CN108018307B - Application of AtNIA1 gene in improving heavy metal pollution resistance and oxidation resistance of Hangzhou white chrysanthemum seedlings - Google Patents
Application of AtNIA1 gene in improving heavy metal pollution resistance and oxidation resistance of Hangzhou white chrysanthemum seedlings Download PDFInfo
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Abstract
The invention providesAtNIA1The gene is applied to improving the heavy metal pollution resistance and the antioxidant activity of Hangzhou white chrysanthemum seedlings. After heavy metal treatment for 20 days, the treatment is carried outAtNIA1The non-enzymatic antioxidant capacity of the Hangzhou white chrysanthemum seedling leaves is remarkably increased: DPPH free radical clearance rate, OH free radical clearance capacity and total reducing power are respectively improved by 104.8%, 87.5% and 114.3% compared with wild chrysanthemum morifolium ramat seedlings; the enzymatic antioxidant activity is also obviously improved: SOD activity, CAT activity, POD activity and GPX activity are respectively increased by 87.4%, 102.4%, 78.0% and 97.4% compared with wild chrysanthemum morifolium ramat seedlings; rotating shaftAtNIA1The damage rate of the cell membrane of the young leaves of the Hangzhou white chrysanthemum seedlings is reduced by 57.3 percent compared with that of the wild Hangzhou white chrysanthemum seedlings, and the cell activity is improved by 44.5 percent; rotating shaftAtNIA1The weight of a single plant of the gene Hangzhou white chrysanthemum seedling is increased by 21.3 percent compared with that of a wild Hangzhou white chrysanthemum seedling.
Description
Technical Field
The invention relates toAtNIA1The gene is applied to improving the heavy metal pollution resistance and the antioxidant activity of Hangzhou white chrysanthemum seedlings.
Background
In recent years, with the rapid development of industrialization, heavy metal pollution is increasingly serious. Research reports show that heavy metal pollution represented by cadmium, lead, mercury and the like becomes one of the main environmental stress factors in plant growth. The heavy metal pollution stress has general toxic action on plants, not only influences the growth and development, yield and quality of the plants, but also limits the cultivation and popularization of the plants. The over-high content of heavy metals such as cadmium, lead and the like in soil becomes one of important factors for restricting the growth and yield of plants in many areas of China. Because the heavy metal property is very stable, once the soil is polluted by the heavy metal, the soil is difficult to remove in a short time, so that the soil becomes a persistent pollutant, and the condition that the environment such as the soil is polluted by the heavy metal becomes more and more severe along with human activities such as the global industrialization process and the like. Therefore, improving the resistance of crops to heavy metals plays an increasingly important role in maintaining high and stable agricultural yield and sustainable development of agriculture.
Research reports show that when plants are subjected to abiotic stress such as heavy metal and the like, a large number of reactive oxygen Radicals (ROS) are generated in cells, so that oxidative stress, membrane lipid peroxidation and membrane protein polymerization are caused, the permeability of a plasma membrane is increased, and the normal structure of the membrane is damaged, so that the activity of the plant cells is reduced, and finally toxic symptoms are caused. During the long-term evolution of plants, a complex antioxidant protection system is formed to eliminate or reduce the damage of ROS, and comprises a non-enzymatic protection system and an enzymatic protection system. The non-enzymatic antioxidant protection system mainly improves the antioxidant capacity of plants by synthesizing some metabolites with reducing capacity, such as flavone, polyphenol secondary metabolites, reducing glutathione, carotenoid, ascorbic acid (AsA) and the like; enzymatic antioxidant protection systems include superoxide dismutase (SOD), Catalase (CAT), Peroxidase (POD), Ascorbate Peroxidase (APN), and the like. Researches show that plants with higher non-enzymatic protective capability and enzymatic protective capability often have stronger capability of resisting heavy metal pollution. Therefore, in previous studies, attempts have been widely made to improve the resistance of plants to heavy metals by introducing various key genes of enzymatic antioxidant protection systems and non-enzymatic antioxidant protection systems into plants by genetic transformation. For example, SOD is the first key antioxidant enzyme to scavenge ROS, and can scavenge superoxide radicals and their derivatives produced by stressed cells. Researches show that the tolerance of a transformed plant to the stress of heavy metals such as cadmium is enhanced by transforming the cDNA of the Mn-SOD of the tobacco into the tobacco; tomato thylakoid ascorbate peroxidase gene Let-1P5 was also over-expressed in tomato by researchers, and the results indicated that: under the lead stress condition, the APN activity, GSH content, chlorophyll content, net photosynthetic rate and maximum photochemical efficiency of the transgenic tomato are improved, the hydrogen peroxide content, ion leakage rate and MDA content are reduced, and the over-expression of Let-1P1 relieves the photoinhibition of cadmium to the transgenic plant and improves the heavy metal resistance of the transgenic plant; the turnip DIDK-1R gene is overexpressed in arabidopsis, so that the contents of ASA, GSH and chlorophyll of transgenic arabidopsis are increased, the content of MDA is reduced, and the aluminum resistance of transgenic plants is improved.
The research results show that the transgenic technology is utilized to over-express various enzymatic and non-enzymatic antioxidant factors in plants, so that the oxidative stress damage of heavy metals to plant cells can be reduced, and the heavy metal resistance of the plants can be improved. However, since the development of heavy metal resistance in plants is a result of the combined action of a plurality of enzymatic and non-enzymatic antioxidant factors, most of the current researches use transgenic technology to introduce one or two factors into the plants, although the resistance of the plants can also be improved, the action effect is often limited because the enzymatic and non-enzymatic cold-resistant protection systems in the plants cannot be comprehensively activated.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to design and provideAtNIA1Genes or containingAtNIA1The technical scheme of the application of the gene recombinant vector in improving the heavy metal pollution resistance and the antioxidant activity of Hangzhou white chrysanthemum seedlings. The invention utilizes transgenic technology toAtNIA1Gene transfer into recipient plants, useAtNIA1The gene activates various enzymatic and non-enzymatic antioxidant factors in plants, thereby improving the heavy metal resistance of the plants.
The technical scheme adopted by the invention is as follows:
saidAtNIA1Genes or containingAtNIA1The recombinant vector of the gene is applied to improving the heavy metal pollution resistance and the antioxidant activity of Hangzhou white chrysanthemum seedlings.
The use of which is characterized in thatAtNIA1Genes or containingAtNIA1The recombinant vector of the gene is transferred into Hangzhou white chrysanthemum cells to obtain the transfer with both improved heavy metal pollution resistance and oxidation resistanceAtNIA1Gene hangAnd (5) white chrysanthemum.
The method for improving the heavy metal pollution resistance and the antioxidant activity of the chrysanthemum morifolium seedlings is characterized in thatAtNIA1Genes or containingAtNIA1The recombinant vector of the gene is transferred into Hangzhou white chrysanthemum cells and stably expressed.
The method for improving the heavy metal pollution resistance and the antioxidant activity of the chrysanthemum morifolium seedlings specifically comprises the following steps:AtNIA1constructing a gene expression vector by DNA recombinationAtNIA1(not limited to onlyAtNIA1And also the NIA1 gene sequence in other plants) to an expression vector plasmid having 35S promoter (not limited to the expression vector plasmid of 35S promoter but also other expression vector plasmids), and constructingAtNIA1A gene recombinant plasmid; the method of infecting with Agrobacterium EHA105 (not limited to EHA105, but also includes other Agrobacterium which can mediate plant gene transfer)AtNIA1The gene is transferred into Hangzhou white chrysanthemum cell and is determined by antibiotic screening, PCR and RT-PCR detectionAtNIA1The gene can be stably expressed in Hangzhou white chrysanthemum, and further stable expression can be obtained by tissue culture regeneration technologyAtNIA1Genetically modified chrysanthemum morifolium ramat.
Preferred cell culture conditions are: MS minimal medium, pH 5.8, 0.25 mg/L NAA, 0.35 mg/L6-BA, 0.20 mg/L2, 4-D, 30 g/L sucrose. Culturing at 28 + -2 deg.C in dark.
The preferable regeneration conditions of the transgenic chrysanthemum morifolium ramat plants are as follows: 1/2MS minimal medium + 1.5 mg/L NAA induces for 5 days, then transfers to 1/2MS minimal medium + 0.5 mg/L BA to continue culturing for a week, induces adventitious buds, transfers to 1/2MS minimal medium + 0.15 mg/L IBA to continue culturing for a week, induces adventitious roots, and moves to hormone-free fresh MS medium for expanding propagation when the plantlet grows to 5 cm.
The invention has the beneficial effects that: providing a stable rotationAtNIA1Hangzhou white chrysanthemum plant. The experimental result shows that the over-expression is carried out in Hangzhou white chrysanthemum seedlingsAtNIA1The gene can obviously improve the enzymatic and non-enzymatic antioxidant activity of the chrysanthemum morifolium ramat seedlings, reduce the oxidative damage of heavy metal pollution to the seedlings and improve the heavy metal pollution resistance of the chrysanthemum morifolium ramat seedlings, and the heavy metal (I) is added in500 mg/Kg lead + 5 mg/Kg cadmium) for 20 days, and then the treatment is carried outAtNIA1The DPPH clearance rate of Hangzhou white chrysanthemum seedling leaves and the OH free radical clearance capacity and the total reducing power are respectively improved by 104.8 percent, 87.5 percent and 114.3 percent compared with the non-transgenic Hangzhou white chrysanthemum; SOD activity, CAT activity, POD activity and GPX activity are respectively increased by 87.4%, 102.4%, 78.0% and 97.4% compared with wild chrysanthemum morifolium ramat seedlings; rotating shaftAtNIA1Compared with non-transgenic chrysanthemum morifolium ramat, the damage rate of the chrysanthemum morifolium ramat seedling young leaf cell membranes is reduced by 57.3%, and the cell activity is improved by 44.5%; rotating shaftAtNIA1The weight of a single plant of the gene Hangzhou white chrysanthemum seedling is increased by 21.3 percent compared with that of a wild Hangzhou white chrysanthemum seedling. The method has important application significance for improving the heavy metal pollution resistance and the antioxidant activity of the chrysanthemum morifolium seedlings.
Drawings
FIG. 1 shows transgenic Hangzhou white chrysanthemum seedlingsAtNIA1Detecting the expression result by RT-PCR;
FIG. 2 shows the downturn of heavy metal treatmentAtNIA1Comparing the DPPH clearance rate, OH free radical clearance capacity and total reducing power of Hangzhou white chrysanthemum seedlings and Hangzhou white chrysanthemum wild seedlings;
FIG. 3 shows the downturn of heavy metal treatmentAtNIA1Comparing the activities of SOD, CAT, GPX and CPX in the Hangzhou white chrysanthemum seedlings and Hangzhou white chrysanthemum wild seedlings;
FIG. 4 shows the downturn of heavy metal treatmentAtNIA1Comparing the cell permeability and cell activity of the gene Hangzhou white chrysanthemum seedling and Hangzhou white chrysanthemum wild type seedling;
FIG. 5 shows the downturn of heavy metal treatmentAtNIA1Comparing the weight of each plant of the gene Hangzhou white chrysanthemum seedlings with that of wild Hangzhou white chrysanthemum seedlings.
Detailed Description
The invention is further described below with reference to specific embodiments, but the scope of protection of the invention is not limited thereto:
example 1: rotating shaftAtNIA1Gene chrysanthemum morifolium ramat construction
（1）AtNIA1Gene expression in public databases such as GeneBankAtNIA1Amplification from Arabidopsis thaliana with Gene sequences as templatesAtNIA1Gene or consignment professional bioengineering technology company (Shanghai Bioengineering services Co., Ltd.)Synthesis ofAtNIA1The gene(s) is (are),AtNIA1the nucleotide sequence of the gene is shown in SEQ ID NO. 1.
(2) To be provided withEcoRⅠ+BamH I enzyme digestionAtNIA1The gene, the recovered restriction fragment and plasmid pBIN19 were processedEcoRⅠ+BamThe large fragment after the H I cleavage was ligated at 16 ℃ overnight to give the novel plasmid pBIN19-AtNIA1。
The reaction system is as follows:
TABLE 1 pBIN19,AtNIA1Ligation reaction System
(3) Plasmid pCHSHindⅢ+XbaRecovering the small fragment containing the CaMV 35S promoter after the I enzyme digestion, and the plasmid pBIN19- AtNIA1Warp beamHindⅢ + XbaThe large fragment after the I enzyme digestion is connected overnight at 16 ℃ to obtain a new plasmid pBIN-35S-AtNIA1。
（4）pBIN-35S- AtNIA1Agrobacterium EHA105 was introduced. The preparation of the agrobacterium competence and the extraction of the plasmid are carried out according to the molecular cloning experimental instruction. The pBIN-35S- AtNIA1Agrobacterium EHA105 was introduced. The operation method comprises the following steps: add about 0.1. mu.g of purified plasmid DNA into 100. mu.L of EHA105 competent cells, mix well, stand on ice at 0 ℃ for 10 min, place in liquid nitrogen and quick-freeze for 5 min, immediately heat shock with 28 ℃ water bath for 5 min, add 500. mu.L of LB liquid medium, shake culture at 28 ℃ for 2 h. 100 μ L of the bacterial suspension was applied to LB + 50 mg/L Km (kanamycin) + 50 mg/L Str (streptomycin) solid medium, and cultured at 28 ℃ for about 48 hours to select resistant colonies.
（5）Agrobacterium EHA105 mediated genetic transformation
EHA105/pBIN-35S- AtNIA1The resistant bacteria are cultured in LB + 50 mg/L Km + 50 mg/L Str liquid culture medium at 28 ℃ and 200 r/min overnight, 5 mL of bacterial liquid is transferred into 50 mL of MS + 50 mg/L Km + 50 mg/L Str liquid culture medium at 28 ℃ and 200 r/min until the resistant bacteria are culturedOD 600Centrifuging at 5000 r/min and 4 deg.C for 10 min to obtain thallus, and collecting thallusAfter the MS culture medium is cleaned for 3 times, the bacterial liquid is diluted by 10 times by using the MS and acetosyringone 20 mg/L culture medium to be used as an infection liquid.
Taking 0.2-0.4 g of chrysanthemum morifolium leaf callus, putting the chrysanthemum morifolium leaf callus into an invasive dye solution for 10 min, taking out the callus, sucking dry bacterial liquid by using sterile filter paper, putting the filter paper into an MS culture medium for co-culture for 3 d, transferring the culture medium into the MS culture medium with 500 mg/L Cef (cefamycin) + 50 mg/L Km culture medium for culture, and screening to obtain resistant cells; meanwhile, non-infected chrysanthemum morifolium ramat leaf callus is cultured on the same culture medium as a negative control.
(6) Rotating shaftAtNIA1PCR identification of gene chrysanthemum morifolium ramat cell
To be provided withAtNIA1And (3) performing PCR amplification on the positive cells screened by the antibiotics by using the DNA as a template, wherein the cells with positive electrophoresis results of amplification products are transgenic cells, and the cells with negative electrophoresis results are negative cells.
(7) Rotating shaftAtNIA1Gene chrysanthemum morifolium ramat plant regeneration
Will obtain a rotorAtNIA1The gene chrysanthemum morifolium ramat positive cells are inoculated into an MS basic culture medium (pH 5.8) + 0.25 mg/L NAA + 0.35 mg/L6-BA + 0.20 mg/L2, 4-D + 30 g/L sucrose, dark culture is carried out for 20 days at 28 +/-2 ℃, then the cells are transferred into 1/2MS basic culture medium + 1.5 mg/L NAA for induction for 5 days, then the cells are transferred into 1/2MS basic culture medium + 0.5 mg/L BA for continuous culture for one week, adventitious buds are induced, then the cells are transferred into 1/2MS basic culture medium + 0.15 mg/L IBA for continuous culture for one week for inducing adventitious roots, and when the seedlings grow to 5 cm, the cells are transferred into a hormone-free fresh MS culture medium for amplification propagation.
(8) Transgenic chrysanthemum morifolium ramat plantAtNIA1Gene expression level detection
RT-PCR technology is adopted to transfer the transgenic chrysanthemum morifolium ramat plantsAtNIA1The expression level of the gene is detected, and the result shows that compared with wild chrysanthemum morifolium ramat, the transgenic chrysanthemum morifolium ramat plantAtNIA1The gene can be stably expressed at a high level (FIG. 1).
Example 2:AtNIA1inhibition effect of gene on heavy metal stress damage of chrysanthemum morifolium ramat seedlings and improvement effect of seedling growth
(1) Subjecting the thus obtained rotor toAtNIA1Gene chrysanthemum morifolium ramatExpanding and propagating the seedlings, putting the seedlings into a closed bottle at room temperature for hardening after 20 days, opening a bottle cover for hardening after 3 days, opening a culture container after 2 days, putting the seedlings under indoor natural illumination for 3 days, taking out the seedlings, washing the nutrient medium on the root system by using tap water, and then planting the seedlings into a prepared medium which is disinfected according to the following method before use. And (3) properly shading before transplanting, and keeping high air humidity (about 98 percent of relative humidity).
The matrix proportion is as follows: river sand: humus soil: vermiculite = 1: 1: 1.
the substrate disinfection method comprises the following steps: packaging the matrix tightly, placing into a sterilizing pot, and sterilizing at 121 deg.C for 45 min.
Rotating shaftAtNIA1After the genetic chrysanthemum morifolium ramat seedlings grow in the matrix for 30 days, the seedlings with consistent growth are selected and transplanted into the matrix containing heavy metals (500 mg/Kg of lead and 5 mg/Kg of cadmium) to continue growing (heavy metal pollution treatment).
(2) Taking chrysanthemum morifolium ramat callus cells which are not subjected to genetic transformation as a material, culturing and regenerating according to the method to obtain chrysanthemum morifolium ramat wild type regeneration seedlings, and culturing according to the culture conditions which are completely the same as those in the step (1).
(3) By growing in heavy metal contaminated matrix for different periods of timeAtNIA1Respectively measuring DPPH clearance, OH free radical clearance, total reducing power, SOD, CAT, POD and GPX activity, cell ion permeability and cell activity of first unfolded leaves of gene Hangzhou white chrysanthemum seedlings and Hangzhou white chrysanthemum wild type regenerated seedlings, and examining heavy metal pollution stressAtNIA1The dynamic change curve of cell damage under the stress of comprehensive antioxidant activity and heavy metal pollution of Hangzhou white chrysanthemum seedlings and Hangzhou white chrysanthemum wild type regenerated seedlings is obtained. The results show that compared with wild chrysanthemum morifolium ramat seedlings, the chrysanthemum morifolium ramat seedlings are subjected to heavy metal pollution stressAtNIA1The enzymatic and non-enzymatic antioxidant capacity of the gene chrysanthemum morifolium ramat seedlings is obviously improved. After 20 days of heavy metal treatment, the mixture is turned intoAtNIA1The gene Hangzhou white chrysanthemum seedling young leaf DPPH clearance rate, OH free radical clearance capacity, total reducing power, SOD activity, CAT activity, POD activity and GPX activity are respectively improved by 104.8%, 87.5%, 114.3%, 87.4% compared with wild Hangzhou white chrysanthemum seedling,102.4%, 78%, and 97.4% (fig. 2, fig. 3); to turn intoAtNIA1Compared with the wild chrysanthemum morifolium ramat seedling, the gene chrysanthemum morifolium ramat seedling young leaf cell ion permeability is reduced by 57.3%, and the cell activity is improved by 44.5%. (FIG. 4).
(4) The experimental result shows that under the stress of heavy metal pollutionAtNIA1The gene can comprehensively improve the enzymatic and non-enzymatic oxidation resistance of Hangzhou white chrysanthemum seedlings, reduce the heavy metal stress damage of the seedlings and improve the heavy metal pollution resistance of the Hangzhou white chrysanthemum seedlings.
(5) To rotateAtNIA1Gene chrysanthemum morifolium ramat seedling and wild chrysanthemum morifolium ramat seedling are used as materials, the materials are cultured according to the methods (1) to (3), the weight of each seedling is measured by sampling after the seedlings grow for 20 days in a heavy metal pollution matrix, and the result is shown in figure 5. The experimental result shows that after being treated by heavy metal for 20 days, the heavy metal is converted into the metalAtNIA1The weight of a single plant of the gene chrysanthemum morifolium ramat seedling is increased by 21.3 percent compared with that of a wild chrysanthemum morifolium ramat seedling, which indicates that the overexpression is performed in the chrysanthemum morifolium ramat seedlingAtNIA1The gene can improve the growth of seedlings in heavy metal polluted environment.
Sequence listing
<110> university of teachers in Hangzhou
The invention provides application of AtNIA1 gene in improving heavy metal pollution resistance and antioxidant activity of Hangzhou white chrysanthemum seedlings
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3475
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 1
cccctataaa tacaacctct catttactca ttcagaaacc aaaaaatttc acaaaccaaa 60
ccaaaaccaa aaaaatagct tatctctctc ttatcaccgg ttcaatcact aaaccatggc 120
gacctccgtc gataaccgcc attatcccac catgaacggc gtccgtcacg ccttcaaacc 180
tcctctcgtt ccttctcctc gctcattcga ccgccaccgt caccaaaacc aaactctcga 240
cgtcatcctc accgaaacta aaatcgtcaa agaaaccgaa gtcatcacca ccgtcgtaga 300
cagttacgac gactcctcaa gcgacgacga agacgagagc cataaccgta acgtccctta 360
ctacaaagag ctggttaaaa aatcaaacag cgatttagaa ccgtcgattt tagatccgag 420
agatgaatca acggctgata gttggattca acgtaactca tccatgctcc gtctcaccgg 480
aaaacatcca ttcaacgccg aagcacctct tcctcgtctt atgcaccatg gattcatcac 540
tccagttcca ctccattacg tccgcaacca cggtgcagtt cccaaagcga attggtcaga 600
ctggtcaatc gaaattaccg gactcgttaa acgtccggct aaattcacca tggaagagct 660
aatctccgag ttccccagcc gcgaattccc ggtgacactc gtctgcgccg gtaatcgccg 720
caaggaacag aacatggtga aacaaacgat cggattcaat tggggatccg ccggagtatc 780
tacttctcta tggaaaggta ttccgttaag cgaaatcctc cgtcgatgcg ggatctatag 840
ccggagagga ggcgcgttaa acgtttgctt cgaaggagcg gaggatcttc ccggcggcgg 900
cggatctaaa tacggaacaa gtattaagaa ggagatggcg atggatccgg ctagagatat 960
tatcttagct tatatgcaaa acggcgagct tttaacgccg gatcatgggt ttccggttcg 1020
ggtcattgta ccgggtttca tcggtggtcg gatggttaaa tggttaaaga gaatcatcgt 1080
cacgcctcaa gaatctgaca gttactatca ctataaagac aatcgagtcc ttccttctct 1140
cgttgatgct gagctggcaa attccgaagg taaactatta acttaccctt gtcttctttt 1200
acattttagt taccgatgct aatattattt acttgttttt gttacagctt ggtggtataa 1260
gcctgaatac ataatcaacg agcttaatat aaactcggtg attacaacac ctggtcacgc 1320
agagatttta ccgattaatg cattcactac tcagaagccg tacacattaa aaggctacgc 1380
ttattctggt aatttatttt aactttgctt tagttaaaag ttaaaaggat tctttactat 1440
gtaattaaaa tcctgatcgg tgtattgatg aaataaacag gaggaggtaa gaaggtaacg 1500
agggttgagg tgactctaga cggaggagac acgtggagtg tttgtgagct tgaccaccag 1560
gagaaaccga acaagtacgg taaattctgg tgctggtgtt tctggtcact tgacgttgag 1620
gttcttgatc tgctcagtgc taaagacgtc gcggttcgag cctgggacga gtctttcaat 1680
actcagcctg ataaactcat ctggaacctc atggtaataa tcctctctct tgatattttt 1740
aaattataga attaattagt ttactttatt ctttactata tgatttaaat agtttaatct 1800
tgtttttgag taaactattc gattttgata tttgtattcg tcctacaaag ttggaactac 1860
tgatgatatt ttcttttgaa cgtgatacct accaatacta atcttacgga atcttttaat 1920
agagcactaa tcaacatgga actaaagacc aattctttaa gtgtctctgt tgtacagttc 1980
attttagtag tgcgtttaag tattataatc tcccttcatg cggggcaatt atgtagatta 2040
aaatcgaaat tatataaaat ttacataagt ctaagtctag ggtctccagc taattgttat 2100
ttttttaacg atgttgacta aagcaataac gacgttgact tgtgttaaac aggggatgat 2160
gaacaattgc tggttcagga ttaggaccaa cgtgtgcaag cctcatagag gagagatagg 2220
gatagttttc gaacacccga ccagacccgg aaaccagtcg ggtgggtgga tggcaaagga 2280
gcgtcagctt gagatttcct cagaatccaa caataccttg aagaaatctg tctcatctcc 2340
tttcatgaac actgcctcga agatgtattc aatctctgaa gttagaaaac acaacactgc 2400
tgactctgca tggatcatcg tccacggtca catctatgac tgtacacgtt tcttgaaaga 2460
tcatccagga ggcacagatt ctatcctgat aaacgcaggt acagattgca ccgaagagtt 2520
tgaagccatt cactctgaca aagccaagaa gcttttggaa gattaccgta tcggtgaact 2580
catcaccact ggctacgact cttcccctaa cgtctcagtt catggtgcct caaactttgg 2640
tcctttgtta gctccaatca aagagctaac tcctcaaaag aacattgctt tggtcaaccc 2700
acgtgagaaa atcccggtta ggctcattga gaagacttcg atctcgcacg acgttcgtaa 2760
gttccgattc gcattaccat cagaagatca acagcttggt ttacccgtgg ggaagcacgt 2820
tttcgtttgc gccaacataa acgacaaact ctgtctcaga gcttatactc ccaccagcgc 2880
catcgacgcg gttggtcata tcgacctcgt cgtcaaagtt tacttcaaag acgttcatcc 2940
aaggtttccc aatggtggac tcatgtctca acacttagac tcgttaccaa tcgggtcaat 3000
gatagatatc aaaggtccac tagggcacat cgagtacaaa ggcaaaggca acttcctggt 3060
cagcggcaaa cctaagtttg ccaagaaact agcaatgctc gccggaggaa caggcatcac 3120
tcctatctac cagatcattc aatctatatt gagtgatcca gaggacgaaa ccgagatgta 3180
tgtggtttac gcaaacagaa ccgaggatga cattcttgtg agggaagagc tagaaggatg 3240
ggctagtaag cataaggaga ggctaaagat ttggtacgtc gttgaaatcg caaaggaagg 3300
ttggagttac agtaccgggt ttataactga agctgtgctt agggaacata tccctgaagg 3360
tttggaaggc gaatcgctag cactcgcatg cggaccaccg cctatgattc agtttgcgtt 3420
gcagccaaat ctagagaaga tgggttacaa cgtgaaggag gatctcttaa tcttc 3475
Claims (3)
1.AtNIA1Genes or containingAtNIA1The recombinant vector of the gene is applied to improving the heavy metal lead and cadmium pollution resistance and the antioxidant activity of the chrysanthemum morifolium ramat seedlings,AtNIA1the nucleotide sequence of the gene is shown in SEQ ID NO. 1.
2. Use according to claim 1, characterized in thatAtNIA1Genes or containingAtNIA1The recombinant vector of the gene is transferred into Hangzhou white chrysanthemum cells to obtain the transfer with improved heavy metal lead and cadmium pollution resistance and oxidation resistanceAtNIA1Genetic chrysanthemum morifolium ramat.
3. A method for improving the heavy metal lead and cadmium pollution resistance and the antioxidant activity of Hangzhou white chrysanthemum seedlings is characterized in thatAtNIA1Genes or containingAtNIA1The recombinant vector of the gene is transferred into Hangzhou white chrysanthemum cells and stably expressed,AtNIA1the nucleotide sequence of the gene is shown in SEQ ID NO. 1.
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Citations (2)
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|---|---|---|---|---|
| EP0389457A3 (en) * | 1989-03-20 | 1991-09-25 | Technological Institute Of Iceland | Isolation of a novel thermophilic bacterial species thermus anaerobicus, production and preparation of a novel formate linked nitrate reductase enzyme system from it for analytical and commercial use |
| CN105263965A (en) * | 2013-03-15 | 2016-01-20 | 斯波根生物技术公司 | Fusion proteins and methods for stimulating plant growth, protecting plants, and immobilizing bacillus spores on plants |
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| CN105368844A (en) * | 2015-10-23 | 2016-03-02 | 杭州师范大学 | Application of plant NIA1 gene in increasing content of flavone and content of lactone of ginkgoes |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0389457A3 (en) * | 1989-03-20 | 1991-09-25 | Technological Institute Of Iceland | Isolation of a novel thermophilic bacterial species thermus anaerobicus, production and preparation of a novel formate linked nitrate reductase enzyme system from it for analytical and commercial use |
| CN105263965A (en) * | 2013-03-15 | 2016-01-20 | 斯波根生物技术公司 | Fusion proteins and methods for stimulating plant growth, protecting plants, and immobilizing bacillus spores on plants |
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| Optimisation of nitrate reductase enzyme activity to synthesise silver nanoparticles;Baharen Khodashenas等;《IET Nanobiotechnol》;20160630;第10卷(第3期);第158-161页 * |
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