Effect Of CRISPR/Cas9-edited NtNINJA Gene On NtJAZ1 Expression Under Low-temperature Stress

Low temperature at seedling stage may affect the normal growth and development of Nicotiana tabacum,resulting in weak growth potential,reduced leaf number,narrow leaves,and reduced yield and quality of tobacco leaves.Previous studies have revealed increased expression of key genes in the signaling pathway and synthesis of jasmonic acid under low-temperature stress.NINJA protein is an important factor associated with JAZ protein and downstream transcription factors in the signaling pathway of jasmonic acid,and the functional relationship between the NtNINJA gene and the NtJAZ1 gene is not clear.CRISPR/Cas9 genome editing technology can be applied to gene function identification.In this study,the NtNINJA gene in Nicotiana tabacum was edited using CRISPR/Cas9 genome editing technology,the expression of NtJAZ1 in mutant and wild-type plants at the T₀ generation was determined by real-time quantitative PCR at low temperature and room temperature,and the difference of NtJAZ1 gene expression under NtNINJA gene mutation was understood,laying the foundation for further analysis of the effect of NtNINJA mutation on NtJAZ1 expression.The main results are as follows:1.With target site 1?F:5-GTTGCAATCCTTAAGGCGTT,R:5-AACGGGTTA AGGATTGCAAC?and target site 2?F:5-AAAGAACACGTCCTATTAAG,R:5-CTTAATAGGACGTGTTCTTT?as target sites,NtNINJA could be edited in Nicotiana tabacum Hongda,with low editing efficiency?<1%?.One mutant plant was obtained,with a mutation rate of 25%and a deletion of 3 bases at 363 bp-365 bp from the start codon in the CDS sequence of the NtNINJA gene.2.CDS clone of mutant NtNINJA showed that the edited NtNINJA gene had transcriptional activity and could be transcribed to mRNA.CDS in this gene had a length of 942 bp and could encode 314 amino acids.Bioinformatics analysis revealed that the mutant NtNINJA protein was predicted to be a hydrophobic protein,non-signal peptide and transmembrane region,with a kinase domain EAR?TPL-binding EAR repression motif?at the N-terminal end,and putative nuclear localization signal at middle part,and Jas TPL-binding domain at the C-terminal end?246-307?.The prediction of phosphorylation sites demonstrated its protein kinase activity in serine and threonine and tyrosine receptor.The length difference in CDS between mutant NtNINJA gene and wild-type NtNINJA gene CDS was 3 bp.Compared with wild-type sequence,3 bp was deleted in mutant sequence,with a deletion of GGC.The deletion in the sequence changed the protein sequence from L120-R121-R122-L123 to L120-S121---L122.The normal protein sequence was changed from 2 arginines to 1 serine in putative nuclear localization signal,which resulted in a significant change in the tertiary structure of mutant and wild-type NtNINJA.This mutation had the potential to cause changes in protein function,and change the ability to combine with JAZ and TPL.3.The mutant and wild-type plants were treated at low temperature and normal temperature.Fourteen days after the treatment,the difference of NtJAZ1 gene expression between mutant and wild-type plants at the T0 generation was detected at different temperatures.NtJAZ1 expression in wild-type and T0-generation plants was1.38 and 1.37 at room temperature,respectively,with a difference of 0.01.NtJAZ1expression in wild-type and T0-generation plants at low temperature was 2.34 and 2.59,respectively,with a difference of 0.25,which was higher than the difference under normal temperature.It was suggested that the NtNINJA gene in mutant plants at the T₀generation induced by low temperature at seedling stage could influence the expression of NtJAZ1 gene,and increase the expression higher than wide-type NtJAZ1 gene.