enriched in the plant hormone signal transduction pathway.Based on the co-expression grid analysis of WGCNA and Cytoscape genes,it was found that the key genes of plant hormone signaling were mainly associated with RNA binding,cytokinin,gibberellin,auxin and TCP protein domains.2 Genome identification and expression analysis of BoTCP family in Chinese kaleIn order to clarify the function of TCP family related genes in Chinese kale in leaf development,based on cabbage genome data,this study identified 40 members of TCP gene family in Chinese kale.Referring to the annotation of homologous TCP genes in Arabidopsis and the relative expression of genes in Arabidopsis database,BoTCP21 and BoTCP25 genes were preliminarily selected and analyzed by q RT-PCR experiment.The results showed that BoTCP21 and BoTCP25 were widely expressed in leaves,but their expression patterns were different in different parts of true leaves.In order to find out the genes involved in leaf development in TCP gene family,the BoTCP family was comprehensively analyzed by bioinformatics and q RT-PCR experiment.The results showed that 40 BoTCP were non-secreted hydrophilic proteins,which were located on 8 chromosomes respectively.The members of BoTCP in Chinese kale are classified into three categories by phylogenetic tree construction and phylogenetic analysis,among which there are nineteen members in PCF subfamily,eight members in CYC subfamily and thirteen members in CIN subfamily.The results of q RT-PCR showed that BoTCP21 and BoTCP25 had higher expression levels in true leaves and bolting leaves,BoTCP14 had higher expression levels in the roots of flowering and podding plants,while BoTCP16 had the highest expression levels in the roots of bolting plants,which indicated that the expression of BoTCP family members in the tissue parts was spatio-temporal specific and widely participated in the morphogenesis and organ development of plants.3 The function of BoTCP25 in the regulation of leaf development of Chinese kaleWe have identified the high expression of BoTCP25 in Chinese kale leaves in the previous study.To understand the function of BoTCP25 in the leaves,we have cloned BoTCP25 and constructed related vectors.Subcellular localization analysis showed that BoTCP25 was localized in the nucleus,and GUS staining of BoTCP25 promoter-transformed Arabidopsis plants showed that it was widely expressed throughout the growth period.We have also obtained overexpressed BoTCP25 transgenic Arabidopsis thaliana and Chinese kale plants.Phenotypic analysis showed that heterologous overexpression of TCP25 promoted an increase in leaf number and leaf area in Arabidopsis,while overexpression of TCP25 in kale affected the location and number of metamorphic leaf-bearing.Analysis of gene expression in Chinese kale overexpression plants showed that BoNGA3 has a similar expression pattern to BoTCP25 and is likely to be involved in the regulation of leaf development by BoTCP25.Yeast one-hybrid experiments verified that BoTCP25 can bind to the promoter of BoNGA3.Moreover,the expression pattern of pre-mi R319 is opposite to that of BoTCP25 and BoNGA3,suggesting that they may form a mi R319a-BoTCP25-BoNGA3 module involved in Chinese kale leaf development.In addition,BoTCP25 expression responds to hormonal conditions such as ethylene and auxin.In conclusion,our study suggests that the mi R319a-BoTCP25-BoNGA3 module plays an important role in plant leaf development and morphogenesis.4 Functional analysis of the mutant tcp16 and mir319 a of Chinese kaleThrough bioinformatics analysis,BoTCP16 and BoTCP25 and mi R319 a were targeted and BoTCP protein and BoARF4 protein may have interaction by protein prediction analysis.By constructing the related knockout vector and transforming Arabidopsis and XG,we found that the Arabidopsis tcp16 mutant leaves decreased and there were no main bolt.On the contrary,the bolting time of mir319 a mutant was delayed,At the same time,the phenotype of the mutant tcp16 XG found that the number of leaves and metamorphic leaves was reduced,and the plant height,plant amplitude and internode length were significantly reduced.The q RT-PCR analysis showed that the down-regulated expression of BoTCP16 in the mutant leaves,which was similar to the expression pattern of BoTCP25 and BoARF4.The interaction between BoTCP16 and BoARF4 proteins was confirmed by bimolecular fluorescence complementation,and exogenous addition of auxin inhibitor NPA indicated that auxin is essential for leaf development and induction of variable leaves.In conclusion,the tcp16 mutant has similar functions in Arabidopsis and XG,BoTCP16 is negatively regulated by mi R319 while possibly directly regulated by auxin,in addition to BoTCP16 interacting with the auxin response factor BoARF4 protein.The above results show that the Bomi R319-BoTCP16/BoARF4 module regulates the distribution of hormone content in the plant,thus jointly participating in the regulation of leaf development,and then affecting the plant type. |
