Metabolic Process Related To Cadmium Detoxification And Key Gene Cloning Of Sedum Alfredii

Plant bioremediation is a new technology with a wide range of application prospect,which has great potential in the restoration of heavy metal contaminated soil.Sedum alfredii Hance is a native Zinc(Zn)/Cadmium(Cd)hyperaccumulator in China and it is a potential beneficial material of plant bioremediation.In this thesis,we globally studied the function of glycine betaine and sulfur(S)metabolism pathway in the Cd hyperaccumulation process,using hyperaccumulating ecotype(HE)and non-hyperaccumulating(NHE)of S.alfrediiHance in hydroponic culture and applying the techniques of real-time Polymerase Chain Reaction(RT-PCR),high performance liquid chromatography(HPLC),laser confocal microscopy and Inductively Coupled Plasma-Mass Spectrometry(ICP-MS).The main results are shown as below:1.We studied the effects of exogenous glycine betaine addition to the Cd tolerance and accumulation of two ecotypes of S.alfredii Hance.The results of hydroponic experiments showed that exogenous glycine betaine promoted the plant growth of S.alfredii Hance.For HE,partly leaves showed chlorosis symptoms under 400 ?M Cd level,but they recovered after application of exogenous glycine betaine.For NHE,the plant showed lodging,leaves abscission symptomsunder 20 ?M Cd level.However,the application of exogenous glycine betaine maintained the normal growth of NHE and increased the shoots biomass.Besides,exogenous addition of glycine betaine increased Cd content of HE and NHE.The Cd content of HE increased 16.8%,33.2%,6.3%and 119.9%in shoots and increased 161.9%,17.6%,2.7%and 58.6%in roots under 10,20,100 and 400 ?M Cd levels,respectively.The Cd content of NHE increased 6.3%,62.3%and 128.3%in shoots and decreased 5.5%,but increased 7.8%and 61.3%in roots under 2,10,2.0?M Cd levels,respectively.The content of glycine betaine in both ecotypes increased as exogenous Cd concentration increasing.The concentration of glycine betaine in HE shoots with 400?M Cd was 6.2 times higher than control treatment.Theconcentration of glycine betaine in NHE shoots with 400 ?M Cd was 1.8 times higher than control treatment.The content of glycine betaine in HE was twice in NHE under 20 ?M Cd.Applying exogenous glycine betaine significantly increased the glycine betaine content in leaves of both ecotypes.All results suggested that the synthesis of glycine betaine was one of the response mechanisms to Cd stress of S.alfredii,but the content of glycine betaine in S.alfrediiwas not enough,hence exogenous glycine betaine addition was an effective approach to increase the Cd accumulation and tolerance.2.We studied the expression of betaine aldehyde dehydrogenase(BADH),antioxidase and transporter genes,the oxide content and the Cd distribution in stem of S.alfredii under Cd and glycine betaine treatments.The results showed that both the BADH activity and BADH expression level of HE increased significantly responding to Cd stress.In NHE,the activity of BADH increased in short time but BADH expression was not affected by Cd stress.In HE,the expression of two kinds of superoxide dismutase(SOD)genes were not influenced by 20?M Cd and glycine betaine application,but catalase(CAT)gene expression was induced.In NHE,exogenous glycine betaine promoted the gene expression of two kinds of SOD and CAT,in the meanwhile lowered their upregulation extent after Cd stress.For both HE and NHE,the gene expression of P1B-type ATPase(HMA2)was induced significantly but Metal Tolerance Protein 1(MTP)was not affected by exogenous glycine betaine.The content of MDA,H2O2 and O2-resulting from Cd stress decreased significantly under glycine betaine treatment.Besides,the Cd distribution showed a trend of accumulating more in stele.All the results indicated that glycine betaine enhanced Cd tolerance of S.alfredii through improving oxidation resistance and Cd regular distribution,and that glycine betaine improved transporters expression to increase Cd accumulation.3.We searched the amino acid sequence of enzyme related to S metabolism pathway in other species and designed degenerate primers of ATP sulfurylase(ATPS),Serine acetyltransferase(SAT),gama-glutamyl-cysteine synthetase(GSH1),glutathione synthetase(GS)and phytochelatins(PCs)by using online software iCODEHOP.We cloned the above genes,S-adenosylmethioninesynthase(SAMS)and nicotianamine synthase(NAS),and detected their relative expression level under Cd treatment by RT-PCR.The results showed that ATPS,PCS and NAS responded to Cd treatment more severely of HE than NHE.For HE,the relative expression level of PCS was highest under 400?M Cd level,but it did not varyobviously under 10 and 100?M Cd levels.HE Roots NAS was induced after 24 hour with Cd treatments and increased sharply in roots and shoots after eight days.Under 100?M Cd treatment,the relative expression level of NAS was 19.5 times higher than the control in HE roots,but for NHE,the NAS expression decreased in roots and increased in shootssignificantly.The expression pattern of NAS in S.alfredii shoots was significantly different from in roots,showing that the expression level in roots was higher than in shoots.For HE,roots NAS expression level was 3171 times higher than shoots after eight days under Cd 100?M Cd treatment.For NHE,it was 45.3 times of roots higher than shoots.Under 10?M Cd treatment,NAS expression level of HE was 141 times higher than NHE in roots,but there was no significant difference in shoots.All the results suggested that the enzymes related to S metabolic pathway were activated responding to Cd treatment and NA may be the important factor for Cd detoxification rather than PCs.4.We detected the S content,S-containing free amino acid and glutathione of both ecotypes after 8 days under 10?M Cd treatment.The results showed that the HE S content of shoots and roots under Cd treatment were 36.7%and 32.6 higher than under control,respectively,but there was no significant difference in NHE.The content of cysteine(Cys)in HE shoots and roots increased after Cd treatment and roots Cys content was 46%significantly higher than control.However,the NHE Cys only increased a little in shoots and decreased 78%in roots comparing to control.In the meanwhile,the Cys content was higher in shoots than roots in HE,but it was higher in roots than shoots in NHE.The trend of methionine(Met)was similar with Cys and the content of Met in HE was higher than in NHE.The glutathione content increased in both ecotypes.In HE,reduced glutathione(GSH)content increased 34%and 142%in shoots and roots,respectively,comparing to control,but oxidized glutathione(GSSH)content did not vary.However,in NHE,GSSG content increased 38%and 84%in shoots and roots significantly comparing to control,and GSH content did not vary.For both ecotypes of S.alfredii,the content of GSH were higher than GSSG in both shoots and roots,and total glutathione content were higher in roots than shoots.All the results suggested that HE assimilated S to synthesize more S-containing compounds responding to Cd stress.