Cloning And Functional Identification Of Cd Stress Response Gene SlUDP In Tomato(Lycopersicon Esculentum Mill.)

In recent years,the land area polluted by the heavy metal cadmium（Cd）has been increasing in China.Cd is a highly toxic heavy metal and one of the biological non-essential elements,which has been listed as one of the primary environmental risk control elements by international organizations such as IARC.Tomato（Lycopersicon esculentum Mill.）,a plant of the Solanaceae family,is widely grown in the world and occupies an important position in vegetable crops.However,with the increasing application of chemical fertilizers and pesticides,the problem of heavy metal residues in soil has become increasingly prominent.Tomato showed physiological disorders such as slow growth and yellow leaves after being poisoned by heavy metals.Heavy metal stress environment seriously restricts the production safety of tomato,so it is particularly important to excavate genes related to heavy metal stress in tomato.Glycosyltransferase is the key enzyme in polysaccharide synthesis,and also the enzyme that catalyzes glycosylreaction.It plays an important role under abiotic stress,mainly regulating hormone homeostasis,secondary metabolism,stress resistance,etc.Plant glycosyltransferase plays an important role in the study of plant stress.It plays an important role in plant resistance to abiotic stress through a series of protein modifications.In our lab,a tomato glycotransferase gene was screened by RNA-seq sequencing technology and up-regulated under Cd stress.Therefore,further study on this gene was conducted in this experiment,and the results are as follows:（1）Cloning and bioinformatics analysis of SlUDP gene in tomatoFirstly,the UDP c DNA sequence was obtained by homologous cloning method and bioinformatics analysis was performed.The results showed that SlUDP protein secondary structure was mainly composed ofα-helical,β-folded and extended chains.Phylogenetic tree analysis showed that SlUDP was the most closely related to sweet pepper,followed by potato and Mosaic tobacco,with the support rate of 98%.（2）Expression pattern analysis of SlUDP gene in tomato under Cd stressThe expression pattern of SlUDP gene under different time and concentration of heavy metal Cd stress was analyzed by real-time quantitative PCR.The results showed that SlUDP gene was expressed in all tissue parts of tomato,and the highest expression level was found in leaves,which was 8.4 times of that in main stem.（3）Functional identification of tomato SlUDP gene in Saccharomyces cerevisiae.Will INVSC1 SlUDP gene carrier into yeast feeling state of recombinant yeast,through carries on the different concentrations of Cd,found that the recombinant yeast improved tolerability of Cd,in 100μmol/L processing control colony found no growth under diluted 1000 times,and INVSC1 p YES2-SlUDP in colony can still grow,according to the results SlUDP gene can improve the recombinant yeast tolerance to heavy metal Cd stress.The function of SlUDP gene and heavy metal Cd stress was preliminarily identified,which provided a certain basis for studying the role of SlUDP gene in Arabidopsis thaliana.（4）Functional identification of over-expression of SlUDP gene in Arabidopsis thaliana under heavy metal Cd stress.The T3 generation plants of Arabidopsis thaliana overexpressing SlUDP gene were subjected to stress treatment.The seed germination rate,adult phenotype,root length,stress-related physiological indexes and gene expression were analyzed.Under different concentrations of Cd Cl₂（40μmol/L、60μmol/L、80μmol/L）,the germination rate of Arabidopsis overexpression reached 35%at the third day under 80 mol/LCd Cl₂treatment,while that of wild-type Arabidopsis was only close to 15%.The survival rate of overexpressed plants in soil-cultured Arabidopsis thaliana was more than 50%,while that of WTArabidopsis thaliana was less than 10%.The results showed that the contents of soluble sugar,MDA、POD and SOD in transgenic Arabidopsis thaliana were significantly higher than those in wild type,indicating that transgenic Arabidopsis thaliana could respond to stress by regulating the contents of soluble sugar,MDA、POD and SOD in vivo,and improve the tolerance of plant to Cd stress.In order to further study the role of SlUDP gene in Cd stress,genes related to heavy metal transport were selected to detect its expression level.The results showed that the expression levels of ZIP1、IRT1 and CSD1 genes in overexpressed Arabidopsis thaliana were significantly higher than those in WT.Therefore,we further speculated that SlUDP gene could improve plant tolerance to Cd stress through reactive oxygen scavenging system and metal ion transport related pathways.