Construction and transformation of CRISPR/Cas9 vector of ALB3 gene in Eucalyptus urophylla×E. grandis

Eucalyptus has a wide range of uses and a complex genetic background. Genetic engineering research can accelerate the genetic improvement of eucalyptus and meet the market demand for excellent eucalyptus varieties. Establishing an efficient method for identifying positive transformants is a prerequisite for carrying out efficient genetic transformation and gene editing in Eucalyptus. ALB3 gene is a commonly used target gene for studying gene editing. The RUBY reporter system is a novel, convenient system that allows the detection of the reporter gene by the naked eye under natural light. It is a commonly used target gene for verifying the efficiency of plant gene editing.

Polymerase chain reaction (PCR) was used to amplify the gRNA fragment of the ALB3 gene in Eucalyptus urophylla×E. grandis. The linearized PHEE401E vector was obtained by BsaI enzyme digestion, and the recombinant plasmid pHEE401E-35S-RUBY-ALB3 was connected with the recombinant enzyme. The recombinant plasmid was transformed into Escherichia coli DH5α sensitivity analysis, PCR identification and sequencing analysis of heavy particles, and transformation of Eucalyptus urophylla × E. grandis callus using Agrobacterium tumefaciens transformation method to identify the expression of reporter genes in eucalyptus tissue and the editing of target genes.

The pHEE401E-35S-RUBY-ALB3 expression vector was constructed and transformed into Agrobacterium tumefaciens. PCR amplification and sequencing analysis were performed on the genome of Eucalyptus grandis callus with a red phenotype after infection by Agrobacterium tumefaciens. The results showed that the RUBY reporter gene had been successfully transferred into the cell and expressed, and some base sequences of Eucalyptus urophylla × E. grandis ALB3 gene was successfully knocked out between two targets. The HEE401E-35S-RUBY-ALB3 expression vector with RUBY screening marker gene constructed using homologous recombination method was successfully introduced into callus cells, and the target gene was successfully edited.

This study provides a certain reference for the establishment of an efficient gene editing system for Eucalyptus urophylla × E. grandis.