Cloning And Functional Analysis Of SaPCR1 And SaPCR2 From Zn/Cd Hyperaccumulator Sedum Alfredii

Phytoremediation is an efficient and economical soil remediation method,and the hyperaccumulator is considered as a representative phytoremediation material for its impressive ability to extract heavy metals.Therefore,the heavy metals accumulation mechanism of hyperaccumulator have drawn considerable attention in the field of heavy metal remediation.Sedum alfredii Hance is a Chinese native hyperaccumulator belonging to the Crassulaceae family,which could accumulate a large amount of heavy metals such as zinc and cadmium.The study of the hyperaccumulation and hypertolerance mechanism of S.alfredii could not only provide a theoretical basis for phytoremediation and bioremediation,but also enriches our understanding of plant adaptability mechanisms.Currently,there have been many researches on the physiological and biochemical mechanism of heavy metal accumulation in S.alfredii,and researches on molecular biology have also been carried out,including the transcriptional sequencing,heavy metal transport related genes cloning and their related functions.However,our understanding of the molecular mechanism of S.alfredii is still limited.The main results achieved in this study are listed as follows:1.Firstly,we cloned two PCR family genes(SaPCR1,SaPCR2)from the hyperaccumulating ecotype(HE)of S.alfredii,and analyzed their sequence as well as the transport function of heavy metals.We found that SaPCRl,SaPCR2 had the closest genetic relationship with ARPCR1,ARPCR2,and ARPCR3 by phylogenic tree alignment.In addition,SaPCR1 and SaPCR2 both contained a CCXXXXCPC structure,which is a cysteine rich domain highly conserved in PCR family and shown to play a important role in Cd detoxification in plants.Relative quantitative results showed that the expression of SaPCR1 and SaPCR2 genes in the HE S.alfredii was significantly higher than those in its non-hyperaccumulate ecotype(NHE).Moreover,SaPCR1 is expressed in all parts with the highest expression in the root,followed by the stem and leaves.SaPCR2 was almost exclusively expressed in roots of the HE S.alfredii.These implied that these two genes,especially SaPCR2,might play an important role in roots of the hyperaccumulator.The subcellular localization of tobacco leaves suggested that both SaPCR1 and SaPCR2 were located on the cell membrane.In addition,the expression of SaPCR1 and SaPCR2 protein in Zn/Cd-sensitive yeast ?zrcl significantly increased its tolerance to Cd stress by decreasing the Cd content in yeast.In contrast,no siginicant variation was observed for the yeast under Zn stress with or without expression of SaPCR1/SaPCR2 protein.The gene overexpression of SaPCRl in Arabidopsis thaliana significantly increased the Cd contents in the plant roots when compared with the controls,indicating that the transformation of SaPCR1 increased the transport of Cd into root cells of A.thaliana.On the contrary,overexpression of SaPCR2 in A.thaliana significantly reduced the Cd content in roots compared with that of the wild type,suggesting that the main function of SaPCR2 is to mediate the Cd efflux out of root cells in plants.2.After successful construction of the NHE S.alfredii transgenic system,we further verified the function of SaPCR2 in Cd accumulation and detoxification of S.alfredii.Firstly,we compared three different plant materials(1-2 cm mature stems with one axillary bud,young stem segments,and S.alfredii seeds)for constructing seedlings for the transgenic system,and found that seedlings germinated from seeds are the best choice due to its fast growth,less variation and easy to culture.Considering the variability and distortion in the process of aseptic culture,we chosed the first generation of seed-germinated seedlings as material for transgenic experiments.The tissue culture system of non-hyperaccumulate S.alfredii was established,with the optimum parameters for the culture medium and hormone ratio of the transgenic system as follows,(1)Callus induction medium:MS+0.5 mg/L 6-BA+1.0 mg/L 2,4-D;(2)Callus differentiation medium:MS+0.5 mg/L 6-BA+0.1 mg/L NAA;(3)Rooting medium:1/2 MS.Afterwords,we successfully obtained the overexpression strains and analyzed the effects of SaPCR2 overexpression on Cd accumulation and detoxification in the NHE S.alfredii.The results showed that the Cd content in roots of the transgenic NHE S.alfredii plants was significantly lower than that of its wild types,while no such effects were observed in either stems or leaves,suggesting that overexpression of SaPCR2 in NHE S.alfredii may play a important role in promoting root Cd efflux in the transgenic lines.Taken together,the results of this study suggest that the high expression of SaPCR2 in roots of the hyperaccumulator HE S.alfredii plays important role in the efflux process of Cd out of the root cells,and thereby alleviates the Cd biotoxicity in root cells to improved the plant tolerance of HE S.alfredii under Cd stress.This study also provide a useful experience for the subsequent construction of transgenic system for the HE S.alfredii.