Study On The Morphology And Internal Cytoskeleton Of Trichones In Tomato

Tomato(Solarium lycopersicum Mill.)is an important economic crop and is cultivated worldwide.Trichomes,covering the surface of tomato plant,are the first line of defense against environmental stresses,which play a critical role in broad-spectrum resistance.Plants use trichomes to deter the spread of viruses and pathogens and the insects’ attacks through space barriers,physical trapping and chemical poisoning.Trichomes are also important in enhancing palnt resistance to abiotic stresses.Along with the development of cell biology,the dynamic changes of cytoskeleton in the development of trichomes has been well illustrated in Arabidopsis thaliana.Kinesin-like calmodulin-binding protein(KCBP),a member of the Kinesin-14 family,has been known as a hub protein,which can interact with both microtubules(MTs)and actin filaments(AFs).Functional studies of KCBP in Arabidopsis thaliana have shown its involvement in trichome morphogenesis,but the studies of KCBP and the cytoskeleton in tomato are still rarely reported.Therefore,exploring the the internal cytoskeleton array of tomato trichomes,identifying the key genes and investigating the molecular mechanism of trichome development are meaningful in improving the broad-spectrum resistance of tomato in both theory and practice.To elucidate the cytoskeleton arrangement patterns,we constructed some transgenic plants,of which the MTs and AFs were labeled with the enhanced green fluorescent protein(EGFP)respectively.Besides,genome-edited tomato plants with the disruption of SIKCBP was created by using CRISPR/Cas9 system,so as to determine the function of SlKCBP in trichome development.Laser scaning confocal microscope(LSCM)was employed to observe the cytoskeleton arrangement in tomato epidermal cells and trichomes.Following are the main results:1.Two marker lines,of which the MTs and AFs were labeled with EGFP respectively,were constructed and named as SlTub6-EGFP and Lifeact-EGFP respectively.The cytoskeleton arrangement in tomato epidermal cells and trichomes was successfully observed by LSCM,which established the foundation for deep research on the cytoskeleton array in tomato.2.The type and the morphology of trichomes in SlTub6-EGFP and Lifeact-EGFP had no significant difference with those in wild type,which provided a feasible basis for the extensive utilization of the cytoskeleton markered lines.By LSCM,it was found that the MTs were arranged transversely,while the AFs showed a bundled longitudinal array.3.The KCBP in tomato was identified and analyzed by bioinformatics method.Moreover,genome-edited tomato plants with the disruption of SlKCBP were created by using CRISPR/Cas9 system.Results showed that there was no significant difference in the type and morphology of trichomes between SlKCBP-CRISPR and the wild type.Moreover,we obtained the cytoskeleton labeled SIKCBP-CRISPR lines,of which the MTs and AFs were labeled with EGFP respectively through hybridization with SlTub6-EGFP or Lifeact-EGFP,which laid a foundation for further study on the role of SlKCBP gene in cytoskeleton arrangement in tomato.