| Tomato(Solanum lycopersicum)is an important horticultural crop with high economic value.In the protected cultivation,however,tomato plant is susceptible to low temperature and induces multi-locule malformation fruits and affects yield and quality.Studies have shown that exogenous plant growth regulators play a role in regulating multiventricular malformations.For example,2,4-D can reduce the occurrence of multiventricular malformations,while GAs increases the incidence,but the role of cytokinin in regulating the number of tomato locule is unclear.Therefore,in this experiment,the multi-locule tomato line LA2006 and the less-locule tomato line Micro-Tom,which are prone to deformed fruits,were used as the test materials for the treatment of low night temperature(28/10℃)at various stages of flower bud differentiation(early,middle,late stages of flower bud differentiation)effects on the number of tomato locule and the changes in CTK(cytokinin)content of tomato stem tips;As a control,spraying clear water with LA2006 as a control,exogenously applying CTK and its inhibitor YZJ(lovastatin)for treatment,through transcriptome analysis,to explore the relevant metabolic pathways of CTK regulating tomato locule formation;CRRSPR technology was used to construct a cyp735a knockout mutant of CTK synthesis key enzyme gene,and to explore the molecular mechanism of CTK regulation of locule.The main research results of the paper are as follows:The early stage of tomato flower bud differentiation was placed in a light incubator for low night temperature and exogenous CTK treatment.The CTK content in the shoot tip and the number of fruit locule were measured,the results showed that low temperature significantly increased the number of fruit locule of the multi-locule line LA2006,but the increase of Micro-Tom with less-locule was not significant;treatment at low temperature of 10℃ for 20 days significantly increased the contents of i P(prenyl adenine)and i PT(prenyl adenosine)in the shoot tip of tomato,but Zt(zeatin)and t Z(trans zeatin))no significant change in content;both exogenous i P and ZR treatments significantly increased the number of fruit locule,but the treatment effect on LA2006 was stronger,and the number of locule increased significantly with the increase of treatment concentration,and the treatment effect of i P was significantly higher than that of ZR treatment;The expression of CYP735 A,a key enzyme of cytokinin synthesis,was determined to be higher than that of less-locule strain in the mid-phase of flower bud differentiation in multi-locule strains.Analysis of tomato stem tip transcriptome data by KEGG showed that differentially expressed genes were significantly enriched in carbohydrate metabolism,lipid metabolism,amino acid metabolism,plant hormone synthesis and signal transduction pathways,we found that after exogenous application of i P,glucosyltransferase synthesis gene,linoleate synthesis gene,glutathione transferase gene,cytokinin oxidative dehydrogenase gene and ARR8 and ARR9 genes were up-regulated,β-Glucosidase and β-mannosidase genes,phospholipase synthesis genes,zein O-glucosyltransferase and other related differential genes were down-regulated.In order to verify the role of CTK in the formation of tomato locule,a cyp735a eukaryotic expression vector was constructed and transformed into Agrobacterium.The Agrobacterium-mediated transformation method was used to obtain the T0 generation plants of cyp735a knockout mutants,and the number of locule was observed after colonization.The results showed that the tomato malformation rate of the knockout mutant plants was reduced from 28.95% to 11.11%,and the fruit was significantly reduced,indicating that CTK is regulating the number of tomato locule,and provides important materials for the next study of cytokinin regulation of tomato locule formation. |
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