Cloning And Functional Verification Of Cd Stress Response Gene SlUPS In Tomato

Excessive cadmium in soil has long been a worldwide environmental problem,It is harmful to the growth of plants and threatens the health of human and livestock through the food chain.Tomato?Lycopersicon esculentum Mill.?belong to the nightshade family?Solanaceae?,is one of the most important crops in the world.When the tomato was poisoned by Cd,it showed some problems such as yellowing and curling of leaves,dwarfing of plants and slow growth and development.Therefore,it is urgent to study the molecular mechanism of response of tomato to Cd stress,SlUPS is a U-box type E3 ubiquitin ligase,the mechanism of the gene response to phosphorus deficiency in rice has been studied by foreign researchers,but the response to cadmium stress in plants has not been reported.In this paper,the gene SlUPS was cloned from tomato,and the genetic transformation of saccharomyces cerevisiae and arabidopsis thaliana was carried out,the results may be useful for studying the function of E3 ubiquitin ligase SlUPS in Cd tolerance in plants,and may provide a theoretical basis for revealing the role of E3 ubiquitin ligase gene in abiotic stress in plants.The main findings are as follows:?1?Cloning and sequence analysis of the SlUPS geneThe gene SlUPS was cloned from tomato and the sequence analysis of the gene showed that,The SlUPS gene is 873bp in length,and the structure and function of the protein encoded by the SlUPS gene are predicted by bioinformatics analysis,the results showed that the protein translated by the gene encodes 223 amino acids,it is composed of?-helix??-fold?extended chain and irregular crimp,it has signal peptide and transmembrane structure,serine,threonine and tyrosine phosphorylation sites.?2?Expression pattern analysis of SlUPS gene in tomato under Cd stressThe expression of the gene SlUPS in tomato roots and leaves was analyzed by qRT-PCR,The results showed that the SlUPS gene was expressed in both leaves and roots,and the expression level in leaves was slightly higher than that in roots.The seedings were treated with different concentrations of Cd?25?50?75?100?mol/L?,at the same time in 3d,the results of expression pattern showed that the level of SlUPS gene expression in leaves increased firstly and then decreased with the increase of concentration,when the concentration of Cd was 25?mol/L,the expression level of Cd in the roots increased continuously,when the concentration of Cd was 50?mol/L,the maximum expression level was obtained.The seedings were treated with Cd 50?mol/L,according to different time?0?3?6?9?12h?to take samples.The results of expression pattern showed that the expression amount of SlUPS gene changed periodically,and reached the maximum at 3h in leaves,in the root,the amplitude of the change was the highest at 9h and the lowest at 6h.The results showed that the gene SlUPS was expressed in tomato roots and leaves,in response to Cd stress,the expression pattern changed with the change of treatment conditions.?3?Stress resistance analysis of SlUPS gene in yeastConstruction of yeast expression vector pYES2-SlUPS and transformation of INVSC1 natural competence,the survival of recombinant yeast and control yeast under different concentrations of CdCl₂?15?20?25?mol/L?stress was compared.The results showed that the recombinant yeast showed a certain tolerance to Cd stress compared with the control yeast,and there was a concentration effect.?4?Stress resistance analysis of SlUPS gene in arabidopsis thalianaConstruction of plant overexpression vector pBI121-Sl UPS,three positive plants were obtained by genetic transformation of wild arabidopsis thaliana with inflorescence infection and identified by molecular markers,the results showed that all the positive plants expressed at transcription level.The growth of WT and OE plants under Cd stress was compared to analyze the stress tolerance of OE-SlUPS arabidopsis thaliana.The results showed that under Cd stress,the growth of OE strain was higher than that of WT strain,the results showed that the SlUPS gene enhanced the tolerance of arabidopsis thaliana to Cd stress at both stages.