Effect Of SlMAPK4 Knockout On Tomato Cold Tolerance

Tomato is a cold-sensitive plant,which is extremely vulnerable to low temperature damage regardless of plant growth and development or transportation or storage of post-harvest tomato fruits.MAPK family genes are key genes in the plant cold resistance signal transduction pathway,and the MAPK cascade pathway is an important link in the plant cold resistance regulation process.In this paper,using“Lichun”tomato as experimental material,combining modern molecular biology and transgenic theory,and using CRISPR/Cas9 gene editing technology,an attempt was made to cultivate SlMAPK4 gene mutant tomato plants and explore the effect of SlMAPK4 gene knockout on cold resistance of tomato fruits.The main experimental results of this paper are as follows:?1?Two targets were constructed in the 5?-ORF region and the gene exon functional domain.After analyzing the position of the target sequence in the SlMAPK4 locus,it was verified that both targets were in the exon sequence.?2?According to the design principles of target adapters,the primers required for the first round of Overlapping PCR were designed,and after the second round of PCR,the addition of the sgRNA expression cassette Bsa?digestion site was completed.The pYLCRISPR/Cas9-SlMAPK4 vector sequencing and Agrobacterium colony PCR verification confirmed that the pYLCRISPR/Cas9-SlMAPK4 vector was successfully introduced into the Agrobacterium genome.?3?After PCR detection of the PHT?hygromycin?gene,a total of 40 positive plants were obtained,and the target rate of the T₀ generation was about 70%after sequencing.According to the sequencing results of the T₁ generation plants,L7 has three target gene mutation editing methods,L10 and L30 each have four target gene mutation editing methods,and more homozygotes appear in the T₁ generation.?4?Tomato seedlings in the?d6,d6?editing mode of the L7 line,?wt,i1?gene editing mode seedlings in the L10 line,and L30 line?d4,d4?gene mutation editing mode having highest cold sensitivity among the three mutant lines were compared with the wild type.It was found that L30 plants and fruits?d4,d4?showed high cold sensitivity.SO fruits of T₁generation from d4 and d4 gene editing methods piants were used as the best experimental materials for subsequent physical and chemical indicators of cold resistance.?5?After cold treatment,compared with WT and L30 tomato,the contents of MDA and H₂O₂ of the end products of cell membrane peroxidation were higher than those in the wild control group.Cell membrane permeability increased,cell membrane fluidity decreased,membrane lipid phase transition temperature increased,and unsaturated fatty acid content decreased.?6?SlMAPK4 gene can induce tomato ethylene biosynthesis,increasing antioxidant enzyme activity and GSH-AsA cycle ability,and then improve active oxygen scavenging ability.SlMAPK4 gene can increase Protein content to increase intracellular osmotic pressure and inhibit extravasation of cell contents due to cell membrane peroxidation.In addition,the SlMAPK4 gene can activate the CBF cold resistance pathway,thereby completing the cold stress response mediated by the Ca²⁺signaling pathway.