Study On The Function Of Iron Transporter MbNramp1 Gene

Iron nutrition is extremely important not only for human health,but also for plant growth and development.The content of iron is second only to the content of oxygen,silicon and aluminum,ranking fourth in the earth's crust.However,the bioavailability of iron is very low,especially in calcareous soil,most of the iron exists in the form of Fe(OH)₃,which cannot be directly absorbed by plants.Iron deficiency in crops generally occurs,which affects human intake of iron nutrition.Therefore,improving the iron nutritional status of plants and improving the nutritional quality of crops is of great significance for improving the iron nutritional status of humans.Nramp proteins in plants are widely involved in the transport of divalent metal ions,especially the transport of iron ions.MbNramp1 was cloned from the apple plant in the early stage.The analysis of the gene family phylogenetic tree shows that the gene has the closest relationship with Le Nramp1 in tomato,and the homology is 66%.It was found that the yeast iron nutrition mutant transformed with this gene could restore the mutation.In this thesis,the protein expressed by the MbNramp1 gene is subcellularly localized in the model plant Arabidopsis thaliana,and then the phenotypic characteristics,nutrient absorption and physiology of the homozygous plants transformed with the MbNramp1 gene under iron deficiency conditions are studied.The main test results obtained are as follows:1.Using gene gun technology,the recombinant plasmids pSuper1300-MbNramp1::GFP and pSuper1300-GFP(control)were transformed into onion epidermal cells,and the subcellular localization was observed under a laser confocal microscope.The results showed that only the transformed pSuper1300-MbNramp1::GFP plasmid green fluorescence was emitted on the cytoplasmic membrane of onion epidermal cells,and green fluorescence was observed on the cell membrane and nucleus of onion epidermal cells transformed with the control plasmid.This indicates that the MbNramp1::GFP fusion protein is located on the plasma membrane.2.The recombinant plasmid pSuper1300-MbNramp1 was transformed into Arabidopsis thaliana,and the positive seedlings were screened using MS medium containing hygromycin,and then transferred to nutrient soil for cultivation.A total of 26 T1generation seeds were harvested per plant.Then,the T2 generation seedlings were sown on the medium containing hygromycin,and the ratio of positive seedlings/negative seedlings(ungerminated seeds are not counted)was calculated.The ratio of 3:1 indicates that the target gene has been inserted into the arabidopsis chromosome in a single copy.The positive seedlings with a 3:1 ratio were transplanted into nutrient soil,and T2 seeds were harvested.The T2 seeds were sown on the medium containing hygromycin,and all the positive seedlings were homozygous.A total of 5 homozygotes were harvested.3.After iron deficiency treatment,compared with wild-type arabidopsis(WT,control),the main root length of arabidopsis transformed with MbNramp1 gene increased significantly,and the Arabidopsis transformed with MbNramp1 gene increased by 35.51%compared with the control.There was no obvious change in wild type plants.Under normal iron supply conditions(40?mol/L Fe),there was no significant difference in fresh weight between pSuper1300-MbNramp1 and wild-type arabidopsis.However,under iron-deficient conditions,the fresh weight of the seedlings of all plants was reduced compared with normal iron-supply conditions.The iron deficiency treatment of pSuper1300-MbNramp1 Arabidopsis thaliana was 0.8 times that of normal iron-supply conditions.However,the fresh weight of wild-type Arabidopsis thaliana is only 0.33 times the normal iron supply condition.After 1 month of iron deficiency solution incubation,the dry weight of Arabidopsis transformed into pSuper1300-MbNramp1 was twice that of the control group.The chlorophyll content of the iron-deficient plants was significantly higher than that of the normal iron-treated plants,and the plants transformed with the MbNramp1gene had the largest chlorophyll content,which was more than 1.77 times higher than that of wild-type Arabidopsis,the difference was very significant.Transferred MbNramp1 gene Arabidopsis thaliana root trivalent iron reductase activity was significantly enhanced,indicating that the transformation of MbNramp1 gene causes the plant rhizosphere to secrete a large amount of reductase to alleviate the impact of arabidopsis iron deficiency;4.After 7 days of iron deficiency treatment,wild-type Arabidopsis under normal iron conditions showed no significant difference in reductase activity under normal iron supply compared with transgenic Arabidopsis,all at 10.89?mol Fe(?)h^-1g^-1FW.However,under iron-deficient conditions,the reductase activity of wild-type Arabidopsis thaliana was 21.89 times that of normal iron conditions.The reductase activity of pSuper1300-MbNramp1 Arabidopsis was significantly higher than that of normal iron supply,37.98 times of normal iron conditions,and the reductase activity was 1.58 times of wild-type arabidopsis.After 14 days of iron deficiency treatment,the activity of wild-type Arabidopsis thaliana root trivalent iron reductase was significantly different from that under normal iron supply,the reductase activity increased by 89.68%,and the pSuper1300-MbNramp1 Arabidopsis reductase activity was significantly higher than normal Iron supply treatment is 93.69%higher than normal iron supply conditions.Compared with wild-type Arabidopsis thaliana,transgenic Arabidopsis pSuper1300-MbNramp1 is 1.92 times of it.Comparing the results of 7 days and 14 days,it was found that the wild-type Arabidopsis did not change significantly under normal iron treatment.Under iron-deficient conditions,the reductase activity of the wild-type Arabidopsis on 14 days was reduced compared with the wild-type Arabidopsis on 7 days38.06%,transgenic pSuper1300-MbNramp1 Arabidopsis only decreased by 24.41%.5.After Perl staining,the pSuper1300-MbNramp1 Arabidopsis roots turned darker blue,indicating that after transformation of the MbNramp1 gene,the Fe enrichment in the plant roots increased.Normal iron(40?mol/L Fe)has no significant difference in trace element content(Fe,Mn,Cu,Zn)compared with wild-type Arabidopsis after iron deficiency treatment.Compared with mustard,the contents of iron,manganese,copper and zinc in roots increased significantly,the iron content increased significantly by 51%,the manganese content increased significantly by 33%,the copper content increased significantly by 49%,and the zinc content increased significantly by 37%.