Cloning And Functional Analysis Of SlEGY2 Gene In Tomato

With the impact of global warming,high temperature has become one of the major environmental factors affecting plant growth,development,yield and geographical distribution.Heat stress can cause a series of physiological and biochemical reactions in plants,including the accumulation of reactive oxygen species,the increase of cell membrane lipid peroxidation,the loss of water,the inhibition of photosynthesis and the loss of enzyme activity.As a thermophilic vegetable originated in the tropics,tomato is very sensitive to high temperature.Continuous high temperatures affect the yield and quality of tomatoes and cause serious economic losses.Therefore,it is of great scientific and practical significance to study the mechanism and molecular basis of tomato to heat stress tolerance.Zinc metalloproteases are a wide variety of proteases with complex functions and play an important role in plant growth and stress response.Three EGY zinc metalloproteases were found in Arabidopsis thaliana.The function of EGY1 gene was mainly studied,but there were few studies on the function of EGY2.In this study,a gene named SlEGY2 was isolated from the tomato genome,and the antisense SlEGY2 transgenic tomato plants were used to analyse the effect of inhibiting SlEGY2 gene on tomato growth and resistance to heat stress.The major results are as follows:?1?We obtained the full-length cDNA of SlEGY2 gene by using PCR reaction and tomato leaf cDNA as a template,as well as specific primers designed based on the SlEGY2 gene sequence?Gen Bank number:XM₀10323653?found on NCBI.The full gene is 1524bp in length and the ORF is 1335bp.SlEGY2 gene encodes 444 amino acids,and has an estimated isoelectric point of 6.19 and a protein molecular weight of 48.56 kDa.This gene is located on chromosome 4.?2?The SlEGY2-GFP fusion protein was constructed and transiently expressed in tobacco.The observation was performed using a laser scanning confocal microscopy.We found the SlEGY2 protein was located in chloroplast.Analysis of the expression pattern of SlEGY2gene in tomato by qRT-PCR and GUS staining showed that the expression level of SlEGY2gene was highest in leaves and flowers,and SlEGY2 gene was induced by high temperature,PEG,ABA and MeJA.?3?The SlEGY2 suppression expression vector was constructed and transformed into tomato using Agrobacterium-mediated leaf disc transformation.The transgenic plants were screened by real-time quantitative PCR.Three antisense transgenic tomato lines?A1,A3,and A33?were selected for further experiments.?4?After heat stress,the leaves of antisense transgenic and WT plants exhibited different degree of wilting.Compared with WT,leaf wilting was much more severe in antisense transgenic plants,and the accumulation of H₂O₂ and O₂^·-in antisense tomato plants was significantly higher than that of WT.The level of REC and MDA were also significantly higher than those of WT,indicating that the damage of the antisense tomato cell membrane was more severe,and suppression of SlEGY2 reduced tomato heat stress resistance.After heat stress,the Fv/Fm,Pn and D1 protein of tomato plants were decreased,but the antisense tomato had higher decreased degree than WT,which indicated suppression of SlEGY2increased the photoinhibitory of transgenic tomatoes under heat stress.?5?Under normal condition,the antisense transgenic tomato seedlings were shorter than WT.Seeds of antisense and WT strains were planted on MS medium and grown in darkness for a week,their cotyledons were unfoled and they all showed yellowing phenomenon with the apical hook curved,but the bending degree of the apical hook of antisense lines were more severe than that of WT.The ethylene content in antisense and WT were detected by gas chromatograhpy.It was found that the ethylene emission in antisense lines was significantly higher than that of WT.Interestingly,the height difference became almost disappeared when antisense lines and WT were adult plants.