| Cunninghamia lanceolata is one of the most timber trees in southern China due to its excellent material and fast growth..In recent decades,some important breakthroughs had been made in the molecular regulation of plant regeneration,such as somatic embryogenesis.Some important genes,such as Baby Boom(BBM)and WUSCHEL(WUS),had been identified and used to improve the efficiency of somatic embryogenesis and genetic transformation.However,in gymnosperms such as Cunninghamia lanceolata,the functions and regulatory mechanisms of these important genes are not clear.In this study,based on the transcriptome database of C.lanceolata,the AP2/ERF transcription factor and the WOX transcription factor family were identified by bioinformatics method,and ClBBM,ClWOX5 and ClWOX6 genes were cloned.The expression patterns of these genes in different stages of embryo development,callus formation,adventitious root formation and different parts of the root were analyzed.At the same time,the functions of Arabidopsis thaliana,tobacco and poplar were studied by heterologous transformation,which provided an important basis for further analysis of ClBBM,ClWOX5 and ClWOX6 genes regulating somatic embryo and root formation.The main findings are as follows:1.Based on full-length transcriptome database of C.lanceolata,95 AP2/ERF genes were identified,including 11 AP2 subfamily,4 RAV subfamily,34 DREB subfamily,45 ERF subfamily and 1 Soloist,and most of the members of the AP2/ERF family showed strong tissue specificity.Further,ClBBM was identified by phylogenetic analysis and multi-sequence comparisons,and the sequence analysis showed that the gene had an important characteristic motif.2.Based on full-length transcriptome database of C.lanceolata,6 ClWOX genes were identified,Among which,ClWOX1-4 belongs to the ancient clade,and ClWOX5 and ClWOX6 belong to the WUS clade showing closer homologous relationship with Wus.Tissue specificity results showed that the expression of ClWOX2 was low in all tissues,while the expression of other genes showed strong tissue specificity.For example,the expression of ClWOX3 was high in root and xylem and ClWOX5 was higher in the stem than in other organs.3.Based on the results of sequence analysis,ClBBM,ClWOX5 and ClWOX6 genes were cloned.Further analysis of expression pattern showed that the expression of ClBBM was high mainly in the early stage of embryo development,ClWOX5 showed high in the middle stage of embryo development,and ClWOX6 was high in the late stage of embryo development..During callus formation,the expression of ClBBM,ClWOX5 and ClWOX6 were induced by 2,4-D.In the process of adventitious root formation,the expression of ClBBM was upregulated and induced by auxin.The expression of ClWOX5 and ClWOX6 were high in the early(middle)stage of adventitious root formation,but their expressions were inhibited by auxin treatment.In addition,ClBBM was mainly expressed in the root tip,while ClWOX5 and ClWOX6 were mainly expressed in the middle and end of the root.These results showed that ClBBM,ClWOX5 and ClWOX6 played different roles in embryo development and adventitious root formation.4.Using model plants as materials,function of ClBBM,ClWOX5 and ClWOX6 were studied by Agrobacterium-mediated transformation.The results showed that overexpression of ClBBM plants bolted early,low seed setting rate and longer root system;ClWOX5 and ClWOX6 transgenic Arabidopsis plants showed early bolting and decreased seed setting rate.ClWOX5 and ClWOX6 transgenic could promote more bud differentiation and regeneration efficiency in tobacco experiments,but there was no significant difference in the ClBBM.In addition,ClBBM promoted the production of more adventitious roots in poplar(84k)leaves without exogenous hormones.These results further suggest that ClBBM,ClWOX5 and ClWOX6 may have important functions in embryo development,but play different roles.ClWOX5 and ClWOX6 have potential applications in genetic engineering.ClBBM plays a more important role in adventitious root formation(initiation). |
