Using Transporter To Enhance Heavy Metal-Uptake Ability In Arabidopsis And Tobacco And Screening AmiRNA Mutant Library For Heavy Metal Related Phenotype

With the increase of mining activity and consumption of rare metals,heavy metal contamination has become a serious problem which is also threatening human health.Heavy metals can be finally accumulated in organisms through food chain,and affect the normal metabolic activity of plants,animals and humans.Therefore,understanding the uptake,transportation,restoration and detoxification mechanisms in plants,and identification of related genes and use them to geneticly modify the plants would help to increase the efficiency of soil phyto-remediation.In the first part of this research,we have over-expressed yeast metal transporters SpAbc2,ScYCF1 and bacteria glutathione synthetase(ECs)or co-expressed two of the above genes to increase the metal-sequstretion ability in Arabidopsis and tobacco.Heterogeneous expression of heavy metal compound transporting related genes is aimed to avoid gene silencing when over-expressing their own genes.We used Atomic Absorption Spectroscopy(AAS)to measure Cd contents in shoot and root after treated with Cd.The result shows that Cd contents of shoot and root in Arabidopsis SpAbc2 transgenic plants are significantly higher than that in Col-0.And the Cd content of shoot in ECs or SLIM1 expression lines are significantly higher than that in WT tobacco.But Cd content in the transgenic lines expressing SpAbc2 and ScYCF1,SpAbc2 and ECs has no difference with that in WT neither in Arabidopsis nor in tobacco.Some unknown feedback inhibition regulation may lead to the result that the effect of dual-genes expression is weaker than single gene.The second part of this research focused on screening mutants with heavy metal sensitive or tolerant phenotypes.There are many functional redundancy genes in plant genome,which makes it hard to identify phenotype in single-gene-mutant.So a genome-wide Arabidopsis amiRNA mutant libraries targeting many redundant genes are used to screen As(Ⅲ),Cd tolerant or sensitive mutant.And PCR-amplification,DNA sequencing are used to identify targeted genes to find heavy metal related transporters,transcription factors or kinases.And two transporter-related mutants have been confirmed,which showed tolerance to heavy metal.Mutant 10-18-2 is more tolerate to As(Ⅲ)than Col-0 and its amiRNA targets to four genes which belong to MATE family.Mutant10-45-2 is more tolerate to Cd than Col-0 and its amiRNA targets to four genes which belong to MFS and Mito families.And we also isolated some mutants targeting transcription factors.And this research provides powerful materials and basis in finding heavy metal transporting and tolerant genes.