Transcriptomic Analysis Of Topping-induced Axillary Shoots Outgrowth And Functional Research Of Related Genes In Tobacco(Nicotiana Tabacum)

Apical dominance is a phenomenon of many plant species that governs shoot tip production and suppresses the outgrowth of axillary shoots.In response to the loss of their apical shoot buds,plants rapidly release axillary shoots.Topping is an important agronomic practice in tobacco production used to maximize yield.However,dormant axillary shoots become active after topping and must be periodically removed to obtain the f?L l benefits of topping.In this study,in order to advance our understanding of how to control axillary shoot growth,transcriptome sequencing,candidate gene cloning and analysis,mutant screening and identification of axillary bud and gene editing of axillary bud development-related genes were used to study the growth-related genes of tobacco axillary bud.The res?L ts are as follows:1.After topping of tobacco plants at early flowering stage,axillary buds of tobacco at the same leaf position were sampled at 1,3 and 5 days,and three biological repeats were set up for transcriptome sequencing.The Gene Ontology(GO)enrichment analysis revealed that the cell?L ar carbohydrate metabolic process and the disaccharide metabolic process,which may contribute to starch accum?L ation and stress/defense,were overrepresented terms for the DEGs.Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis revealed that many DEGs were involved in starch and sucrose metabolism,glycolysis/gluconeogenesis,pyruvate metabolism,and plant hormone signal transduction,among other processes.Prediction of transcription factors from transcriptome data showed that GRAS family,MYB family and LOB family,which were involved in the development of plant branching,were more numbers.The expression levels of LS genes in GRAS family,RAX and MYB44 genes in MYB family and LOB genes in LOB family were significantly different before and after topping.The transcriptome analysis of roots before and after topping showed that there were significant differences in the expression levels of MAX3 and MAX4 genes.MAX3 and MAX4 were all involved in the biosynthesis of strigolactones.2.Using Genome editing technology,two transcription factors,MYB44 and LOB were carried out for gene editing.Three editing target loci were selected for each target gene.At present,T0 generation positive seedlings have been obtained,and the screening and identification of homozygotes for gene editing of T1 generation is still in progress.3.Exogenous addition of strigolactones analogues GR24,GR24 can significantly reduce the length,fresh weight and stem circumference of tobacco branches after topping.The results indicated that strigolactones might participate in the regulation of axillary bud development and have the effect of inhibiting axillary bud growth.4.The genes Nt MAX3 and Nt MAX4 related to the synthesis of strigolactones were cloned by RACE.The full length of Nt MAX3 is 2253 bp,ORF1188 bp,translation 395 aa;the full length of Nt MAX4 is 2135 bp,ORF1671 bp,translation 556 aa.For these two genes,their RNAi and overexpression vectors were constructed respectively to obtain genetic transformation plants.The res?L ts showed that the RNAi of Nt MAX3 and Nt MAX4 showed a significant decrease in plant height and an increase in thenumber of branches.However,the overexpression plants did not show significant changes in traits..5.The compiler traits of 1011 EMS mutant populations of M2 generation were investigated and axillary bud(branching)mutants were screened.586 mutant lines of M2 generation were found.The mutant rate was 57.96%.The mutation rate of 2876 mutant single plants was 19.88%.Fifteen axillary bud(branching)mutants were obtained after successive screening to M5 generation.Eight of the mutants were genetically stable.The phenotype of mutant mbd3 polybranched mutant was significantly different from that of wild type."Zhongyan 100","mbd3","F1","F2" and "BC1F1" were planted in the field.The axillary bud(branching)phenotypes of each generation were recorded after budding stage.The results of genetic analysis showed that the segregation ratio of wild type and mutant in F2 population was 3:1,and that of wild type and mutant in BC1F1 population was 1:1,indicating that the inheritance of mbd3 mutant was controlled by a pair of recessive nuclear genes.