| Cadmium(Cd)is a kind of non-essential heavy metal element for natural organisms and is harmful to plants and animals.However,the cultivated soil in China is seriously polluted by Cadmium,which accounts for 16.67%of the total area of cultivated land in China,accounting for about 1/6 of the total area of cultivated land in China.Therefore,it is urgent to control the soil Cd pollution.Phytoremediation is considered to be an effective and economic method.Due to the small biomass of super-enriched plants and their difficulty in survival,the application of high-biomass plants to the treatment of soil heavy metal pollution has attracted widespread attention.As a food crop,biomass Sorghum(Sorghum Dochna(Forssk)Snowden)can be used in biofuel production.Compared with other Sorghum varieties,biomass Sorghum has the characteristics of high stem,stem diameter and high biomass.Inositol 1-phosphate synthase(MIPS),the rate-limiting enzyme for inositol synthesis,catalyzes the conversion of glucose-6-phosphate to L-inositol 1-phosphate,which is then catalyzed by inositol monophosphatase(IMP)to inositol.Inositol and its derivatives play an important role in the growth and development of organisms and in the process of stress resistance.In this study,the physiological and biochemical responses of biomass sorghum to cadmium stress,gene cloning,prokaryotic expression and qRT-PCR were used to explore the physiological response and molecular mechanism of biomass sorghum to cadmium stress.The main findings are as follows:(1)The hydroponic experiments on biomass sorghum seedlings with different concentrations of Cd stress showed that biomass sorghum seedlings showed high tolerance to Cd concentration lower than 30μmol/L,and Cd had obvious toxic effect on biomass sorghum seedlings when the concentration was higher than that.The five antioxidant enzymes in plants,superoxide dismutase(SOD),peroxidase(POD),catalase(CAT),ascorbate peroxidase(APX)and glutathione reductase(GR),responded positively to Cd stress,but the activity of antioxidant enzymes decreased significantly under high Cd stress.(2)Screening and gene cloning full-length gene and bioinformatics analysis to get:the 1533bp SoMIPS2 gene sequences,encoding 510 amino acids,the protein of transmembrane protein,belonging to members of the family of MFS super,the theory of molecular weight of 53.91 kD,theory of isoelectric point 8.78,to cluster analysis showed that the amino acid sequence homology with the sweet sorghum and maize.(3)After sequencing pUCm-T-SoMIPS2 recombinant plasmid by Hind III and Xho I after double enzyme,connected to the pET-21b expression vector,and then identified and enzyme digestion,PCR constructed prokaryotic expression vector of pET-21b-SoMIPS2.The successfully constructed prokaryotic expression vector was transformed into BL21 expressing host bacteria,and the inducer IPTG was used to induce the expression.It was found that a large amount of fusion protein pET-21b-SoMIPS2 was induced at 25℃ and 100mM IPTG for 24 h.It was found that the sample containing the recombinant protein had a high light absorption value at 820nm,which indicated that the recombinant protein had the ability to synthesize L-inositol 1-phosphate in vitro.(4)Through the qRT-PCR analyses SoMIPS2 gene expression in different time and the concentration of cadmium stresses difference show that SoMIPS2 genes expressed in different times and different tissues were,the aboveground and underground parts SoMIPS2 gene expression quantity presents ascendant trend,with the increase of the stress concentration at low concentrations under Cd stress increased with the increase of stress time expression,and in 30μmol/L express quantity highest 24 h under cadmium stress.It was preliminarily speculated that the protein might be involved in the detoxification process of cadmium in biomass sorghum as an early response protein to cadmium stress. |
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