Construction Of LAZY And TAC Gene Editing Vector And Optimization Of 717 Poplar Transformation System

IGT gene family is a key family that affects the angle of plant branching and plays an important role in plant growth and plant type change,but its function is not clear.Plant branching angle is an important part of plant type,which is regulated by plant genes,Endogenous hormone and external environment.The angle of plant branching affects the spatial distribution ability of plant,and the spatial distribution ability directly affects the light,temperature and growth of plant.IGT gene family mainly includes LAZY subfamily and TAC subfamily.The study on the function of IGT gene is very important for understanding the formation of branch angle.In this study,using 717-1B4(Populus tremula×alba 717-1B4)Poplar as research material,using 717 genome database and recommended gene editing targets,gene editing targets were designed for three genes of LAZY subfamily and two genes of TAC subfamily.The gene editing expression vector of the above genes was constructed and transformed into poplar 717-1B4 by agrobacterium-mediated leaf disk method.The research results are as follows:(1)The study we completed the design of gene editing targets of LAZY and TAC sub-families.LAZY gene targets are:LAZY1-1?LAZY1-2?LAZY2-1?LAZY2-2?LAZY3-1?LAZY3-2;TAC gene targets are as follows:TAC1-1?TAC1-2?TAC2-1?TAC2-2.(2)The gene editing expression vectors of LAZY and TAC subfamilies were successfully constructed.The expression vectors of LAZY gene were as follows:pDE-CAS9-NPTII::LAZY1-1?pDE-CAS9-NPTII::LAZY1-2?pDE-CAS9-NPTII::LAZY2-1?pDE-CAS9-NPTII::LAZY2-2?pDE-CAS9-NPTII::LAZY3-1?pDE-CAS9-NPTII::LAZY3-2;The ex-pression vectors of TAC gene were as follows:pDE-CAS9-NPTII::TAC1-1?pDE-CAS9-NPTII::TAC1-2?pDE-CAS9-NPTII::TAC2-1?pDE-CAS9-NPTII::TAC2-2.(3)The genetic transformation system of 717-1B4 poplar was optimized.The best conditions for genetic transformation were:the best preculture time was 9 days,the best infection concentration OD₆₀₀ was 0.5 and the best time was 10 minutes.(4)Transgenic lines were obtained.A total of 195 resistant strains were obtained.PCR analysis showed that 31 strains of pde-cas9-nptii::lazy-1-2 and 15 strains of pde-cas9-nptii::tac-1-2 were positive.In this thesis,LAZY and TAC single gene editing vectors were successfully constructed and transformed into 717-1B4 hybrid poplar.The transgenic plant is an important research material for analyzing IGT gene family function.This study elucidates the function of LAZY and TAC genes in the IGT gene family in woody plants and provides a basis for identifying the functional differences between the LAZY and TAC homologous genes,it enriches IGT gene family function and lays a foundation for the application and research of IGT gene family molecular breeding.