Editing Of Nicotiana Tabacum NtBRI1 Gene By CRISPR/Cas9 And Study On Biological Effects

Tobacco is an important economic crop in China.The growth of plants directly affects the quality of tobacco leaves.Brassinosteroids?BRs?is an important hormone and have many regulatory effects on plant growth and development.Brassinosteroid receptor BRI1 is a single transmembrane leucine-rich repeat-like receptor-like protein kinase?Leu-rich repeat-like receptor-like protein kinase,LRR-RLKs?,which is the key components of BR signal transduction pathways,there is less research on NtBRI1 gene in Nicotiana tabacum,and the functional mechanism of BRI1 isn't yet clear.Therefore,this experiment used CRISPR/Cas9 technology to edit the NtBRI1 gene of common tobacco.The NtBRI1 gene of the T₀ mutant was subjected to bioinformatics analysis,selection of key domain-changing mutants to determine its NtBRI1 gene expression and observation of its phenotype,to analyze the biological effects of the NtBRI1 gene in tobacco and create a new genetic material for follow-up research.The results of the study are as follows:?1?CRISPR/Cas9 site-directed mutagenesis of NtBRI1 gene.Using CRISPR/Cas9 technology to edit the NtBRI1 gene,26 mutants were obtained with an editing efficiency of 25.7%?26/101?.The mutation efficiency of target site 1 was 43.8%?91/208?;the mutation rate of target site 2 was 71.2%?148/208?.Mutations are based on the deletion,insertion or replacement of short fragments of 1-5bp.There were also 2 clones missing 33bp and 68bp respectively.The mutation at position 1 is mainly based on the substitution of bases?75/208?;There are many types of mutations at position 2 and single-base insertions are dominant.?2?Bioinformatics analysis of T₀ mutant NtBRI1 gene.The bioinformatics analysis of the NtBRI1 gene was performed by screening the T0 mutants B-2,B-3,B-4,B-10,B-20,B-21,and B-24 with different mutation types.The results showed that the mutant NtBRI1 gene had the same functional domains and contained a leucine-rich repeat at the N-terminus?52-87?.The intermediate position?194-207,243-301,312-372?contains a repeating sequence of leucine Compared to the wild type,the intermediate leucine repeats are reduced,there is no classical subtype leucine repeats,and there is no protein kinase domain at the C-terminus.The protein secondary structure of each mutant NtBRI1 gene is dominated by random coils,including alpha helix and extension strands.The tertiary structure of the mutant NtBRI1gene protein is similar,and compared with the mutant,the tertiary structure of the wild-type NtBRI1 gene protein is relatively different.Editing of the T₀ mutant NtBRI1 gene leads to the deletion of its key domain,the protein kinase domain,which makes its tertiary structure significantly different from the wild type.?3?Differential expression analysis and phenotype observation of NtBRI1 gene expression in T₀ mutants.Through the analysis of gene expression differences in the T₀ mutant NtBRI1,we found that the mutant NtBRI1 gene expression was lower than the wild type,indicating that editing of NtBRI1 gene affects the expression of NtBRI1 gene.The most obvious decrease in NtBRI1 gene expression was observed in B-4,B-20,and B-24,with the same mutation sites,indicating that mutations at the same mutation site had similar effects on NtBRI1 gene expression.Observing the T₀ mutant phenotype,we found that the height and pitch of the mutants were lower than those of the wild type,and the gap between the mutant and the wild type increased with the growth of the plants.There were differences in the appearance of different mutants,including increased number of leaves,delayed budding stage,and more tillers in low leaf position.The phenotypes of the obtained T₀ mutants are different,creating a rich genetic material for subsequent research.