CN109608528B

CN109608528B - Method for improving heavy metal Cd transport capacity and resistance of plants by transforming Salix matsudana SmZIP protein

Info

Publication number: CN109608528B
Application number: CN201811513859.7A
Authority: CN
Inventors: 邹金华; 薛文秀; 尚晓硕; 李崇豪; 王嘉玥; 刘祥君
Original assignee: Tianjin Normal University
Current assignee: Tianjin Normal University
Priority date: 2018-12-11
Filing date: 2018-12-11
Publication date: 2021-05-25
Anticipated expiration: 2038-12-11
Also published as: CN109608528A

Abstract

The invention discloses a method for improving the transport capacity and resistance of heavy metal Cd by transforming a SmZIP protein of salix matsudana, which transfers the gene into wild tobacco by a genetic engineering technology to ensure that the gene is specifically expressed in plant cells, so that the transport capacity and resistance of the gene to the heavy metal Cd can be obviously improved; the method has wide practicability, and can obtain novel plant varieties with high Cd transferring capacity and resistance.

Description

Method for improving heavy metal Cd transport capacity and resistance of plants by transforming Salix matsudana SmZIP protein

The invention obtains: tianjin City Natural science fund (17JCYBJC 22500); science and technology development project (20110603) of higher schools in Tianjin; the foundation of the doctor fund at Tianjin teacher university and the animal and plant resistance key experiment in Tianjin City are funded by open fund.

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a method for improving the transport capacity and resistance of heavy metal Cd in plants by transforming Salix alba SmZIP protein.

Background

Heavy metal Cd pollutes the ecological environment and human health seriously, and the method for treating heavy metal pollution is divided into physical remediation, chemical remediation and biological remediation, and the phytoremediation technology has become a hot field of research and development at home and abroad due to the characteristics of no damage to the ecological environment of soil, safety, low price, no secondary pollution and the like. To date, over-enriched accumulation plants of more than 450 heavy metals have been found. However, the super-enriched plants which are found at present are mostly herbaceous plants, the biomass of overground parts is small, the heavy metal enrichment and transfer amount is small, the restoration time is long, the efficiency is low, and the restoration effect of the plants is directly limited, so the heavy metal super-enriched plants are generally only used as materials for researching the heavy metal absorption and tolerance mechanism. The enrichment capacity of woody plants on heavy metals is not as good as that of herbaceous super-enrichment accumulation plants, but perennial and high-speed woody plants have more obvious superiority in treating soil polluted by heavy metals due to the characteristics of large biomass, developed root systems, no connection with food chains and the like, and have the potential of becoming phytoremediation candidate plants.

Zinc-iron transport protein (ZIP) is detected in plants, animals, fungi and bacteria, mainly absorbs and transports divalent metal ions such as Fe, Zn, Mn, Cd, Co, Ni and the like, belongs to transmembrane protein, is positioned on cytoplasmic membranes, is also positioned on chloroplast membranes and vacuolar membranes, has an amino acid sequence of 309-476, contains a plurality of hydrophobic groups in the amino acid composition, has 8 transmembrane regions, and transports the divalent metal ions from the outside of the cell to the inside of the cell by the N-end and the C-end.

Zinc-iron transport protein (ZIP) is transformed or overexpressed by a biotechnology, so that the heavy metal transport capacity and resistance of fast-growing woody plants are improved, and the phytoremediation efficiency is improved. Therefore, the invention provides a theoretical basis for plant repair practice.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for improving the transport capacity and resistance of heavy metal Cd in plants by transforming Salix alba SmZIP protein.

The invention is realized by the following technical scheme:

use of a protein in any one of the following a1) -a 2):

a1) the transport capacity and resistance of the heavy metal Cd in plants are improved;

a2) breeding a plant variety with high heavy metal Cd transport capacity and high resistance;

the protein has an amino acid sequence shown in a sequence 1 in a sequence table.

The application of a protein coding gene or a recombinant vector containing the coding gene in any one of a1) -a2) as follows:

the coding gene has a nucleotide sequence shown as a sequence 1 in a sequence table.

The method comprises the following steps:

a) introducing a coding gene of a protein into a target plant to obtain a transgenic plant expressing the coding gene;

b) obtaining a transgenic plant with improved heavy metal Cd transport capacity and resistance compared with the target plant from the transgenic plant obtained in the step a);

the SmZIP protein consists of 350 amino acids, and in the method, the coding gene is introduced into the target plant through a recombinant expression vector containing the coding gene of the protein.

The recombinant expression vector can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like, such as pCAMBIA3301, pCAMBIA2300, pCAMBIA2301, pCAMBIA1300, pCAMBIA1301, pWM101, pGreen0029, pBI121, pBin19, pCAMBIA1301-UBIN and the like or other derivative plant expression vectors.

In the present invention, the recombinant expression vector is specifically as follows:

the DNA fragment shown in the sequence 2 in the sequence table is inserted into a vector pMD18 containing restriction enzyme sites KpnI and BamHI to obtain a recombinant plasmid pMD 18-T-SmZIP.

Wherein, the coding DNA fragment of the sequence 2 is a full sequence of SmZIP gene amplified by a designed primer (SmZIP-F, SmZIP-R) and taking cDNA of salix matsudana as a template, carrying out agarose gel electrophoresis on a PCR amplification product, recovering and purifying a target band, and connecting a T vector, wherein the primer sequence is as follows:

5'-CGGAATTCCCTTCATCTTCAATCCCTTCT-3' in sequence 3 in the sequence table;

5'-CGCGGATCCCTACAGGTTCAAGCCCATTT-3' in sequence 4 in the sequence table;

extracting T vector plasmid containing SmZIP gene sequence by alkaline cracking method, using restriction endonuclease KpnI/BamHI, recovering enzyme digestion product, connecting it with the skeleton fragment of pCAMBIA2300-35S-OCS vector which is subjected to the same double enzyme digestion to construct expression vector pCAMBIA 2300-SmZIP.

In the above method, the introducing the recombinant expression vector carrying the coding gene into the target plant may specifically be: transforming plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, electric shock method/Agrobacterium mediation, etc., and culturing the transformed plant tissues into plants.

In the above applications or methods, the inventive methods are applicable to both dicotyledonous and monocotyledonous plants, and thus the transformed plants can be derived from tobacco, canola, cotton, soybean, poplar, eucalyptus, wheat, rice, corn, alfalfa, and the like.

In the above application or method, the plant is specifically wild type tobacco.

Compared with the prior art, the invention has the beneficial effects that: the gene is transferred into wild tobacco by a gene engineering technology, so that the gene is specifically expressed in plant cells, and the transfer capacity and resistance of the gene to heavy metal Cd can be obviously improved; the method has wide practicability, and can obtain novel plant varieties with high Cd transferring capacity and resistance.

Drawings

FIG. 1 is a diagram of the PCR verification result of the transgenic tobacco genome.

FIG. 2 is a photograph of transgenic tobacco plants grown to flowering stage.

FIG. 3 is a graph of growth of wild type and SmZIP transgenic tobacco leaves on day 28 of Cd stress, wherein SmZIP-ck and WT-ck are leaves of transgenic and wild type tobacco that have not been subjected to Cd stress; SmZIP-1 and WT-1 are transgenic and wild type tobacco and are subjected to 10 mu mol/LCdCl₂Leaves treated with the solution for 28 days; SmZIP-2 and WT-2 are transgenic and wild type tobacco and are subjected to 100 mu mol/LCdCl₂Leaves were treated with the solution at 28 days.

For a person skilled in the art, other relevant figures can be obtained from the above figures without inventive effort.

Detailed Description

In order to make the technical solution of the present invention better understood, the technical solution of the present invention is further described below with reference to specific examples.

The raw materials and various devices used in the invention are conventional commercially available products, and can be directly obtained by market purchase. The sources of the biomaterials used in the examples are as follows: wild type tobacco was kept in the laboratory of the institute of Life sciences, university of Tianjin. Coli (Escherichia coli) DH5 a and Agrobacterium (Agrobacterium) C58 were purchased from Shanghai-Weidi Biotechnology Ltd. In the quantitative experiments in the following examples, three replicates were set up and the results averaged.

The gene of the invention is derived from ZIP gene of Salix matsudana Koidz, the GENBANK number is KU921227, the sequence total length is 1140bp, the invention comprises an open reading frame of 1053bp, 350 amino acids are coded, and the positions of the coding frame are as follows: 71bp-1123bp, 5 'non-coding region 70bp and 3' non-coding region 17 bp. The sequence is (shown as sequence 2 in a sequence table):

CGGATTCCCTTCATCTTCAATCCCTTCTCCTCAACTTTGCAAACAAATTTTCTCTTGATATCATCTCACCATGCAGAGTTCTATCAAATTTTATTTAAAGTTCTTTTGCTTGCTTCTGCTACTCCCTACTCTTGCTTTAGGAGAATGCACATGTGATGCAGCAGGAGGAGAAGACACGAATAAATCTGAGGCCTTGAAATACAAAGCCGCAGCAATTGCTTCTATCCTTTTTGCGGGTGCAGTTGGAGTTTGTATTCCAGTTCTTGGAAAAAAGATCCCTGTTTTAAGCCCTGAAAGGAGTATTTTCTTCATCATCAAAGCTTTTGCGGCCGGTGTTATATTGTCGACAGCCTTTATTCATGTGCTTCCCGATGCTTTTGATAGCTTGACATCGCCATGCCTCGCTGAGAATCCTTGGGGTAAATTTCCCTTCACGGGTTTTGTGGCAATGATGTCGGCAATTGGGACTTTAATGGTGGATTGTCTTGCTAGTTCTTATTTTACACGGTTGCACCTCATCAAGGCTCAACCAGAGGAGAGCGGGGACGAGGAGAAGGCAGCAGGAGAGGCTCATGTTCATACTCATGCCCATTCTCATGGCATAGTTGCGGATAGTTCTGGTTCTGCTCCATCTCCTCAGCTTATTCGCCATCGGGTTATTACTCAGGTTCTCGAGTTGGGAATTGTGGTGCACTCTGTGATTATAGGAGTTTCTCTGGGAGCTTCTTCAAGTCCCAAGACAATAAGACCTCTAGTGGGTGCCCTAAGCTTTCACCAATTTTTTGAGGGTATAGGACTTGGTGGATGCATTACTCAGGCAAAATTCAAGACCAAAACTATGGTGACAATGGGACTCTTCTTCTCTCTAACAACCCCAGTTGGAATTGCAGTCGGGTTAGGCATATCAAATGTCTATAACGAGAGCAGTCCTAATGCTCTTATTGTTGAAGGAATTTTTAATGCCGCATCAGCTGGTATCCTAATCTACATGGCTCTTGTGGATCTTCTGGCAGCTGATTTTATGCATCCAAGAGTGCAAAGTAATGGAGCTCTTCAACTTGGGGTCAACGTTTCTCTTCTTCTAGGAGTTGGCTGTATGTCTCTCATCGCCAAATGGGCTTGAACCTGTAGGGATCCGCG。

the coded amino acid sequence is (shown as sequence 1 in a sequence table):

MQSSIKFYLKFFCLLLLLPTLALGECTCDAAGGEDTNKSEALKYKAAAIASILFAGAVGVCIPVLGKKIPVLSPERSIFFIIKAFAAGVILSTAFIHVLPDAFDSLTSPCLAENPWGKFPFTGFVAMMSAIGTLMVDCLASSYFTRLHLIKAQPEESGDEEKAAGEAHVHTHAHSHGIVADSSGSAPSPQLIRHRVITQVLELGIVVHSVIIGVSLGASSSPKTIRPLVGALSFHQFFEGIGLGGCITQAKFKTKTMVTMGLFFSLTTPVGIAVGLGISNVYNESSPNALIVEGIFNAASAGILIYMALVDLLAADFMHPRVQSNGALQLGVNVSLLLGVGCMSLIAKWA

cloning of SmZIP Gene

(1) Synthesis of first Strand of cDNA of SmZIP Gene

Selecting Salix matsudana Koidz cuttings cultured by the institute of Life sciences of Tianjin university, performing rooting culture for 7 days, taking young roots, performing liquid nitrogen quick freezing, extracting root total RNA by using an EASY spin plant micro RNA rapid extraction kit (Adela, Beijing), and synthesizing a First Strand of cDNA by using the Salix matsudana total RNA as a template by using a Trans Script First-Strand cDNA Synthesis kit (20 mu L system), wherein the reverse transcription system is as follows:

mixing the reactants, and performing PCR at 25 deg.C for 10 min; 30min at 42 ℃; 5min at 85 ℃. The product is used immediately or stored at-20 ℃ for cryopreservation.

(2) Design of primers

The biological software Prime 5.0 is used to design the upstream and downstream primers for amplifying the SmZIP gene:

SmZIP-F:5’-CGGAATTCCCTTCATCTTCAATCCCTTCT-3’；

SmZIP-R:5’-CGCGGATCCCTACAGGTTCAAGCCCATTT-3’；

the full length of the SmZIP gene is amplified by taking the first strand of the Salix matsudana cDNA as a template, and a PCR reaction system is as follows:

the PCR reaction conditions are as follows: 4min at 94 ℃; 30s at 94 ℃, 30s at 56.4 ℃, 1min at 72 ℃ for 20s (34 cycles); 8min at 72 ℃; infinity at 4 ℃. The PCR product was detected by electrophoresis.

(3) Recovery, purification, colony verification and sequencing of SmZIP target gene

And (3) after verifying the PCR amplification product by 1% agarose gel electrophoresis, recovering and purifying by a DNA agarose gel recovery kit, inserting the purified SmZIP gene into a pMD18-T vector, transforming the recombinant plasmid into an escherichia coli competent cell by a heat shock transformation method, selecting a positive colony for colony PCR verification, re-shaking the bacterial strain containing the target band, and sending the bacterial strain to Huada gene sequencing.

Construction of SmZIP Gene plant expression vector

(1) Extracting a T vector plasmid containing a SmZIP gene sequence by using a plasmid extraction kit, carrying out enzyme digestion by using restriction enzymes KpnI and BamHI, and recovering an enzyme digestion product;

(2) the vector pCAMBIA2300-35S-OCS (stored in the laboratory) is cut by restriction enzymes KpnI and BamHI, and the vector skeleton is recovered;

(3) and (3) connecting the enzyme digestion product in the step (1) with the vector framework in the step (2) by using T4-DNA ligase (TaKaRa), wherein the connection system is as follows:

(4) mixing the reaction systems evenly, centrifuging, and connecting overnight at 16 ℃;

(5) and (3) thermally shocking the product to transform the competence of escherichia coli, culturing overnight at 37 ℃, growing a white colony, and carrying out bacterium selection verification, wherein the verification result shows that the correct recombinant plasmid is obtained.

3. Agrobacterium transformation of plant expression vectors C58

(1) Taking out the agrobacterium tumefaciens C58 competent cells from-80 ℃, thawing at low temperature, sucking 5 mu L of expression vector, adding the expression vector into the thawed competent cells, and uniformly mixing by using a micropipette; quickly adding the uniformly mixed solution into a precooled electric shock cup, and placing on ice for 1min (all the operation steps are finished on the ice); setting the voltage of an electric shock converter to be 1500-2000V;

(2) after electric shock, 0.5mL YEP liquid culture medium is immediately added, and after uniform mixing, the mixture is transferred into a 1.5mL EP tube and cultured for more than 3h at 28 ℃ and 180 rpm;

(3) sucking 50 μ L of the above cultured bacterial liquid, uniformly spreading on YEP solid plate (containing 100mg/L Kan) containing antibiotic, and culturing at 28 deg.C for 48-72 hr until clear single colony is observed;

(4) and (5) carrying out colony PCR verification, wherein the positive clone is used for transforming the tobacco plant.

4. Tobacco leaf disc method transformation using agrobacterium mediation

(1) Taking a proper amount of plump wild type tobacco seeds, sterilizing for 1h by using chlorine (20mL of NaClO and 3mL of concentrated HCl), uniformly spraying the seeds on a culture medium A plate, and inoculating about 50 seeds on each plate; sealing the culture dish by using a sealing film, culturing at 26 ℃ for 16h/8h in a photoperiod, germinating seedlings to grow new leaves, transferring the seedlings into a tissue culture bottle of a culture medium A for continuous culture, and culturing for about 3 weeks, wherein the seedlings can be used for infection experiments;

(2) activating the constructed C58 agrobacterium containing a plant expression vector pCAMBIA2300-SmZIP and the C58 agrobacterium containing a pCAMBIA2300-35S-OCS vector, and carrying out streak culture under the culture condition of 28 ℃ for 48-72 h; picking single colony from the plate, inoculating it into 5mL YEP liquid culture medium (containing 100mg/L Kan), culturing at 28 deg.C and 180rpm overnight; respectively inoculating 400 μ L of the above bacterial liquid into 50mL YEP liquid culture medium (containing 100mg/L Kan), culturing at 28 deg.C in shaking table at 180rpm, and culturing to OD₆₀₀Values above about 0.5; respectively transferring the bacterial liquid into a sterilized 50mL centrifuge tube, centrifuging at 4 ℃ and 4000rpm for 10min, and collecting thalli; respectively re-suspending thalli collected at the bottom of the tube by using 1/2MS liquid culture medium, and then adding AS for a subsequent infection experiment;

(3) selecting good-growth sterile tobacco seedling, cutting leaf into about 1cm on sterilized filter paper²The leaf disc is put into the prepared bacterial liquid, and is infected for 20min, and is slightly shaken twice in the period; taking out the leaves, sucking the bacterial liquid on the leaves with sterile filter paper, placing the leaves with the front face facing downwards on the surface of a culture medium B containing the sterile filter paper, sealing the plate with a sealing film, and culturing in the dark at 25 ℃ for 3 days;

(4) when the resistant bud grows to 1.0cm in the tissue culture bottle containing the screening culture medium, the resistant bud can be cut off and transferred into the tissue culture bottle containing the culture medium D to induce rooting, the culture condition is 16h/8h photoperiod, when the root of the tobacco seedling in the tissue culture bottle grows to be more than 3cm and more roots and the seedling grows to be more robust, the bottle cover of the tissue culture bottle is opened, the seedling is hardened in a greenhouse, and the hardening generally requires 2-3 days. Taking out the tobacco seedling from the tissue culture bottle, slightly removing the culture medium at the root by hands, and cleaning the root by clear water without damaging the root and leaves of the tissue culture seedling. Transplanting the tobacco seedling into a small flowerpot taking vermiculite and nutrient soil as substrates, watering, covering with a preservative film, and culturing in a greenhouse. When the seedling grows to a certain stage, the seedling is moved into a large flowerpot to continue growing.

5.T₀DNA level verification of transgenic tobacco

(1) Extracting genome DNA of the transgenic tobacco by using a CTAB method, and simultaneously extracting DNA of a wild tobacco plant as a control to perform PCR identification;

(2) the CTAB method for extracting DNA comprises the following steps: taking a blade, cleaning, placing the blade in a 2mL Ep tube, and placing a small steel ball; adding 600 μ L preheated CTAB extract (Tris-HCl, EDTA, NaCl, CTAB), grinding with grinder; water bath at 65 deg.C for 1h, and gently oscillating every 15 min; adding equal volume of chloroform, shaking vigorously, centrifuging at room temperature at 12,000rpm for 10min, and collecting supernatant; repeating the fourth step; adding isopropanol with the same volume, and precipitating at room temperature for more than 2 h; centrifuging at 12,000rpm for 10min at room temperature; discarding the supernatant, adding 1mL of 75% ethanol, centrifuging, discarding the supernatant, and repeating twice; drying the precipitate at 37 deg.C, storing in dry powder at-80 deg.C or adding 30 μ LddH2O and RNase to dissolve.

(3) Using tobacco genome as a template, respectively screening positive plants for transforming pCAMBIA2300-SmZIP by PCR;

(4) the reaction system is as follows:

the PCR reaction conditions are as follows: 4min at 94 ℃; 30s at 94 ℃, 30s at 56.4 ℃, 1min at 72 ℃ for 20s (30 cycles); 8min at 72 ℃; infinity at 4 ℃.

(5) The PCR product was detected by electrophoresis, and the results are shown in FIG. 1.

6.T₁Screening of tobacco substitute seeds

(1) Continuously culturing the positive plants screened by the PCR in a greenhouse to the flowering phase (figure 2), bagging, self-pollinating until the seeds are mature, collecting the seeds of each positive plant, and fully drying in the sun in an oven to remove impurities and unsaturated seeds;

(2) taking a proper amount of seeds, sterilizing by chlorine, and inoculating the seeds on a plate of an MS culture medium (containing 100mg/L Kan), wherein each dish contains about 50 seeds;

(3) placing the flat plate with the sowed seeds in a tissue culture room for culture under the conditions of 25 ℃ and the illumination intensity of 300 mu mol/(m)²·s)；

(4) After one week of culture, the seeds all start to germinate, after the seedlings grow roots to a certain stage, the plants are transplanted into a small flowerpot and placed in a greenhouse for culture;

(5) after 4-5 weeks of growth, the assay of the subsequent experiment was performed.

7. Detection of Cd content and Cd resistance observation of different organs of transgenic tobacco subjected to Cd stress with different concentrations

(1) Material culture: selecting wild type and SmZIP transgenic tobacco seeds to germinate and grow on a culture medium for about 2 weeks (24h continuous illumination, constant temperature of 25 ℃ and 30-40% humidity), taking out tobacco seedlings from an MS culture medium when the seedlings grow to 5cm, transferring the tobacco seedlings into a flowerpot to grow for about 5 weeks, randomly dividing the seedlings into 3 groups, pouring 1/2 Hoagland's nutrient solution into a control group, pouring 1/2 Hoagland's nutrient solution with Cd concentration of 10 mu mol/L and 100 mu mol/L into a treatment group, and culturing for 28 days while supplementing the solution at any time.

(2) Material taking: collecting fresh root, stem and leaf materials of wild type and SmZIP transgenic tobacco treated with Cd for 14 days, cleaning, oven drying at 80 deg.C for 2 days, oven drying at 90 deg.C for 2 days to constant weight, grinding, and storing.

(3) Weighing: 0.2g of each sample was weighed into a small beaker using a one-ten-thousandth scale.

(4) Nitration: adding 5mL of concentrated nitric acid into a small beaker, soaking the small beaker at room temperature overnight until the powder turns into earthy yellow, then placing the beaker on a temperature-regulating plate at 80 ℃, heating the temperature gradually to volatilize the concentrated nitric acid, adding 5mL of concentrated nitric acid and 2mL of perchloric acid after the concentrated nitric acid is almost completely volatilized, continuing heating, repeating the process until the solution in the beaker is colorless, heating the beaker to 135 ℃ to volatilize the acid completely, and slowly evaporating the acid until colorless crystals are separated out from the beaker.

(5) And (3) volume fixing: and after the whole beaker is cooled, adding 5mL of 10% dilute nitric acid to dissolve the sample sufficiently overnight, fixing the volume to 25mL again the next day, and measuring after standing for 1 day.

(6) And detecting the content of Cd in each sample by an atomic absorption spectrometer, calculating the content of Cd in different organs of wild type and transgenic tobacco after the Cd is treated for 14 days, and calculating the transfer rate of Cd from underground to overground part, wherein the results are shown in Table 1.

TABLE 1 Cd content in organs of wild type and transgenic tobacco after being stressed with Cd at different concentrations for 14 days

(7) Leaves of wild-type and SmZIP transgenic tobacco 28 days after Cd stress were observed and recorded, and the results are shown in FIG. 3.

Results and analysis

1. As shown in table 1, with increasing treatment concentration, the Cd content increased in the roots, stems and leaves of both wild-type and SmZIP transgenic tobacco, and the rate of increase in Cd content was: root > stem > leaf; not subjected to Cd treatment and at 10. mu. mol/L CdCl₂Solution and 100. mu. mol/L CdCl₂After 14 days of treatment in the solution, the underground to overground part transport coefficients of Cd in the wild type tobacco are 0.62, 0.36 and 0.10 respectively. Under the same treatment conditions, the content of Cd in roots, stems and leaves of SmZIP transgenic tobacco is higher than that of wild tobacco, and the underground-to-overground part transport coefficients of Cd in untreated transgenic tobacco and transgenic tobacco treated at different concentrations are 0.60, 0.41 and 0.16 through calculation. Therefore, at 10 and 100. mu. mol/L CdCl₂After the solution is stressed for a long time, the transport capacity of SmZIP transgenic tobacco to Cd is stronger than that of wild tobacco, and SmZIP can promote Cd to be transported to the overground part.

2. As shown in FIG. 3, the growth status of both wild-type and SmZIP transgenic tobacco was affected after 28 days of Cd stress. Comparing the growth states of the leaves of the two tobaccos in different concentration gradients when Cd is stressed for 28 days, and knowing that the shapes of the leaves of the SmZIP transgenic tobaccos are approximately the same along with the increase of the treatment concentration, and the color of the leaves begins to be yellow only after the high-concentration treatment; however, the leaf shape of wild tobacco changed greatly, at 10. mu. mol/L CdCl₂The leaves become long and narrow under the solution treatment, and the concentration of CdCl is 100 mu mol/L₂Solutions ofAfter the treatment, large area of leaves turns yellow, the leaf edges are curled, and a plurality of leaves also have brown spots. Therefore, the growth activity of SmZIP transgenic tobacco plants is stronger than that of wild tobacco under the same Cd stress condition, which shows that the SmZIP gene enhances the Cd tolerance of the tobacco.

In conclusion, experiments prove that the SmZIP protein-containing transgenic tobacco obtained by the method disclosed by the invention has the advantages that under the condition of Cd stress with different concentrations, the Cd content in each organ is increased, and the transfer rate of Cd to the overground part is increased; meanwhile, the transgenic plant shows stronger Cd resistance in the Cd treatment process than the wild plant.

The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.

SEQUENCE LISTING

<110> university of Tianjin

<120> method for improving heavy metal Cd transport capacity and resistance of plants by transforming Salix alba SmZIP protein

<130> 1

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 350

<212> PRT

<213> Salix matsudana

<400> 1

Met Gln Ser Ser Ile Lys Phe Tyr Leu Lys Phe Phe Cys Leu Leu Leu

1 5 10 15

Leu Leu Pro Thr Leu Ala Leu Gly Glu Cys Thr Cys Asp Ala Ala Gly

20 25 30

Gly Glu Asp Thr Asn Lys Ser Glu Ala Leu Lys Tyr Lys Ala Ala Ala

35 40 45

Ile Ala Ser Ile Leu Phe Ala Gly Ala Val Gly Val Cys Ile Pro Val

50 55 60

Leu Gly Lys Lys Ile Pro Val Leu Ser Pro Glu Arg Ser Ile Phe Phe

65 70 75 80

Ile Ile Lys Ala Phe Ala Ala Gly Val Ile Leu Ser Thr Ala Phe Ile

85 90 95

His Val Leu Pro Asp Ala Phe Asp Ser Leu Thr Ser Pro Cys Leu Ala

100 105 110

Glu Asn Pro Trp Gly Lys Phe Pro Phe Thr Gly Phe Val Ala Met Met

115 120 125

Ser Ala Ile Gly Thr Leu Met Val Asp Cys Leu Ala Ser Ser Tyr Phe

130 135 140

Thr Arg Leu His Leu Ile Lys Ala Gln Pro Glu Glu Ser Gly Asp Glu

145 150 155 160

Glu Lys Ala Ala Gly Glu Ala His Val His Thr His Ala His Ser His

165 170 175

Gly Ile Val Ala Asp Ser Ser Gly Ser Ala Pro Ser Pro Gln Leu Ile

180 185 190

Arg His Arg Val Ile Thr Gln Val Leu Glu Leu Gly Ile Val Val His

195 200 205

Ser Val Ile Ile Gly Val Ser Leu Gly Ala Ser Ser Ser Pro Lys Thr

210 215 220

Ile Arg Pro Leu Val Gly Ala Leu Ser Phe His Gln Phe Phe Glu Gly

225 230 235 240

Ile Gly Leu Gly Gly Cys Ile Thr Gln Ala Lys Phe Lys Thr Lys Thr

245 250 255

Met Val Thr Met Gly Leu Phe Phe Ser Leu Thr Thr Pro Val Gly Ile

260 265 270

Ala Val Gly Leu Gly Ile Ser Asn Val Tyr Asn Glu Ser Ser Pro Asn

275 280 285

Ala Leu Ile Val Glu Gly Ile Phe Asn Ala Ala Ser Ala Gly Ile Leu

290 295 300

Ile Tyr Met Ala Leu Val Asp Leu Leu Ala Ala Asp Phe Met His Pro

305 310 315 320

Arg Val Gln Ser Asn Gly Ala Leu Gln Leu Gly Val Asn Val Ser Leu

325 330 335

Leu Leu Gly Val Gly Cys Met Ser Leu Ile Ala Lys Trp Ala

340 345 350

<210> 2

<211> 1140

<212> DNA

<213> Salix matsudana

<400> 2

cggattccct tcatcttcaa tcccttctcc tcaactttgc aaacaaattt tctcttgata 60

tcatctcacc atgcagagtt ctatcaaatt ttatttaaag ttcttttgct tgcttctgct 120

actccctact cttgctttag gagaatgcac atgtgatgca gcaggaggag aagacacgaa 180

taaatctgag gccttgaaat acaaagccgc agcaattgct tctatccttt ttgcgggtgc 240

agttggagtt tgtattccag ttcttggaaa aaagatccct gttttaagcc ctgaaaggag 300

tattttcttc atcatcaaag cttttgcggc cggtgttata ttgtcgacag cctttattca 360

tgtgcttccc gatgcttttg atagcttgac atcgccatgc ctcgctgaga atccttgggg 420

taaatttccc ttcacgggtt ttgtggcaat gatgtcggca attgggactt taatggtgga 480

ttgtcttgct agttcttatt ttacacggtt gcacctcatc aaggctcaac cagaggagag 540

cggggacgag gagaaggcag caggagaggc tcatgttcat actcatgccc attctcatgg 600

catagttgcg gatagttctg gttctgctcc atctcctcag cttattcgcc atcgggttat 660

tactcaggtt ctcgagttgg gaattgtggt gcactctgtg attataggag tttctctggg 720

agcttcttca agtcccaaga caataagacc tctagtgggt gccctaagct ttcaccaatt 780

ttttgagggt ataggacttg gtggatgcat tactcaggca aaattcaaga ccaaaactat 840

ggtgacaatg ggactcttct tctctctaac aaccccagtt ggaattgcag tcgggttagg 900

catatcaaat gtctataacg agagcagtcc taatgctctt attgttgaag gaatttttaa 960

tgccgcatca gctggtatcc taatctacat ggctcttgtg gatcttctgg cagctgattt 1020

tatgcatcca agagtgcaaa gtaatggagc tcttcaactt ggggtcaacg tttctcttct 1080

tctaggagtt ggctgtatgt ctctcatcgc caaatgggct tgaacctgta gggatccgcg 1140

<210> 3

<211> 29

<212> DNA

<213> Artificial Synthesis

<400> 3

cggaattccc ttcatcttca atcccttct 29

<210> 4

<211> 29

<212> DNA

<213> Artificial Synthesis

<400> 4

cgcggatccc tacaggttca agcccattt 29

Claims

1. The application of the protein in any one of the following a1) -a2), which is characterized in that:

a1) the transport capacity and resistance of heavy metal Cd in tobacco are improved;

a2) breeding tobacco varieties with high heavy metal Cd transferring capacity and high resistance;

2. The application of a coding gene of protein or a recombinant vector containing the coding gene in any one of a1) -a2) is characterized in that:

the coding gene has a nucleotide sequence shown in a sequence 2 in a sequence table.

3. Use according to claim 2, characterized in that: the recombinant vector is a recombinant plasmid pMD18-T-SmZIP obtained by inserting a DNA fragment shown in a sequence 2 in a sequence table into a vector pMD18 containing restriction enzyme sites KpnI and BamHI.

4. Use according to claim 2, characterized in that: the coding gene of the protein is obtained by amplification by taking cDNA of salix matsudana as a template through the following primers:

SmZIP-F:5’-CGGAATTCCCTTCATCTTCAATCCCTTCT-3’；

SmZIP-R:5’-CGCGGATCCCTACAGGTTCAAGCCCATTT-3’。

5. a method for improving the transport capacity and resistance of heavy metal Cd in plants by transforming a SmZIP protein of salix matsudana is characterized by comprising the following steps:

a) introducing a coding gene of protein into target tobacco to obtain transgenic tobacco expressing the coding gene, wherein the coding gene has a nucleotide sequence shown as a sequence 2 in a sequence table;

b) obtaining the transgenic tobacco with improved heavy metal Cd transport capacity and resistance compared with the target tobacco from the transgenic tobacco obtained in the step a).

6. The method of claim 5, wherein: the coding gene is introduced into the target tobacco through a recombinant expression vector containing the coding gene of the protein.

7. The method of claim 6, wherein: the recombinant expression vector is constructed by adopting the existing plant expression vector, and the plant expression vector comprises a binary agrobacterium vector and a vector which can be used for plant microprojectile bombardment.

8. The method of claim 7, wherein the binary Agrobacterium vector and the vector useful for microprojectile bombardment of plants are one of the following: pCAMBIA3301, pCAMBIA2300, pCAMBIA2301, pCAMBIA1300, pCAMBIA1301, pWM101, pGreen0029, pBI121, pBin19, pCAMBIA 1301-Ubin.

9. The method according to claim 5, wherein the method for introducing a gene encoding a protein into the tobacco of interest in step a) comprises: transforming plant cells or tissues by using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, electric shock method/agrobacterium mediation and other conventional biological methods, and culturing the transformed tobacco tissues into plants.