Study On The Effect Of Raffinose Family Oligosaccharides In Low Temperature Resistance Of Tomato

Tomato(Solanum Lycopersicum L.)is a kind of thermophilic vegetable,which can grow normally at 13-33 °C.Low temperature stress will reduce the yield and quality of tomato fruit.Studies have shown that raffinose oligosaccharide plays an important role in plant resistance to low temperature stress.A.nanus,a key enzyme in the metabolism of raffinose oligosaccharide,ginitol galactosidase(An Gol Ss)overexpresses tomato The cold resistance of the plants was significantly improved.This experiment analyzed the expression level of tomato inositol galactosidase synthase(Sl Gol Ss)under low temperature stress,the effects of the expression of key enzymes of Sl Gol Ss and other raffinose metabolism are studied by exogenous application of raffinose oligosaccharide metabolism related substances,and the effect of raffinose family oligosaccharide on tomato low temperature resistance is clarified;the prokaryotic expression of Sl Gol Ss protein in E.coli,the role of inositol galactosidase synthetase under low temperature stress;The identified Sl Gol Ss were overexpressed in tomato in order to increase the low temperature resistance of tomato.The main conclusions of the paper are as follows:(1)The changes of Sl Gol S1,2,3,4 gene expression in tomato roots,stems and leaves under low temperature stress are analyzed by q RT-PCR.The results showed that Sl Gol S2,4 is highly expressed in tomato roots,stems and leaves under low temperature treatment,and gradually increased with the treatment time.(2)Exogenous spraying of raffinose,sucrose,inositol,inositol Golactoside(Gol),DGJ(inhibitor,inhibition of alpha-Golactose hydrolysis),Gol+DGJ are used to determine the MDA,CAT,GSH,POD,SOD,H2O2,relative ion leakage rate of each treatment.The changes of gene expression of Sl Gol S in Tomato under low temperature stress are analyzed by q RT-PCR.Inositol Golactoside,DGJ and DGJ+Gol significantly increased the stress resistance of Tomato under low temperature stress than inositol,sucrose and raffinose.Sl-Gol S2,4,Sl-RS1/2/3,Sl Stas,Sl?-ga1,Sl?-ga2,Sl?-ga3 are up-regulated under low temperature stress,but Sl-Gol S2,4 is much higher in magnitude than other Genes indicate that these genes are regulated by low temperature stress,and Sl-Gol S2,4 plays a major role in low temperature response,the Gol content is regulated by high-efficiency expression to resist low temperature stress,and the external application of Gol has the same effect.(3)In order to verify the cold-resistant function of Sl Gol S2,4 gene,we transfer the recombinant plasmid into the competent cells of E.coli BL21.IPTG is add to induce the expression of Sl Gol S2,4 protein in E.coli.The number of colonies of pet32a-Sl Gol S2,4 is significantly higher than that of pet32a-Sl Gol S1,3 and pet32 a blank control after 6 hours of treatment at-20 ?,which prove that Sl Gol S2,4 is a cold-responsive gene and its protein expression could be increased.The low temperature resistance of strong E.coli can improve the survival rate of colonies.(4)According to the requirements of transgenic technology,the recombinant plasmid is constructed and transformed into Agrobacterium tumefaciens.The cotyledon of tomato is infected by Agrobacterium tumefaciens mediated method,and the callus is cultured in resistant medium to obtain transgenic tomato plants.The positive transgenic tomato seedlings are identified by PCR amplification.Finally,17 Sl Gol S2 overexpressing transgenic tomato T0 plants and 10 Sl Gol S4 overexpressing transgenic tomato T0 plants are obtained.One step study on cold resistance of tomato provides important experimental materials.