Cloning And Expressing Analysis Of The PCS Gene To Cadmium Stress In Tagetes Patula L.

Phytochelatins(PCs)play a vital role in the process of balance,detoxification and accumulation of metal ion in plants,and thus show a wide application prospect.PCs are synthesized,as a mixture of peptides of different length,from reduced glutathione(GSH)by phytochelatin synthase(PCS),which is activated by a range of heavy metals.Therefore,exploration of PCS gene and their functions has been a topic issue in biology.Tagetes patula has gained much attention for its excellent potential of heavy metal accumulation.Here,we expect to complete full-length clone of PCS gene in Tagetes patula L.Also,we hope to reveal the expression pattern and function of PCS gene in Tagetes patula L,and provide an important proof of mechanism at the molecular level that how plant responses to Cd stress.The current results are summarized as follows:1.In cloning the homologous full-length PCS genes,a conserved sequence(311bp)was first obtained by homologous primer amplification,which is highly similar to SaPCS1 and LsPCS1(both of the similarities are more than 90%).Based on the result above,a 3' sequence(1401 bp)and a 5' sequence(568 bp)in PCS genes conserved sequence were further gained via rapid amplification of cDNA ends(RACE).The full-length PCS genes cDNA sequence(1970 bp)was then split with MEGA6.0software.Using ORF analysis,we showed that the length of coding sequence in PCS genes is 1764 bp,which encodes a protein with 587 amino acid residues.The gene was named as PCS1(TpPCS1)with an accession NO.of KY007159.2.By analyzing TpPCS1 using bioinformatics methods,we indicated that the full length of TpPCS1 is 1970 bp with an ORF length of 1764 bp.The gene encodes a protein with 587 amino acid residues,in which 9 cysteine residues are included in N terminal of the protein,and in total of 34 restriction enzyme sites are also included.The theoretical molecular weight of the protein is 65.23 KDa,the isoelectric point is8.01,the instability coefficient is 47.72,and the average hydrophobicity index is-0.171.All the properties above indicate that the protein may be a hydrophilic unstable protein.Moreover,the signal peptide prediction results didn't show any signal peptide in the protein,which indicated that the protein is not a secreted protein.Also,the transmembrane prediction results didn't show any transmembrane sequence in the protein.Combine the predicted results above and the functional domain predicted results,we concluded the protein encoded with TpPCS1 is phytochelatins synthase with a potential of chelating intracellular heavy metal.In addition,the predicted secondary structure showed the protein has a mixed structure,in which helical structure and turn structure are dominating secondary structures.The predicted secondary structures of the protein are matched well with its tertiary structures.Finally,conclude from the phylogenetic tree,we showed that TpPCS1 is similar to SaPCS1 and LsPCS1.3.Subsequently,TpPCS1 relative expression in Tagetes patula stressed with Cd at different concentrations were detected using real-time quantificative PCR.18 S rRNA in Tagetes patula and ?-actin mRNA in Lactuca sativa were employed as reference genes.The results indicated that compared with relative expressions in leaf tissue and seed tissue respectively,the TpPCS1 relative expression in root tissue was much higher,which may due to the Cd contents were different from tissue to tissue under the stress of Cd at the same concentration.In general,Cd content in root tissue was much higher than that in other tissues.However,TpPCS1 relative expressions in root,stem and leaf were upregulated with the stress of rising Cd concentration,while the TpPCS1 relative expression in seed was downregulated.With the time going on,both of the TpPCS1 relative expressions in Cd stressed root tissue and stem tissue were upregulated(the expression in root was higher).The TpPCS1 relative expression in leaf tissue was high at relative low concentration of Cd,but it was reduced at relative high concentration.4.MSAP technique was employed to analyze the changes of DNA methylation inTagetes patula under different concentrations of cadmium(Cd)in a pot experiment.The results showed that the genomic MSAP rate of Tagetes patula was 24%,30%,35% and 41%,and the total methylation rate of Tagetes patula was 20%,23%,25%and 27%,respectively,when treated with the different concetrations of Cd2+(0,50,250,500 mg·kg-1).The results indicated that the total methylation level of DNA was improved with the increase of Cd2+ concentration.Moreover,as regard to the change of DNA methylation pattern,the denove methylation rates were 10%,10%and 11%,respectively,under stress of 50,250 and 500 mg·kg-1 Cd2+,which is indicating that the denove methylation was the main methylation pattern.