Phenotypic Identification Of SpsTACs Gene Overexpression And Homology Knockout In Poplars

Tree’s architecture is an important specific trait of trees which is affected by many factors,and the architecture differences would have great impacts on wood development,forest biomass,ecological value and landscape benefits.Salix psammophila is an important native shrub species with important ecological and economic value in western China,and its plant architecture are extremely diverse,which is a good material for studying the development of different trees architecture.Therefore,in this thesis,TAC1 and TAC2 genes regulated to tree’s architecture were isolated from S.psammophila,multi-sequence alignment and physicochemical characteristics were analyzed,and the overexpression vectors of Sps TAC1 and Sps TAC2 genes was constructed,and the cas9 guide RNA,which were selected from 84 K poplars(Populus alba × P.glandulosa ’84K’)TAC genes by our research group,were used to construct gene co-knockout vectors of Pag TAC1 and Pag TAC2.All of these vectors was transformed into 84 K poplars.Transgenic 84 K poplars were obtained and phenotypic changes were indentified.This study lays foundation on the subsequent verification of the TAC gene’s function and the further exploration for its molecular regulatory mechanism,and enriches the molecular genetic knowledge related to the regulatory angle of branching in the IGT gene family.The main findings are as follows:1.Multiple Sps TAC1 and Sps TAC2 genes were cloned from S.psammophila with different architectures,which encode 272 and 260 amino acids,respectively.The sequence alignment results showed that SNP sites existed between Sps TAC1 sequences and between Sps TAC2 sequences,but no significant differences,and only one SNP site in the Sps TAC1 gene which led to differences in amino acid sequences.2.According to comparison of the Sps TAC1 and Sps TAC2 CDS sequences,the results showed that Sps TAC2 gene lost the domain I compared with Sps TAC1 gene,and the evolutionary tree analysis showed that Sps TAC1 and Sps TAC2 converged into different branches.3.The overexpression vectors of Sps TAC1 and Sps TAC2 were successfully constructed,and both of them were transformed into 84 K poplar,and a total of 10 lines of overexpressing Sps TAC1 and 22 lines of overexpressing Sps TAC2 were obtained,and RT-q PCR analysis showed that the target genes expressed highly in different degrees.4.The phenotypic identification results of transgenic lines overexpressing Sps TAC1 and Sps TAC2 showed that the leaf growth angle,plant height,leaf length,leaf width,leaf circumference and leaf area were significantly increased than those of the non-transgenic84 K,indicating that the overexpression of Sps TAC1 and Sps TAC2 promoted the increase of leaf growth angle,plant height growth and leaf elongation growth of 84 K poplar,respectively.5.The Pag TAC1 and Pag TAC2 gene co-knockout vectors of 84 K poplar were successfully constructed,and 3 transgenic lines were obtained.Target sequences analysis showed that the target genes of Line-1,Line-4 and Line-5 were successfully mutated,and the two homologous chromosomes of the target gene were successfully edited.6.The phenotypic indentification results of Pag TAC1 and Pag TAC2 knocked out lines showed that the transgenic lines showed significant differences compared with the control 84 K plants,with significant curvature in the lower part of the stem,significantly reduced leaf landing angle,significantly shorter plant height and significantly smaller leaves.The slice analysis of the bending stem indicated that the anatomical structure of the bending stem showed typical characteristics of reaction wood,which indicated its gravity response was affected.The above results show that there are domain differences in the Sps TAC1 and Sps TAC2 gene sequences.The sequencing results of gene editing lines showed that both of two target genes were successfully edited,and the results showed that the two target genes were successfully knocked out.The Sps TAC1 and Sps TAC2 genes can promote the increase of leaf growth angle,plant height growth and leaf elongation growth,with positive regulatory effects.The TAC gene affects the stems gravity response of 84 K poplar and closely related to their gravity response.This thesis provides research materials for further exploring the function of TAC genes in woody plants,and lays a research foundation for elucidating the function of TAC gene and exploring its molecular regulation mechanism.