Screening And Functional Identification Of Cd Stress Response Gene SlPLA₂?in Tomato ?Lycopersicon Esculentum Mill.?

With the frequent activities of humans and the discharge of pollutants from factories,the problem of heavy metal pollution is becoming more and more serious.Cadmium?Cd?iswidely used as a highly toxic heavy metal in the natural environment.It can be dissolved in water and can remain in the soil for a long time and cannot be degraded by organisms.Therefore,when the plant absorbs cadmium,it will have a toxic effect on the animal and the human body through the accumulation of the food chain.Tomato?Lycopersicon esculentum Mill.?belongs to the family?Solanaceae?.It is widely cultivated for its fresh food or processing and is one of the most important worldwide vegetable crops.Tomatoes are mainly poisoned by heavy metals,which are characterized by short stature,slow development,and yellowing of leaves.Phospholipase A₂?PLA₂?is a class of enzymes that catalyze the hydrolysis of phospholipids and plays an important role in the perception of external stimuli and their signaling.At present,compared with a large number of reports in animals,PLA₂ has relatively few reports on plants,so studying its specific mechanism of action has become one of the fastest growing fields in plant molecular biology in recent years.Therefore,this study used tomato phospholipase gene SlPLA₂?as the research object,and carried out preliminary research on the expression and function of this gene,aiming to provide a theoretical basis for exploring the molecular mechanism of SlPLA₂?gene involved in tomato response to Cd stress.The results show that:?1?The structure and function of the protein encoded by SlPLA₂?gene were predicted by cloning and bioinformatics analysis.The results showed that the protein translated by the open reading frame of this sequence has 156 amino acids,mainly composed of?-helix,?-sheet,and extended chain.The random coiled composition is presumed to be a non-soluble extracellular protein,a signal peptide exists,and has a threonine phosphorylation site.The predictive analysis of the promoter of the homeopathic potential indicates that the gene is involved in stress regulation.?2?The expression pattern of SlPLA₂?gene in roots and leaves of different tissues and organs under different stress conditions was analyzed by real-time PCR.The results showed that the expression of SlPLA₂?gene was significantly different in different tissues of tomato.Under stress treatment,SlPLA₂?gene could respond to CdCl₂ and2,4-epitheloside?EBR?treatment,and with the treatment conditions.Different expression patterns have also changed.?3?The SlPLA₂?gene was constructed into the yeast expression vector PYES2 and transformed into yeast INVSC1 competent cells.The survival of non-recombinant yeast and recombinant yeast under different concentrations of CdCl₂?5,10,15,20,25?mol/L?was compared.The results showed that the SlPLA₂?gene significantly increased the tolerance of yeast to CdCl₂.?4?The SlPLA₂?gene was constructed into the plant expression vector pCAMBIA23002300,and the Arabidopsis thaliana genetic transformation was carried out by Agrobacterium tumefaciens-mediated genetic transformation.Six Kana-resistant Arabidopsis thaliana were obtained.PCR detection showed that the genes were integrated into the pseudo-Southern Mustard genome.Three strains of Arabidopsis thaliana were treated with different concentrations of CdCl₂?25,50,75?mol/L?,and the germination rate of each strain were compared to analyze the ability of transgenic Arabidopsis to resist Cd stress.The results showed that under the different concentrations of CdCl₂,the germination rate of the three transgenic Arabidopsis lines was higher than that of the wild type,and the#4 strain was the most significant.It indicated that the transgenic SlPLA₂?gene Arabidopsis thaliana increased its tolerance to Cd stress during seed germination.