Screening And Function Investigation Of Candidate Genes Related To Adventitious Root Formation In Plants

Malus domestica,a deciduous tree belonging to the the Rosaceae,apple family,apple genus.In recent years,the dwarf dense planting has gradually become the main cultivation method of the apple planting industry due to its many advantages such as fast forming,high yield,convenient management and so on,and has been promoted in advanced apple production countries in the world.However,since adventitious root formation of dwarf rootstock is difficult,the induction of adventitious roots has become a key step in the asexual reproduction of apple rootstocks.Therefore,this study is devoted to cloning more genes related to adventitious root formation and further exploring the molecular mechanism of adventitious root formation,which is very important for solving the problem of difficult rooting of many excellent species in asexual reproduction,and also provides theoretical basis for revealing the growth and development of plants.The main content of this study are as follows:using euphylla of wild-type Arabidopsis thaliana and the apple rootstock“M26”tissue culture seedling stem discs as experimental material to determine the optimal conditions for inducing adventitious root generation,collecting samples for transcriptome sequencing,analyzing the sequencing results for data analysis,screening significantly differentially expressed genes,purchasing and identifing T-DNA mutants of the corresponding genes,observing the main roots and adventitious roots of the mutants,and select mutant genes with obvious adventitious root formation phenotypes as candidate genes in further exploration,construct transgenic complementary lines and overexpression lines of candidate genes to further analyze the function of the candidate genes.The research results are as follows:?1?The optimal IBA concentration for inducing adventitious root formation in euphylla of Arabidopsis thaliana is 10 mg/L,and the optimal induction condition was:after dark culture in medium containing IBA for 3 days,then transfer to hormone-free 1/2 MS medium,vertical cultivation under light,and the root phenotype was calculated after 5-7 days.The optimal IBA concentration for inducing adventitious root formation of apple rootstock“M26”tissue culture seedling stem discs was 2.5 mg/L,and the optimal induction condition was:after dark culture in medium containing IBA for 3 days,then transfer to hormone-free 1/2MS medium,and the root phenotype was calculated after three weeks under light.?2?Analyse the results of RNA-seq,significantly differentially expressed genes were screened,GO enrichment analysis to find that the biological process involved in differentially expressed genes in Arabidopsis thaliana are concentrated in cellular process,metabolic process,single-organism process,response to stimulus,and the biological process of differentially expressed genes in Malus domestica are concentrated in cellular process,single-organism process,metabolic process,biological regulation,response to stimulus;the cellular component of differentially expressed genes in Arabidopsis thaliana and Malus domestica are mostly distributed in cell,cell part,membrane and organelle;the molecular fuction of differentially expressed genes in Arabidopsis thaliana and Malus domestica are mostly enriched in binding,catalytic activity and nucleic acid binding transcription factor activity.?3?After the preliminary data analysis of the sequencing results,we selected 12 genes in Arabidopsis thaliana for follow-up research,at the same time,we performed the amino acid sequence of the selected genes and their homologous genes in Malus domestica through DNAMAN8 software,the results showed that the sequence similarity was mostly more than55%,which provided a theoretical basis for us to study the corresponding genes in Arabidopsis thaliana.?4?We purchased the T-DNA mutant seeds of the corresponding gene through the search of the Tair website,and obtained the corresponding homozygous mutant plants through the identification of DNA and RNA levels.Through phenotypic analysis,the mutant genes ROOT1?whose function is unknown and is temporarily named ROOT1?and ROOT2?whose function is unknown and is temporarily named ROOT2?with obvious root formation phenotypes were selected as candidate genes for further exploration.?5?Through semi-quantitative PCR analysis,it was found that the RNA expression of ROOT1 in root2 was significantly down-regulated.Therefore,we obtained double mutants of root1root2 by crossing.?6?Through in vitro DNA recombination technology,we constructed the plant binary expressed vectors of 35S::ROOT1-HA-PRI101-AN and 35S::ROOT2-HA-PB003,which were transformed into Col-0 and corresponding T-DNA mutants respectively.The T₂generation transgenic plants have been screened to provide materials for further phenotypic analysis.?7?We constructed the fusion expression vectors of ROOT1,ROOT2 and GFP driven by35S,expressed it transiently in tobacco,the results of subcellular localization showed that both ROOT1 and ROOT2 were located in the membrane and nucleus.Meanwhile,Col-0 was transformed to obtain stable transgenic plants,which provided materials for subsequent fine localization.?8?We constructed the plant binary expression vectors p ROOT1::GUS and p ROOT2::GUS,which were transformed into Col-0,The T₂generation transgenic plants have been screened,which provides materials for the subsequent observation of gene specific expression.In summary,the acquisition of various mutant materials and transgenic plants in this experiment provides convenience for subsequent experiments and is conducive to further explore the functional mechanism the adventitious roots of plants.