CRISPR/Cas9 Gene Editing Of Tobacco NtBSK3 And Analysis Of Biological Effects Of NtBSK Gene Family

Tobacco is one of China's important economic crops.The steric climate in mountainous regions can lead to early flowering of tobacco,reducing the number of effective leaves and affecting the yield and quality.Based on the detection and analysis of previous tobacco gene expression profiles,the BR signal pathway was selected as a candidate signal pathway for the induction of low temperature in the corresponding seedling stage of early flowering of tobacco.In order to understand the characteristics and functions of the key gene family NtBSK in the BR pathway in the tobacco,homologous NtBSK1,NtBSK2 and NtBSK3 genes were found by homology comparison with AtBSK1,AtBSK2 and AtBSK3 in the Chinese tobacco genome database,and their structural characteristics were predicted by bioinformatics methods.The NtBSK3 gene was selected and the CRISPR/Cas9 site-specific knockout system was used to test the efficiency of this technique for editing the NtBSK3 gene and the bioinformatics analysis of the mutant gene.Meanwhile,the feasibility of CRISPR/Cas9 in tobacco-mediated gene-directed knockout was explored as well.Exogenously administered BR was used to detect the difference in expression levels of NtBSK1,NtBSK2,NtBSK3 and frameshift mutation NtBSK3 during treatment by qRT-PCR.The main findings are as follows:1.Based on Arabidopsis AtBSK1,AtBSK2 and AtBSK3 gene sequences and protein sequences,the genes with the highest homology were named as NtBSK1,NtBSK2 and NtBSK3 by homology comparison in the tobacco genome database.By bioinformatics analysis,the NtBSK1/NtBSK2/NtBSK3 ORFs were 1464bp/1467bp/1488bp respectively,and the numbers of encoded amino acids were 487/488/495 respectively.All the three have a kinase domain PKC at the N-terminus and a triple Trp repeat TPR domain at the C-terminus,i.e.there is no signal peptide or transmembrane region,but they all contain kinase recognition sites and the hydrophilicity is strong.These are the typical structures of the receptor-like cytoplasmic kinase subfamily gene members.In the evolutionary tree,the homologous relationship between NtBSK1 and NtBSK2 is closer,indicating that the functions of the two may be more similar.2.Using CRISPR/Cas9 technology to edit the gene of NtBSK3,the mutation rates at site 1 and site 2 were 55.0%?33/60?and 63.5%?33/52?respectively,and the editing efficiency reached 75.6%.There was a 32bp deletion of the longer fragment in the NtBSK3 mutant,but the modification mode was mainly single-base insertion and small fragment deletion.For all the mutations detected,it was found that almost all of the mutations occurred at the position immediately upstream of the PAM sequence at the base of the 4th base,which was in complete accord with the position of the Cas9enzyme to cleave the DNA duplex.A further bioinformatic analysis of the NtBSK3 gene mutated at different sites revealed that the types of frame shift mutations disrupted the TPR domain,and that theoretically those without frame shift mutation did not destroy the protein domain.Site 1 mutation did a greater damage to the protein domain than site2,so when designing the target site,as far as specificity is good,it's best to select the former exons.The observation of the T0 plants revealed that the transgenic plants were significantly shorter than the wild type.3.By comparison of exogenous spraying BR and distilled water for 6 leaves of wild tobacco seedlings,the results showed that the relative expression levels of NtBSK1,NtBSK2,and NtBSK3 genes were up-regulated during the 8-hour period after BR application and the amount of expression rose relatively slow during the first 6 hours.However,the expression increased rapidly during the 6th-8th hour and reached a maximum at the 8^th hour.It can be seen that exogenous application of BR effectively affects the BR signaling pathway in plants;and NtBSK1,NtBSK2,and NtBSK3 genes all participate in the BR signaling pathway,of which NtBSK1 has the largest change.During the 8 hour of spraying the distilled water control,the relative expression levels of the genes NtBSK1,NtBSK2 and NtBSK3 showed a normal distribution,and reached a maximum at the 6^th hour.And the maximum value of the sprayed BR expression was greater than the maximum value of the sprayed distilled water control expression.By comparison of exogenously spraying BR and distilled water on the frame shift mutation plants and false-positive plants,it can be found that the real-time quantitative PCR results of the false positive plants basically accorded with those of the wild type,but the relative expression levels of the frame shift mutation plants were significantly lower than the relative expression levels of the false positive plants.Furthermore,the relative expression levels of the BR were increased by exogenous spraying but still significantly lower than the expression of the false positive plants.