| Heavy mental copper is a necessary trace element in plant.Toxicity stress will appear in plant when it absorbs and accumulates excess copper,which will decrease the chlorophyll content,inhibit the growth of root,induce oxidative stress and finally damage the normal growth of plant.As a kind of sulfur amino acids,methione(Met)can easily be oxidized to methionine sulfoxide(MetSO),effecting the normal process of cell life activities.MetSO can be reduced back to Met by methionine sulfoxide reductase(MSR),while the protein activity can be recovered.MSR exists in free-type and dabsyl-type in plant cell.Proteins of this family can be divided into Methionine sulfoxide reductase A(MSRA)and Methionine sulfoxide reductase B(MSRB).MSRA can specifically reduce Met-S-SO,While MSRB reduces Met-R-SO.Nine methionine sulfoxide reductases have been found in rice,while 5 of them belong to MSRA and the other 4 belong to MSRB.However,physiological function of MSR under heavy mental stress remains unclear.In this thesis,firstly cupric sulfate(CuSO4)and methyl viologen(MV)of gradient concentration were used to treat wild type rice(Dongjin)respectively.The result showed that CuSO4 of 20 μM and MV of 1 μM can obviously inhibit the normal growth of rice,decrease the chlorophyll content in rice leaves and excess copper will increase the permeability of root tip plasma membrane observably.Secondly,with transient expression,subcelluar localization of OsMSRA2 and OsMSRB3 were analysed in the thesis.The result of confocal laser scanning demonstrated that OsMSRA2 is located in cytoplasm and OsMSRA2 is located in plastid.Since the TDNA knock-out rice mutant osmsra2、osmsrb3 and osmsra4 have been prepared,the OsMSRA4.1 knock-out rice mutant through Crispr-Cas9 was constructed.In normal growth conditions,the mutants grew more slowly and had lighter thousand-grain weight compared with the wild-type.After treating rice with copper,the mutant rice got more obvious injury of plasma membrane in root tips and more significant accumulation of H2O2 in leaves.The activities of antioxidative enzymest increased as well.Meanwhile,osmsra2 showed the most serious injury of plasma membrane in root-tips.The concentration of copper in root was higher in osmsra2 and osmsrab3 than in wild type.Measurement of total-MSR activity in rice leaves showed that MSR activity of rice was lower in the mutant than in the wild-type.And the MSR reductive activity to Met-R-SO in osmsrab3 was lower than other rice materials,which was probably related to that MSRB reduces Met-R-SO specifically.Moreover,to verify the influence of MSR Cys-active site to its antioxidant capacity,the experiment replace the 159st and 212st Cys-active site bases in OsMSRB3 CDS and set prokaryotic expression of OsMSRB3 and its activity site mutation protein(OsMSRB3C159,C212).In experiment of tolerance observation of Escherichia Coli,the OsMSRB3C159,C212 showed higher growth activity than the negative control,but lower growth activity compared with Escherichia Coli expressing OsMSRB3 under 1.0mM Cu.This result confirmed the function of the Cys-active site in OsMSRB3.Meanwhile,the result also showed the possibility that there is other method of OsMSRB3 to play its role against oxidative stress.The conjecture needs more verification experiment.Finally,for further research on the mechanism of OsMSR under Cu stress,the experiment found an interact protein of OsMSRA2.1 from cytoplasmic protein library through yeast two hybrid system,OsMYBAS1-3.The interact protein was cloned and constructed into vector to hybrid with OsMSRA2.1,and the hybridization result showed positive.Subcelluar localization in onion cell demonstrated that OsMYBAS1-3 is located in nucleus,which coincided with its feature as a transcription factor.Meanwhile,Cu or MV can cause upregulation of OsMYBAS1-3 of transcriptional level in wild-type rice.Succeeding research is expected to verify the function of this interact protein and the mechanism of its interaction with OsMSRA2.1. |
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