Tolerance Mechanism Preliminary Study Of Carex Putuoshan Sp. Under Pb-Zn Stress

This study based on the early research of our team in Putuo Mountain Pb-Zn mine of Hanyuan county in Ya’an. taking the Carex putuoshan sp. which a suspected new species in the Carex genus as the research object.Study the heavy metal tolerance mechanism under single and combined Pb-Zn treatment on the impact of Carex putuoshan sp. through the sand culture experiment, including:subcellular distribution of Pb and Zn, antioxidant system response and Chelation. Cloned the conservative fragment of two heavy metal tolerance gene with the method of homology-based cloning of Carex putuoshan sp.named type 2 metallothionein(CpM72) and gamma-glutamylcysteine synthetase(CpGCS); Analyzed the expression of CpMT2 and CpGCS Under the stress of Pb and Zn with the method of real-time flurescent quantitive polymerase chain reaction(QRT-PCR).Provided the reference for the enrichment mechanism of plants of the composite heavy metals soil pollution, enriched the gene bank of hyperaccumulation plants, provided the theory basis for heavy metal contaminated soil in biological engineering management.The main results are summarized as follows:(1) Under the different concentration stress of Pb and Zn, the subcellular distribution of Pb and Zn content in roots were greater than leaves. The Pb distribution in various subcellular components of leaves and roots are presented:The cell wall constituents(F1)>Soluble constituents(F3)>Cell membranes and organelles constituents(F2), and the Zn distribution in various subcellular components of leaves and roots are presented:Soluble constituents(F3)>the cell wall constituents(F1)>Cell membranes and organelles constituents (F2). Under the Pb and Zn composite stress, the higher Pb concentrations is, the higher Pb concentration of subcellular leaves components are. The Zn content of soluble constituents reduced with the concentration of Pb increased, but the Zn content of the cell wall components increased.Different heavy metal ions accumulated in different subcellular components in different ways, and Pb maybe a certain extent inhibited Zn into cells to reduce the toxicity to cells.(2) Under the different concentration stress of Pb and Zn, The Superoxide Dismutase (SOD) activity of C. putuoshan sp. is more higher than Catalase(CAT). Due to the different reactive oxygen removal function, SOD and CAT plays different functions.in C. putuoshan sp. oxidation process. Both glutathione reductase (GR) and Ascorbate peroxidase (APX) activity changes with the increase of the concentration. They presents a trend of going upward then downward, but the change of GR activity was significantly higher than APX. GSH in oxidation process plays a more important role.The change of Ascorbic acid(AsA) is the same as glutathione (GSH). They both induced synthesis under the stress of low concentration, and a large number of consumption under the stress of high concentration, but GSH content changed significantly than AsA content.(3) With the homology cloning method, based on the SorMT2 gene of Solanum nigrum degenerate primers for PCR amplification, received a conservative CpMT2 gene of C. putuoshan sp. fragments, length of 237 bp. BLAST the tmino acid sequence fragments in NCBI, founded that the tmino acid sequence homology with most plants were all above 70%.Based on four GCS amino acid sequence(Registration number in GenBank are: ACK38262.1、CAD48599.3、CAC83005.1、CAC83005.1) designed 12 degenerate primer, using PCR screening out a pair of optimal primers, received the CpGCS conservative fragment, length of 438 bp. BLAST the tmino acid sequence fragments in NCBI, founded that the tmino acid sequence homology with most plants were all above 90%.(4) Used the method of real-time flurescent quantitative polymerase chain reaction (QTR-PCR), established the fluorescence quantitative system of CpMT2 gene, measured the CpMT2 gene expression under different concentration of Pb and Zn stress. Founded that:when the Pb concentration was 600 mg/L, CpMT2 expression reached to the highest,was 3.49 times stronger than the control group; when the Zn concentration was 250 mg/L, CpMT2 expression reached to the lowest, was 0.15 times weaker than control group. When Pb600mg/L and Zn250mg/L stress compared with Pb600mg/L, CpMT2 gene expression decreased by 22.3 times. Pb can significantly promote the expression of CpMT2; the low concentrations of Zn can promote CpMT2 expression, but high concentrations of Zn can significantly inhibit CpMT2 expression.(5) Used the method of real-time flurescent quantitative polymerase chain reaction(QTR-PCR),established the fluorescence quantitative system of CpGCS gene.Founded that:when the Pb concentration was 600 mg/L, CpGCS expression reached to the highest,was 29.5 times stronger than the control group; when the Zn concentration was 250 mg/L, CpGCS expression reached to the lowest,was 0.23 times weaker than the control group; under the stress of Pb600mg/L and Zn250mg/L, CpGCS expression increased 6.53 times compared with Zn concentration of 250 mg/L. Under the single stress of Pb and Zn, the expression of CpGCS increased at first and then decreased with the increase of concentration, under the combined stress, the addition of Pb increased CpGCS expression compared with the single Zn stress.