Construction And Transformation Of Knockout Vectors For LAZYs And TACs Genes In 84K Poplar

The branching angle is an important component of plant spatial structure and has a significant impact on plant productivity,biomass,carbon sequestration and landscape effects.The formation of plant branching angle is a complex biological phenomenon,which is regulated by a serious of factors including its own genes,endogenous hormones and external environment.Among them,genes’ regulation is an important internal factor,and it was found that the IGT gene family has a particularly significant role in the regulation of branching angle.Therefore,the study of the IGT gene family function can help to reveal the mechanism of branching development,growth angle,and spatial structure formation in trees.In this thesis,based on the genomic sequence information of84 K poplar(Populus alba × P.glandulosa ’84K’),Populus trichocarpa and Salix purpurea,as well as the known sequence information of LAZYs and TACs of Salix,peach and Arabidopsis,multiple sequence alignment was performed and phylogenetic tree was constructed.And the two alleles of these genes from 84 K poplar were cloned,in which specific spacer sequences were selected.Then single gene knockout expression vectors were constructed,and transformed to 84 K poplar by Agrobacterium-mediated transformation.The main findings are as follows.(1)Multiple sequence alignment and phylogenetic tree analysis with LAZY and TAC amino acid sequences from different species showed that PagLAZY had high homology with Sps LAZY and Potri.LAZY,both containing six amino acid homologous sequence regions;PagTAC had high homology with Sps TAC and Potri.TAC,both containing four amino acid homologous sequence regions.(2)The genomic sequences of two alleles of PagLAZYs and PagTACs were cloned,in which the first three exons of the above genes were mainly containing.(3)The two allele sequences of PagLAZY1 a,PagLAZY1 b,PagLAZY1 c,and PagTAC1 and PagTAC2 genes were compared and analyzed.Two gene-specific spacer sequences were selected for each gene,and five single-gene editing expression vectors were successfully constructed.Each vector contains 2 gene-specific spacer sequences for the same gene.(4)The above gene editing vector was transformed into 84 K poplar,and the resistant strains were obtained from transforming of p De-CAS9 : : LAZY1 b,p De-CAS9 : : TAC1 editing vectors.The resistant strains of 84 K poplar were identified by PCR.In this thesis,the phylogenetic relationships of LAZY and TAC genes were preliminarily revealed,LAZY and TAC gene editing vectors were successfully designed and constructed,and resistant and transgenic strains were obtained.This study provides important research materials for analyzing the functions of IGT gene family,and lays a research foundation for elucidating the functions of LAZY and TAC genes and the synergistic effects of homologous genes,which can enrich the functional studies of IGT gene family and lay the foundation for the application and research of IGT gene family in molecular breeding of woody plants.