Screening And Functional Study Of Non-specific Lipid Transporters (nsLTPs) Related To The Synthesis Of Lycopersicon Esculentum Epidermal Cuticle

Tomato is a model plant to study the problem of fruit softening and water loss.The study of the molecular regulation mechanism in the process of fruit softening and water loss is of great significance for improving the quality of fruit storage.Previous studies have shown that plant non-specific lipid transfer proteins(nsLTPs)are related to many biological processes,including the formation of the stratum corneum and wax biosynthesis(Fang et al.,2015).An important factor in fruit dehydration and softening.At present,there are few studies on the function and mechanism of tomato nsLTPs,and how it affects the fruit dehydration and softening has not been clarified.Therefore,by constructing an overexpression vector,this paper uses transgenic technology to analyze the function of tomato nsLTPs—SlLTPg1,in order to lay the foundation for future exploration and improvement of the shelf life of other cash crops such as tomatoes.(1)The SlLTPg1 overexpression vector was constructed,and 18 positive overexpression plants(over expression,OE)were obtained by using the leaf disc method mediated by Agrobacterium.Real-time fluorescent quantitative PCR(Real-time fluorescent quantitative PCR,qRT-PCR)was used to obtain 18 positive overexpression plants.)The expression level of SlLTPg1 was analyzed on 18 positive overexpression plants.?-actin was used as the internal control,and the wild type(WT)plant was used as the control.The expression level of SlLTPg1 in all positive transgenic plants was up-regulated by 3-4 times.Seeds were harvested and Plant the T1 generation and proceed to the next experiment.(2)The relative water content,transpiration water loss rate,fruit compression resistance and fruit compression resistance during the mature green period(MG)and red ripe period(Red rip,RR)were measured by dry weight method and texture analyzer analysis.Peel puncture resistance.In the RR period,the water content of OE fruit was significantly higher than that of WT.The water loss rate of OE fruit was much lower than that of WT fruit within20 days,and the rising trend was slow,reaching only 30%at 20 days and WT reaching 60%.In the RR stage,the compression resistance of OE fruit is about 300 N/mm/s,and that of WT fruit is only 180 N/mm/s;similarly,the puncture resistance of OE fruit skin is more than 100N/mm/s,and WT is only 50 N/mm./s,there is no significant difference in MG period;(3)The thickness of the stratum corneum of the peel was measured by the Sudan IV staining and the oil red O slice scanning electron microscope.Including the MG stage and the RR stage,the thickness of the stratum corneum of OE fruit increased significantly.In the RR stage,the stratum corneum thickness of the OE fruit peel is about 15?m,and the WT is about 12?m.Analyzing the structure of the stratum corneum,there are obvious anticline nails between the WT and OE epidermal cells,but in the RR stage,a large amount of keratin deposits appear under the epidermis of the OE fruit;(4)GC-MS analysis of stratum corneum wax and keratin monomer composition changes.There is no difference in the composition of keratin monomers and waxes of WT and OE peels.?,(?)-hydroxyalkanoic acid derivatives and alkanoic acid derivatives account for the main components of keratin monomers.In the MG phase,the total amount of OE keratin monomers increased by 30%;alkanes and triterpenoids accounted for the main components of stratum corneum wax,and C₃₁ alkanes increased significantly.The MG and RR phases increased by about 76%and 39%,respectively.(5)Detect the expression of wax synthesis-related genes by qRT-PCR.In the MG stage,the expression levels of all genes in the OE peel were higher than those in the WT plants.In the RR stage,except for LACS1,the expression levels of all genes decreased,but the APX1,LACS1,and CER6 gene expression levels were still higher in the OE plants than in the WT plants,among which GDSL2 and GPAT4 The amount of gene expression of the virus declined rapidly,and there was no significant difference.