Cloning And Functional Verification Of Petiole Development-related Genes BrEXPA30 And BrEXPLA2 In Chinese Cabbage

Chinese cabbage(Brassica rapa ssp.Pekinensis)is a Brassica leaf vegetable.The leaf is one of the most important organs of the plant.The leaves of plants not only serve as vegetative organs,but even serve as edible organs in some plants.Ripe Chinese cabbage leaves are large and wide,the leaves curl inward to form a leaf ball,the petioles are thicker and whiter with more water content.For this phenomenon,the inbred line PHL was used as material,and three petioles at different developmental stages were selected for transcriptome sequencing.Taking Pet1 in the first growth stage of the petiole as a reference,the transcriptomes were compared with Pet2 in the second stage and Pet3 in the third stage,to obtain differentially expressed genes during petiole development.The dominantly expressed genes of the Chinese cabbage Expansin gene family were selected for cloning and functional verification.The experimental results are divided into four parts:1.Transcriptome sequencing of chinese cabbage petioles at different developmental stages showed that,7500 genes expressed different in the Pet2 with Pet1 comparison group,14153 genes expressed different in the Pet3 with Pet1 comparison group,and there were 2914 genes expressed different in the two comparison groups.The results of GO enrichment and KEGG signaling pathway enrichment analysis showed that,there were 541 GO significantly enriched terms and 20 significant metabolic pathways in the Pet2 with Pet1 comparison group respectively.There were 496 GO significantly enriched terms and 17 significant metabolic pathways in the Pet3 with Pet1 comparison group respectively.2.Summary of differently expressed genes associated with Expansin gene family,re-identification of family members using biological analysis of the updated cabbage genome protein sequence,finally,56 members of the cabbage Expansin genes were identified.According to the phylogenetic relationship,it is divided into four subfamilies Br EXPA,Br EXPB,Br EXPLA,Br EXPLB.The physicochemical properties analysis showed that,the amino acid length of Br EXPs gene was between 191 and 371,the relative molecular mass was between 19839.14 Da and 62936.42 Da,and the isoelectric point was between 4.83 and 10.74.The number of introns in the Br EXPs gene was between 1 and 4.Br EXPs genes were unevenly distributed on 10 chromosomes,and there were tandem repeats.3.The petiole dominant expression genes Br EXPA30 and Br EXPLA2 of the Chinese cabbage Expansin gene family were selected for expression pattern analysis.Subcellular localization results showed that the protein products of both genes were distributed in the nucleus.QRT-PCR analysis showed that,the expression levels of genes Br EXPA30 decreased during petiole development,while the Br EXPLA2 increased first and then decreased during petiole development.GUS histochemical staining showed that the promoter of the gene Br EXPA30 was only expressed in petioles.4.The results of transgenic Arabidopsis thaliana showed that,compared with the wild type ’Col-0’,the transgenic Br EXPA30 plants showed the phenomenon of petiole elongation and increased stress resistance.The mutant AT3g29030 showed shrinking petioles and early bolting;transgenic Br EXPLA2 plants showed leaf enlargement and growth faster.Therefore,it is speculated that the genes Br EXPA30 and Br EXPLA2 are genes related to plant petiole development.