Cloning And Functional Analysis Of SaPCS Gene From Sedum Alfredii Hance

Global soil is facing the threat of heavy metal pollution,and one of the great technologies to solve this problem is the plant repair technology.Super-accumulation of Sedum alfredii was discovered by Zhejiang University Yang Xiao'e in an ancient lead-zinc mine in Zhejiang Province,and had strong super-accumulation effect on Cd/Pb/Zn.Research has been shown that PCS gene plays an important role in the process of plant involvement in heavy metal chelation,this paper takes hyperaccumulating Sedum alfredii Hance as experimentalnmaterial to clone the PCS gene which function will be studied.Conclusion as below: 1.According to a cadmium resistant related gene fragment from S.alfredii transcriptome database,we amplified the SaPCS length by PCR.The 1668 bp opening reading frame?ORF?,encoding 555 amino acids and 62.078 kD of protein.Through the sequence analysis,SaPCS protein was found to contain PCS domain,and no transmembrane structure.Analysis of SaPCS genomic DNA shows the gene containing 8 introns.2.We performed Real-time quantitative PCR?qRT-PCR?test to analyze tissue specificity and time-space expression patterns.The results show,under normal conditions,the highest SaPCS expression in the roots.After cadmium?Cd?stress,that of root was increased,and the expression level at 96 hours was 13 times higher than that of initial value.SaPCS expression in leaives was declined first and then increased,reaching a maximum at 48 hours,about 1.5 times that of zero hour.It is suggested that the SaPCS is mainly worked in roots than in stems and leaives.3.The cadmium-sensitive yeast strain ycf1 which expressed the SaPCS gene Was carried out growth status,growth curve determination and determination of yeast cadmium accumulation.The results showed that the growth of turn the empty yeast and SaPCS gene on cadmium-free plates were no different.On the cadmium-containing plate,the growth of SaPCS yeast transformants was significantly better than that of the transgenic plants.This experiment shows that SaPCS gene can improve cadmium tolerance.The results of the growth curve also showed that the SaPCS gene could improve the resistance to cadmium.The accumulation of cadmium in the SaPCS yeast was higher than that in the transgenic yeast,indicating that the SaPCS gene could enhance the accumulation of Cd.4.The SaPCS gene was overexpressed and screened in Arabidopsis thaliana,and we obtained three positive and high transgenic lines.The three lines and wild-type Arabidopsis plants were treated with cadmium,and the phenotypic changes were observed.The physiological indexes of roots and leaves were measured.The results showed that the growth status of transgenic Arabidopsis was better than that of wild type.And the accumulation of cadmium was the result that the accumulation of transgenic lines was significantly higher than that of wild type.We determined Cd²⁺ flow rate and direction in the roots of Arabidopsis thaliana seedlings,and found that Cd²⁺ was always flowing into the cells,and the transgenic plants flow rate was significantly higher than that of wild type plants.This suggests that the gene contributes to Cd²⁺ uptake and chelation.These three experiments showed that SaPCS gene could enhance the resistance and accumulation of cadmium.