Gene Editing And Functional Analysis Of BpCCR1 And BpCCR2 In Betula Platyphylla

Lignin is an important secondary metabolite in the process of plant growth and development which plays a crucial role in regulation of plant growth,development and response to biotic and abiotic stresses.Recent studies have shown that the synthesis of lignin is induced by the ABA pathway and provided evidence that lignin synthesis response to drought stress.Cinnamoyl-Co A reductase(CCR)is an important limiting enzyme that catalyzes lignin synthesis in plants.Birch(Betula platyphylla)is a valuable pioneer species widely distributed in the northeast of China,with fast growth,high yield,strong sprouting ability.We are facing with the increasing trend of global warming,the continuous expansion of arid and semi-arid areas in China and difficult sites for forest lands.To achieve the long-term breeding goals for fast-growing,high-quality,and high-resistance of trees,it is important to obtain tree germplasm resources with strong drought resistance and normal growth,which is valuable for ecological restoration of difficult sites and the selection of resistant resources.In this study,we analyzed the gene structure,conserved functional domains,expression patterns of the BpCCRs gene family members in birch.Mature seeds of Betula family strain 1-34 were used as test material,we construct mutants of the BpCCRs gene family with gene editing methods,and screened the phenotypes of mutants according to the growth parameters and physiological index.The role of BpCCR1 and BpCCR2 in regulating growth,development,and drought resistance.This study provides the foundation for exploring the gene function of BpCCR1 and BpCCR2 and obtaining high-quality stress-resistant birch germplasm resources through gene editing technology.The main research results are as follows:1.A total of 23 BpCCR genes were found in birch through homologous sequence alignment.Furthermore,gene structure,phylogenetic analysis,and conserved domain analysis were performed.All BpCCRs contain the NADP-binding domain.BpCCR2 shares the highest amino acid similarity with At CCR1 and PPCCR2.There are 6 stress or hormone responsive elements in the BpCCR1 promoter region,and 2 stress or hormone responsive elements in the BpCCR2 promoter region.2.The expression of BpCCRs gene family members has tissue specificity.BpCCR1 and BpCCR2 are highly expressed in roots,stems,and leaves.3.Two sg RNAs were designed for each of BpCCR1 and BpCCR2 genes,and four gene editing vectors were constructed successfully.Mutant plants were obtained via Agrobacteriummediated zygotic embryo induction callus way,with a total of 263 positive transformed plants detected out of 485 infected explants.The average rate of genetic transformation was 54.23%,and the average efficiency of gene editing was 26.52%.4.Compared to the wild type,bpccr1 mutants were significantly taller and with higher chlorophyll content.The lignin content in the stem increases,while the lignin content in the leaves decreases significantly.The photosynthetic efficiency is significantly higher than that of the wild type,exhibiting Fast-growing phenotype.5.Compared to the wild type,bpccr2 mutant grows normally with significantly higher chlorophyll content.The lignin content in the stem and leaves decreases significantly.The water loss in leaf is significantly higher than that of the wild type,and the stomatal aperture significantly increases,indicating a decrease in drought tolerance.