Mining Of Rooting Related Genes In Larch Cutting And Functional Identification Of Rooting Genes

Larch is a major afforestation tree species in Northeast China,which is difficult to root by cutting.The rooting rate of cutting is low.Therefore,it is difficult to obtain a large number of excellent clonal seedlings through cutting propagation.In order to understand the rooting mechanism of larch cuttings,the critical period of adventitious rooting in larch cuttings was determined through physiological and biochemical indicators measurement and transcriptome sequencing.Based on transcriptome sequencing results,genes related to larch rooting were mined and screened,and gene functions were analyzed through transgenic methods,providing assistance to the research on larch genome and promoting root growth.The main findings are as follows:(1)The physiological and biochemical indexes of the base of cutting stem segments were measured at five stages: rooting start stage(s0),callus formation stage(s1),early adventitious root induction stage(s2),late adventitious root induction stage(s3),and adventitious root growth stage(s4).The activity changes of POD,IAAO and PPO showed that the metabolic activity in cuttings was intense in these two periods and would lead to the change of IAA content in cuttings,which could induce the formation of adventitious root,and nutrients also accumulated in these two periods to provide energy for the elongation and growth of adventitious root in the later period.(2)The KEGG data analysis of transcriptome sequencing results showed that there were obvious pathways related to rooting and development during s1 and s2 periods.Comprehensive GO enrichment and KEGG enrichment analysis revealed that plant hormones also played an important role in the adventitious root development process of larch.Using the GEINE3 software,a comprehensive analysis of the differential gene sequences was conducted,and 14 genes most likely related to larch cutting rooting were selected.By analyzing the expression levels of these 14 genes in the leaves,stems,taproots,and lateral roots of larch seedlings and their expression patterns during the rooting process of larch seedlings,it was determined that LBD and b HLH family genes may have regulatory effects on the root development of larch seedlings.(3)Based on the regulatory map of gene interaction network and the analysis results of rooting gene expression patterns,the LBD family gene c149623.graph＿c0 was selected as the follow-up research object and named La LBD1.The target gene of 702 bp was obtained by cloning La LBD1.La LBD1 was constructed on a GFP labeled overexpression vector(VB191104-2204tvd),and the subcellular structural mapping results showed that the gene was located in the nucleus.(4)The heterologous overexpression of La LBD1 in poplar 84 K by agrobacterium mediated method showed that the transgenic strain took root earlier than the wild type,with a higher rooting rate,and the number and length of adventitious root were higher than the wild type,indicating that the gene could promote the formation and growth of adventitious roots.