CN112575005A - Method for improving heavy metal cadmium stress resistance of tobacco and reducing cadmium enrichment - Google Patents
Method for improving heavy metal cadmium stress resistance of tobacco and reducing cadmium enrichment Download PDFInfo
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- CN112575005A CN112575005A CN202110004305.XA CN202110004305A CN112575005A CN 112575005 A CN112575005 A CN 112575005A CN 202110004305 A CN202110004305 A CN 202110004305A CN 112575005 A CN112575005 A CN 112575005A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
Abstract
The invention relates to the technical field of tobacco transgenic engineering breeding, and particularly discloses a method for improving heavy metal cadmium stress resistance and reducing cadmium enrichment of tobaccoPn14‑3‑3The protein gene and the base sequence of the protein gene are transferred into tobacco with SEQ ID NO.1, and the application of enhancing cadmium stress resistance of transgenic tobacco and reducing heavy metal cadmium enrichment is obtained. The invention provides a method for improving heavy metal cadmium stress of tobaccoThe method for resisting and reducing cadmium enrichment provides a new gene resource for green sustainable development genetic breeding of the tobacco industry.
Description
Technical Field
The invention relates to tobacco transgenic technologyThe technical field of breeding, in particular to pseudo-ginsengPn14-3-3The method improves the resistance of the tobacco to heavy metal cadmium stress and reduces cadmium enrichment after the protein is transferred into the tobacco.
Background
Cadmium (Cd) in soil2+) Mainly comes from the mining, smelting, heavy metal tailings, smelting waste slag, slag heaps and the like of lead-zinc mines. Cadmium is the most common and highly carcinogenic metal element in farmland soil pollution, can cause toxic reaction even under low concentration, has toxic action on soil and plants, is easy to convert into nutrient coverage, and poses great threat to food safety and human health. According to incomplete statistics, the farmland polluted by heavy metal cadmium in China exceeds 20 kilohm2The yield of agricultural products with excessive cadmium content reaches 14.6 hundred million kg every year.
Tobacco is one of important economic crops in China, is a plant easy to enrich cadmium, is mainly accumulated in leaves after absorbing cadmium through root systems, and cadmium pollution of soil is to cause tobacco Cd2+The main factor of the increase of the content. Research and analysis show that the transfer rate of cadmium in the cigarettes can reach 10% on average, and research and development also provide that cadmium can be used as an index for evaluating environmental smoke. In recent years, the cadmium pollution problem of tobacco planting soil in China is serious, and when the cadmium content of soil in main tobacco areas in China is measured, the average value of the cadmium pollution problem is found to exceed the total cadmium content of a sample by 12.4 percent compared with the soil quality standard (GB-15618-.
The research on the safety of tobacco has become a hot spot, and the cadmium resistance mechanism of tobacco also draws extensive attention. Cadmium not only affects the growth and development of tobacco and the coordination of chemical components of tobacco leaves, but also enters a human body through mainstream smoke in the smoking process of cigarettes and can not be discharged, so that heavy metal cadmium is gradually accumulated in the human body, and the health of smokers is harmed.
The 14-3-3 protein is a highly conserved regulatory protein which is ubiquitous in eukaryotic cells, and in the process of plant growth and development, the interaction of the 14-3-3 protein and other proteins participates in various regulation processes such as metabolic regulation, substance transportation, light signal response, phytohormone signal transduction, heavy metal stress and the like, but notThe 14-3-3 protein isoforms of the same type are expressed in different tissues of plants and are very different, and they specifically regulate target proteins, participate in a series of signal transduction and metabolic processes in plant cells and influence the growth and development of plants. In the previous research, the 14-3-3 protein is found to reduce the generation of NO induced by cadmium through the interaction with nitrate reductase under the cadmium stress, and prevent the accumulation of cadmium in the root of pseudo-ginseng. Although the CN201810790482.3 tobacco 14-3-3 protein, the coding gene and the application thereof in the low potassium response of tobacco in the prior art, the plant expression vector of the CN201710974873.6 tobacco 14-3-3c gene and the application thereof have made a certain research on the 14-3-3 protein in tobacco, whether the 14-3-3 protein can reduce the accumulation of cadmium in transgenic tobacco is not involved. On the basis of earlier stage research, pseudo-ginseng is obtained by cloningPn14-3-3Protein gene and obtaining the over-expressed pseudo-ginseng by transgenic technologyPn14-3-3Transgenic tobacco of protein gene. The results of the study show that the overexpressionPn14-3-3The resistance of the transgenic tobacco to cadmium stress is obviously improved, and the activity of nitrate reductase, NO content and cadmium content in roots are obviously reduced compared with wild plants.
Disclosure of Invention
The invention aims to provide a new application of a gene, namely pseudo-ginsengPn14-3-3The protein gene is applied to enhancing cadmium stress resistance of transgenic tobacco and reducing heavy metal cadmium enrichment so as to obtain tobacco seeds with cadmium stress resistance.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a pseudo-ginsengPn14-3-3The protein gene has the base sequence of SEQ ID NO.1 and is applied to enhancing the cadmium stress resistance of transgenic tobacco and reducing the enrichment of heavy metal cadmium.
A method for improving heavy metal cadmium stress resistance and reducing cadmium enrichment of tobacco comprises the following steps:
(1) according to the pseudo-ginsengPn14-3-3Amplifying the gene sequence SEQ ID NO.1 to synthesize a whole gene sequence;
(2) mixing Notoginseng radixPn14-3-3The protein gene is connected with an expression vector to form the pseudo-ginseng-containing productPn14-3-3Plant expression vectors for protein genes;
(3) will contain notoginsengPn14-3-3Transforming the plant expression vector of the protein gene into agrobacterium tumefaciens to obtain agrobacterium tumefaciens engineering bacteria containing the pseudo-ginseng Pn14-3-3 protein gene plant expression vector for transforming tobacco;
(4) transforming tobacco callus by using the constructed agrobacterium engineering strain to obtain F1 generation seeds of the transgenic tobacco plant detected by quantitative PCR;
(5) culturing the obtained transgenic tobacco seed, measuring the root elongation of the seedling, the cadmium content, the NO content and the NR activity in the plant, and verifying the pseudo-ginsengPn14-3-3The resistance of the gene tobacco to cadmium stress and cadmium enrichment.
Preferably, the expression vector in the step (2) is pCAMBIA1300-35S plant overexpression vectorBamHI andSali pairs of pseudo-ginsengPn14-3-3Respectively performing double enzyme digestion on protein gene and pCAMBIA1300-35S vector, and then subjecting Notoginseng radix toPn14-3- 3The protein gene is connected to pCAMBIA1300-35S vector to obtain Notoginseng radixPn14-3-3Plant over-expression vector pCAMBIA1300-35S-Pn14-3-3。
Preferably, the Agrobacterium tumefaciens in step (3) is the GV3101 strain.
The invention has the advantages that:
the invention adopts gene engineering operation technology to separate pseudo-ginsengPn14-3-3The protein gene is transferred into tobacco, the cadmium stress resistance of the obtained transgenic tobacco is obviously improved, when the transgenic tobacco is subjected to cadmium stress with different concentration gradients, the elongation of the young root of the transgenic tobacco is obviously improved compared with that of non-transgenic tobacco, and the cadmium content, NO content and NR activity in the transgenic tobacco are obviously reduced compared with that of the non-transgenic tobacco. The invention provides a method for improving heavy metal cadmium stress resistance of tobacco and reducing cadmium enrichment, and provides a new gene resource for green sustainable development genetic breeding of tobacco industry.
Drawings
FIG. 1 is a technical flow diagram of the present invention;
FIG. 2 is a schematic representation of the vector construction of the present invention;
FIG. 3 shows the result of the quantitative PCR assay of the present invention;
FIG. 4 is a schematic diagram of a transgenic tobacco of the present invention, in which COKER is a wild type, and 14-3-3-2 is a transgenic type;
FIG. 5 is a statistical plot of the resistance of tobacco seedling root system to cadmium stress (root elongation) after 10 days of growth;
FIG. 6 is a statistical graph of the difference in cadmium content between wild type and transgenic tobacco roots;
FIG. 7 is a statistical plot of the differences in NO content between wild-type and transgenic tobacco roots;
FIG. 8 is a statistical graph of the differences in NR activity between wild-type and transgenic tobacco roots.
Detailed Description
The invention is explained in more detail below with reference to the figures and examples, without limiting the scope of the invention. In the examples, the procedures were carried out in accordance with the usual procedures unless otherwise specified, and all the reagents used were either conventional commercially available reagents or reagents prepared in accordance with the conventional procedures unless otherwise specified.
Example 1Pn14-3-3Protein gene plant expression vector pCAMBIA1300-35S-Pn14-3-3Construction of and transformation of tobacco
One, gene sequence
Amplifying and synthesizing a whole gene sequence according to the Pn14-3-3 gene sequence, adding CDS at both ends of the geneBamHI andSali enzyme cutting site, and synthesizing sequence SEQ ID NO. 1.
Second, vector construction
BamHI andSali double enzyme digestion pCAMBIA1300-35S carrier and recovery purification,BamHi andSali double enzyme digestionPn14- 3-3The fragment, the carrier after enzyme digestion and the fragment are mixed evenly, and the pCAMBIA1300-35S-Pn14-3-3And (3) a carrier.
The digestion reaction (30 min at 37 ℃) was as shown in Table 1:
the ligation reaction (60 min at 22 ℃) was as shown in Table 2:
after completion of the reaction the ligation product was placed on ice for the conversion step:
1. adding the ligation product into 100 mu L DH5 alpha competent cells, mixing evenly, and carrying out ice bath for 30 min;
2. performing heat shock at the constant temperature of 42 ℃ for 90 s in a water bath kettle, and immediately placing in an ice bath for 2 min after the heat shock is finished;
3. adding 700 μ L LB liquid medium without antibiotics, and shake culturing at 37 deg.C and 220 rpm for 60 min;
4. centrifuging at room temperature 8000 rpm for 1 min, discarding the supernatant to obtain 100 μ L liquid suspension bacteria, uniformly spreading on LB solid agar medium containing 50 mg/L kanamycin, and culturing at 37 deg.C for 12-16 h by inversion.
Extracting plasmids by shaking and sequencing:
and (3) checking and analyzing the sequence obtained after sequencing by using Chromas and DNASTAR software, manually checking the base interpretation result, and reducing the error of a sequencer.
The invention adopts high-fidelity DNA polymerase, and the experimental result is credible;
3. agrobacterium transformation
1. Melting Agrobacterium rhizogenes strain at-80 deg.C at room temperature, and inserting into ice when in ice-water mixed state;
2. adding 0.01-1 μ g plasmid DNA per 100 μ L competence, and mixing with hand-made tube bottom;
3. standing on ice for 5 minutes, using liquid nitrogen for 5 minutes, using a water bath at 37 ℃ for 5 minutes, and carrying out ice bath for 5 minutes in sequence;
4. adding 700 mu L of LB or YEB liquid culture medium without antibiotics, and carrying out shake culture at 28 ℃ for 2-3 hours;
5. centrifuging at room temperature of 6000 rpm for 1 min, collecting supernatant of about 100 μ L, lightly blowing and beating the heavy suspension bacterium block, coating on LB or YEB plate containing corresponding antibiotics, and culturing in 28 deg.C incubator for 2-3 days;
6. infecting tobacco by a leaf disc method, transforming a target gene into wild tobacco, detecting the gene expression level by quantitative PCR, and obtaining F1 generation seeds of the transgenic tobacco.
The main reagents and consumables used in this experiment are shown in table 3.
Example 2 resistance of tobacco seedling root systems to cadmium stress
Seeding wild type and transplanting notoginseng simultaneouslyPn14-3-3Protein tobacco seed, transnotoginsengPn14-3-3The protein tobacco seed has a faster germination time by 4-5 days than wild tobacco seeds, and has a better growth vigor and a higher germination rate than wild tobacco seeds.
Root of pseudo-ginsengPn14-3-3After the protein tobacco seeds are sown for 10 days, after the Hoagland culture solution water culture transgenic seedlings are adapted to the wild tobacco seedlings for 1d, the root length is measured, and the culture solution is added with CdCl with the concentration gradient of 0 mu M, 25 mu M, 50 mu M, 75 mu M and 100 mu M2After 24h of culture, the root length is measured again, and the root elongation is calculated by two measurements. The result shows that the pseudo-ginseng is changedPn14-3-3Root elongation of protein tobacco seedlings, especially14-3-3-2Compared with wild type (COKER) tobacco, the transgenic tobacco is obviously improved and has significant difference.
First, measuring cadmium content in tobacco root system
Sample treatment:
1. after 60 days of tobacco growth, a concentration gradient of 0. mu.M, 25. mu.M, 50. mu.M, 75. mu.M, 100. mu.M CdCl was used2Treating and growing 60d tobacco for 24 h;
2. soaking 5-6g tobacco root in pure water for 3min, washing for 3-5 times, and sucking water with filter paper;
oven drying at 3.65 deg.C for 4 d.
Sample detection:
after drying, 1g of sample is sent to analytical test research center of Kunming science university, and the content of cadmium in the root system is measured by inductively coupled plasma emission spectrometer ICP-OES.
And (3) detection results:
after gradient treatment of different cadmium concentrations, pseudo-ginseng is transferredPn14-3-3The cadmium content in the protein tobacco root system is reduced compared with that of a wild type (COKER): when the cadmium concentration is 25 mu M, the wild type cadmium-containing strain is transformedThe cadmium content in the gene type tobacco root system is reduced by 28.3 percent; when the cadmium concentration is 50 mu M, the cadmium content in the transgenic tobacco root system is reduced by 18.5 percent; when the cadmium concentration is 75 mu M, the cadmium content in the transgenic tobacco root system is reduced by 5.7 percent; when the cadmium concentration is 100 mu M, the cadmium content in the transgenic tobacco root system is reduced by 2.3 percent, and the cadmium content difference under each concentration gradient has obvious difference.
Second, measuring NO content in tobacco root system
Sample treatment:
1. after 60 days of tobacco growth, a concentration gradient of 0. mu.M, 25. mu.M, 50. mu.M, 75. mu.M, 100. mu.M CdCl was used2Treating and growing 60d tobacco for 24 h;
2. soaking 0.1g tobacco root hair in pure water for 3min, washing for 3-5 times, and drying with filter paper;
3. preparing a homogenizing medium;
4. fully grinding with liquid nitrogen for 4-5 times, and mixing with a homogenizing medium;
5. standing for 2h, and fully extracting;
6.10000 g, centrifuging at 4 ℃ for 20min, and taking the supernatant to ice for testing.
Sample detection:
the NO content in the root system is determined by using Nanjing built nitric oxide determination kit (A012-1).
And (3) detection results:
after gradient treatment of different cadmium concentrations, pseudo-ginseng is transferredPn14-3-3The content of NO in the protein tobacco root system is reduced compared with that of a wild type (COKER), and the reduction of 14-3-3-2 tobacco is most obvious: compared with the wild type, when the cadmium concentration is 25 muM, 50 muM and 75 muM, the NO content in the transgenic tobacco root system is reduced by 20 percent; when the cadmium concentration is 100 mu M, the NO content in the transgenic tobacco root system is reduced by 16.3 percent, and the cadmium content difference under each concentration gradient has obvious difference.
Third, the determination of the NR activity of the tobacco root system
Sample treatment:
1. after 60 days of tobacco growth, a concentration gradient of 0. mu.M, 25. mu.M, 50. mu.M, 75. mu.M, 100. mu.M CdCl was used2Treating and growing 60d tobacco for 24 h;
2. soaking 0.1g tobacco root hair in pure water for 3min, washing for 3-5 times, and drying with filter paper;
3. soaking the materials in an inducer in a Nanjing-built nitrate reductase activity assay kit (A096-1-2) for 2h, and sucking water by filter paper;
4. according to root system mass (g): extract volume (mL) 1: 10 (0.1 g root added with 1mL extract) to be homogenized in ice bath;
5. standing for 2h, and fully extracting;
6.10000 g, centrifuging at 4 ℃ for 20min, and taking the supernatant to ice for testing.
Sample detection:
using Nanjing built nitrate reductase activity determination kit (A096-1-2) to determine NR activity in root system;
the protein concentration in the root system was determined using the solibao BCA protein concentration detection kit (PC 0020).
And (3) detection results:
after gradient treatment of different cadmium concentrations, pseudo-ginseng is transferredPn14-3-3The activity of NR in the protein tobacco root system is reduced compared with that of a wild type (COKER): compared with the wild type, when the cadmium concentration is 25 muM, 50 muM, 75 muM and 100 muM, the NR activity in the transgenic tobacco root system is respectively reduced by 31.7 percent, 20.5 percent, 21.9 percent and 25.2 percent, and the NR activity difference under each concentration gradient has significant difference.
Sequence listing
<110> university of Kunming science
<120> method for improving heavy metal cadmium stress resistance of tobacco and reducing cadmium enrichment
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ttggctgagt ttaagattgg agctgagcgg aaagaagcag ctgagagtac tcttactgca 480
tataaagctg ctcaggatat tggaaataca gaacttgctc caacacaccc aattcggctt 540
ggactggctc ttaacttctc tgtgttttac tatgagattt tgaactctcc cgaccgtgca 600
tgtaatcttg caaaacaggc ttttgatgaa gcaattgctg agttggatac attaggcgag 660
gaatcctaca aagatagcac gttgattatg caacttcttc gtgacaacct taccctctgg 720
acatccgata tgcaggatga tgggacagat gaaattaagg aagctgctgc tcccaagcct 780
gatgatgaac aacagtgagt cgac 804
Claims (4)
1. Notoginseng radixPn14-3-3The protein gene has the base sequence of SEQ ID NO.1 and is applied to enhancing the cadmium stress resistance of transgenic tobacco and reducing the enrichment of heavy metal cadmium.
2. A method for improving heavy metal cadmium stress resistance and reducing cadmium enrichment of tobacco is characterized by comprising the following steps:
(1) according to the pseudo-ginsengPn14-3-3Amplifying the gene sequence SEQ ID NO.1 to synthesize a whole gene sequence;
(2) mixing Notoginseng radixPn14-3-3The protein gene is connected with an expression vector to form the pseudo-ginseng-containing productPn14-3-3Plant expression vectors for protein genes;
(3) will contain notoginsengPn14-3-3The plant expression vector of the protein gene is transformed into agrobacterium tumefaciens to obtain the gene containing pseudo-ginseng Pn14 for transforming tobacco-3-3 protein gene plant expression vector of agrobacterium tumefaciens engineering bacteria;
(4) transforming tobacco callus by using the constructed agrobacterium engineering strain to obtain F1 generation seeds of the transgenic tobacco plant detected by quantitative PCR;
(5) culturing the obtained transgenic tobacco seed, measuring the root elongation of the seedling, the cadmium content, the NO content and the NR activity in the plant, and verifying the pseudo-ginsengPn14-3-3The resistance of the gene tobacco to cadmium stress and cadmium enrichment.
3. The method for improving heavy metal cadmium stress resistance and reducing cadmium enrichment of tobacco according to claim 2, wherein the expression vector in the step (2) is pCAMBIA1300-35S plant overexpression vectorBamHI andSali pairs of pseudo-ginsengPn14-3-3Respectively performing double enzyme digestion on protein gene and pCAMBIA1300-35S vector, and then subjecting Notoginseng radix toPn14-3-3The protein gene is connected to pCAMBIA1300-35S vector to obtain Notoginseng radixPn14-3-3Plant over-expression vector pCAMBIA1300-35S-Pn14-3-3。
4. The method for improving heavy metal cadmium stress resistance and reducing cadmium enrichment of tobacco according to claim 2, wherein the agrobacterium tumefaciens in step (3) is the GV3101 strain.
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