CN103184237A - Method for improving plant heavy metal tolerance and regulating heavy metal oriented distribution - Google Patents

Abstract

The invention relates to a method for improving the tolerance of plants to heavy metals and regulating the directional distribution of heavy metals. The invention expresses the tonoplast membrane transporter HMT1 gene in plants for the first time, and improves the tolerance of plants to heavy metals. And through the tissue-specific expression of the gene, the directional distribution of heavy metals in plants can be realized. The gene of the present invention can be applied to the improvement of plant varieties, including: improving the resistance of plants to heavy metals; regulating the content of heavy metals in different tissues and organs of plants; reducing the content of heavy metals in edible parts of plants; promoting phytoremediation; Chlorophyll content of plants under heavy metal stress. The invention provides a very valuable gene directional expression operation method for cultivating new plant varieties by using molecular breeding techniques such as transgene.

Description

A method of improving plant tolerance to heavy metals and regulating the directional distribution of heavy metals

technical field

The invention belongs to the field of biotechnology; more specifically, the invention relates to the action mechanism of improving heavy metal tolerance and regulating heavy metal directional distribution by using tonoplast membrane transporter HMT1 and its application.

Background technique

In recent years, due to the development of modern industry, more and more heavy metals, such as cadmium, arsenic, lead, etc., have been discharged into the biosphere. The pollution of heavy metals such as cadmium not only leads to crop yield reduction, but also poses a threat to food safety. Cadmium is one of the most toxic heavy metals, and it is known as a class IA carcinogen that is most likely to accumulate in the body. Excessive cadmium in food and water accumulates in human tissues and organs through the food chain, causing various hazards, including damage to the kidneys, liver, and even cancer.

Therefore, the remediation of soil contaminated by heavy metals such as cadmium is an environmental problem that needs to be solved urgently. Due to the high cost of traditional methods, phytoremediation, as an emerging green environmental treatment technology, has received great attention and has been widely used. However, in some countries with serious pollution, such as China, by the end of the last century, the area of farmland polluted by heavy metal cadmium had reached 20,000 hectares, and the annual production of agricultural products with excessive cadmium content reached 1.46 billion kilograms. Recently, according to the research report of Nanjing Agricultural University, more than 10% of the rice in the Chinese market is polluted by cadmium, which has already constituted a huge potential threat to human health. Therefore, while performing phytoremediation, reducing the migration of toxic heavy metals such as cadmium to edible parts may be a more realistic option for countries with large-scale pollution.

The method of using molecular breeding to reduce the content of cadmium in edible parts such as crop grains has good and broad development prospects, but there are still many problems in the research on the absorption, transport and resistance mechanism of heavy metal cadmium in plants, especially in the edible parts such as grains. The molecular mechanism of site migration is still poorly understood.

Therefore, in-depth research is needed in this field to develop plants with good tolerance to heavy metals; further, to develop methods and products that can effectively reduce the content of toxic heavy metals in edible parts such as crop grains.

Contents of the invention

The purpose of the present invention is to provide a tonoplast membrane transporter HMT1 which can be successfully overexpressed in plants and can improve the tolerance and accumulation ability of plants to heavy metals.

The object of the present invention is also to provide a method for reducing the content of heavy metals (especially cadmium) in edible parts such as crop grains.

In the first aspect of the present invention, there is provided a use of tonoplast membrane transporter HMT1, which is used to improve the tolerance of plants to heavy metals, regulate the directional distribution of heavy metals or increase the chlorophyll content of plants under heavy metal stress.

In a preferred example, the tonoplast membrane transporter HMT1 is used to transport heavy metals into the vacuoles of plant cells.

In another preferred example, the heavy metal includes: cadmium (Cd), arsenic (As), copper (Cu) or zinc (Zn).

In another preferred example, the HMT1 is:

(a) a protein having the amino acid sequence shown in GenBank accession number CAA78419; or

(b) passing the amino acid sequence shown in GenBank accession number CAA78419 through one or more (such as 1-30; preferably 1-20; more preferably 1-10; more preferably 1-5) amino acid residues A protein formed by substitution, deletion or addition of a group and having the protein function defined in (a);

(c) A protein having a sequence identity higher than 70% with the protein of the amino acid sequence shown in GenBank accession number CAA78419 and having the protein function defined in (a).

In another aspect of the present invention, there is provided a method for improving the tolerance of plants to heavy metals, regulating the degree of accumulation of heavy metals in plant tissues, or increasing the chlorophyll content of plants under heavy metal stress, the method comprising: expressing exogenous The tonoplast membrane transporter HMT1.

In a preferred example, the method includes: transferring the expression cassette of the tonoplast membrane transporter HMT1 into the plant, so as to express the exogenous tonoplast membrane transporter HMT1 in the plant.

In another preferred example, the method includes:

(1) providing an Agrobacterium carrying an expression vector containing an expression cassette of the tonoplast membrane transporter HMT1;

(2) contacting the plant cell or tissue or organ with the Agrobacterium in step (1), so that the expression cassette of the tonoplast transporter HMT1 is transferred into the plant cell and integrated into the chromosome of the plant cell;

(3) selecting plant cells or tissues or organs that have been transferred into the expression cassette of the tonoplast membrane transporter HMT1; and

(4) Regenerating the plant cells or tissues or organs in step (3) into plants.

In another aspect of the present invention, a plant that is tolerant to heavy metals or has different accumulation levels of heavy metals in tissues is provided, and its genome includes an expression cassette of tonoplast membrane transporter HMT1.

In another aspect of the present invention, there is provided a method for regulating the degree of accumulation of heavy metals in plant tissue, the method comprising:

Transferring the expression cassette of the tonoplast membrane transporter HMT1 into the plant, the expression cassette of the tonoplast membrane transporter HMT1 includes an operably linked promoter and a polynucleotide encoding the tonoplast membrane transporter HMT1;

Wherein, the promoter is a plant-specific tissue-specific expression promoter, thereby driving the expression of the tonoplast membrane transporter HMT1 in a plant-specific tissue, promoting the transport of heavy metals into the plant-specific tissue (preferably, reducing the concentration of heavy metals in other plant tissues) content in).

In a preferred example, in the expression cassette, the 3' end of the polynucleotide encoding tonoplast membrane transporter HMT1 further includes a terminator.

In another preferred example, the specific plant tissue is selected from (but not limited to): roots, stems, leaves, seeds, and seed coats.

In another preferred example, the plant is a plant including edible tissues, and the promoter is a non-edible tissue-specific expression promoter of the plant, so as to transport heavy metals into the non-edible tissue of the plant (preferably , to reduce the content of heavy metals in edible plant tissues).

In another preferred example, the non-edible tissue-specific expression promoter is a plant root-specific expression promoter. The root-specific expression promoter is selected from but not limited to: Adh promoter, NT1 promoter, ZmGLU1 promoter and the like.

In another preferred example, the promoter is a specific promoter for aboveground parts of plants.

In another preferred example, the above-ground plant tissue-specific promoter is a CAB2 promoter.

In another preferred example, the method includes:

(1) providing an Agrobacterium carrying an expression vector, the expression vector containing the expression cassette of the tonoplast membrane transporter HMT1;

(2) contacting the plant cell or tissue or organ with the Agrobacterium in step (1), so that the expression cassette of the tonoplast transporter HMT1 is transferred into the plant cell and integrated into the chromosome of the plant cell;

(3) selecting plant cells or tissues or organs that have been transferred into the expression cassette of the tonoplast membrane transporter HMT1; and

(4) Regenerating the plant cells or tissues or organs in step (3) into plants.

In another aspect of the present invention, there is provided an expression cassette of the tonoplast membrane transporter HMT1, which includes an operably linked promoter and a polynucleotide encoding the tonoplast membrane transporter HMT1; wherein the promoter is a plant-specific Tissue-specific expression promoter.

In another aspect of the present invention, the use of the expression cassette of the tonoplast membrane transporter HMT1 is provided, which is used to regulate the accumulation degree of heavy metals in plant tissues, promote the transport of heavy metals into specific plant tissues, and reduce the concentration of heavy metals in other plant tissues. content.

In another aspect of the present invention, there is provided a plant with different levels of accumulation of heavy metals in tissues, the genome of which includes an expression cassette of tonoplast membrane transporter HMT1, and the expression cassette of tonoplast membrane transporter HMT1 includes an operably linked promoter 1. A polynucleotide encoding the tonoplast membrane transporter HMT1; wherein, the promoter is a plant-specific tissue-specific expression promoter, which drives the expression of the tonoplast membrane transporter HMT1 in a specific plant tissue, and promotes the transport of heavy metals into the plant Plant-specific tissues (preferably, reducing the content of heavy metals in other plant tissues).

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1. The modified SpHMT1 can complement the cadmium-sensitive phenotype of the yeast mutant Δhmt1.

A. Yeast cadmium resistance plate experiment. The Δhmt1 mutant yeast (LK100) was transformed into the empty vector pART1 (V/LK100), the SpHMT1 (HMT1/LK100) containing the 5'utr of the yeast itself and the modified Kozak-HMT1 (KHMT1/LK100), and the wild Type yeast (V/Sp223) was used as a positive control.

B. Yeast growth curve under cadmium concentration gradient.

Fig. 2. RT-PCR analysis of the expression of SpHMT1 in transgenic Arabidopsis.

A, 35S:SpHMT1/Col-04 transgenic SphMT1 expression in shoots and leaves of Arabidopsis thaliana. W2-11, W3-13 and W7-3 respectively represent three independent lines obtained after transforming Col-0 with 35S/SpHMT1:cmyc-pBI121. The expression of SpHMT1 in transgenic Arabidopsis roots is the same as the figure.

B, 35S:SpHMT1/cad1-34 transgenic SpHMT1 expression in shoots and leaves of Arabidopsis thaliana. M10-8 and M1-12 respectively represent two independent strains obtained after transforming cad1-3 with 35S/SpHMT1:cmyc-pBI121. The expression of SpHMT1 in transgenic Arabidopsis roots is the same as the figure.

C. The expression of SpHMT1 in the roots of Adh:SpHMT1/Col-04 transgenic Arabidopsis. A5-9 and A26-4 respectively represent two independent lines obtained after Adh/SpHMT1:cmyc-pBI121 transformed into Col-0.

D. The expression of SpHMT1 in the shoots and leaves of Adh:SpHMT1/Col-04 transgenic Arabidopsis.

Figure 3. Overexpression of SpHMT1 in wild-type Arabidopsis can enhance plant resistance to heavy metals.

AD and seeds were grown vertically on 1/4×MS plates supplemented with 50 μM CdCl ₂ (A), 150 μM KH ₂ AsO ₄ (B), 40 μM CuSO ₄ (C) or 150 μM ZnSO ₄ (D) for 4 weeks after surface disinfection .

E, Fresh weight of plants in panels A-D. Among them, 50 in Cd50 means that the Cd content in the medium is 50 μM, As150 means that the As content in the medium is 150 μM, and so on.

F, Corresponding heavy metal content of plants (whole plant seedlings) in AD. Data are means±SD of three independent experiments, each experiment including >100 plants, ^** P<0.01.

W2-11 and W7-3 respectively represent two independent strains obtained after transforming Col-0 with 35S/SpHMT1:cmyc-pBI121.

Figure 4. BSO inhibited the resistance and accumulation of heavy metals in SpHMT1 transgenic plants.

AB, seed surface sterilized and grown vertically for 20 days on 1/4×MS plates added with 10 μM CdCl ₂ and 0.5 mM BSO (A) or 0.5 mM BSO (B) respectively.

Fresh weight of plants in panels C, A and B.

D. Detection of cadmium content in each tissue of transgenic plants. Among them, S represents the stem, L represents the rosette leaf, and R represents the root.

Data are mean±SD of three independent experiments, each experiment including >100 plants.

W2-11 and W7-3 respectively represent two independent strains obtained after transforming Col-0 with 35S/SpHMT1:cmyc-pBI121.

Figure 5. Overexpression of SpHMT1 in Arabidopsis PCs deletion mutant cad1-3 fails to enhance heavy metal resistance and accumulation.

A. Seeds were grown vertically on 1/4×MS plates for 7 days after surface disinfection.

B. Seeds were grown vertically on 1/4×MS plates supplemented with 5 μM CdCl ₂ for 20 days after surface disinfection.

Fresh weight of plants in panels C, A and B.

Cadmium concentrations in plants in panels D, A, and B. CK represents control condition (common 1/2×MS plate culture).

Data are mean±SD of 3 independent experiments, each experiment including >100 plants.

M10-8 and M1-12 respectively represent two independent strains obtained after transforming cad1-3 with 35S/SpHMT1:cmyc-pBI121.

Figure 6. Partitioning of cadmium between vacuoles and protoplasts.

A. The content of cadmium in Col-0, cad1-3 and their corresponding transgenic plants W2-11 and M10-8 leaf protoplasts and vacuoles. Plant acid phosphatase (ACP) activity was used as the conversion standard for cadmium content. Data are mean ± standard error of three independent experiments.

B, Partition ratio of cadmium in cytosol and vacuole. Values represent the ratio of cadmium content in the vacuole to the whole protoplast.

Figure 7. Content of phytochelatins (PCs) in vacuoles. Plant acid phosphatase (ACP) activity was used as the conversion standard of PCs content. Data are mean ± standard error of three independent experiments.

PC2, PC3, and PC4 respectively represent PC molecules with different chain lengths, and the number is the value of n in the common structure (γ-Glu-Cys) _n -Gly of PC molecules.

Figure 8. Specific expression of SpHMT1 in roots increases plant resistance to Cd ²⁺ .

The chlorophyll content of rosette leaves was extracted from plants grown in hydroponics for 4 weeks and treated with 10 μM CdCl ₂ for 7 days for comparison. Data are mean ± SD, and >8 plants were included in each experiment. Control is the normal culture condition.

A5-9 and A26-4 respectively represent two independent lines obtained after Adh/SpHMT1:cmyc-pBI121 transformed into Col-0.

Figure 9. The specific expression of SpHMT1 in roots can reduce the heavy metal content in plant aerial parts and seeds.

AC, 4-week-old plants were treated with 10μM CdCl ₂ (A), 100μM KH ₂ AsO ₄ (B) or 20μM CuSO ₄ (C) for 3 days, and the corresponding heavy metal content.

DF and plants grown in water for 2 weeks were treated with 5 μM CdCl ₂ (D), KH ₂ AsO ₄ (E) or CuSO ₄ (F) until the seeds matured, and the corresponding heavy metal content in the seeds was measured.

Data are mean±SD of three independent experiments, each experiment including >8 plants.

A5-9 and A26-4 respectively represent two independent strains obtained after transforming Col-0 with Adh/SpHMT1:cmyc-pBI121; W2-11 represents the strain obtained after transforming Col-0 with 35S/SpHMT1:cmyc-pBI121 .

Figure 10. The specific expression of SpHMT1 above ground can change the distribution of heavy metal cadmium.

A. Plants cultured in water for 4 weeks were treated with 10 μM CdCl ₂ for 3 days, and the corresponding heavy metal contents in roots (R) and rosette leaves (L) were measured.

B. Plants cultured in water for 2 weeks were treated with 5 μM CdCl ₂ until the seeds matured, and the corresponding heavy metal content in the seeds was measured.

Data are mean±SD of three independent experiments, each experiment including >8 plants.

C20-2 and C23-1 respectively represent two independent strains obtained after transforming Col-0 with CAB2/SpHMT1:cmyc-pBI121; W2-11 represents the strain obtained after transforming Col-0 with 35S/SpHMT1:cmyc-pBI121 .

Detailed ways

After extensive research, the inventors found a gene useful for improving the tolerance of plants to heavy metals and regulating the accumulation of heavy metals in plant tissues—the tonoplast membrane transporter HMT1 gene (HMT1). The gene of the present invention can be applied to the improvement of plant varieties, including: improving the resistance of plants to heavy metals; regulating the content of heavy metals in different tissues and organs of plants; reducing the content of heavy metals in edible parts of plants; improving plant tissue under heavy metal stress chlorophyll content in. The invention provides very valuable gene resources for cultivating new plant varieties by molecular breeding techniques such as transgenes.

In the present invention, there is no particular limitation on the plants (or crops) applicable to the present invention, as long as they are suitable for gene transformation operations, such as various crops, floral plants, or forestry plants, etc. The plant may be, for example (not limited to): a dicotyledonous plant, a monocotyledonous plant, or a gymnosperm. More specifically, said plants include (but are not limited to): wheat, barley, rye, rice, corn, sorghum, sugar beet, apple, pear, plum, peach, apricot, cherry, strawberry, raspberry, blackberry, bean , lentils, peas, soybeans, canola, mustard, poppies, olean, sunflowers, coconuts, castor oil plants, cocoa beans, peanuts, gourds, cucumbers, watermelons, cotton, flax, hemp, jute, citrus, lemons, grapes Grapefruit, spinach, licorice, asparagus, cabbage, Chinese cabbage, bok choy, carrot, onion, potato, tomato, green pepper, avocado, cinnamon, camphor, tobacco, nuts, coffee, eggplant, sugar cane, tea, pepper, grapevine , oyster grass, bananas, natural rubber trees and ornamental plants.

As a preferred manner, the "plant" includes, but is not limited to: Brassicaceae, Poaceae, and Rosaceae. For example, the "plants" include but are not limited to: Brassica Chinese cabbage and Chinese cabbage, Brassicaceae Arabidopsis plants such as Arabidopsis, Poaceae rice, wheat, corn, etc., in addition It also includes tobacco, fruits, vegetables, rapeseed and so on.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state .

As used herein, "isolated tonoplast transporter HMT1", "isolated SpHMT1 protein" or "isolated SpHMT1 polypeptide" means that the SpHMT1 protein is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated . Those skilled in the art can purify SpHMT1 protein using standard protein purification techniques. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels.

As used herein, "edible tissue (organ)" of a plant refers to a plant tissue (organ) derived from a plant body that can be directly used as food or processed into food. In some crops, "edible tissues (organs)" include, for example, the seeds of gramineous crops such as rice and wheat (with or without seed coats), tubers such as sweet potatoes, and leaves of cruciferous plants such as Chinese cabbage.

As used herein, "non-edible tissue (organ)" of a plant refers to plant tissue (organ) derived from a plant body that is not normally used or processed as food. In some crops, "non-edible tissues (organs)" are for example: stems, leaves, and roots of gramineous crops such as rice and wheat, stems and tendrils of potato plants such as sweet potatoes, cruciferous plants such as Chinese cabbage, root.

As used herein, "expression cassette" refers here to a recombinant DNA molecule that contains the desired nucleic acid coding sequence that encodes the SpHMT1 protein; this DNA molecule also contains the necessary or necessary linking coding sequence for transcription in vitro or in vivo. Suitable regulatory elements are expected. "Regulatory element" herein refers to a nucleotide sequence that controls the expression of a nucleic acid sequence. Exemplary regulatory elements include promoters, transcription termination sequences or upstream regulatory regions, which facilitate nucleic acid replication, transcription, post-transcriptional modification, and the like. In addition, regulatory elements may also include: enhancers, internal ribosomal entry sites (IRES), replication origins, polyadenylation signals, and the like.

As used herein, the term "operably linked" or "operably linked to" refers to the condition that certain parts of a linear DNA sequence are capable of modulating or controlling the activity of other parts of the same linear DNA sequence. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.

As used herein, the "promoter" or "promoter region (domain)" refers to a nucleic acid sequence, which usually exists upstream (5' end) of the coding sequence of the gene of interest, and can guide the transcription of the nucleic acid sequence into mRNA . Generally, a promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription. Herein, the promoter or promoter region includes variants of the promoter, which can be obtained by inserting or deleting the regulatory region, performing random or site-directed mutation of the promoter, and the like.

As used herein, "tissue-specific promoters" are also called "organ-specific promoters". Under the regulation of such promoters, genes are often expressed only in certain specific organs or tissue parts, and exhibit developmental regulation characteristics . Generally, a promoter is considered tissue or organ specific if the mRNA is expressed in a tissue or organ at a level at least 10 times higher, preferably at least 100 times higher, more preferably at least 1000 times higher than in other tissues or organs of.

As used herein, the words "comprising", "having" or "comprising" include "comprising", "consisting essentially of", "consisting essentially of", and "Consisting of"; "Consisting essentially of", "Consisting essentially of" and "Consisting of" are "contains" , The subordinate concept of "has" or "includes".

Tonoplast membrane transporter HMT1

Tonoplast membrane transporter HMT1 protein, also known as: PC-metal complex tonoplast membrane transporter, vacuolar metal transporter, PC-transporter, phytochelatin (prime) transporter, heavy metal tolerance factor. Existing studies have found that HMT1 genes encoding HMT1 proteins have been found in various organisms, such as SpHMT1 (Z14055), DmHMT1 (EU571211) and CeHMT1 (AF497513) genes in yeast, Drosophila and nematodes, respectively.

The polypeptide (protein) of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, preferably a recombinant polypeptide. Polypeptides of the present invention may be naturally purified, or chemically synthesized, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. Polypeptides of the invention may or may not include an initial methionine residue.

The present invention also includes fragments, derivatives and analogs of HMT1 protein. As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially maintains the same biological function or activity of the HMT1 protein of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). These fragments, derivatives and analogs are within the purview of those skilled in the art as defined herein.

In the present invention, the term "SpHMT1 protein" refers to a polypeptide having a GenBank accession number CAA78419 sequence that improves plant tolerance to heavy metals such as cadmium or can regulate the accumulation of heavy metals in plant tissues. The term also includes variant forms of the GenBank accession number CAA78419 sequence having the ability to improve plant tolerance to heavy metals such as cadmium. These variations include (but are not limited to): several (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10, and more preferably 1 - 8 or 1-5) amino acid deletions, insertions and/or substitutions, and addition of one or several (usually within 20, preferably within 10, more preferably at the C-terminal and/or N-terminal within 5) amino acids. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding or subtracting one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the SpHMT1 protein.

Variant forms of polypeptides include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with SpHMT1 protein DNA under high or low stringency conditions, And the polypeptide or protein obtained by using anti-SpHMT1 protein antiserum. The present invention also provides other polypeptides, such as fusion proteins comprising SpHMT1 protein or fragments thereof. In addition to the nearly full-length polypeptides, the present invention also includes soluble fragments of the SpHMT1 protein. Typically, the fragment has at least about 20 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the SpHMT1 protein sequence. consecutive amino acids.

The present invention also provides analogs of SpHMT1 protein or polypeptide. The difference between these analogs and the natural SpHMT1 protein may be the difference in the amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modification also includes glycosylation. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, "SpHMT1 protein conservative variant polypeptide" means that compared with the amino acid sequence of GenBank accession number CAA78419, there are at most 30, preferably at most 20, more preferably at most 10, more preferably at most 5 , preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.

Table 1

amino acid residue representative replacement preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

Existing studies have found that homologous genes of SpHMT1 have been found in various other organisms, for example, the homologous genes DmHMT1 (EU571211) and CeHMT1 (AF497513) of SpHMT1 exist in Drosophila and nematode respectively. Obviously, the protein encoded by the homologous gene of SpHMT1 also has the same or close effect of SpHMT1 protein, and can also mediate the resistance and accumulation of Cd ²⁺ . Therefore, these homologous genes of SpHMT1 and their encoded proteins are also included in the present invention, and are used for improving the tolerance of plants to heavy metals and changing the distribution of heavy metals in plant tissues.

The present invention also provides a polynucleotide sequence encoding the SpHMT1 protein of the present invention or its conservative variant polypeptide.

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in GenBank accession number Z14055 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a protein with GenBank accession number CAA78419 but differs from the coding region sequence shown in GenBank accession number Z14055.

A polynucleotide encoding the mature polypeptide of GenBank Accession No. CAA78419 includes: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non- coding sequence.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .

The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 80%, compared Preferably at least 90% or more, more preferably 95% or more before hybridization occurs. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in GenBank accession number CAA78419.

The present invention also relates to nucleic acid fragments that hybridize to the above-mentioned sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides in length, more preferably at least 50 nucleotides in length, most preferably at least 100 nucleotides in length. The nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and/or isolate the polynucleotide encoding the SPHMT1 protein.

The use of HMT1 and its expression in plants

The invention provides the use of HMT1 for improving the tolerance of plants to heavy metals, or for preparing transgenic plants with strong tolerance to heavy metals, or for regulating the degree of accumulation of heavy metals in plant tissues. The HMT1 can transport heavy metals into the vacuole of plant cells, thereby improving the tolerance of plants to heavy metals.

The HMT1 (such as SpHMT1) can improve the tolerance of plants to heavy metals by expressing HMT1 in plants. The present inventors realized the expression (or overexpression) of HMT1 in plants for the first time.

Therefore, the present invention also provides an expression cassette for expressing HMT1 in plants. The expression cassette includes operatively linked regulatory elements and the coding sequence of HMT1, so that when it is transferred into the cell or integrated into the genome, the HMT1 protein can be recombinantly expressed.

The expression cassette includes a promoter operably linked to the HMT1 coding sequence. The promoter can be any promoter that can direct the expression of the HMT1 coding sequence in plant tissues, such as constitutive (such as CaMV35S promoter) or tissue-specific. Driven by the promoter, the expression of HMT1 protein can improve the tolerance of plants to heavy metals, enhance the survival ability of plants in heavy metal environments, and regulate the degree of heavy metal accumulation in plant tissues.

As a preferred mode of the present invention, the promoter is a plant tissue-specific expression promoter, and the expression cassette containing this promoter can regulate the degree of heavy metal accumulation in plant tissues when it is transferred into a plant and starts expression. The embodiments of the present invention have proved that a plant-specific tissue-specific expression promoter can drive the expression of HMT1 protein in a plant-specific tissue, promote the transport of heavy metals into the plant-specific tissue, and reduce the content of heavy metals in other plant tissues.

As a preferred mode of the present invention, the plants are crops, which include edible tissues that can be eaten by humans or non-human mammals and non-edible tissues that cannot be eaten. The promoter is a non-edible tissue-specific expression promoter of plants, thereby driving the expression of HMT1 protein in non-edible plant tissues, transporting heavy metals into non-edible plant tissues, and reducing the content of heavy metals in edible plant tissues . The definition of edible tissues and non-edible tissues of plants varies according to different plants, but it is easy for those skilled in the art or plant growers to judge.

The tissue specific expression promoter of plant can be various tissue specific promoters known in the art, such as plant root specific promoter Adh, NT1, ZmGLU1 promoter etc.; Plant stem specific promoter CAB2, C4H, RBCS1 Promoters, etc.; plant seed coat-specific promoters SCSP, SCFP promoters, etc.

In the present invention, the expression cassette of HMT1 protein can be inserted into the recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or kanamycin or ampicillin resistance for E. coli.

The present invention also relates to a method for improving crops, the method comprising expressing exogenous HMT1 protein in the plant. The protein transports heavy metals into the vacuole in cells, so that the plant has better tolerance to heavy metals, or increases the chlorophyll content in plant tissues under the stress of heavy metals.

Methods for HMT1 gene expression are well known in the art. Usually, plants can express HMT1 by introducing an expression cassette carrying a gene encoding HMT1.

As a preferred mode of the present invention, the method for obtaining a plant expressing HMT1 is as follows:

(1) providing an Agrobacterium carrying an expression vector, the expression vector containing an expression cassette of the HMT1 protein;

(2) contacting the plant cell or tissue or organ with the Agrobacterium in step (1), so that the expression cassette of the HMT1 protein is transferred into the plant cell and integrated into the chromosome of the plant cell;

(3) selecting plant cells or tissues transferred into the HMT1 protein expression cassette; and

(4) Regenerating the plant cells or tissues in step (3) into plants.

Wherein, any appropriate conventional means, including reagents, temperature, pressure conditions, etc., can be used to implement the method.

The main advantage of the present invention is that: HMT1 is successfully overexpressed in plants for the first time, and can improve the resistance and accumulation ability of plants to heavy metals such as cadmium. The tissue-specific expression of the HMT1 gene in the present invention can be excellently applied to the improvement of plant varieties, reduce the content of heavy metals such as cadmium in the edible parts of plants, increase the content of heavy metals such as cadmium in the aboveground parts of plants, and improve the quality of plants under heavy metal stress. Chlorophyll content in tissues. The invention provides very valuable gene resources and operation methods for developing new varieties of crops with higher food safety or developing new varieties of phytoremediation.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods that do not indicate specific conditions in the following examples, usually according to the conditions described in J. Sambrook et al., Molecular Cloning Experiment Guide, Science Press, 2002, or according to the conditions suggested by the manufacturer . Percentages and parts are by weight unless otherwise indicated.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can also be applied in the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

Example 1. Cloning and Transformation of Tonoplast Membrane Transporter Gene SpHMT1

The present inventors used fission yeast (S. pombe) cDNA as a template, through SpHMT1 cloning primers (SpHMT1(+)-PCR and SpHMT1(-)-PCR), and KOD-plus reaction system to amplify the 5'-UTR in SpHMT1 full-length cDNA within. At the same time, primers (K-SpHMT1(+)-PCR and K-SpHMT1(-)-PCR) were designed to clone the cDNA that replaced the 5'-UTR of yeast itself with the Kozak sequence conserved in higher plants. Primers were designed to mutate the stop codon of cDNA (from sequence TAA to TCA) and introduce SpeI restriction site. The cDNA was constructed into the 35S/TaPCS1:cmyc-pBI121 expression vector using the SpeI restriction site (references Jiming GONG et al., Long-distanceroot-to-shoot transport of phytochelatins and cadmium in Arabidopsis. (2003); PNAS, vol.100 , no.17, 10118-10123; constructed as follows: introduce wheat TaPCS1 containing BamHI/SpeI sites at both ends into pBluescript II SK, and introduce 3×myc tags into the SpeI/SacI sites of the vector; the above After the plasmid was digested with BamHI and SacI, the fusion DNA fragment was obtained, and inserted into the pBI121 vector to obtain 35S/TaPCS1:cmyc-pBI121) and to achieve fusion expression of the cymc protein tag thereafter.

Then it was tested whether the modified protein was still active in the yeast complementation experiment.

Gene cloning primer sequences:

SpHMT1(+)-PCR:

AGGATCCAAATTAGCAATTGAAGCAGTAACAA (SEQ ID NO: 1);

SpHMT1(-)-PCR:

AACTAGTGAATGAGTTTCAGCAGAAGTTTTT (SEQ ID NO: 2);

K-SpHMT1(+)-PCR:

AGGATCCACCATGGTTCTACGTTACAACAGCCC (SEQ ID NO: 3);

K-SpHMT1(-)-PCR (SEQ ID NO: 4):

AACTAGTGAATGAGTTTCAGCAGAGAGTTTTTT;

The primers for cloning SpHMT1 and K-SpHMT1 were synthesized by Invitrogen, USA. The KOD-plus high-fidelity enzyme (Toyobo) system was used for RT-RCR reaction and sequencing was provided by Shanghai Sunny Company.

Extraction method of yeast RNA:

Cultivate yeast to (1-5)×10 ⁸ or OD600=0.5 bacterial liquid. Cells were collected by centrifugation at 12000 g and suspended in 400 μL of AE buffer. Add 1/10 volume of 10% SDS, Vortex, plus an equal volume of phenol. Vortex, 65°C, 5 minutes. Quick-freeze in liquid nitrogen (phenol crystals appear, 3-5 seconds), centrifuge at 12,000 g for 2 minutes; transfer the supernatant to a new tube, add 1/2 volume of phenol and 1/2 volume of chloroform, and leave at room temperature for 5 minutes. 12000g, 10 minutes, transfer the supernatant to a new tube, add 1/10 volume of 3M sodium acetate (pH4-5) and 2.5 volumes of absolute ethanol, -20°C, 1 hour. 13000g, 15 minutes, discard the supernatant. Wash once with 70% ethanol, air dry, dissolve in 20 μL DEPC-treated water, and set aside.

High-fidelity PCR reaction and gel recovery:

KOD-plus (Toyobo) PCR reaction system is 50μL: 10×KOD-plus buffer 5μL, 25mM MgSO4 4μL, 2mM dNTPs 5μL, 10μM Primer(+) 2μL, 10μM Primer(-) 2μL, cDNA 5μL, KOD-plus 1μL , 26 μL of sterile deionized water. The reaction conditions of PCR: 95° C., 2 minutes (pre-denaturation); 95° C., 30 seconds; 58° C., 30 seconds; 68° C., 2 minutes and 30 seconds (34 cycles).

The PCR product was subjected to 1% agarose gel electrophoresis, under ultraviolet light, the gel containing the target DNA band was cut with a scalpel, and the DNA was recovered using a gel recovery kit (Axygen) (see the instructions of the Axygen kit for the recovery method).

Ligation reaction, heat shock transformation of E. coli competent and identification of positive clones:

The obtained DNA was recovered, and A addition reaction was performed. The system is 10 μL, DNA 7.9 μL, 2mM dATP 1 μL, 10×Ex Taq ^TM buffer (containing 2mM MgSO ₄ ) 1 μL, Ex Taq ^TM 0.1 μL. Add A reaction conditions: 72°C, 20 minutes.

The recovered product after adding A was ligated with pGEM-T Easy (Promega) vector. Ligation system: 7 μL of the recovered product after adding A, 1 μL of pGEM T-Easy, 1 μL of 10×T ₄ DNA ligation buffer, and 1 μL of T ₄ DNA ligase. 4°C, 12 hours.

The ligation product was heat-shock transformed into Escherichia coli competent cell TOP10, and the transformation solution was spread on an LB plate containing 100 μg/mL ampicillin at 37°C for 12 hours. Pick a single clone on the LB plate and transfer it to 2 mL of LB culture medium containing 100 μg/mL ampicillin, at 37° C., 200 rpm, for 12 hours. Take 1 mL of the culture product, extract the plasmid by alkaline lysis, and identify it by restriction digestion with BamHI. The positive clones were sent to the company for sequence determination (Shanghai Sunny Company). The sequence obtained by sequencing was compared with the SpHMT1 sequence in the NCBI database.

Construction of recombinant vectors for yeast expression and yeast transformation:

Recombinant pGEM-T Easy plasmid (Progema) with the correct sequence was digested with BamHI and SpeI, the target DNA was recovered, and ligated into the plant binary expression vector 35S/TaPCS1:cmyc-pBI121 and Adh/TaPCS1:cmyc-pBI121 (Adh promoter was used to replace promoter 35S in 35S/TaPCS1:cmyc-pBI121 to obtain Adh/TaPCS1:cmyc-pBI121). 35S/SpHMT1: cmyc-pBI121, 35S/Kozak-SpHMT1: cmyc-pBI121 and Adh/SpHMT1: cmyc-pBI121, Adh/Kozak-SpHMT1: cmyc-pBI121 obtained after identification (by using BamHI and SpeI restriction sites to SpHMT1 cDNA or Kozak-SpHMT1 replaced the TaPCS 1 cDNA in the vector, thereby realizing the fusion expression of cymc) the plasmid was double-digested with BamHI and SacI, and connected into the same-cut yeast expression vector pART1 (obtained from South China Botanical Garden, Chinese Academy of Sciences). The ligation product was heat-shock transformed into Escherichia coli competent TOP10, and the transformation solution was spread on an LB plate containing 100 μg/mL ampicillin at 37°C for 12 hours. Therefore, obtain the recombinant pART1 vector with SpHMT1 cDNA+5'UTR (abbreviated as HMT1), SpHMT1 cDNA+kozak (abbreviated as KHMT1) respectively.

Pick a single clone on the LB plate and transfer it to 2 mL of LB culture medium containing 100 μg/mL ampicillin, at 37° C., 200 rpm, for 12 hours. Take 1mL of the culture product, and extract the plasmid by alkaline lysis method. The recombinant plasmid is identified by BamHI and SacI double enzyme digestion, and the positive recombinant plasmid is transformed into the Δhmt1 mutant LK100 (cadmium-sensitive phenotype, see the literature Heavy metal tolerance in the fission yeast requires an ATP -binding cassette-type vacuolar membrane transporter (1992)) and the corresponding wild-type Sp223 (see literature above). Yeast transformation method refers to the literature A Simple and Efficient Procedure for Transformation of Yeasts.BioTechniques.13 (1992).

Single clones were picked from yeast transformants and cultured in liquid at 28°C, 200 rpm, for 12 hours, and the OD ₆₀₀ to 1 was measured using a spectrophotometer. Dilute with fresh culture medium to _OD600 of 0.5, culture at 28°C, 200rpm to _OD600 of 1. Take 1 mL of the culture solution, collect the cells by centrifugation, wash the cells with 1 mL of sterile deionized water, and resuspend the cells with 1×TE (pH 7.5). The resuspension was serially diluted to OD ₆₀₀ =0.1, OD ₆₀₀ =0.01. Take 10 μL of the resuspension solution and sow it on yeast culture plates with different metal concentrations, and culture at 28°C for 2-4 days.

The experimental results are shown in Figure 1. On the 50 μM CdCl ₂ plate, the growth of the Δhmt1 mutant LK100 (V/LK100) transformed with the empty vector has been significantly inhibited, while the growth of the Δhmt1 mutant LK100 (KHMT1/LK100, HMT1 /LK100) could still grow on 200μM CdCl ₂ plates and the growth was similar to that of the wild-type yeast (V/Sp223) transformed with the empty vector (Fig. 1A). The yeast growth curve experiment (Fig. 1B) under the more sensitive concentration gradient cadmium stress also obtained similar results.

Therefore, the inventors believe that both the SpHMT1 cDNA with the 5'-UTR and the kozak sequence can complement the cadmium-sensitive phenotype of the Δhmt1 mutant, wherein the SpHMT1 gene containing the 5'-UTR of the yeast itself is resistant to Cd ²⁺ closer to wild type. It also proved that the c-myc protein tag did not affect the function of the fusion protein, which laid the foundation for the overexpression of SpHMT1 in higher plants.

Example 2, the successful overexpression of yeast gene SpHMT1 in Arabidopsis

The preparation method of transgenic plants (35S:SpHMT1/Col-0; 35S:SpHMT1/cad1-3; Adh:SpHMT1/Col-0) is as follows:

The plasmid 35S/SpHMT1:cmyc-pBI121 constructed above was used to transform wild-type C01-0 and AtPCS1 deletion mutant cad1-3, respectively. The previously constructed plasmid Adh/SpHMT1:cmyc-pBI121 was transformed into wild-type Col-0. Arabidopsis transformation method refers to the literature Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. (1998)

In the shoots, leaves and roots of transgenic Arabidopsis plants 35S:SpHMT1/Col-0, Adh:SpHMT1/Col-0 and 35S:SpHMT1/cad1-3, the expression of SphMT1 was analyzed by RT-PCR.

RT-PCR primer sequences:

SpHMT1 (+) RT-PCR: TATCTTAAGCAAGAGCGGAAGG (SEQ ID NO: 5);

SpHMT1(-) RT-PCR: CTTTAAGCCTCTTTCTCCGACA (SEQ ID NO: 6);

AtACT1 (+) RT-PCR: CCCTGTTTCTTCTTACCGAG (SEQ ID NO: 7);

AtACT1(-) RT-PCR: CCACATCTGCTGGAATG (SEQ ID NO: 8).

Primers were synthesized by Invitrogen, USA. AtACT1 (AtActin1), a constitutively expressed control gene, is used for normalization purposes.

RT-PCR method:

Plant material was ground with liquid nitrogen, and total RNA was extracted using the Trizol kit (Invitrogen). For the specific extraction method, refer to the instructions of the Trizol kit.

Total RNA was treated with DNaseI at 37°C for 30 minutes. Extract with equal volume of phenol-chloroform-chloroform, precipitate with 0.1 volume of 3M sodium acetate (pH5.2) and 2 volumes of absolute ethanol, at -20°C for 30 minutes. 12000g, 4°C, 15 minutes. Wash the precipitate once with 1 mL of 70% ethanol, air-dry for 5 minutes, and dissolve the precipitate with 30 μL of DEPC-treated water to obtain DNA-free total RNA.

3 μg of total RNA samples were taken and reverse-transcribed using the reverse transcription system of M_MLV (Promega), respectively. For specific methods, refer to the instructions of the M_MLV kit of Promega Company.

RT-PCR reaction system is 20 μL: Ex Taq ^TM buffer (10×) (Takara) 2 μL, 25 mM dNTPs 1.6 μL, Ex Taq ^TM En 0.1 μL, 20 μM Primer (+) 0.5 μL, 20 μM Primer (-) 0.5 μL, cDNA Template 0.5 μL, sterile deionized water 14.8 μL.

RT-PCR reaction conditions: 95°C, 2 minutes (pre-denaturation); 95°C, 15 seconds; 58°C, 30 seconds; 72°C, 40 seconds (25 cycles for Atactin, 30 cycles for SpHMT1 gene).

PCR instrument: GeneAmp PCR System 9700 (Applied Biosystems).

The results are shown in Figure 2: the SpHMT1 gene was not detected in the stems, leaves and roots of the control wild-type Col-0. The SpHMT1 gene was expressed in the stems, leaves and roots of 35S:SpHMT1/Col-0 and 35S:SpHMT1/cad1-3 transgenic plants (Figure 2A, B); it was expressed in the roots of Adh:SpHMT1/Col-0 transgenic plants but Not expressed in shoots and leaves (Fig. 2C, D). The addition of Kozak sequence has nothing to do with whether the gene is expressed or not.

Therefore, the present inventors successfully expressed the yeast gene SpHMT1 in Arabidopsis, which is also the first time that the tonoplast membrane transporter HMT1 gene was successfully expressed in higher plants. The CaMV 35S promoter and Adh promoter were used to achieve the global and root-specific overexpression of the gene, respectively, which laid the foundation for the application of the gene in higher plants.

Example 3, SpHMT1 gene can enhance the resistance and accumulation of wild-type Arabidopsis to heavy metals, and its function depends on phytochelatin PCs

Heavy metal resistance plate test:

The T3 generation of transgenic plants and the corresponding control were vertically cultured on the 1/4×MS plate with different concentrations of heavy metals after the seed surface was sterilized. After about 4 weeks of growth, they were photographed and observed, and the fresh weight was counted.

Determination of heavy metal content:

After the heavy metal treatment, the plant material was washed twice with deionized water for 4 minutes each time, then washed twice with 25mMCaCl ₂ (PH4-5) for 4 minutes each time, and finally cleaned in Milli-Q water. 80°C, 48 hours. Weigh the dry weight, add 1 mL of 70% nitric acid (MOS grade), and let stand for 12 hours. Nitrolysis at 99°C for 2 hours, cooled to room temperature, diluted to 14 mL with Milli-Q water. ICP-MS (Perkin-Elmer ELAN DRC-e) was used to determine the content of heavy metals in the samples.

In order to study whether the overexpression of SpHMT1 in plants can enhance their resistance to heavy metals, the identified 35S:SpHMT1/Col-0 transgenic plant seeds were surface-sterilized and cultured vertically on a plate supplemented with 50 μM CdCl ₂ , and observed after about 4 weeks It was found that the transgenic plants grew better than wild-type Col-0 (Fig. 3A): their leaves were larger and greener, their roots were longer and their fresh weight was significantly higher than that of the control (Fig. 3E). In addition, the chlorophyll content of the transgenic plants was significantly higher than that of the control after the seedlings cultured in water for 4 weeks were treated with 10 μM CdCl ₂ for 7 days. However, the phenotype and fresh weight of the transgenic plants had no significant difference from the control when the seedlings grew for 7 days on the common 1/2×MS plate. It proved that overexpression of SpHMT1 in Arabidopsis can indeed enhance its resistance to heavy metal cadmium. The resistance of transgenic plants to other heavy metals was detected by the same method, and it was found that on the heavy metal plates added with 150 μM KH ₂ AsO ₄ (Figure 3B), 40 μM CuSO ₄ (Figure 3C) or 150 μM ZnSO ₄ (Figure 3D), the Growth and fresh weight (Fig. 3E) were better than the control. Therefore, it can be seen that overexpressing the yeast gene SpHMT1 in wild-type Arabidopsis can enhance the plant's resistance to some heavy metals including cadmium.

In order to study the mechanism of action of SpHMT1 in plants, the present inventors detected the content of heavy metals in plants in the resistant plate shown in Figure 3 . When converted to fresh weight, the cadmium concentration in 35S:SpHMT1/Col-0 transgenic seedlings was significantly higher than that in the control. In the two transgenic lines tested, the concentration of Cd ²⁺ was 139% and 142% of the control, respectively (Fig. 3F). As shown in Figure 3E, the fresh weight of the transgenic plants under Cd ²⁺ stress was higher than that of the control, so the Cd ²⁺ content in the seedlings of the transgenic plants was also significantly higher than that of the control, about 1.7-1.9 times that of the control. Similar to the case of cadmium, the concentrations and contents of arsenic, copper and zinc in 35S:SpHMT1/Col-0 transgenic seedlings were also significantly higher than those in the control (Fig. 3F). Therefore, it can be seen that SpHMT1 can enhance the resistance and accumulation of heavy metals in plants, not by efflux of heavy metals, but by transporting heavy metals or their complexes into organs such as vacuoles for storage.

In order to further study whether the detoxification mechanism of SpHMT1 is related to glutathione (Glutataione, GSH for short) or phytochelatins (Phytochelatins, PCs for short), the inventors used the GSH inhibitor buthionine-sulfoximine (L -buthionine sulfoximine, referred to as BSO) to treat transgenic plants. 35S:SpHMT1/Col-0 transgenic plants were vertically cultured on 1/4×MS plates supplemented with 0.5mM BSO for 20 days, and it was found that the plant growth was somewhat inhibited, but there was no significant difference between the transgenic plants and the control (Col-0) ( Figure 4B,C). On the 1/4×MS plate supplemented with 10 μM CdCl ₂ and 0.5 mM BSO at the same time, the phenotype and fresh weight of the transgenic plants were also similar to those of the control (Figure 4A, C), that is, the resistance of the transgenic plants to the heavy metal cadmium was lost. In addition, the present inventors also tested the seedlings at the mature stage: after the seedlings were hydrocultured for 4 weeks, they were treated with 10 μM CdCl ₂ and 0.25 mM BSO for 3 days, and the cadmium content in the roots, stems and rosette leaves were measured respectively. After conversion to dry weight, the cadmium concentration in each part of the transgenic plants was not significantly different from that of the control (Fig. 4D). These results indicate that the ability of SpHMT1 to enhance the resistance and accumulation of heavy metal cadmium is inhibited by BSO, suggesting that the role of SpHMT1 is related to GSH or PCs that rely on it as a substrate for synthesis.

Because BSO inhibits the synthesis of both GSH and PCs, in order to further study whether the function of SpHMT1 depends on GSH or PCs, or which one is the main transport substrate, the inventors overexpressed SpHMT1 in the PCs deletion mutant cad1-3. According to the reference Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient (1995), the cad1-3 mutant is a loss-of-function mutant of the Arabidopsis AtPCS1 gene, in which the content of PCs cannot be detected. As described above, the inventors observed the resistance and accumulation of heavy metal cadmium in 35S:SpHMT1/cad1-3 transgenic plants. It was found that the phenotypes of the transgenic plants 35S:SpHMT1/cad1-3 were different whether the seedlings grown for 7 days on the ordinary 1/2×MS plate or the seedlings grown for 20 days on the resistant plate added with 5 μM CdCl ₂ (Fig. 5A, B ) and fresh weight (Fig. 5C) were not significantly different from the control plant cad1-3, indicating that the overexpression of SpHMT1 in the Arabidopsis PCs deletion mutant cad1-3 could not enhance heavy metal resistance. The cadmium content of the plate seedlings in Figure 5A and B was measured, and it was found that the cadmium content of the 35S:SpHMT1/cad1-3 transgenic plants in the cadmium plate was not significantly different from that of the control plants (PCs deletion mutant cad1-3) (Figure 5D ).

In summary, the expression of SpHMT1 in wild-type Arabidopsis can enhance the plant's resistance and accumulation of heavy metals such as cadmium, copper, zinc and arsenic, while the overexpression of SpHMT1 in cad1-3 has no effect, so it is suggested that The action of SpHMT1 is dependent on PCs.

Example 4, PCs play a central role in the transport of heavy metal cadmium across the tonoplast membrane

In this example, in order to further determine whether the enhanced heavy metal resistance mediated by SpHMT1 is due to the transfer and storage of more PCs-Cd complexes in the vacuole, and to study the role of PCs-mediated cadmium transport in Arabidopsis thaliana resistance to cadmium Whether it plays a dominant role in the detoxification mechanism, the inventors isolated Col-0 and cad1-3, and their corresponding protoplasts and vacuoles of SpHMT1 overexpressed plants, and detected the concentration and presence of cadmium in them.

Vacuole extraction method:

The seedlings cultivated in soil for 2 weeks were treated with CdCl ₂ for a week, that is, the seedlings were poured twice with 500ml of 40 μM CdCl ₂ solution. Protoplasts were extracted from rosette leaves of unbolted seedlings. The obtained protoplasts were divided into two equal parts, one was used for the detection of cadmium and PCs content, and the other was used to extract vacuoles according to the method of reference Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplasts (2007). The extracted vacuoles were observed under a light microscope, and it was found that the purity was >95% and there was no protoplast contamination. The chlorophyll content and the enzyme activities of cytochrome C oxidase (COX) and acid phosphatase (ACP) (enzyme activity assay kit from GenMed Sci Inc) were detected in protoplasts and vacuoles to identify the purity of the vacuoles.

Determination of PCs content:

The contents of PCs and GSH in the vacuoles of Cd-treated plants were determined by fluorescent HPLC (Agilent 1200 series), using the reference Separation and quantification of monothiols and phytochelatins from a wide variety of cell cultures and tissues of trees and other plants using high performance liquid chromatography (2008) The reversed-phase C18 column (Agilent 300Extend-C18 4.6×150mm, 3.5μm particle size) used in the experiment. The thiol group was fluorescently labeled with monobromobimane according to the method of reference Long-distance root-to-shoot transport of phytochelatins and cadmium in Arabidopsis (2003). The standard PCs PC2, PC3 and PC4 were synthesized from AnaSpec. GSH standard was purchased from sigma company. The cadmium, PCs and GSH contents in the vacuole were all normalized by the enzyme activity of plant acid phosphatase (ACP).

The results are shown in Figure 6, whether in 35S:SpHMT1/Col-0 transgenic plants or in wild-type Col-0, most of the cadmium is stored in the vacuoles, and the ratio of cadmium content in the vacuoles and protoplasts (V /P ratio) were 101.39%±1.02% and 97.49%±1.53%, respectively. However, the cadmium content was significantly higher in the protoplasts and vacuoles of 35S:SpHMT1/Col-0 transgenic plants than in Col-0.

On the other hand, the content of PCs (including PC2, PC3 and PC4) in the vacuoles of 35S:SpHMT1/Col-0 transgenic plants was higher than that of wild-type plants, as shown in Figure 7. Therefore, although the expression of SpHMT1 hardly increased the V/P ratio, it enhanced the PCs-mediated ability to transport and accumulate Cd to the vacuole, thereby enhancing the resistance and accumulation of heavy metals in plants.

On the other hand, there was no significant difference in the cadmium content in the protoplasts and vacuoles of the cad1-3 mutant and its transgenic plants, as shown in Figure 6. Although the distribution ratio of Cd inside and outside the vacuole was similar in cad1-3 and 35S::SpHMT1/cad1-3 transgenic plants and far lower than that of wild-type Col-0, it was still 35.09%±0.21% and 35.14%±0.41%, respectively. % of cadmium is stored in vacuoles, possibly in the form of GSH-Cd or Cd-S. This ratio also suggested that although plants could still transport cadmium into vacuolar storage in the absence of PCs, the ratio was low, which may be the reason for the hypersensitivity of cad1-3 to cadmium.

Therefore, the inventors believe that in the Arabidopsis cadmium detoxification mechanism, the pathway mediated by cadmium in the form of PCs-Cd to the vacuole plays a major role, that is, the role of PCs is not limited to chelation, but more importantly, the role of Cd to the vacuole transport and storage. Although the expression of SpHMT1 hardly increased the V/P ratio, it enhanced the ability to transport and accumulate cadmium to the vacuole, thereby enhancing the resistance and accumulation of heavy metal cadmium in plants.

The root-specific expression of embodiment 5, SpHMT1 can reduce the cadmium content in aerial part and seed

Because the global expression of the 35S promoter limits the practicability of transgenic plants, the inventors constructed the SpHMT1 gene into a vector under the regulation of the root-specific expression promoter Adh and transformed it into Col-0 (Adh:SpHMT1/Col-0). As shown in Figure 8, under the treatment of CdCl ₂ , the content of chlorophyll in the rosette leaves of Adh:SpHMT1/Col-0 transgenic plants was higher than that of Col-0, that is, the root-specific expression of the transgene also enhanced the tolerance of plants to Cd ²⁺ . As shown in Figure 9, after 4 weeks of hydroponic seedlings were treated with 10 μM CdCl ₂ for 3 days, compared with the control, the Adh:SpHMT1/Col-0 transgenic plants accumulated more cadmium in the root, while the cadmium in the aerial part was much lower than that of the control ( Figure 9A). In order to detect the cadmium content in the seeds, the inventors treated the newly bolted hydroponic seedlings with 5 μM CdCl _for a long time until the seeds matured, and found that the cadmium content in the 35S:SpHMT1/Col-0 transgenic plant seeds was higher than that of the control Col-0, while The cadmium content in seeds of Adh:SpHMT1/Col-0 transgenic plants was significantly reduced (Fig. 9D). The same method was used to treat the transgenic seedlings with copper and arsenic, and it was found that the contents of copper and arsenic in the aerial parts and seeds of the transgenic seedlings were also significantly lower than those of the wild type (Fig. 9B, C, E, F). These results indicated that the root-specific expression of SpHMT1 transgenic plants could limit more heavy metals such as cadmium, copper and arsenic to the roots, thereby reducing the heavy metal content in the aerial parts and seeds.

As shown in Figure 8, after 10 μM CdCl ₂ treatment for 7 days, the chlorophyll content in the rosette leaves of the plants all decreased, while the chlorophyll content of the root-specific expression transgenic plants was significantly higher than that of the wild type (Control, Col-0), indicating that it is consistent with the global Consistent with the results of sexual overexpression, the root-specific expression of SpHMT1 can also enhance the tolerance of Col-0 to the heavy metal cadmium. Since Cd stress will reduce chlorophyll content, chlorophyll content is recognized as an important indicator for detecting plant tolerance to Cd (references: Van Assche F and ClijstersC. (1990) Effects of metals on enzyme activity in plants. Plant Cell Environ .13: 195-206; Somashekaraiah, BV, Padmaja, K., Prasad, ARK (1992) Phytotoxicity of cadmium ions on germinating seedlings of mung bean (Phaseolus vulgaris): involvement of lipid peroxides in chlorophyll degradation. Plant Physiol. 85: 85 -89.).

On the other hand, the inventors specifically expressed SpHMT1 in the aerial parts of Arabidopsis thaliana under the drive of the CAB2 promoter (the preparation of transgenic plants is the same as above, the difference is that the promoter 35S in the 35S/SpHMT1:cmyc-pBI121 plasmid was replaced CAB2 promoter, CAB2 promoter reference An improved grafting technique for mature Arabidopsis plantsdemonstrates long-distance shoot-to-root transport of phytochelatins in Arabidopsis.Plant Physiol (2006)), obtain CAB2:SpHMT1/Col-0 transgenic Arabidopsis mustard. As shown in Figure 10, after 4 weeks of hydroponic seedlings treated with 10 μM CdCl ₂ for 3 days, compared with the control, the CAB2:SpHMT1/Col-0 transgenic plants accumulated more cadmium in the rosette leaves, while the cadmium in the roots was significantly lower than that of the control (FIG. 10A). In order to detect the cadmium content in the seeds, the inventor treated the newly bolted hydroponic seedlings with 5 μM CdCl _for a long time until the seeds matured, and measured the cadmium content in the seeds of CAB2:SpHMT1/Col-0 and 35S:SpHMT1/Col-0 transgenic plants Both were higher than the control Col-0, and there was no significant difference in the cadmium content in the seeds of the two transgenic plants (Fig. 10B). The specific expression of SpHMT1 in aerial parts may play a role in phytoremediation.

In phytoremediation, it is hoped to obtain plants that are highly tolerant to heavy metals and can accumulate more heavy metals in the above-ground parts, while in the production of food crops, it is hoped that more heavy metals can be controlled in the roots, thereby reducing the number of fruits on the ground. and content in seeds. Therefore, the root-specific expression of the SpHMT1 gene has practical application value, and can be applied to the improvement of plant varieties, reducing the content of heavy metals such as cadmium in edible parts of plants, and improving new gene resources for the development of new varieties of crops with higher safety. and how to do it. The specific expression of the above-ground part of the SpHMT1 gene also has practical application value and can be used for phytoremediation and the like.

In conclusion, the present inventors studied the function of SpHMT1 in plants by ectopically overexpressing the yeast vacuolar membrane transporter SpHMT1 gene in Arabidopsis, and explored the position and role of PCs transport pathway in the mechanism of cadmium detoxification. The resistance and accumulation ability of transgenic plants to cadmium, zinc, copper, arsenic and other heavy metals were significantly enhanced, and this effect was inhibited by BSO. However, overexpression of SpHMT1 in the PCs-deleted mutant cad1-3 has no effect, indicating that the function of SpHMT1 in plants depends on the synthesis of PCs, that is, the main transport substrate of SpHMT1 in the presence of PCs pathway should be PCs-Cd instead of GS ₂ -Cd.

In addition, in the wild type, almost 100% of the cadmium in the protoplast accumulates in the vacuole, while the ratio in cad1-3 is only about 35%, indicating that the hypersensitive phenotype of cad1-3 to cadmium is mainly because the cadmium complex cannot are efficiently transported into the vacuole. At the same time, it also shows that both PCs and GSH play a role in the transport of heavy metals across the tonoplast membrane in higher plants, but PCs occupy the core position. At the same time, the inventors have shown through genetic engineering methods that more heavy metals such as cadmium can be retained in the roots by using the directional expression of SpHMT1, which can effectively reduce the migration of heavy metals to edible parts, thereby reducing the content of heavy metals such as cadmium in edible parts of plants , providing new genetic resources and operation methods for the development of new crop varieties with higher safety.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (12)

1. the purposes of a vacuole skin translocator HMT1 is used for improving plant to the chlorophyll content of plant under the tolerance of heavy metal, the fixed direction allocation of regulating heavy metal or the raising heavy metal stress.

2. purposes as claimed in claim 1 is characterized in that, described vacuole skin translocator HMT1 is used for transporting heavy metal in the vacuole of vegetable cell.

3. purposes as claimed in claim 1 is characterized in that, described heavy metal comprises: cadmium, arsenic, copper or zinc.

One kind improve plant for the tolerance of heavy metal, regulate heavy metal accumulation degree in the plant tissue or improve the method for the chlorophyll content of plant under the heavy metal stress, it is characterized in that described method comprises: in plant, express vacuole skin translocator HMT1.

5. method as claimed in claim 4 is characterized in that, described method comprises: the expression cassette of vacuole skin translocator HMT1 is changed in the plant, thereby express vacuole skin translocator HMT1 in plant.

6. method as claimed in claim 5 is characterized in that, described method comprises:

(1) provide the Agrobacterium of carrying expression vector, described expression vector contains the expression cassette of vacuole skin translocator HMT1;

(2) vegetable cell or tissue or organ are contacted with Agrobacterium in the step (1), thereby make the expression cassette of vacuole skin translocator HMT1 change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;

(3) select vegetable cell or tissue or the organ of the expression cassette that has changed vacuole skin translocator HMT1 over to; With

(4) vegetable cell in the step (3) or tissue or neomorph are become plant.

7. method as claimed in claim 4 is characterized in that, heavy metal accumulation degree methods comprises in the described adjusting plant tissue:

The expression cassette of vacuole skin translocator HMT1 is changed in the plant, and the expression cassette of this vacuole skin translocator HMT1 comprises the promotor of operability connection, the polynucleotide of coding vacuole skin translocator HMT1;

Wherein, described promotor is plant particular organization specific expressing promoter, expresses in plant particular organization thereby drive vacuole skin translocator HMT1, promotes heavy metal to transport into this plant particular organization.

8. method as claimed in claim 7 is characterized in that, described plant is the plant that comprises edible tissues, and described promotor is the non-edible tissue specific expressing promoter of plant, thereby heavy metal is transported non-edible tissue into plant.

9. method as claimed in claim 8 is characterized in that, described non-edible tissue specific expressing promoter is the roots of plants specific expressing promoter;

Preferably, described root-specific expression promotor comprises: Adh promotor, NT1 promotor, ZmGLU1 promotor.

10. method as claimed in claim 7 is characterized in that, described promotor is the specificity promoter that plant shoot divides tissue.

11. the expression cassette of a vacuole skin translocator HMT1, it comprises the promotor of operability connection, the polynucleotide of coding vacuole skin translocator HMT1; Wherein, described promotor is plant particular organization specific expressing promoter.

12. the purposes of the expression cassette of the described vacuole skin translocator of claim 11 HMT1 is used for regulating plant tissue heavy metal accumulation degree, promotes heavy metal to transport into this plant particular organization.

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