CN114032245A

CN114032245A - Gene VLNHX3D in regulating plant cell Na+And/or K+Application in concentration

Info

Publication number: CN114032245A
Application number: CN202110367995.5A
Authority: CN
Inventors: 刘伟; 马宗斌; 马兴立; 朱伟
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2021-04-06
Filing date: 2021-04-06
Publication date: 2022-02-11
Anticipated expiration: 2041-04-06
Also published as: CN114032245B

Abstract

The invention relates to the field of plant molecular biology, in particular to a gene VLNHX3D for regulating plant cell Na⁺And/or K⁺Use in concentration. The invention provides a gene VLNHX3D for regulating plant cell Na⁺And/or K⁺The application of concentration, the gene VLNHX3D has the nucleotide sequence shown in SEQ ID NO 1. The invention constructs a transgenic vector by cloning gene VLNHX3D, reduces expression of VLNHX3D by VIGS technology, and silences Na in roots, stems and leaves of plants under the condition of salt stress⁺The accumulation is increased remarkably, and K is in the root⁺The content is obviously reduced, the salt tolerance of cotton is obviously reduced, and the VLNHX3D is shown to participate in early salt stress response and regulate intracellular Na⁺And K⁺To improve the salt tolerance of cotton.

Description

Gene VLNHX3D in regulating plant cell Na+And/or K+Application in concentration

Technical Field

The invention relates to the field of plant molecular biology, in particular to a gene VLNHX3D for regulating plant cell Na⁺And/or K⁺Use in concentration.

Background

Salt stress can cause the plants to grow and develop slowly, yellow or wither leaves, and when the plants are severe, the whole plants are dry and dead. The harm of salt stress mainly comprises three aspects of cell osmotic stress, ion toxicity and nutrient imbalance.

Osmotic stress: after the plant is damaged by salt, the original water balance of plant cells is broken. The water absorption capacity of the plant is reduced, so that the plant is subjected to physiological drought, and the plant production is inhibited. Osmotic stress rapidly reduces cell expansion of the root tip and young leaves, resulting in stomatal closure. The increase of the content of sodium ions in the soil not only quickly reduces the effectiveness of soil moisture, but also slowly accumulates the sodium ions on the overground part; this spatial and temporal distribution result indicates that early salt stress responses are caused by osmotic or drought stress, and specific ion stress responses are caused later. Low water potential induces ABA production and signal transduction, leading to depolarisation of guard cells, reduction in stomatal pore size and conductivity. As the time of salt stress progresses, the reduction of cell elongation and cell division will result in smaller and thicker leaves thereby reducing photosynthesis, further affecting the growth and development of the plant.

Ion stress: na (Na)⁺And Cl^-The inorganic ions are necessary for plant life activities, but when excessive, they produce ion poisoning, damage the selective permeability of plasma membrane, and cause extracellular salt dissociationThe large amount of the seed enters the cell to destroy the ion balance. Na can be extracted by transpiration⁺The Na is transferred from the root to the upper part of the ground and accumulated in the blade, but only a small part of the Na in the blade⁺Can migrate through the phloem to the root, which can lead to increased ion content in the plant leaves and thus cause damage. Excessive absorption of certain salts by plants reduces the absorption of other salts, and the plants develop symptoms of nutrient deficiency or ion toxicity.

Nutrient unbalance: when plants grow in NaCl stress environment, the plants absorb a large amount of Na⁺To result in K⁺Absorption is reduced, resulting in potassium ion deficiency and affecting Ca²⁺And Mg²⁺Further influences the nutrient absorption of the plants, disturbs the metabolism of the plants and inhibits the growth of the plants. Research proves that under the condition of salt stress, more Na is accumulated in the oleaster⁺At the same time reduce K⁺And Ca²⁺Content (c); a large amount of Na is accumulated in the root system of the oleaster variety with stronger salt tolerance⁺Thereby reducing Na in the leaves⁺Accumulation, less loss of K in salt-tolerant narrow-leaved oleaster than in salt-intolerant species⁺And Ca²⁺. Plant uptake of Cl^-And SO₄ ^2-Too much affects HPO₄ ^2-Absorption of (2); too much phosphate will cause Zn²⁺Deficiency results in a nutritional imbalance in the plant.

The response of plants to salt stress is divided into two phases: firstly, the rapid osmotic stress stage of young leaf growth is inhibited, and secondly, the slow ion poisoning process of mature leaf senescence is accelerated.

Na⁺/H⁺Reverse transporter (Na)⁺/H⁺NHX) is a reverse transporter widely present in higher plants, and plays an important role in regulation of intracellular pH and ion balance. Researchers find the protein in plants such as barley, rice, arabidopsis thaliana, atriplex maritima, beet and soybean in sequence. At present, Na of different species has been cloned⁺/H⁺Reverse transporter gene, study of the expression properties of NHX. The results indicate that some NHX proteins are only in salt stress barsThe transfer activity is shown under the induction of the element, and the transfer activity is not shown under the non-stress environment. Some NHX genes are expressed at lower levels under normal environmental conditions, but their expression levels are induced to increase as salt stress levels increase. There are also some plant NHX genes that are not detectable under salt stress conditions.

6 Na were found in Arabidopsis thaliana⁺/H⁺Reverse transporter (AtNHX1-6), phylogenetic analysis divided these 6 AtNHX into two subgroups, where AtNHX1-4 belongs to subgroup I and AtNHX5-6 belongs to subgroup II. Studies have shown that NHX in subgroup I is located on the vacuolar membrane, vs Na⁺And K⁺Have the same affinity; they remove Na accumulated in the cytoplasm⁺And (or) K⁺Isolated in vacuoles, maintaining the turgor pressure of the cells. NHX of subgroup II is located in the intracellular membrane system and primarily regulates intracellular K⁺By accumulating more K in the cell under salt stress⁺To alleviate the harm caused by the stress.

The vacuolar membrane-localized NHX protein is Na⁺/H⁺The field of antiporter protein research is hot. The research shows that the insertion of the NHXs gene of the coding tonoplast into the plant can effectively improve the salt tolerance of the plant. Transgenic Arabidopsis overexpressing AtNHX1 can be grown at 200 mmol/LNaCl. Na from transgenic Arabidopsis plants compared to wild type⁺Increased content of Na⁺/H⁺Enhanced antiporter activity. AtNHX1 leads to intracellular Na after heterologous expression in sweet potato, tartary buckwheat, tall fescue and other plants⁺Increased concentration, K⁺/Na⁺The ratio is increased and the tolerance of the plant to salt stress is improved. Overexpression of BnNHX1 improves the salt tolerance of the transgenic tobacco. Therefore, we speculate that the plant vacuole type Na⁺/H⁺The reverse transport protein can specifically recognize Na⁺And separating the plants, thereby improving the salt tolerance of the plants. However, for vacuolated Na⁺/H⁺The deep research analysis of the antiporters shows that the salt tolerance mechanism of the antiporters possibly relates to the mediation of K⁺Accumulation and maintenance of low Na⁺/K⁺A ratio. In previous studies, overexpressionTransgenic tomato of AtNHX1 can convert more K⁺Dispensing into the vacuole by increasing K in the vacuole⁺Content feedback inhibition of Na⁺Thereby improving the salt tolerance of the plants. Transgenic rice overexpressing PgNHX1 survived and completed its life cycle, successfully flowering and seed setting. Wild type plant upper leaf Na⁺Higher content, and the upper leaf K of the transgenic plant⁺The content is higher. Expression of the NHXS1-IRES-TVP1 gene in transgenic tobacco resulted in leaf Na⁺Reduced content of K⁺The content is increased. These findings indicate that the mechanism by which vacuolated NHX proteins regulate salt tolerance may differ from plant to plant.

In addition, studies have also found that different NHXs may be distributed on different structures of the cell, some on the vacuolar membrane and some on the endomembrane system of the cell. And Na identified in different organisms⁺/H⁺The reverse transporters differ somewhat in structure.

China is a world where cotton is produced and consumed, and cotton is an important economic crop and a national strategic material in China. As the cultivated land area of China is gradually reduced, the competitive strength of grain crops and cotton to the cultivated land is continuously increased. China has wide saline-alkali land, and the cotton planting in the saline-alkali land can solve the problem that grains and cotton struggle for land, promote the healthy development of the cotton industry and realize sustainable development of agriculture. Therefore, the method has great significance for the research of the salt-tolerant genes of cotton.

Today, although genomic sequence sequencing of cotton has been completed, the function of some genes is unknown and requires further exploration by researchers.

Disclosure of Invention

The invention clones a Na from upland cotton genetic standard line TM-1⁺/H⁺The reverse transporter gene VLNHX3D, is located in the vacuolar membrane. Real-time fluorescence quantification results show that VLNHX3D is induced to be up-regulated in cotton leaves at the initial stage of salt stress, and the expression level of VLNHX3D is gradually increased along with the increase of the salt stress degree. When VLNHX3D was expressed in yeast mutant ATX3, yeast transformed with VLNHX3D gene was fermented in comparison with control (no-load transformed)The mother material has higher salt tolerance. After the expression of VLNHX3D is reduced by using VIGS technology, Na in roots, stems and leaves of plants is silenced under salt stress⁺The accumulation amount of (A) is remarkably increased, and K in the root⁺The content is obviously reduced, the salt tolerance of cotton is obviously reduced, and the VLNHX3D is shown to participate in early salt stress response and regulate intracellular Na⁺And K⁺To improve the salt tolerance of cotton.

The first aspect of the invention provides the following technical solutions:

gene VLNHX3D in regulating plant cell Na⁺And/or K⁺The application of the gene VLNHX3D in concentration, the gene VLNHX3D has the nucleotide sequence shown in SEQ ID NO 1.

The invention constructs a transgenic vector by cloning gene VLNHX3D, reduces the expression of VLNHX3D by VIGS technology, and silences Na in roots, stems and leaves of plants under salt stress⁺The accumulation amount of (A) is remarkably increased, and K in the root⁺The content is obviously reduced, the salt tolerance of cotton is obviously reduced, and the VLNHX3D is shown to participate in early salt stress response and regulate intracellular Na⁺And K⁺To improve the salt tolerance of cotton.

Further, the Na in the roots, stems and leaves of the gene VLNHX3D plants is silenced compared with wild plants⁺Increase in the accumulation of (A), K in the stem and leaves⁺The accumulated amount of (A) is constant, K in the root⁺The content is reduced.

The second aspect of the invention provides the application of gene VLNHX3D in cultivating or detecting salt-tolerant transgenic plants, wherein gene VLNHX3D has the nucleotide sequence shown in SEQ ID NO 1.

The invention discovers that the expression level of the gene VLNHX3D in leaves is gradually increased along with the increase of salt stress time and reaches a peak value 6h after 200mM salt stress treatment, and the expression level of the gene VLNHX3D in leaves is also gradually increased along with the increase of salt concentration, which indicates that the salt stress induces the change of the expression level of VLNHX 3D. After further expressing the gene VLNHX3D in the yeast mutant ATX3, the yeast transformed with VLNHX3D gene has higher salt tolerance than the yeast of the control group. The gene VLNHX3D is proved to have the function of improving the salt tolerance of plants. Therefore, the gene can be used for cultivating transgenic plants and detecting the salt tolerance of the transgenic plants.

Further, the overexpression or the transfer of the gene VLNHX3D is detected, and the salt tolerance of the plant is increased;

the gene VLNHX3D was not detected or silenced, and the salt tolerance of the plants was weakened.

In the present invention, the plant includes monocotyledons and dicotyledons;

the monocotyledon comprises rice, corn and wheat;

the dicotyledonous plants comprise soybean, cotton, arabidopsis thaliana and tobacco.

Furthermore, the protein expressed by the gene VLNHX3D is located in the vacuolar membrane.

Namely, the gene VLNHX3D provided by the invention expresses protein located on the vacuolar membrane, and the gene VLNHX3D plays a role through the protein expressed on the vacuolar membrane.

The third aspect of the invention provides a method for detecting the salt tolerance of plants, which detects the existence or expression condition of a gene VLNHX3D of a sample to be detected to judge the salt tolerance of the sample;

the gene VLNHX3D has the nucleotide sequence shown in SEQ ID NO. 1.

Detecting the existence of the gene VLNHX3D of the sample to be detected, if so, detecting whether the gene VLNHX3D exists; or the expression condition of the gene VLNHX3D to judge the salt tolerance of the target plant.

The detection of whether the gene VLNHX3D is contained in the sample to be detected can be performed in various ways, for example, whether the gene VLNHX3D itself is contained can be directly detected, or a product produced by the gene VLNHX3D can be detected, wherein the product includes a direct product, an indirect product, a secondary product and the like, and the product can be mRNA, protein, a certain compound and the like.

The gene VLNHX3D can be directly detected by adopting a specific primer pair of the gene VLNHX3D, or by adopting a probe or a chip designed aiming at the gene VLNHX 3D. Furthermore, the sample to be detected is detected by a primer pair or a probe or a chip of the gene VLNHX 3D.

The primer pair, probe or chip for gene VLNHX3D of the present invention may be designed through conventional process.

Further, the nucleic acid sequence of the primer pair is shown as SEQ ID NO: 2 and SEQ ID NO: 3 or SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

Namely, the primer pair SEQ ID NO: 2 and SEQ ID NO: 3 can be used to detect the gene VLNHX 3D; primer pair SEQ ID NO: 4 and SEQ ID NO: 5, and has higher sensitivity.

The manner of detecting the gene VLNHX3D itself according to the invention is not limited thereto, and any biologically achievable detection manner is within the scope of the invention.

Likewise, detection of the product produced by the gene VLNHX3D can be carried out by various means, such as ELISA detection kit and the like.

Further, the sample to be tested comprises a material suitable for tissue culture of sexually reproducing, asexually reproducing or regenerable cells.

These samples to be tested may be materials suitable for sexual reproduction, such as selected from pollen, embryo sacs, ovules, ovaries, etc.;

materials suitable for vegetative propagation may be selected from roots, cuttings, stems, protoplasts, and the like;

suitable materials for tissue culture of regenerable cells may be selected from, for example, seeds, embryos, cotyledons, leaves, pollen, meristematic cells, roots, root tips, hypocotyls and stems, etc.

Specifically, further, the sample to be detected comprises any one of the following materials: leaf, root, stem, radicle, germ, seed.

Wherein the plant from which the sample to be detected is taken comprises monocotyledons and dicotyledons; monocotyledons include rice, corn, wheat, and the like; dicotyledonous plants include soybean, cotton, Arabidopsis, tobacco, and the like.

The fourth aspect of the present invention also provides a method for conferring salt tolerance to a plant by preparing a transgenic plant comprising or overexpressing gene VLNHX 3D;

the gene VLNHX3D has the nucleotide sequence shown in SEQ ID NO. 1.

The present invention adopts conventional biological method to prepare transgenic plant containing or over-expressing gene VLNHX 3D. Transgenic plants containing gene VLNHX3D can be obtained by various methods, such as the common vector-mediated transformation method, in which the target gene is inserted into a vector molecule, such as DNA of plasmid or virus of Agrobacterium, and the target gene is introduced into the plant genome along with the transfer of the vector DNA; for another example, the gene direct introduction method is a method of directly introducing an exogenous target gene into the genome of a plant by a physical or chemical method, the physical method includes a gene gun transformation method, an electric excitation transformation method, an ultrasonic method, a microinjection method, a laser microbeam method and the like, and the chemical method includes a PEG-mediated transformation method, a liposome method and the like; also, germplasm systems, including pollen tube pathway methods, germ cell invasion methods, embryo sac and ovary injection methods, etc. Transgenic plants overexpressing the gene VLNHX3D can also be produced in a number of ways, essentially as well, except that a promoter that enhances gene transcription is added to the vector.

The fifth aspect of the invention also provides the application of the gene VLNHX3D in the research of the genetic diversity of plant populations.

Compared with the prior art, the beneficial effects of the invention at least comprise the following aspects:

(1) the invention provides a gene VLNHX3D for the first time to regulate plant cell Na through systematic research⁺And/or K⁺Silencing Na in roots, stems and leaves of plants under salt stress⁺The accumulation amount of (A) is remarkably increased, and K in the root⁺The content is obviously reduced.

(2) After the gene VLNHX3D is expressed in the yeast mutant ATX3, the yeast transformed with the gene VLNHX3D has higher salt tolerance than the yeast of a control group, which shows that the gene VLNHX3D has the function of improving the salt tolerance of plants.

(3) The expression product of the VLNHX3D gene provided by the invention is positioned on a vacuolar membrane, and the gene can be applied to the aspect of cultivating or detecting the salt tolerance of plants, wherein the plants comprise rice, corn, wheat, soybean, cotton, arabidopsis thaliana, tobacco and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is an electrophoretogram of a PCR product of gene VLNHX3D in example 1 of the present invention;

FIG. 2 is the alignment chart of the structure of VLNHX3D protein and its amino acid sequence with other plant NHX in example 1 of the present invention;

FIG. 3 is a diagram showing the phylogenetic analysis of the VLNHX3D protein and NHX proteins of other species in example 1 of the present invention;

FIG. 4 is a map of the subcellular localization of the VLNHX3D protein in Arabidopsis protoplasts according to example 1 of the invention;

FIG. 5 is a diagram showing the expression pattern of gene VLNHX3D under NaCl stress in example 2 of the present invention;

FIG. 6 is a picture of the enhanced salt tolerance of yeast transformed with VLNHX3D gene in example 2 of the present invention;

FIG. 7 is a graph of albino phenotype of TRV CLA cotton seedlings and the silencing efficiency of VLNHX3D at TRV VLNHX3D in example 2 of the present invention;

FIG. 8 is a bar graph of expression levels of GhNHX3A in TRV VLNHX3D silenced plants according to example 2 of the present invention;

FIG. 9 is a graph showing the salt tolerance of plants after silencing of VLNHX3D gene in example 2 of the present invention;

FIG. 10 shows Na in TRV: VLNHX3D and TRV:00 plants under salt stress in example 2 of the present invention⁺And K⁺Content and Na⁺/K⁺Bar graph.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Materials and processes

The cotton material is upland cotton genetic standard system TM-1(Gossypium hirsutum cv TM-1).

Planting cotton materials: plump cotton seeds are selected, soaked in distilled water and placed in an incubator at 30 ℃ overnight to promote germination. And (3) selecting seeds which germinate and expose white in the next day, planting the seeds into a square plastic pot containing vermiculite, covering a mulching film, and placing the square plastic pot in a greenhouse at the temperature of 23 ℃, wherein the illumination time is 16h illumination/8 h dark cycle, and the relative humidity is 60%. And (3) removing the mulching film after cotyledons of the seeds are exposed, selecting cotton seedlings with consistent growth vigor after the cotyledons are unfolded, washing the cotton seedlings clean by using tap water, wrapping the stem base part by using foam cotton, and putting the wrapped stem base part into a water culture box (containing Hoagland nutrient solution) for later-stage culture and treatment.

Treatment of cotton material: and performing salt stress treatment when the cotton seedlings grow to have two leaves and one heart. 50, 100, 150 and 200mmol/L NaCl is respectively added into the Hoagland nutrient solution. Sampling is carried out 0, 1, 3, 6 and 12 hours after treatment, roots, stems and true leaves (at least 3 plants) are separately sampled, wrapped by tinfoil and marked, quickly placed in liquid nitrogen for quick freezing, and then stored in a refrigerator at the temperature of 80 ℃ below zero for subsequent experiments.

II, related primers

Primer sequences referred to in Table 1

Example 1

First, Gene cloning

1. Total RNA extraction: the roots, stems and leaves of the cotton materials which are frozen and preserved at the temperature of minus 80 ℃ are respectively ground into powder in liquid nitrogen, and 100mg of sample powder is respectively put into a 2mL RNase-Free centrifuge tube. Subsequent experimental procedures were performed according to the instructions of the Tiangen RNA extraction kit (DP 441).

2. And (3) cDNA synthesis: cDNA Synthesis was performed using the HiScript III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) (R312-01) reverse transcription reagent from Novonop.

3. Gene cloning (RT-PCR): the first strand cDNA obtained by reverse transcription was used as a template, and the desired gene was amplified using the high fidelity enzyme KOD-Plus-Neo (Code No. KOD-401) of TaKaRa.

TABLE 2 Gene cloning PCR System

TABLE 3 Gene cloning PCR reaction procedure

5. After the PCR reaction, the PCR products were detected by 1.5% agarose gel electrophoresis, and the results are shown in FIG. 1, in which the PCR products of VLNHX3D were all located between 1000-2000bp of Maker.

The band of interest was recovered using the FastPure Gel DNA Extraction Mini Kit (DC301) from Novowed.

6. Connecting the PCR product with a cloning vector: mu.L of PCR gel recovery product 4. mu.L and 1. mu.L of pEASY-Blunt Zero Cloning Vector were added to a 200. mu.L clean centrifuge tube, and reacted at 25 ℃ for 30min (temperature controlled by PCR instrument) after gentle mixing. After the reaction was complete, the reaction mixture was placed on ice.

7. Transforming escherichia coli competence, picking single clone in 500 mu L LB + Kan liquid culture medium in sterile environment, culturing at 37 ℃ and 200rpm for 6h until the bacterial liquid is turbid. PCR and sequencing were performed using the bacterial solution as a template (sequence shown in SEQ ID NO: 1). A positive strain containing VLNHX3D was obtained.

II, VLNHX3D protein structure prediction and amino acid sequence alignment

The conserved domain of VLNHX3D protein was revealed using DOG 2.0 software, TMHMServerv.2.0 (http:// www.cbs.dtu.dk/services/TMHMM /), for prediction and analysis of the transmembrane domain of VLNHX 3D.

Other species NHX protein sequences and VLNHX3D were subjected to multiple sequence alignments using DNAMAN 9.0 software, other species NHX including VLNHX3D (GH _ D02G0494), GhNHX1A (GhA11G2132), GhNHX1D (GhD11G2440), AtNHX1(AT5G27150.1), GmNHX1(AEA07714.1), PeNHX3 and OsNHX1(BAA 83337.1).

By constructing a protein structure diagram of VLNHX3D (FIG. 2), it was shown that the amino acid sequence encoded by VLNHX3D contains Na which is a characteristic feature of NHX⁺/H⁺Conserved domain of transport proteins, i.e. Na⁺_H⁺Exchanger, and has 11 transmembrane structure conserved regions (fig. 2A). The amino acid sequence of VLNHX3D was subjected to multiple sequence alignment with the amino acid sequences of other plant vacuole-type NHXs using DNAMAN 9.0 software. (FIG. 2B) shows that the VLNHX3D protein sequence has higher homology with other phytovacuole type NHX proteins. They all have an identical aminopyrazine amidine binding site (Aminopyrazine amidine binding sites) which is complementary to Na⁺Is involved in competitive inhibition and has a conserved CaM binding site at the C-terminus.

Construction of evolutionary tree

1. Construction of the evolution tree of VLNHX3D and other species NHX

To further analyze the relationship between the VLNHX3D protein and NHX in other plants, Arabidopsis thaliana (At), Populus diversifolia (Pe), rice (Os) and maize (Zm) were used as research targets, in combination with Na in upland cotton, which has been confirmed in previous studies⁺/H⁺GhNHX members with the function of reverse transport protein together construct a phylogenetic tree.

NHX protein sequence information of upland cotton, Arabidopsis, Populus euphratica, rice and maize (Table 3.8) was collected, multiple sequence alignments were performed using MEGA X software, a phylogenetic tree was constructed using a Neighbor-joining method, and a bootstrap value was set to 1000.

Table 4 other species information

As shown in fig. 3, a total of 28 NHX proteins of 5 species were classified into 2 subfamilies, vacuolar NHX and intranuclear NHX in the phylogenetic tree. Vacuolar NHX comprises 20 NHX genes and the endosomal type comprises 8 NHX genes. VLNHX3D was closer to the vacuolated NHX family members PeNHX4 and AtNHX4 in the evolutionary tree, indicating that they have a closer relationship; distant relativity to members of the nuclear endosomal NHX family of Arabidopsis, Populus diversifolia, rice, maize and Gossypium hirsutum. These results indicate that VLNHX3D is a member of the vacuolar NHX family.

Four, gene subcellular localization

1. Vector construction

(1) And (3) vector linearization: the pCAMBIA2300(35S: GFP) vector was linearized with BamHI and EcoRI restriction enzymes.

(2) After the enzyme digestion reaction of the vector is finished, the vector is checked by 1.5 percent agarose gel electrophoresis, and a target band is cut under an ultraviolet lamp for gel recovery.

(3) According to the vector and gene sequence, a vector homologous primer containing an enzyme cutting site sequence but not containing a gene stop codon is designed, and is shown as a subcellular localization primer in the table 1, and then PCR amplification reaction and gel recovery of a target band are carried out.

(4) Homologous recombinase of Novozam (C.), (

Ultra One Step Cloning Kit) the amplified product was ligated to pCAMBIA2300(35S: GFP) vector, 3. mu.L of the product was recovered from the gene amplification gel, 2. mu.L of the product was recovered from the vector gel, 5. mu.L of homologous recombinase was gently mixed and reacted at 50 ℃ for 5 min.

(5) The ligation products were transferred to E.coli in a freeze-thaw state.

(6) The other steps are the same as the above escherichia coli transformation and positive clone detection. The upstream primer is HP158, and the downstream primer is a downstream carrier connecting primer of the gene.

(7) The VLNHX3D-GFP fusion expression vector can be obtained after the sequencing is correct.

2. Plasmid extraction of Escherichia coli from VLNHX3D-GFP Using Novonoprazan Plasmid extraction Kit (FastPure Endo Free Plasmid Maxi Kit, DC 202-01).

3. Preparation and transformation of arabidopsis protoplasts: the preparation of Arabidopsis protoplasts and transformation kit (PPT101) from Coolaber was used.

4. Subcellular localization results observations: the protoplasts were observed and photographed using a confocal laser microscope.

The VLNHX3D-GFP expression fusion vector was obtained by fusing GFP to VLNHX3D using the tonoplast-tagged fusion protein delta-TIP-RFP as a positive control. The delta-TIP-RFP and VLNHX3D-GFP plasmids were co-transformed into Arabidopsis protoplasts for transient expression, with the empty vector 35S, GFP and delta-TIP-RFP plasmid co-transformed protoplasts, as a negative control. It was observed by confocal laser microscopy (FIG. 4) that, in Arabidopsis protoplasts, after transfer of 35S fusion expression vectors of GFP and. delta. -TIP-RFP, the green fluorescence generated by GFP was dispersed throughout the cytoplasm, while the red fluorescence generated by. delta. -TIP-RFP was distributed over the vacuolar membrane, and their fluorescence signals did not completely overlap. When the delta-TIP-RFP and VLNHX3D-GFP fusion vector were expressed in Arabidopsis protoplasts, the complete superposition of the green fluorescence generated by the VLNHX3D-GFP fusion protein and the red fluorescence generated by the delta-TIP-RFP on the tonoplast produced yellow fluorescence, and the yellow fluorescence signal formed a circle on the tonoplast, these results visually indicate that the VLNHX3D protein was on the tonoplast.

Example 2

First, real-time fluorescent quantitative PCR of VLNHX3D

1. The cotton material was subjected to RNA extraction for roots, stems and leaves at various time points after salt treatment.

2. And (3) cDNA synthesis: HiScriptII Q RT Supermix for qPCR (+ gDNA wiper) (R233-01) reverse transcription reagent from Novozan was used.

3. Designing a specific fluorescent quantitative primer: primermier 5 was used to design specific primers for VLNHX3D and GhNHX3A, with GhHIS3 as the reference gene and the primer sequences shown in Table 1.

4. Real-time fluorescent quantitative PCR (RT-qPCR): the following reaction mixtures were prepared using ChamQ Universal SYBR qPCR MasterMix.

Three biological replicates per sample were performed, and results were used 2^-△CtAnd (5) carrying out analysis calculation.

The expression level of VLNHX3D in upland cotton leaves without salt stress treatment (0, 1, 3, 6 and 12h) was used as a control (Mock), and the expression level of VLNHX3D in leaves at the same time point under NaCl stress treatment was used as an experimental group. The fluorescent quantitation result shows that the expression level of VLNHX3D in leaves gradually increased with the increase of salt stress time and reached a peak 6h after 200mM salt stress treatment. When the treatment time was extended to 12 hours, the expression level of VLNHX3D decreased. These results indicate that salt stress induced changes in the expression level of VLNHX 3D.

Further analysis of the expression pattern of VLNHX3D on upland cotton leaves treated with different concentrations of NaCl cotton seedlings at the two-leaf one-heart stage were treated with 0, 50, 100, 150 and 200mM NaCl. Since the expression level of VLNHX3D was highest at 6h, the expression level of VLNHX3D was analyzed at 6h of leaves treated with NaCl at different concentrations. As shown in FIG. 5, from the results of fluorescence quantification, the expression level of VLNHX3D in leaf discs increased with the increase of NaCl concentration. This further verifies that salt stress can induce and modulate the expression level of VLNHX 3D.

Second, yeast function complementation experiment

1. Constructing a yeast expression vector:

(1) the plasmid containing VLNHX3D colibacillus is used as a template, and primers respectively contain BamH I and Sac I enzyme cutting site sequences (VLNHX3D-Y-F/R) for PCR amplification and gel recovery.

(2) The yeast expression vector pYES2 plasmid was vector linearized with BamH I and SacI restriction enzymes and then gel recovered.

(3) And (3) connecting the glue recovery product of VLNHX3D containing the enzyme cutting sites with the carrier recovery product in the step (2) by using homologous recombinase to construct a carrier, wherein the resistance of the LB culture medium is Amp. The yeast expression vector plasmid pYES2-VLNHX3D was obtained.

2. Yeast competence preparation: the Coolaber yeast kit (SK2401-200T) was used.

3. Yeast plasmid transformation: yeast expression vectors pYES2-VLNHX3D and pYES2 (empty, control) were each transferred into a prepared yeast competence.

4. And (3) positive strain verification: the monoclonal strains were selected and cultured overnight in YNB + Ade + Try liquid medium at 30 ℃ and 200 rpm. And (3) carrying out PCR by taking the yeast liquid as a template to verify the positive strains. Since yeast contains cell wall, the pre-denaturation time is extended to 5 min.

5. Yeast function complementation experiment: yeast salt stress functional screening was performed with different APG media. Five nutrient components of Ade, Ura, Try, Leu and His are added on the basis of an APG culture medium, and three culture conditions of 0 (contrast), 40mM NaCl and 50mM NaCl are set.

(1) 200. mu.L of the positive clone strain obtained in step 5 above (pYES 2-VLNHX3D or pYES2, respectively) was added to a glass flask containing 10mL of YNB + Ade + Try medium, and 200. mu.L of wild type W303200 (see yeast competent preparation for activation) was added to a glass flask containing 10mL of YPD medium.

(2) Culturing the above bacterial liquid at 30 deg.C and 200rpm in incubator to OD₆₀₀＝1.2。

(3) 10 mu L of the bacterial liquid is taken and diluted by 20, 200 and 2000 times respectively.

(4) Each 8. mu.L of the diluted four gradient solutions was spotted on APG medium containing 0, 40mM NaCl and 50mM NaCl.

(5) The cell culture was inverted at 30 ℃ for 5 days to observe the outer row and photographed with a camera.

The pYES2-VLNHX3D yeast fusion expression vector is transferred into a yeast mutant AXT3, a transgenic yeast transferred with an empty vector pYES2 is used as a negative control, a wild-type yeast W303 is used as a positive control, and the pYES2-VLNHX3D transgenic yeast is subjected to salt tolerance analysis. The results are shown in FIG. 6: in an APG medium without NaClIn the wild-type yeast W303, the empty vector pYES2 transgenic yeast and the pYES2-VLNHX3D transgenic yeast all can grow normally and have basically consistent growth conditions. In APG medium containing 40mM NaCl, W303 still grows normally after being diluted 2000-fold. Due to deletion of endogenous Na in mutant AXT3⁺Transporter, AXT3, is exposed to Na⁺When poisoned, the growth was inhibited, so that the yeast transfected with pYES2 was inhibited in the medium containing 40mM NaCl and was not able to grow substantially. The growth of pYES2-VLNHX3D transgenic yeast was slightly inhibited after 2000-fold dilution compared to the wild type yeast W303. When the NaCl concentration was increased to 50mM, the growth of W303 was not significantly inhibited, whereas the growth of pYES2 and pYES2-VLNHX3D both showed different degrees of inhibition. Compared with the pYES2 transgenic yeast negative control, the growth of the pYES2-VLNHX3D yeast is slightly inhibited, better growth conditions are shown, a small amount of bacterial plaque can still be seen after 200-fold and 2000-fold dilution, and the pYES2 transgenic yeast after 200-fold dilution can hardly grow. The results of these experiments show that pYES2-VLNHX3D can not only partially recover Na of yeast mutants⁺The transfer function, and the transgenic yeast shows higher salt tolerance.

Third, virus-induced silencing of VLNHX3D in cotton

1. Constructing a VIGS recombinant expression vector: see specifically the above steps; the restriction sites are EcoRI and Xho I. The VIGS vector of TRV VLNHX3D is finally obtained.

(1) VIGS vector TRV VLNHX3D transformed Agrobacterium (GV3101),

(2) positive clone detection

(3) And carrying out PCR verification by taking the bacterial liquid as a PCR template.

(4) And (4) after amplification culture of the bacterial liquid corresponding to the correct sequencing, adding 50% glycerol with the same volume for preserving bacteria, and storing at-80 ℃.

2. And (3) planting upland cotton TM-1 under a water culture condition. After two cotyledons of cotton are completely unfolded, VIGS bacterial liquid injection is carried out.

3. Preparing a heavy suspension: to 500mL of deionized water were added 5mL of MES solution, 1mL of AS solution and 5mL of MgSO₄The solution is mixed evenly (ready for use).

4. Resuspending the bacterial liquid:

(1) and (3) taking 40mL of overnight cultured agrobacterium liquid containing the VIGS vector to be placed in a 50mL clean centrifugal tube, centrifuging at 5000rpm for 5min, and collecting thalli.

(2) Suspending the thallus with the prepared re-suspension, and adjusting OD₆₀₀＝1.2。

(3) Standing at room temperature for 3h under dark condition.

5. Cotton injection:

(1) the resuspended pTRV1 (helper plasmid) was mixed in equal volumes with pTRV2 (empty), TRV: VLNHX3D and TRV: CLA (positive control), respectively.

(2) The back of the cotton cotyledon was gently lacerated with the needle of a 1mL sterile syringe (care was taken not to cut through the leaf), and then the bacterial solution was injected from the wound to soak the leaf.

(3) And after infection, growing for 24 hours in a dark place, and then growing under normal conditions.

6. Detecting the gene silencing efficiency: after 10 days of injecting the bacteria liquid, the true leaves of the TRV CLA (positive control) plants have whitening character. Randomly taking 3 target gene silencing plants and injecting roots, stems and leaves of the unloaded plants for RNA extraction and reverse transcription. The silencing efficiency of the gene is quantitatively detected by fluorescence.

To further verify that VLNHX3D has the function of enhancing salt tolerance, VLNHX3D was analyzed for its effects under cotton salt stress using VIGS technology. Virus Induced Gene Silencing (VIGS) technology can infect plants with viruses or bacterial solutions containing target gene fragments, induce plant endogenous gene silencing to cause corresponding physiological and morphological changes, and then study gene functions through the changes. The constructed silent expression vectors TRV: VLNHX3D, TRV:00 (negative control) and TRV: CLA (positive control) are respectively injected to the back of cotyledons of cotton seedlings after germination for 10 days after the auxiliary plasmids are mixed in equal volume. The true leaves of the positive control showed whitening after 12 days (fig. 7).

The silencing efficiency of VLNHX3D was tested in TRV VLNHX3D plants after the positive control appeared albino phenotype. RNA of roots, stems and leaves of TRV: VLNHX3D and TRV:00 cotton plants were extracted, and then expression level of VLNHX3D was detected using fluorescent quantitation technique. The results showed that root, stem and leaf expression of VLNHX3D was significantly lower in TRV: VLNHX3D than in TRV:00 plants (fig. 7). To ensure specificity of the gene silencing effect, the corresponding expression level of the homologous gene GhNHX3A of VLNHX3D was also examined. The results in FIG. 8 show that there is no significant difference in the expression level of GhNHX3A in the leaves, stems and roots of TRV VLNHX3D and TRV 00 plants. This indicates that VLNHX3D is specifically silenced in TRV VLNHX3D silenced cotton plants with high efficiency.

7. And (3) silent plant salt treatment: after VIGS injection, when cotton seedlings grow to have two leaves and one heart, 200mM NaCl treatment is respectively carried out on TRV:00 (no load) and plants with silent target genes. NaCl of the corresponding mass was added to the Hoagland nutrient solution, while the Hoagland nutrient solution without NaCl was used as a control treatment. Phenotypic observations and photographs of the gene silenced and control plants were taken 10 days after treatment.

The treatment group was applied with 200mM NaCl using a control group containing 0mM NaCl, and the salt tolerance of cotton seedlings was observed 10 days after the treatment. FIG. 9 results show that under normal growth conditions, TRV: VLNHX3D silenced plants and TRV:00 grow consistently; TRV: VLNHX3D leaves were significantly yellow and wilted and the plant height was shorter than TRV:00 after 200mM NaCl salt stress treatment. It shows that when the expression of VLNHX3D is silenced, the sensitivity of the plant to salt stress is increased.

8. And (3) ion content determination: respectively taking 9 TRV:00 and TRV: VLNHX3D plants (sampling for the treated group and the control group), separating the roots, stems and leaves, labeling, deactivating enzyme at 105 deg.C for 5min, oven drying at 75 deg.C to constant weight, and grinding into powder.

And (3) measuring the content of sodium ions and potassium ions: the measurement was performed by using an atomic absorption method. And (5) measuring the content of sodium ions and potassium ions.

And taking the dried sample powder and sieving the sample powder through a 100-mesh nylon sieve. Weighing 0.1g of the sieved plant powder, drying second true leaves, stems and roots of TRV:00 and TRV: GhNHX3D plants at 90 deg.C, and grinding into powder. 0.05g of a sample of the powder was dissolved in 5mL of concentrated HNO₃(nitration). Diluted 12 times with deionized water and centrifuged. Collecting supernatant, and measuring sodium ion by atomic absorption methodSeed and potassium ion content.

The salt tolerance of plants is related to the intracellular ion content. Too high a concentration of ions in the cells can poison the cells, thereby reducing the salt tolerance of the plants. This study determined Na in the roots, stems and leaves of the TRV VLNHX3D and TRV 00 plants⁺And K⁺Content and calculation of Na⁺/K⁺The salt tolerance mechanism of VLNHX3D was further explored. FIG. 10 shows the results indicating Na in the roots, stems and leaves of the TRV VLNHX3D and TRV 00 plants in the control group⁺And K⁺Content and Na⁺/K⁺The ratios all showed the same level. TRV VLNHX3D plant root, stem and leaf Na after 200mM NaCl treatment⁺The content is obviously higher than TRV: 00; TRV VLNHX3D root and Stem K⁺Has no difference in the content of (A), but K is present in VLNHX3D roots⁺Is significantly lower than TRV: 00.

TRV VLNHX3D silences plant roots, stems and leaves for Na after salt stress as compared to TRV 00 plants⁺/K⁺The ratio is significantly higher. Shows that the TRV VLNHX3D plants accumulate more Na in vivo after being stressed by salt⁺Especially K in root⁺The content is obviously reduced, which results in that the TRV VLNHX3D cotton plant has higher Na⁺/K⁺Sensitivity to salt stress is increased.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Sequence listing

<110> Henan university of agriculture

Application of <120> gene VLNHX3D in regulating concentration of Na + and/or K + in plant cells

<130> 2021

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1608

<212> DNA

<213> Gossypium hirsutum

<400> 1

atggcgatcg ggatcttaaa ctctctttta gcctctgatc atagctccat agtttcaatg 60

aacttattcg tggcgcttct ttgcggttgt attgtgattg gtcatttact agaggaaagc 120

cgatggatga acgagtccat tactgctctt gtcattgggg tgtgcactgg agttgtaatt 180

ttgcttacaa caggaggaaa aagctctcac ctgttagttt tcagtgaaga cttgttcttc 240

atttatttgc ttcctcctat tatttttaat gcgggattcc aagtgaagaa gaagcaattt 300

ttccgcaact ttatgactat catgctgttt ggtgcagttg gtactttaat atcatttggc 360

atcatatctg caggtgccat acagtttttc aaggaattgc atattggtga tctgcagata 420

ggggactatc ttgcaattgg ggcaatattt tctgcaacag attctgtttg cactttgcaa 480

gttcttaatc aggacgagac acctttgttg tacagtctgg tttttgggga gggagttgtg 540

aatgatgcca catcagtggt tcttttcaat gcaatccaga gctttgacct taatcacatc 600

aactctacca ttgccttgaa atttgtcgga aatttttttt atttgttcat ctcaagtact 660

ttgctaggag ttgtgactgg actgctcagt gctttcatta ttaaaaagct gtatttcgga 720

aggcattcaa ctgatcgcga ggttgctctt atgatcctca tggcttacct ctcatacatg 780

ctcgctgaac ttttctattt aagcggaatt cttacagtat tcttttgtgg gattgttatg 840

tctcactata catggcataa tgttacagaa agttcaagag tgacaacaaa gcatgctttt 900

gctactctat catttgttgc tgagatcttt atcttcctct atgttggtat ggatgctttg 960

gacatcgaga agtggagagt tatcagtgat agccccggaa aatcagttgg ggtgagttcg 1020

attctactgg gcttgattct tgttggaaga gcagcctttg ttttcccctt gtcgttcata 1080

tccaacttga caaagaaagc tcctcatgag aaaattgaat tcaaacagca agttaccatt 1140

tggtgggctg gtcttatgcg cggtgctgtc tcaatggcac ttgcttataa tcagtttact 1200

agtttagggc atactcaagt gcgagggaat gcgatgatga taaccagcac aatcacggtt 1260

gttcttttca gcacagtggt tttcggattg atgactaaac cattagttag gatcttgctt 1320

ccttctccaa aacatctctc gagaatgctt tcgtccgagc caactactcc taaatcattc 1380

ttcctaccac ttctcaacaa tgggcaagaa tctgaggctg aacaaggcaa ccgaagcgtg 1440

atccggccgt ccagcttaag aatgctcttg accactcctt cccacaccgt gcactattat 1500

tggagaaaat tcgatgatgc cttcatgcga cctgtattcg gtggaagggg tttcgtacca 1560

tttgttcccg gatcacccac tgaacaaaac ggtcctcagt ggcaatga 1608

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 2

atggcgatcg ggatcttaaa 20

<210> 3

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 3

tcattgccac tgaggacc 18

<210> 4

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 4

tactcaagtg cgagggaatg cg 22

<210> 5

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 5

ggttgccttg ttcagcctca ga 22

Claims

1. Gene VLNHX3D in regulating plant cell Na⁺And/or K⁺The application of the gene VLNHX3D in concentration, the gene VLNHX3D has the nucleotide sequence shown in SEQ ID NO 1.

2. Use according to claim 1, characterized in that the Na is silenced in the roots, stems and leaves of plants of the gene VLNHX3D compared to wild plants⁺Increased accumulation, K in stems and leaves⁺Constant accumulation, K in the root⁺The content is reduced.

3. Application of gene VLNHX3D in cultivating or detecting salt-tolerant transgenic plants, wherein the gene VLNHX3D has a nucleotide sequence shown in SEQ ID NO 1.

4. The use according to claim 1, characterized in that the overexpression or the transfer of the gene VLNHX3D is detected, and the salt tolerance of the plant is increased;

5. The use according to any one of claims 1 to 4, wherein the plant comprises a monocotyledonous plant and a dicotyledonous plant;

the monocotyledon comprises rice, corn and wheat;

the dicotyledonous plants comprise soybean, cotton, arabidopsis thaliana and tobacco;

6. A method for detecting the salt tolerance of plants is characterized in that the salt tolerance of the plants is judged by detecting the existence or expression condition of a gene VLNHX3D of a sample to be detected;

the gene VLNHX3D has the nucleotide sequence shown in SEQ ID NO. 1.

7. The method for detecting the salt tolerance of plants according to claim 6, wherein the sample to be detected is detected by a primer pair or a probe or a chip of gene VLNHX 3D.

8. The method for detecting the salt tolerance of a plant according to any one of claims 5 to 7, wherein the sample to be detected comprises a material suitable for tissue culture of sexually reproducing, asexually reproducing or regenerable cells;

further, the sample to be tested comprises any one of the following materials: leaf, root, stem, radicle, germ, seed.

9. A method for conferring salt tolerance to a plant, characterized in that a transgenic plant containing or overexpressing the gene VLNHX3D is prepared;

the gene VLNHX3D has a nucleotide sequence shown in SEQ ID NO. 1;

further, the plant includes monocotyledons and dicotyledons;

the monocotyledon comprises rice, corn and wheat;

the dicotyledonous plants comprise soybean, cotton and arabidopsis thaliana.

10. The gene VLNHX3D is applied to the research of the genetic diversity of plant populations.