CN120966789A - A cadmium-tolerant protein in Dendrobium officinale, its encoding gene, and its applications. - Google Patents

Abstract

The invention discloses a dendrobium candidum cadmium-resistant protein, a coding gene and application thereof, wherein the protein has an amino acid sequence shown as SEQ ID NO.2, the gene has a nucleotide sequence shown as SEQ ID NO.1, and the cadmium-resistant protein or the coding gene thereof is used for improving cadmium resistance of plants or microorganisms. The invention provides a multifunctional dendrobium candidum cadmium-resistant protein and a coding gene thereof, which can obviously improve cadmium-resistant capability of plants and microorganisms, reduce cadmium accumulation, cultivate low-cadmium accumulation plant varieties, regulate and control a plant antioxidant system to reduce cadmium-induced active oxygen accumulation, strengthen plant root growth and biomass, and express in yeast to strengthen cadmium resistance, thus providing brand-new gene resources and application directions for cadmium pollution treatment and low-cadmium plant variety cultivation.

Description

Dendrobium candidum cadmium-resistant protein, and encoding gene and application thereof

Technical Field

The invention relates to the technical fields of plant molecular biology and genetic engineering, in particular to a cadmium-resistant protein DoGST derived from dendrobium candidum (Dendrobium officinale) and a coding gene thereof, and application thereof in improving cadmium resistance of plants or microorganisms, reducing cadmium accumulation and cultivating low-cadmium varieties. In addition, the interaction mechanism of the protein and heat shock protein S5M5N9 and the function verification method thereof in cadmium stress response are also related.

Background

Cadmium (Cd) is a non-essential and extremely toxic heavy metal element that is easily absorbed by plant roots and enriched in vivo, causing multiple hazards to plant growth and development, including:

a. Root damage, namely inhibiting root tip cell division, preventing root elongation and interfering mineral element absorption;

b. The photosynthetic system is destroyed, namely the chloroplast structure is destroyed, so that the stomata are closed, and the photosynthetic efficiency is reduced;

c. Oxidative stress, which induces excessive accumulation of Reactive Oxygen Species (ROS), leading to membrane lipid peroxidation and apoptosis;

d. Agricultural product safety risks that cadmium is transmitted through the food chain, threatening human health (e.g. "pain").

Traditional cadmium pollution treatment depends on soil restoration or agricultural measures (such as passivating agent application), but has the problems of high cost, unstable effect and the like. Therefore, the development of cadmium-resistant and low-cadmium accumulation crop varieties through molecular breeding becomes a key strategy for agricultural sustainable development.

Glutathione S-transferase (GST) is a core enzyme of plant detoxification systems, and can reduce heavy metal toxicity by catalyzing the combination of Glutathione (GSH) and electrophilic compounds. Studies have shown that GST is involved in cadmium stress response in plants such as rice and Arabidopsis, but the functional mechanism is mostly limited to model plants, and the functional differences of GST family members among different species are obvious. Dendrobium officinale is used as a rare medicinal orchid plant, the problem of cadmium pollution of a culture medium is increasingly prominent, and the industrial healthy development is severely restricted.

However, no functional research on dendrobium candidum GST family members in cadmium detoxification exists at present, and especially the system mining and mechanism analysis of cadmium-resistant key genes of dendrobium candidum GST family members is lacking.

Disclosure of Invention

In view of the above, the invention aims to clone and functionally identify a cadmium stress response key gene DoGST from dendrobium candidum, and define the mechanism that the coded protein thereof obviously improves the tolerance of plants and microorganisms to cadmium and reduces cadmium accumulation by reducing active oxygen accumulation, enhancing root growth, forming a compound with heat shock protein S5M5N9 and the like, thereby utilizing the gene and the interaction protein thereof to develop new varieties and biological preparations of cadmium resistance and low cadmium, and providing core gene resources and technical means for safe production and ecological restoration of heavy metal contaminated area medicinal materials.

The inventor continuously reforms and innovates through long-term exploration and trial and repeated experiments and efforts, and in order to solve the technical problems, the technical scheme provided by the invention is that the dendrobium candidum cadmium-resistant protein is provided, and the protein has an amino acid sequence shown as SEQ ID NO. 2.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides the cadmium-resistant protein DoGST from dendrobium candidum for the first time, can be directly used for improving the cadmium resistance of plants/microorganisms and reducing cadmium accumulation, and breaks through the limitation that the prior art only depends on model plants and lacks medicinal plant gene resources.

The invention also provides a gene for encoding the cadmium-resistant protein, which has a nucleotide sequence shown as SEQ ID NO. 1.

Compared with the prior art, the invention has the beneficial effects that:

The invention provides the dendrobium candidum DoGST gene which can be directly used for genetic transformation and can efficiently improve the cadmium resistance of plants for the first time, fills up the blank of the cadmium resistance gene resource of medicinal plants, and breaks through the limitation of lacking effective low-cadmium breeding targets in the prior art.

The invention also provides application of the cadmium-resistant protein or the coding gene thereof, which is used for improving cadmium resistance of plants or microorganisms.

The invention also provides application of the cadmium-resistant protein or the coding gene thereof, which is used for reducing cadmium accumulation in plants or microorganisms.

The invention also provides application of the cadmium-resistant protein or the coding gene thereof in cultivating plant varieties with low cadmium accumulation.

The invention also provides application of the cadmium-resistant protein or the coding gene thereof, which is used for regulating and controlling a plant antioxidant system and reducing Cd-induced ROS accumulation.

The invention also provides application of the cadmium-resistant protein or the coding gene thereof, which is used for enhancing plant root growth and improving biomass under Cd stress.

The invention also provides application of the cadmium-resistant protein or the coding gene thereof, which is used for expressing in yeast to enhance the Cd stress tolerance.

Compared with the prior art, the invention has the beneficial effects that the multifunctional dendrobium candidum cadmium-resistant protein and the coding gene thereof are provided, the cadmium-resistant capability of plants and microorganisms can be obviously improved, the cadmium accumulation is reduced, the low-cadmium accumulation plant varieties are cultivated, the plant antioxidation system is regulated and controlled to reduce the cadmium-induced active oxygen accumulation, the plant root growth and biomass are enhanced, and the expression in yeast is carried out to enhance the cadmium resistance, so that brand-new gene resources and application directions are provided for cadmium pollution treatment and low-cadmium plant variety cultivation.

The invention also provides a method for improving cadmium tolerance of plants, which comprises introducing and expressing the genes into plants.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a method for improving cadmium tolerance of plants with high efficiency and a definite mechanism, which remarkably enhances the tolerance of the plants under cadmium stress, reduces the accumulation of cadmium and improves the growth performance (such as root growth and biomass) of the plants by introducing and expressing DoGST genes into the plants. The method not only provides a direct technical means for cultivating plant varieties with low cadmium accumulation, but also provides a new solution for the agricultural production and ecological environment restoration for coping with cadmium pollution.

The invention also provides a protein complex, which comprises the cadmium-resistant protein and heat shock protein S5M5N9.

Compared with the prior art, the invention has the beneficial effects that:

The invention not only enriches the understanding of the plant cadmium stress response mechanism, but also provides a brand new target point for developing a cadmium-resistant strategy based on protein interaction. The discovery of the compound provides a more accurate molecular tool for cultivating cadmium-resistant plant varieties and reducing cadmium accumulation, and has important theoretical and application values.

The invention also provides an expression vector, which comprises the gene, wherein the vector is any one of a plant expression vector, a yeast expression vector or a prokaryotic expression vector.

Compared with the prior art, the invention has the beneficial effects that:

The multifunctional expression vector provided by the invention can be compatible with plants, yeasts and prokaryotic expression systems, greatly expands the application range of the cadmium-resistant gene DoGST, can be used for plant cadmium resistance research and variety improvement, can be used for microbial cadmium resistance mechanism exploration and related biotechnology application, and remarkably improves the practicability and economic value of the gene.

The invention also provides a host cell which comprises the expression vector, wherein the host cell is one of plant cells, yeast cells, escherichia coli or agrobacterium.

Compared with the prior art, the invention has the beneficial effects that:

The multifunctional host cell system provided by the invention covers plant cells, yeast cells, escherichia coli and agrobacterium, greatly expands the application potential of the gene in different biological systems, provides a diversified experimental platform for cadmium-resistant mechanism research, gene function verification and subsequent genetic transformation and biotechnology application, and remarkably improves the utilization efficiency and transformation value of the gene.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows DoGST protein characterization and structural analysis. In FIG. 1, panel A shows the predicted result of DoGST protein secondary structure, panel B shows the predicted protein signal peptide of DoGST, and Panel C shows the analysis of DoGST transmembrane domain.

FIG. 2 is a diagram showing the expression pattern of DoGST18 in response to Cd stress in different tissues of Dendrobium officinale. In FIG. 2, CK, C2, C5, C9 and C14 represent cadmium treatments for 0, 2, 5, 9 and 14 days, respectively, and S1-S15 represent 15 samples.

FIG. 3 is a DoGST subcellular localization analysis. In fig. 3, panel a shows the subcellular localization of DoGST-GFP in tobacco leaves, panel B shows 35s: GFP empty control in tobacco leaves, scale ratio = 20 μm.

FIG. 4 is a DoGST over-expressed Arabidopsis thaliana Cd-resistant phenotype. In FIG. 4, the difference in phenotype was observed and photographed after culturing WT and overexpressing Arabidopsis on 1/2MS solid medium of 0 and 60. Mu.M CdCl ₂ for 14 days, scale 1 cm.

FIG. 5 is a graph showing the root length and fresh weight measurements of DoGST A.thaliana overexpressed under Cd stress. In FIG. 5, the measured data were sampled after incubation of WT and overexpressing Arabidopsis on 1/2MS solid medium at 0 and 60. Mu.M CdCl ₂ for 14 days, with the different lower case letters representing significant differences and P <0.05.

FIG. 6 is DoGST that overexpression of Arabidopsis thaliana reduced Cd stress-induced excessive ROS levels. In FIG. 6, A and B are H ₂O₂ and.O ^2- levels, respectively, of DoGST18 overexpressing and WT plants under normal growth and Cd treatment. WT and overexpressing Arabidopsis were cultured on 1/2 MS solid medium of 0 and 60. Mu.M CdCl ₂ for 14 days and the assay data were sampled. * For significant differences, P <0.01, P <0.001.

FIG. 7 is a graph showing the result of cadmium tolerance analysis of recombinant Saccharomyces cerevisiae. In FIG. 7, spots of control and recombinant Saccharomyces cerevisiae were determined on SG-Ura plates and SG-Ura plates with CdCl ₂ (10. Mu.M, 20. Mu.M, 30. Mu.M, 40. Mu.M, 50. Mu.M).

FIG. 8 is a SDS-PAGE analysis. In FIG. 8, lane M is Maker, lane 1 is pMAL-DoGST protein, lane 2 is cadmium treated Dendrobium candidum total protein, lane 3 is MBP control and cadmium treated Dendrobium candidum total protein, lane4 is pMAL-DoGST protein and cadmium treated Dendrobium candidum total protein.

FIG. 9 is a yeast two-hybrid assay DoGST for interaction with S5M5N 9.

Fig. 10 is the interaction of DoGST and S5M5N9 in tobacco.

Detailed Description

The following description is made with reference to specific embodiments.

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention. Thus, the following detailed description of the embodiments of the invention is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention.

In the present invention, all the equipment and materials are commercially available or commonly used in the industry, and the methods in the examples described below are conventional in the art unless otherwise specified.

Based on the transcription group data of the dendrobium candidum root system and the response of the transcription level of GST family members to Cd in leaves, the inventor selects GST family gene DoGST (the gene sequence is shown as Seq ID NO.1, the protein sequence is shown as Seq ID NO. 2) which is obviously induced to express by Cd in the leaves and the root system, and the expression quantity is firstly increased and then decreased to the Cd response, as a research object, analyzes the characteristics and the space-time expression mode of the gene, further analyzes the Cd tolerance function and mechanism on the basis, and the research result can lay a theoretical foundation for creating new varieties of dendrobium candidum with low-cadmium absorption and accumulation through genetic improvement means in the future.

In this example, the primer information used is as follows:

fluorescence quantification pcr DoGST-QPCR-F/DoGST-QPCR-R, doACTIN-QPCR-F/DoACTIN-QPCR-R.

Arabidopsis thaliana transgene DoGST-OX-F/DoGST-OX-R.

Yeast double hybrid S5M5N9-AD-F/S5M5N9-AD-R DoGST-BD-F/DoGST-BD-R.

In this example, a conventional primer design method was used to design a specific primer to amplify a target gene based on the SEQ ID NO. 1 sequence and the cleavage site of the expression vector (e.g., bamHI/SalI sites of pCAMBIA 1300), and ligate to the corresponding vector.

In this example, selfing seeds of three-year pig iron dendrobium candidum are collected from a biological industrial base of mountain country dendrobium candidum (28.37 degrees North latitude and 121.03 degrees east longitude; qing, zhejiang province, china) of Yan Liang Zhang, uniformly sown on an MS culture medium containing 100 mL for cultivation (0.5 g.L ^-1NAA,7 g·L^-1 agar and 30 g.L ^-1 sucrose, pH value is 5.8-6.0), and a tissue culture chamber (14/10 h light/dark cycle, 60 mu mol.m ⁻²·s⁻¹, humidity 75%, 25+ -1 ℃). After 180 d growth, seedlings were transferred to MS medium supplemented with different concentrations of CdSO ₄, with CdSO ₄ concentrations set to 0 (Control), 2, 5, 9 and 14 mg.L ^-1.

Protein characterization of glutathione transferase was predicted using ProtParam tool (http:// us. Expasy. Org/tools/ProtParam. Html).

Protein secondary structure prediction was performed using NPS SOFMA server (http:// npsa-pbil. Ibcp. Fr/cgi-bin/npsa _automation. Plpage= npsa _sopma. Html).

Protein tertiary structure prediction was performed using SWISS MODEL (http:// npsa-pbil. Ibcp. Fr/cgi-bin/npsa _automation. Plpage= npsa _sopma. Html).

Signal peptide prediction was performed using SignalP 5.0 server (SignalP-5.0-Services-DTU HEALTH TECH).

The transmembrane structure prediction used TMHMM 2.0 server (TMHMM-2.0-Services-DTU HEALTH TECH).

Subcellular localization prediction was performed using WOLFPSORT prediction (Wolf PSORT: protein Subcellular Localization Prediction (hgc. Jp)).

ProtParam results showed that DoGST18 had a number of amino acids of 197, a molecular weight of about 22. 22 kDa, an isoelectric point of 8.70, a theoretical molecular formula of C ₁₀₆₁H₁₆₃₂N₂₆₆O₂₈₉S₂, an absorbance Abs of 0.1% (1 g. L ^-1) of 1.669 measured against water at 280 nm, an instability index (instability index II) of 33.28, indicating stable properties of the protein, an aliphatic coefficient of 102.33, a total average hydrophilicity value of-0.354, a total number of GST negative charges (Asp+Glu) of 29, and a total number of positive charges (Arg+Lys) of 32. Further systematic analysis of DoGST18 revealed that DoGST a-helix 56.85%, random coil 28.93%, extended strand 10.15%, no signal peptide, no transmembrane structure (A, B, C in fig. 1).

RNA extraction

The Trizol method is adopted to extract the total RNA of the dendrobium candidum, and the method is as follows:

(1) Precooling the mortar with liquid nitrogen, putting a plant sample into the mortar, adding liquid nitrogen, grinding, and transferring to a 1.5 mL RNase-free EP tube;

(2) Adding Trizol into the EP pipe according to 50-100 mg.L ^-1 Trizol, shaking and mixing uniformly, and standing at room temperature for 5min to fully crack;

(3) Adding chloroform, specifically 200 mu L/mL ^-1 Trizol, shaking to mix, and standing at room temperature for 10: 10 min;

(4) Centrifugation was performed in a4 ℃ environment at 12000 rpm rpm for 15min hours. A new centrifuge tube was selected and the upper surface of the water phase was aspirated. Then adding a certain amount of isopropanol with the specific specification of 500 mu L.mL ^-1 Trizol to precipitate, and then placing the precipitate in an environment of-20 ℃ for 30min;

(5) Centrifugation was performed in a 4 ℃ environment at 12000 rpm rpm for 10min hours. After the operation is finished, RNA is precipitated at the bottom of the tube;

(6) Adding 75% ethanol according to 1 mL 75% ethanol/mL trizol, gently oscillating the centrifuge tube, suspending and precipitating, centrifuging at 4 ℃ at 8000rpm for 5min, and discarding the supernatant as much as possible;

(7) Repeating the step 6 once;

(8) And (3) airing at room temperature or drying in vacuum for 5-10 min, adding a proper amount of RNase-free ddH ₂ O to dissolve the RNA sample, and sucking 2 mu L to perform agarose electrophoresis detection.

RT-qPCR analysis

Genomic DNA was removed by adding 5X GDNA ERASER Buffer 2. Mu.L, GDNAERASER. Mu.L, total RNA 800 ng, RNase-free dH ₂ O to 10. Mu.L to 200. Mu.L of RNase-free EP tube, and placing the tube in a PCR apparatus to perform a reaction at 42℃for 2 min.

Reverse transcription of RNA by adding 5×PrimeScript Buffer2 4 μL,PrimeScript RT Enzyme Mix 1 μL,RT Primer Mix 1 μL,RNase-free dH₂O 4 μL, to the reaction solution, placing the mixture into a PCR instrument at 37 ℃ for 15min and at 85 ℃ for 5 s. The cDNA was stored at-20℃until use.

The expression difference of DoGST in the root, stem and leaf of dendrobium candidum under the treatment of 0 and 14 mg.L ^-1 Cd is identified by using RT-qPCR. As a result, as shown in FIG. 2, doGST18 had its expression gradually increased in roots with the increase of Cd treatment time, and DoGST18 had an expression level in roots 7 times that of the control on the 14 th day of treatment, and slightly induced expression under Cd stress in stems and leaves, respectively. These results indicate that DoGST < 18 > may play an important role in the detoxification of Cd in the root of dendrobium candidum, thereby helping plants relieve the stress of Cd.

Subcellular localization vector construction

The existing carrier of target gene in laboratory is used as template, specific primer is designed according to the enzyme cutting site of the carrier, rTaq enzyme amplification is carried out, doGST is amplified and the target gene is respectively constructed on pBWA (V) HS-GLosgfp carrier.

Subcellular localization

(1) Selecting Arabidopsis seedlings which are cultured for 7-15 days at the temperature of about 25 ℃;

(2) Taking a plurality of blades, spreading the blades on a flat plate, cutting She Chengtiao by a surgical knife, transferring the blades into a conical flask which is added with 10 mL enzymolysis liquid in advance, and soaking the blades completely. At room temperature, 45 rpm is slowly vibrated, and enzymolysis is carried out for 4-6 hours;

(3) Filtering the protoplast solution by a filter screen with the specification of 40 μm, centrifuging at a rotation speed of 100 g for 5min, and discarding the supernatant;

(4) Performing 2 washing operations by 10 mL of W5 solution subjected to pre-cooling treatment in advance, performing centrifugal operation at a rotating speed of 100 g in a 4 ℃ environment for 5 min, and discarding the supernatant;

(5) From the experimental demand level, MMG solution was added to suspend the samples with a capacity of 500. Mu.L, protoplasts were selected for each sample, and the capacity was 200. Mu.L. Performing microscopic examination, namely performing operation according to a 40-time mirror, wherein each view is approximately 20 to 40;

(6) The protoplast suspension and DNA were selected to have a capacity of 200. Mu.L and 10. Mu.L, respectively, and the purified plasmid reached more than 500 ng, and then the PEG solution with the same volume was selected to be mixed uniformly. Wherein the marker is added during positioning for cotransfer, and the capacity is 10 mu L. Standing in room temperature environment for 30 min hours;

(7) The reaction was stopped with 1 mL of W5, centrifuged at 5 min at 100g at 4℃and the supernatant was discarded;

(8) Adding 1 mL of W5 for washing 2 times, centrifuging at 4 ℃ at 100 g for 5min, and discarding the supernatant;

(9) Adding 1 mL of W5 to suspend, and culturing for 24-48 h at 28 ℃ in weak light;

(10) And (5) observing by a fluorescence microscope or a laser confocal microscope.

GFP fusion protein DoGST is expressed in tobacco leaves by constructing pBWA (V) HS-DoGST-GLosgfp vector and utilizing tobacco leaf transient transformation technique. Expression of the GFP protein control alone was observed in tobacco leaf cells by laser confocal, whereas DoGST 18:GFP was co-localized with cytoplasmic membrane markers, indicating its specific localization to the cytoplasmic membrane (FIG. 3). The above results indicate that DoGST a.sup.18 plays a role in the cytoplasmic membrane.

Arabidopsis transgenes

The super-expression vector uses pCAMBIA1300, the enzyme cutting site is selected from BamHI and SalI, and the E.coli is connected and transformed, and the E.coli GV3101 is transformed after the plasmid is extracted. LB liquid medium is selected for culture, wherein the medium comprises rifampicin and kana, the concentration is 50 mug.mL ^-1, and the capacity is 2 mL. The Agrobacterium (containing the plasmid of interest) is then cultivated. Then, the bacterial liquid was taken out, the bacterial liquid was put in a fresh medium with a volume of 1mL, and the fresh medium was cultured in a shaking table of 200 rpm at 28℃to a volume of about OD ₆₀₀ to 0.8, the bacterial liquid was centrifuged at room temperature 8000rpm for 5 min, the supernatant was discarded, and the bacterial liquid was resuspended in a heavy suspension (1/2 MS liquid medium, 5% sucrose, 0.03% Silwet L-77,0.01. Mu.g.mL ^-16-BA,20 mg·L^-1 AS, finally pH was adjusted to 5.7 with KOH) to a concentration of 0.8.

The arabidopsis is firstly subjected to drought treatment for 3 days before starting, so that the aim of experiments is better achieved. Under the state, the flowering degree of the arabidopsis is good, and then the resuspension in the last step is taken out, and the arabidopsis is soaked in 5 min. When the fungus liquid is adhered to the neck and the leaves, the fresh-keeping bag needs to be cleaned by paper towels in time, and the arabidopsis thaliana is wrapped by the fresh-keeping bag, so that the aim of moisture locking is fulfilled. Then the fresh-keeping bag is placed in a dark place for 1 day, and the fresh-keeping bag is taken out and then normally cultured. The transformation was performed once again at intervals of one week. After the seeds were ripe, the seeds were harvested and sterilized 5 min with 5% sodium hypochlorite and washed 3 times with sterile water. Positive seedlings are screened on a solid medium containing 50 mug.mL ^-1 hygromycin 1/2MS, after the seedlings are dried in the air, the seedlings are sealed and cultured for three days at 4 ℃, then the seedlings are put into an incubator for growth, after the seedlings grow into two true leaves, the seedlings are transplanted into a seedling pot and marked as T ₁ generation. And (3) taking the T ₁ generation until the seedling grows to bolting, collecting leaves, extracting DNA, identifying, continuously screening the T ₁ generation seeds under the conditions, and selecting a strain which does not sprout and has a germination ratio of 1:3, thus obtaining the T ₂ generation seeds. And then screening the seeds according to the conditions, wherein the obtained mature seeds are seeds of the generation T ₃ for later experiments.

Arabidopsis seed spots of wild type and DoGST-overexpressed homozygous lines were grown on 1/2MS solid medium containing 0 and 60. Mu.M CdCl ₂ and placed in plant incubator for 14 days after which their phenotypic differences were observed and photographed, root length and biomass were measured.

As shown in FIG. 4, under the medium conditions containing 60. Mu.M CdCl ₂, the over-expressed DoGST Arabidopsis lines all exhibited a stronger Cd-resistant phenotype than the wild-type. The root length of the specific expression overexpression strain is obviously longer than that of the wild type, and compared analysis is carried out from the fresh weight layer surface, and the root length is larger than that of the wild type. While wild-type and over-expressed DoGST Arabidopsis lines were not significantly different in root length and fresh weight under the medium conditions of 0. Mu.M CdCl ₂ (FIG. 5).

ROS content determination

(1) Hydrogen peroxide (H ₂O₂) content determination

A. And (3) carrying out hydrogen peroxide content measurement on the arabidopsis seedlings by using a Biyun hydrogen peroxide detection kit, grinding the arabidopsis seedlings into powder, adding hydrogen peroxide detection lysate, adding 100-200 mu L of lysate into every 5-10 mg of tissue, fully reacting, centrifuging at a temperature of 4 ℃ 12000 rpm for 5min, and taking the supernatant for subsequent measurement. All of the above operations are performed on 4oC or ice.

(2) Determination of the content of superoxide radical (. O ₂ ^-)

A. And (3) determining the content of the superoxide anions in the arabidopsis thaliana by using a Biyun-Tian superoxide detection kit, and preparing a superoxide detection working solution.

B. Grinding Arabidopsis thaliana seedlings, adding a superoxide detection working solution, fully reacting, centrifuging 12000rpm for 5min, taking 200 mu L of supernatant, adding the supernatant into an ELISA plate, and placing the ELISA plate into an ELISA instrument (TECAN SPARK: tecan, switzerland) A ₄₅₀ to measure absorbance.

The present example further determines ROS content, including H ₂O₂ and O ^2-, of wild-type and overexpressed DoGST A.thaliana lines based on phenotypic results. The results show that the H ₂O₂ and O ^2- contents of the Arabidopsis after Cd treatment are obviously increased compared with the control group, and the H ₂O₂ and O ^2- contents of the Arabidopsis strain of the over-expression DoGST are obviously lower than those of the wild Arabidopsis after Cd treatment (figure 6), which shows that the over-expression DoGST18 can reduce excessive ROS accumulation induced by Cd stress in the plant, thereby reducing the cell damage of the plant and improving the Cd tolerance of the plant.

Verification of cadmium resistance performance in yeast

Yeast competent preparation and transformation

(1) Selecting a delta ycf1 single colony on a YPDA plate, inoculating the single colony into a YPDA liquid culture medium with the capacity of 4 mL, performing centrifugal operation at the ambient temperature of 30 ℃, performing shake culture at the rotating speed of 225-rpm for 18-20 h, and performing overnight treatment until OD ₆₀₀ is more than 1.5;

(2) Transferring YPDA liquid culture medium, and culturing for 50mL hours, so as to promote OD ₆₀₀ =0.2, then performing centrifugal operation at 30 ℃ at 225rpm, and performing shaking culture for 4-5 hours, wherein OD ₆₀₀ =0.6;

(3) Collecting bacteria by centrifugation, and performing centrifugation at 4000 rpm and 5: 5min;

(4) Re-suspending thallus with capacity of 20 mL with sterile water, collecting thallus with centrifugation at 4000 rpm and duration of 5min, and discarding supernatant;

(5) Re-suspending the thallus by 5mL of 0.1M LiAc, uniformly mixing, collecting the thallus by centrifugation, centrifuging at 4000rpm for 5 min min, and discarding the supernatant;

(6) Re-suspending the thalli by 500 mu L of 0.1M LiAc, taking out a centrifuge tube prepared in advance for sub-packaging, wherein the specification is 1.5 mL, and all the conversions are 50 mu L for standby;

(7) Adding the reagents into a centrifuge tube according to a certain sequence, blowing through a gun head to uniformly mix the reagents or carrying out intense shaking for about 1min to fully and uniformly mix the reagents;

(8) Incubating in a water bath at 30 ℃ and 30 min;

(9) Performing heat shock operation in a 42 ℃ water bath for a period of 25 min hours;

(10) Collecting bacteria by centrifugation, centrifuging at 4000 rpm and 5 min for a period of time, and discarding the supernatant;

(11) 3 mL of 2 XYPDA was added to suspend the cells as gently as possible, 30℃and 225 rpm, and 1h was shake-cultured;

(12) Collecting bacteria by centrifugation, centrifuging at 4000 rpm and 5 min for a period of time, and discarding the supernatant;

(13) All transformations were accomplished by sterile aqueous suspension of the cells, 200. Mu.L in volume, gently mixed to homogeneity, and plated on SD-U screening plates. 30. Culturing for 3-5 d at the temperature of C.

Yeast cadmium resistant phenotype

(1) After the plates grow clones, randomly picking 8 plates to carry out yeast colony PCR, and suspending correct clones by ddH ₂ O;

(2) A series of concentration gradient dilutions (10 ⁰、10^-1、10^-2、10^-3、10^-4、10^-5) were performed, followed by spotting separately into SG-U (galactose instead of glucose) containing CdCl ₂;

(3) Culturing for 3-5 d at 30 ℃, observing the result and photographing.

To further verify whether DoGST18 has cadmium-resistant potential, we constructed DoGST18 into the yeast expression vector pYES2 for expression analysis, while using the pYES2 empty vector as a control. And (3) converting pYES2-DoGST and pYES2 BY using a yeast conversion kit to obtain a saccharomyces cerevisiae BY4741 strain, culturing and screening positive clones on a SD-Ura culture medium, and screening the positive clones BY colony PCR. BY4741 (pYES 2) is used as a negative control, galactose is used as a protein expression inducer, and the influence of recombinant BY4741 (pYES 2-DoGST 18) expressed protein on the cadmium resistance of yeast is detected. The results show that the growth of the control bacteria and the recombinant bacteria is reduced, the higher the Cd concentration is, the more obvious the inhibition degree is, the colony density of DoGST in 10 ^-2、10^-3、10^-4、10^-5 dilutions is higher than that of the control bacteria at the concentrations of 20 mu M, 30 mu M, 40 mu M and 50 mu M CdCl ₂, and the growth condition is superior to that of the control bacteria (figure 7), so that DoGST can improve the tolerance of a host to Cd to a certain extent.

Prokaryotic expression and pull down LC-MS

Prokaryotic expression vector construction

The method comprises the steps of designing a specific primer by using a carrier of an existing target gene in a laboratory as a template according to enzyme cleavage sites of the carrier, carrying out rTaq enzyme amplification, amplifying DoGST and 18, and carrying out an amplification procedure of pre-denaturation at 95 ℃ 5min, denaturation at 95 ℃ 30 s, renaturation at 55 ℃ 30 s, extension at 72 ℃ for 2 min and 32 cycles. The PCR product was isolated by 1% Agarose gel and purified using Agarose gel DNA purification kit (TAKARA, japan) recovery. The pMAL vector was linearized with the corresponding restriction enzyme and purified using Agarose gel DNA purification kit (TAKARA, japan), recombined with the fragment using homologous recombinase, and TOP10 competent cells transformed. Colony PCR was performed with vector primer pairs with gene primers to screen positive clones. And selecting correct positive clones, sequencing, shaking the successfully sequenced clones, extracting plasmids, and transforming prokaryotic expression strains.

Prokaryotic expression

(1) Transforming a prokaryotic expression strain Rosetta or BL21 based on the constructed plasmid;

(2) Selecting corresponding single colonies, and shaking the single colonies overnight in a 37 ℃ environment by using 3-4 mL of 2 XYT culture medium;

(3) Selecting the culture medium, wherein the volume of the culture medium is 30-200 mL, the concentration is 1:50-1:100, and the culture medium is expanded in a 37 ℃ environment until OD ₆₀₀ = 0.6-0.8;

(4) Placing the bacterial liquid in a normal temperature environment, and inducing protein by a certain induction condition:

a. 37 Culturing 6h under the condition of C0.1 mM IPTG (the set condition is conventional, only partial condition is applicable)

B. 20 Culturing 12h under conditions of C0.1 mM IPTG (the conditions set are extreme conditions, generally applicable)

C. 37 Culture 3h (protein suitable for very good induction) under C environmental conditions, 1.0 mM IPTG

(5) Centrifugation at 10min was performed at 5000 rpm, washing and sedimentation was performed 2 times with ddH ₂ O, then washing and sedimentation were performed 3 times with 1X PBS phosphate buffer saline, EP tubes were selected, the specification was 2 mL, and sub-packaging was performed, the amount of bacterial liquid per tube was 1 mL. Under the general condition, the bacterial liquid of 100mL can be concentrated to the size of 3 mL, and under the specific condition, the bacterial liquid can be automatically adjusted according to the bacterial amount;

(6) Adding a certain amount of lysozyme into each tube of bacterial liquid, wherein the volume is 20 mu L, the concentration is 20 mg.mL ^-1, the volume is 100 mM PMSF, and the volume is 10 mu L;

(7) Crushing treatment is carried out by ultrasonic waves, the time length of each time is controlled within 10s, and the ice setting time is more than 10 s;

(8) After the treatment, adding a certain amount of Triton X-100 into the bacterial liquid until the concentration is 1%, and incubating for 30 min in a4 ℃ environment through a 360-degree rotor;

(9) Centrifugation at 5min was performed at 8000 rpm f, and the supernatant and pellet were removed and protein expression was detected by SDS-PAGE gel or Western.

To further understand the detoxification mechanism of DoGST18, doGST18 was constructed on a pMAL vector fused with MBP tag, and the protein of interest was obtained by a prokaryotic protein expression system. And incubating the target protein and the dendrobium candidum total protein and performing pull down operation to obtain a protein sample for specifically adsorbing MBP (figure 8). Carrying out protein mass spectrum sequencing on the residual protein sample, obtaining 50 specific peptide fragments according to a sequencing result by DoGST < 18 >, and selecting one candidate target protein according to factors such as coverage (coverage) of the peptide fragments with the unouse score and the credibility of more than 95 percent, the number of the peptide fragments with the confidence interval of more than 95 percent, the research progress of the current cadmium-resistant mechanism of dendrobium candidum and the like, and carrying out the next specific interaction verification, namely S5M5N9/HSP70.

Agrobacterium-mediated transient expression of tobacco and Co-immunoprecipitation (Co-IP)

Agrobacterium injection

(1) Inoculating agrobacterium containing a target plasmid into a3 mL LB liquid culture medium, and performing shake culture at 28 ℃ for 24-36 hours;

(2) Performing 4 min centrifugation at 4000 rpm deg.f at room temperature, collecting thallus, and removing supernatant;

(3) Adding 10 mM MgCl ₂ to resuspend thallus, 1 mL, centrifuging 4000 rpm at room temperature for 4min, and removing the supernatant;

(4) Repeating the above steps for 3 times;

(5) Constant volume with injection buffer, OD ₆₀₀ =0.6, culturing 2 h in dark environment at 28 ℃;

(6) Selecting good-growth Nicotiana benthamiana, specifically 3 rd to 6 th fully developed true leaves from top to bottom, and then infiltrating and inoculating agrobacterium;

(7) Treating the leaf blade, manufacturing a very tiny wound through a needle at the lower epidermis position, injecting agrobacterium (30-50 mu L) into the tobacco leaf blade by using a syringe (with the needle removed), and marking;

(8) And after 36-48 hours, collecting the leaves, and freezing and storing at-80 ℃.

Tobacco leaf protein extraction

(1) Pre-cooling the mortar rod with liquid nitrogen, grinding the blades to be powdery through the mortar, and then putting the powder into a 1.5 mL centrifuge tube;

(2) Taking out 500 mg from the sample, adding 1 mL protein lysate, and shaking and mixing;

(3) Centrifuging at 13000 rpm at 4 deg.C for 15: 15min, taking out supernatant and placing into new EP tube;

(4) Adding Protein loading buffer of corresponding volume, boiling at 100deg.C for 5-7min;

(5) WB detection was performed and the remaining samples were stored frozen at-80 ℃.

Co-immunoprecipitation (Co-IP)

(1) Selecting a certain amount of GFP (green fluorescent protein) tag magnetic beads according to the content of the instruction, storing the GFP tag magnetic beads in a 1.5 mL centrifuge tube, and setting the temperature to be 4 ℃;

(2) Placing the solution on a magnetic rack, and discarding the supernatant after the solution is gradually clarified;

(3) Adding 1 mL wash buffer protein, reversing, mixing, instantly centrifuging, placing in a magnetic rack, clarifying the solution gradually, and discarding the supernatant;

(4) Repeating the above steps for 3 times;

(5) Adding proteins corresponding to the transient expression of tobacco into the obtained magnetic beads, and incubating at 4 ℃ in a rotating way for 4 h;

(6) Centrifuging for a short time, placing on a magnetic rack, gradually clarifying the solution, and discarding the supernatant;

(7) Continuously adding 1 mL wash buffer protein, reversing, uniformly mixing, performing instantaneous centrifugation, then placing on a magnetic rack, gradually clarifying the solution, and discarding the supernatant;

(8) Repeating the above steps for 6 to 8 times;

(9) The supernatant was finally drained, note that the process did not collide with the beads, a proper amount of elution buffer protein was added, and the incubation was rotated for 1h at 4 ℃;

(10) Performing centrifugal operation for a short time, then placing the centrifugal operation on a magnetic rack, and sucking out and transferring supernatant into a new centrifuge tube after the solution is gradually clarified, wherein the specification is 1.5 mL;

(11) Adding Protein loading buffer, boiling for 5-7min;

(12) The final sample can be used in WB assay and the remaining sample stored frozen at-80 ℃.

Western immunoblotting

(1) Protein electrophoresis is carried out on the protein solution 20 mL, the voltage is set to 80V, and the voltage is adjusted to 100V after the laminating adhesive is run out until the electrophoresis is finished;

(2) Transferring film, 100V for 90 min;

(3) Washing the membrane through TBST, performing 1 washing operation for 10 min hours, and then pouring out the TBST;

(4) Blocking with TBST (containing 5% skimmed milk powder) at room temperature on a low speed shaker for 1h;

(5) Diluting the primary antibody by using the sealing solution, placing the primary antibody in a low-speed shaking table, and hybridizing the primary antibody at room temperature by more than 4 h;

(6) Performing 3 times of cleaning operation on the membrane by TBST for 10min hours each time, diluting the secondary antibody by using the sealing liquid, placing the secondary antibody in a low-speed shaking table, and hybridizing for 45 minutes at room temperature;

(7) After ECL exposure, the films were scanned by a machine.

This example demonstrates the interaction between DoGST and S5M5N9 proteins using the tobacco leaf transient expression and co-immunoprecipitation method. From the figure, doGST interacts with S5M5N9, and by selecting the empty-expressed GFP protein as a negative control, it was found that it could not interact with S5M5N9, indicating that the GFP tag did not affect the protein interaction (fig. 9). In FIG. 9, doGST-GFP and S5M5N9-FLAG were transiently co-expressed in Nicotiana benthamiana leaves. Co-IP experiments were performed with anti-GFP antibodies, doGST or GFP empty vector was detected with anti-GFP antibodies, S5M5N9 fusion protein was detected with anti-FLAG antibodies, immunoblotted with isolated proteins, and GFP as negative control.

Yeast double hybrid (Y2H) verification experiment

Yeast competent preparation and transformation

(1) AH109 single colonies were selected on YPDA plates and inoculated in 4mL YPDA broth at 30℃for 18-20 hours shaking culture at 225rpm until OD ₆₀₀ was above 1.5;

(2) Transferring the culture medium into a 50 mL YPDA liquid culture medium, carrying out shake culture for 4-5 hours at the temperature of OD ₆₀₀ = 0.2,30 ℃ at the rotating speed of 225rpm until OD ₆₀₀ =0.6-0.8;

(3) Centrifugally collecting thalli at room temperature, wherein the rotating speed is 4000 rpm, and the duration is 5 min;

(4) Re-suspending the thalli by using 20 mL sterile water, and centrifugally collecting the thalli at room temperature, wherein the rotating speed is 4000 rpm and the duration is 5 min;

(5) Re-suspending the thalli by 5 mL of 0.1M LiAc, and centrifugally collecting the thalli at room temperature, wherein the rotating speed is 4000 rpm and the duration is 5 min;

(6) Re-suspending the thalli by 500 mu L of 0.1M LiAc, sub-packaging the thalli in a 1.5 mL centrifuge tube, wherein all the transformation is 50 mu L, and keeping the thalli for standby;

(7) Adding the reagents into a 1.5 mL centrifuge tube according to a certain sequence, and blowing and uniformly mixing;

TABLE 1 Yeast two hybrid System Components and amounts

50%PEG3350	1 M LiAc	Salmon sperm (20 mg mL -1)	Plasmid DNA
				240 μL	36 μL	5 μL	5 Mu L each

(8) Incubating in a water bath at a temperature of 30 ℃ for 30 min;

(9) Heat shock 25 min in a 42 ℃ water bath;

(10) Resuscitating in a water bath at a temperature of 30 ℃ for 30 min;

(11) Centrifugally collecting thalli, performing at room temperature with the rotating speed of 4000 rpm and the duration of 5min, and discarding the supernatant;

(12) All the transformation is carried out suspension operation on thalli by sterile water, the capacity is 200 mu L, the whole process is gently shaken to be uniform, and a corresponding defect type screening plate is coated;

(13) Culturing at 30deg.C 4 d.

Point plate experiment

(1) Colonies that were successfully verified by colony PCR were resuspended with 2 mL ddh2o, adjusting OD ₆₀₀ to 0.6;

(2) After performing gradient dilution (10 ²、10^-1、10^-2), 10 μl of the spot plate was pipetted onto SD-Trp/-Leu（SD-TL）、SD-Trp/-Leu/-His（SD-TLH）、SD-Trp/-Leu/-His/-Ade（SD-TLHA）、SD-Trp/-Leu/-His/-Ade+X-α-gal（SD-TLHA+X-α-gal）;

(3) Culturing for 3-5 d at 30 ℃, and observing and photographing records.

To verify DoGST interaction with S5M5N9, a multiple control set was set up to perform a yeast double hetero experiment as shown in table 2.

The negative control is pGBKT7-DoGST18+pGADT7, pGBKT7+pGADT7-S5M5N9, pGBKT7-largeT +pGADT7-lam, the positive control is pGBKT7-largeT +pGADT7-p53, the experimental result is shown in figure 10, and the result shows that DoGST18 can directly interact with S5M5N 9.

TABLE 2 cultivation results on SD plates of different auxotrophs in DoGST18 interaction validation

BD	AD	DDO	TDO	QDO	QDO+X-α-gal
						DoGST18	S5M5N9	+++	+++	+++	+++
DoGST18	AD	+++	-	-	-
						BD	S5M5N9	+++	-	-	-
largeT	p53	+++	+++	+++	+++
						largeT	lam	+++	-	-	-

Note that: ++ most growth; -inability to grow.

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.

Claims (6)

1. A dendrobium candidum cadmium-resistant protein is characterized by having an amino acid sequence shown as SEQ ID NO. 2.

2. A gene encoding the cadmium-resistant protein according to claim 1, wherein the gene has a nucleotide sequence shown in SEQ ID No. 1.

3. Use of a cadmium-resistant protein according to claim 1 or a gene encoding it for any of the following applications:

a) Improving cadmium resistance of plants or microorganisms;

b) Reducing cadmium accumulation in plants or microorganisms;

c) Cultivating a plant variety with low cadmium accumulation;

d) Regulating and controlling a plant antioxidant system and reducing Cd-induced ROS accumulation;

e) Enhancing plant root growth and improving biomass under Cd stress;

f) Expressed in yeast to enhance its tolerance to Cd stress.

4. A method for improving cadmium tolerance in a plant comprising introducing and expressing the gene of claim 2 into the plant.

5. An expression vector comprising the gene of claim 2, wherein the vector is any one of a plant expression vector, a yeast expression vector, or a prokaryotic expression vector.

6. A host cell comprising the expression vector of claim 5, wherein the host cell is one of a plant cell, a yeast cell, e.coli, or an agrobacterium.