CN114032245B

CN114032245B - Gene VLNHX D regulates plant cell Na+And/or K+Application in concentration

Info

Publication number: CN114032245B
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: 2024-05-28
Anticipated expiration: 2041-04-06
Also published as: CN114032245A

Abstract

The invention relates to the field of plant molecular biology, in particular to application of a gene VLNHX D in regulating concentration of Na ⁺ and/or K ⁺ in plant cells. The invention provides application of a gene VLNHX D in regulating the concentration of Na ⁺ and/or K ⁺ in plant cells, wherein the gene VLNHX D has a nucleotide sequence shown as SEQ ID NO. 1. According to the invention, through cloning gene VLNHX D, constructing a transgenic vector, reducing VLNHX D expression by using VIGS technology, under salt stress conditions, the Na ⁺ accumulation amount in roots, stems and leaves of silent plants is obviously increased, the K ⁺ content in roots is obviously reduced, and the salt tolerance of cotton is obviously reduced, so that VLNHX D participates in early salt stress response, and the salt tolerance of cotton is improved by regulating the steady balance of intracellular Na ⁺ and K ⁺.

Description

Application of gene VLNHX D in regulating concentration of Na + and/or K + in plant cells

Technical Field

The invention relates to the field of plant molecular biology, in particular to application of a gene VLNHX D in regulating concentration of Na ⁺ and/or K ⁺ in plant cells.

Background

Salt stress can lead to slow growth and development of plants, yellowing or wilting of leaves, and death of whole plants when serious. The hazards of salt stress mainly include 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. Plants have reduced water uptake capacity resulting in plants suffering from physiological drought, resulting in suppressed plant production. Osmotic stress can rapidly reduce cell expansion in root tips and young leaves, resulting in stomatal closure. The increase of the sodium ion content in the soil not only rapidly reduces the effectiveness of soil moisture, but also slowly accumulates sodium ions on the overground part; the spatial and temporal distribution results indicate that early salt stress responses are caused by osmotic or drought stress, but only then are specific ionic stress responses. Low water potential induces ABA production and signal transduction, resulting in guard cell depolarization, and reduced pore size and conductivity. Over time of salt stress, the decrease in cell elongation and cell division will result in smaller and thicker leaves and thus reduced photosynthesis, further affecting the growth and development of the plant.

Ion stress: inorganic ions such as Na ⁺ and Cl ^- are necessary for plant life activities, but when the inorganic ions are excessive, the inorganic ions are harmful to ions, so that the selective permeability of plasma membranes is damaged, and extracellular salt ions enter cells in a large amount to damage ion balance. Transpiration can transfer Na ⁺ from the root to the leaves above the ground and accumulate in the leaves, but only a small portion of Na ⁺ in the leaves can move to the root through the phloem again, which can cause an increase in the ion content in the plant leaves and thus create a hazard. Excessive absorption of certain salts by plants reduces the absorption of other salts and plants produce symptoms of nutrient deficiency or ion poisoning.

Nutritional imbalance: when plants grow in a NaCl stress environment, the plants absorb a large amount of Na ⁺ to reduce the absorption of K ⁺, so that potassium ions are deficient, the absorption of Ca ²⁺ and Mg ²⁺ is influenced, the nutrition absorption of the plants is further influenced, the metabolism of the plants is disturbed, and the growth of the plants is inhibited. Research proves that under the condition of salt stress, more Na ⁺ is accumulated in the oleaster, and the content of K ⁺ and Ca ²⁺ is reduced; a large amount of Na ⁺ is accumulated in the root system of the oleaster variety with stronger salt tolerance, so that Na ⁺ accumulation in the leaves is reduced, and compared with the variety which is not salt tolerant, K ⁺ and Ca ²⁺ are less lost in the oleaster with salt tolerance. Too much plant uptake of Cl ^- and SO ₄ ^2- affects the uptake of HPO ₄ ^2-; excessive phosphate causes a deficiency of Zn ²⁺, which results in unbalanced nutrition of plants.

Plant response to salt stress is divided into two phases: the method comprises the steps of inhibiting the growth of young leaves in a rapid osmotic stress stage and accelerating the slow ion poisoning process of aging of mature leaves.

Na ⁺/H⁺ reverse transporter (NHX) is a reverse transporter widely existing in higher plants, and plays an important role in regulating the maintenance of intracellular pH, ion balance and the like. Researchers find the protein in barley, rice, arabidopsis thaliana, atriplex canescens, beet, soybean and other plants. At present, na ⁺/H⁺ inverse transferrin genes of different species have been cloned, and the expression characteristics of NHX have been studied. The results indicate that some NHX proteins only show transport activity under salt stress conditions and do not have transport activity under non-stress environments. Some NHX genes are expressed at lower levels under normal environmental conditions, but their expression levels are induced to increase as the degree of salt stress increases. There are also some plants in which NHX gene expression is undetectable under salt stress conditions.

6 Na ⁺/H⁺ inverse transport proteins (AtNHX 1-6) were found in Arabidopsis, and phylogenetic analysis divided these 6 AtNHX into two subgroups, with AtNHX1-4 belonging to subgroup I and AtNHX5-6 belonging to subgroup II. Studies have shown that NHX in subgroup I is located on the vacuolar membrane, with the same affinity for Na ⁺ and K ⁺; they sequester the accumulated Na ⁺ and/or K ⁺ in the cytoplasm in the vacuoles, maintaining the turgor pressure of the cells. NHX of subgroup II is located in the endomembrane system of cells, mainly regulating the balance of intracellular K ⁺, alleviating the damage caused by stress by accumulating more K ⁺ in cells under salt stress.

The NHX protein located in the vacuole membrane is a popular research field of Na ⁺/H⁺ antiporters. It has been shown that the insertion of the gene encoding vacuole membrane NHXs into plants can effectively improve the salt tolerance of the plants. Transgenic Arabidopsis thaliana overexpressing AtNHX1 can be grown at 200 mmol/LNaCl. Compared with the wild type, the Na ⁺ content of the transgenic arabidopsis plant is increased, and the Na ⁺/H⁺ antiport protein activity is enhanced. After being heterologously expressed in plants such as sweet potato, tartary buckwheat and festuca arundinacea, atNHX1 causes the increase of intracellular Na ⁺ concentration and K ⁺/Na⁺ ratio, and improves the tolerance of the plants to salt stress. Over-expression BnNHX1 improves the salt tolerance of transgenic tobacco. Therefore, we speculate that the plant vacuolar Na ⁺/H⁺ antiport protein is able to specifically recognize and differentiate Na ⁺, thereby improving the salt tolerance of the plant. However, in-depth analysis of the vacuolar Na ⁺/H⁺ antiporters showed that their salt tolerance mechanism may be involved in mediating K ⁺ accumulation and maintaining lower Na ⁺/K⁺ ratios. In the previous study, transgenic tomatoes which overexpress AtNHX1 can distribute more K ⁺ into vacuoles, and the absorption of Na ⁺ is inhibited by increasing the content of K ⁺ in the vacuoles in a feedback way, so that the salt tolerance of plants is improved. Transgenic rice overexpressing PgNHX a 1 survived and completed its life cycle, successfully flowering and seed setting. The wild type plant upper leaf has higher Na ⁺ content, while the transgenic plant upper leaf has higher K ⁺ content. Expression of the NHXS1-IRES-TVP1 gene in transgenic tobacco results in reduced leaf Na ⁺ content and increased K ⁺ content. These findings indicate that the mechanism by which vacuolar NHX proteins regulate salt tolerance may be different in different plants.

In addition, studies have also found that different NHX may be distributed over different structures of the cell, some on the vacuolar membrane and some on the intimal system of the cell. And the Na ⁺/H⁺ reverse transport proteins identified in different organisms have a certain difference in structure.

China is a large country for cotton production and consumption in the world, and cotton is an important economic crop in China. As the cultivated land area of China is gradually reduced, the competition strength of grain crops and cotton for cultivated land is continuously increased. The method has the advantages that the method has wide saline-alkali soil in China, cotton planting in the saline-alkali soil can solve the contradiction between grain and cotton competing, promote the healthy development of cotton industry and realize sustainable agriculture. Therefore, the method has great significance for researching the salt-tolerant genes of cotton.

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

Disclosure of Invention

The invention clones a Na ⁺/H⁺ inverse transport protein gene VLNHX D from upland cotton genetic standard line TM-1, and is positioned on a vacuole membrane. The real-time fluorescence quantitative result shows that VLNHX D is induced to up-regulate expression in cotton leaves at the early stage of salt stress, and the expression quantity of VLNHX D is gradually increased along with the deepening of the salt stress degree. After VLNHX D was expressed in the yeast mutant ATX3, VLNHX D gene-transferred yeast had higher salt tolerance than control (empty transferred) yeasts. After VLNHX D expression is reduced by using a VIGS technology, the accumulation amount of Na ⁺ in roots, stems and leaves of silent plants is obviously increased under salt stress, the content of K ⁺ in the roots is obviously reduced, the salt tolerance of cotton is obviously reduced, VLNHX D participates in early salt stress response, and the salt tolerance of cotton is improved by regulating the steady-state balance of intracellular Na ⁺ and K ⁺.

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

Use of gene VLNHX D in modulating the concentration of Na ⁺ and/or K ⁺ in a plant cell, said gene VLNHX D having the nucleotide sequence set forth in SEQ ID No. 1.

According to the invention, through cloning gene VLNHX D, constructing a transgenic vector, reducing VLNHX D expression by using VIGS technology, under salt stress, the accumulation amount of Na ⁺ in roots, stems and leaves of silent plants is obviously increased, the content of K ⁺ in roots is obviously reduced, the salt tolerance of cotton is obviously reduced, and VLNHX D participates in early salt stress response, and the salt tolerance of cotton is improved by regulating the steady balance of intracellular Na ⁺ and K ⁺.

Further, silencing the gene VLNHX3D plants increased the amount of Na ⁺ accumulated in the roots, stems and leaves, unchanged the amount of K ⁺ accumulated in the stems and leaves, and reduced the content of K ⁺ in the roots, as compared to the wild plants.

In a second aspect, the invention provides the use of gene VLNHX D in the cultivation or detection of salt tolerant transgenic plants, said gene VLNHX D having the nucleotide sequence shown in SEQ ID NO. 1.

The invention discovers that the expression quantity of the gene VLNHX D in the leaf gradually increases along with the increase of the salt stress time, reaches a peak value after 200mM salt stress treatment for 6 hours, and gradually increases along with the increase of the salt concentration, thereby indicating that the expression quantity of the gene VLNHX D in the leaf changes along with the increase of the salt concentration, and the induction of VLNHX D by the salt stress. After further expressing gene VLNHX D in yeast mutant ATX3, the VLNHX D gene-transformed yeast has higher salt tolerance than the control group. The gene VLNHX D has the effect of improving the salt tolerance of plants. Thus, the gene can be used for breeding transgenic plants and for detecting salt tolerance of transgenic plants.

Further, the 3D overexpression or the transfer of the gene VLNHX is detected, and the salt tolerance of the plant is increased;

The gene VLNHX D was not detected or silenced and the salt tolerance of the plant was weakened.

In the present invention, the plants include monocotyledonous plants and dicotyledonous plants;

The monocotyledonous plants comprise rice, corn and wheat;

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

Further, the protein expressed by the gene VLNHX in 3D is located in the vacuolar membrane.

That is, the protein expressed by the gene VLNHX D provided by the invention is positioned on the vacuolar membrane, and the gene VLNHX D plays a role by the protein expressed on the vacuolar membrane.

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

the gene VLNHX D has a nucleotide sequence shown as SEQ ID NO. 1.

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

The detection of whether the sample to be detected contains the gene VLNHX D can be performed in various modes, for example, whether the sample to be detected contains the gene VLNHX D itself can be directly detected, products generated by the gene VLNHX D can be detected, the products comprise direct products, indirect products, secondary products and the like, and the products can be mRNA, protein, a certain compound and the like.

The gene VLNHX D can be directly detected, and the specific primer pair of the gene VLNHX D can be used for detection, and probes or chips designed for the gene VLNHX D can also be used for detection. Further, the sample to be detected is detected by a primer pair or a probe or a chip of the gene VLNHX D.

The primer pair or the probe or the chip for the gene VLNHX D related in the invention can be designed according to a conventional method.

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: shown at 5.

Namely primer pair SEQ ID NO:2 and SEQ ID NO:3 can be used to detect gene VLNHX3D; primer pair SEQ ID NO:4 and SEQ ID NO:5, and has higher sensitivity.

The method of detecting the gene VLNHX D of the present invention is not limited to this, and any biologically available detection method is within the scope of the present invention.

Similarly, detection of the product produced by gene VLNHX D can be performed by a variety of means, such as ELISA detection kits, and the like.

Further, the sample to be tested comprises 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 for example be selected from roots, cuttings, stems, protoplasts and the like;

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

Specifically, further, the sample to be detected includes any one of the following materials: leaves, roots, stems, radicle, embryo, seeds.

Wherein the plant from which the sample is to be detected is taken includes monocotyledonous plants and dicotyledonous plants; monocots including rice, maize, wheat, and the like; dicotyledonous plants include soybean, cotton, arabidopsis, tobacco, and the like.

In a fourth aspect the invention also provides a method of conferring salt tolerance to a plant, producing a transgenic plant comprising or overexpressing gene VLNHX D;

the gene VLNHX D has a nucleotide sequence shown as SEQ ID NO. 1.

The present invention employs conventional biological methods to prepare transgenic plants containing or overexpressing gene VLNHX D. Transgenic plants containing the gene VLNHX D can be transformed in a variety of ways, such as the usual vector-mediated transformation methods, i.e., insertion of the gene of interest into a vector molecule such as a plasmid of agrobacterium or a DNA of a virus, followed by transfer of the vector DNA to introduce the gene of interest into the plant genome; in another example, the direct gene transfer method is to directly transfer exogenous target gene into plant genome through physical or chemical method, wherein the physical method includes gene gun transformation method, electric excitation transformation method, ultrasonic wave method, microinjection method, laser microbeam method, etc., and the chemical method includes PEG mediated transformation method, liposome method, etc.; also, germplasm systems methods, including pollen tube channel methods, germ cell infection methods, embryo sac and ovary injection methods, and the like. Transgenic plants that overexpress gene VLNHX D can also be made in a variety of ways, essentially as above, except that a promoter that enhances gene transcription is added to the vector.

The fifth aspect of the invention also provides the use of gene VLNHX D in the study of genetic diversity in plant populations.

Compared with the prior art, the invention has the beneficial effects that at least the following aspects are included:

(1) Through systematic research, the invention provides that the gene VLNHX D has the biological function of regulating the concentration of Na ⁺ and/or K ⁺ in plant cells for the first time, specifically, under salt stress, the accumulation amount of Na ⁺ in roots, stems and leaves of silent plants is obviously increased, and the content of K ⁺ in the roots is obviously reduced.

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

(3) The expression product of VLNHX D gene provided by the invention is positioned on a vacuole membrane, and the gene can be applied to cultivation or detection of salt tolerance of plants, wherein the plants comprise rice, corn, wheat, soybean, cotton, arabidopsis, 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 electrophoresis chart of PCR products of gene VLNHX D in example 1 of the present invention;

FIG. 2 is an amino acid sequence alignment of VLNHX D protein structure and other plant NHX in example 1 of the present invention;

FIG. 3 is a phylogenetic analysis of VLNHX D proteins and NHX proteins from other species in example 1 according to the present invention;

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

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

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

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

FIG. 8 is a bar graph showing the expression level of GhNHX3A in a TRV VLNHX 3D-silenced plant according to example 2 of the present invention;

FIG. 9 is a salt tolerance chart of VLNHX D gene-silenced plants according to example 2 of the present invention;

FIG. 10 is a bar graph of Na ⁺ and K ⁺ content and Na ⁺/K⁺ in TRV VLNHX D and TRV 00 plants under salt stress in example 2 of the present invention.

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 for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

1. Material and treatment

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

Planting cotton materials: soaking plump cotton seeds in distilled water, and placing in a 30 ℃ incubator overnight to promote germination. The next day seeds which have germinated and are exposed are selected and planted into a square plastic basin containing vermiculite, covered with a mulching film and placed in a greenhouse, the temperature is 23 ℃, the illumination time is 16h illumination/8 h dark cycle, and the relative humidity is 60%. Removing the mulching film after the cotyledons of the seeds are exposed, selecting cotton seedlings with consistent growth vigor after the cotyledons are unfolded, washing the cotton seedlings with tap water, wrapping the base of the stems with foam cotton, and placing the cotton seedlings into a water culture box (containing Hoagland nutrient solution) for later culture and treatment.

Treatment of cotton material: salt stress treatment was performed during the period of cotton seedling growth to two leaves and one heart. To the Hoagland nutrient solution, naCl was added in an amount of 50, 100, 150, 200mmol/L, respectively. Sampling at 0,1, 3, 6, and 12 hr, sampling root, stem and true leaf (at least 3 plants) separately, wrapping with tinfoil, marking, quick freezing with liquid nitrogen, and storing in-80deg.C refrigerator for subsequent experiment.

2. Related primers

Primer sequences referred to in Table 1

Example 1

1. Gene cloning

1. Total RNA extraction: the roots, stems and leaves of cotton material frozen at-80℃were ground into powder in liquid nitrogen, respectively, and 100mg of each sample powder was loaded into a 2mL RNase-Free centrifuge tube. The subsequent experimental procedure was performed according to the instructions of the Tiangen company RNA extraction kit (DP 441).

2. CDNA synthesis: cDNA synthesis was performed using the reverse transcription reagent of Norflu HISCRIPT III, inc. 1st Strand cDNA Synthesis Kit (+ GDNA WIPER) (R312-01).

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

TABLE 2 Gene cloning PCR System

TABLE 3 Gene cloning PCR reaction procedure

5. The PCR products were detected by 1.5% agarose gel electrophoresis at the end of the PCR reaction, and the result is shown in FIG. 1, wherein VLNHX D PCR products are located between 1000-2000bp Maker.

The target strip was recovered using a glue recovery kit FastPure Gel DNA Extraction Mini Kit (DC 301) from Norwegian corporation.

6. PCR product ligation cloning vector: 4 mu L of the PCR gel recovery product and 1 mu L of pEASY-Blunt Zero Cloning Vector are added into a 200 mu L clean centrifuge tube respectively, and the mixture is gently mixed and reacted for 30min at 25 ℃ (temperature control of a PCR instrument). After the reaction was completed, the mixture was placed on ice.

7. E.coli competent cells were transformed, and the monoclonal cells were picked up in a sterile environment in 500. Mu.L of LB+Kan liquid medium and cultured at 37℃for 6 hours at 200rpm until the bacterial solution became turbid. PCR and sequencing were performed using the bacterial liquid as a template (the sequence is shown as SEQ ID NO: 1). Positive bacteria containing VLNHX D were obtained.

2. VLNHX3D protein Structure prediction and amino acid sequence alignment

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

Other species NHX protein sequences and VLNHX D multiple sequence alignments were performed 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 VLNHX D protein structure schematic (fig. 2), it was shown that the VLNHX D encoded amino acid sequence contains a conserved domain of the Na ⁺/H⁺ transporter, typical of NHX, i.e., na ⁺_H⁺ _exchanger, and has 11 transmembrane structure conserved regions (fig. 2A). The amino acid sequence of VLNHX D was aligned multiple times with the amino acid sequence of other plant vacuolar NHX using DNAMAN 9.0 software. (FIG. 2B) shows that VLNHX D protein sequence has higher homology with other plant vacuole NHX proteins. They all have an identical aminopyrazine amidine binding site (Aminopyrazidine amidine binding sites) which is associated with competitive inhibition of Na ⁺ and a conserved CaM binding site at the C-terminus.

3. Evolutionary tree construction

1. Construction VLNHX of 3D and other species NHX evolutionary trees

In order to further analyze the relationship between VLNHX D protein and NHX in other plants, arabidopsis thaliana (At), populus (Pe), rice (Os) and corn (Zm) are taken as research objects, and GhNHX members with Na ⁺/H⁺ inverse transport protein function in upland cotton proved by previous researches are combined to jointly construct a phylogenetic tree.

NHX protein sequence information (Table 3.8) of upland cotton, arabidopsis, populus, rice and maize was collected, multiple sequence alignment was performed using MEGA X software, and phylogenetic tree was constructed using Neighbor-joining method, with boottrap value set to 1000.

TABLE 4 other species information

As shown in fig. 3, in phylogenetic tree, 28 total NHX proteins of 5 species are divided into 2 subfamilies, vacuolar NHX and endosomal NHX. Vacuolar NHX contains 20 NHX genes and endosomes contain 8 NHX genes. VLNHX3D is closer to vacuole NHX family members PeNHX4 and AtNHX4 in the evolutionary tree, indicating that the members have closer affinity; distant relationship with the endosomal NHX family members of arabidopsis, populus, rice, maize and upland cotton. These results indicate VLNHX D is a member of the vacuolar NHX family.

4. Gene subcellular localization

1. Vector construction

(1) Linearizing a carrier: the pCAMBIA2300 (35S: GFP) vector was linearized with BamHI and EcoRI restriction enzymes.

(2) After the completion of the vector cleavage reaction, the reaction was examined by electrophoresis on a 1.5% agarose gel, and the target band was cut off under an ultraviolet lamp to recover the gel.

(3) The vector homologous primers containing the cleavage site sequence but not the termination codon of the gene were designed based on the vector and gene sequence, see subcellular localization primers in Table 1, followed by PCR amplification and gel recovery of the target band.

(4) Using homologous recombination enzyme of Northenan companyUltra One Step Cloning Kit) the amplified product was ligated to pCAMBIA2300 (35S: GFP) vector, 3. Mu.L of the gene amplification gel recovered product, 2. Mu.L of the vector gel recovered product, 5. Mu.L of homologous recombinase, and reacted at 50℃for 5min after gentle mixing.

(5) The ligation product was transferred into E.coli in a freeze-thaw state.

(6) The remaining steps were identical to E.coli transformation and positive cloning assays described above. The upstream primer is HP158, and the downstream primer is a downstream vector connection primer of the gene.

(7) And obtaining VLNHX D-GFP fusion expression vector after sequencing correctly.

2. Plasmid extraction of VLNHX E.coli for 3D-GFP was performed using the Revozanol plasmid extraction Kit (FastPure Endo FREE PLASMID Maxi Kit, DC 202-01).

3. Preparation and transformation of Arabidopsis protoplasts: the procedure was performed using the Coolaber Arabidopsis protoplast preparation and transformation kit (PPT 101).

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

VLNHX3D-GFP expression fusion vectors were obtained by fusing GFP to VLNHX D using the vacuolar membrane-tagged fusion protein delta-TIP-RFP as positive control. The delta-TIP-RFP and VLNHX D-GFP plasmids were co-transformed into Arabidopsis protoplasts for transient expression, and the protoplasts co-transformed with the empty vector 35S: GFP and delta-TIP-RFP plasmids were used as negative controls. As observed by a laser confocal microscope (FIG. 4), in Arabidopsis protoplasts, after transfer into 35S: GFP and delta-TIP-RFP fusion expression vectors, GFP-generated green fluorescence was dispersed throughout the cytoplasm, while delta-TIP-RFP-generated red fluorescence was distributed over the vacuolar membrane, and their fluorescence signals did not overlap completely. When delta-TIP-RFP and VLNHX D-GFP fusion vector were expressed in Arabidopsis protoplast, the green fluorescence generated by VLNHX D-GFP fusion protein and red fluorescence generated by delta-TIP-RFP on the vacuolar membrane were completely superimposed to generate yellow fluorescence, and the yellow fluorescence signal formed a circle on the vacuolar membrane, which visually indicated that VLNHX D protein was on the vacuolar membrane.

Example 2

1. VLNHX3D real-time fluorescent quantitative PCR

1. Cotton material was extracted with RNA for roots, stems and leaves at various time points after salt treatment.

2. CDNA synthesis: HISCRIPTII Q RT SuperMix for qPCR (+ GDNA WIPER) (R233-01) reverse transcription reagent from Norpraise was used.

3. Designing a specific fluorescent quantitative primer: specific primers for VLNHX D and GhNHX3A were designed using PRIMERPREMIER and GhHIS3 was used as an internal reference gene, the primer sequences are 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 were performed for each sample and the results were calculated using analysis using 2 ^-△Ct.

Wherein, the expression level of VLNHX D in cotton leaves of upland cotton without salt stress treatment (0, 1,3, 6 and 12 h) is used as a control (Mock), and the expression level of VLNHX D in the leaves at the same time point under NaCl stress treatment is used as an experimental group. The fluorescence quantitative result shows that the expression level of VLNHX D in the leaf gradually increases with the increase of the salt stress time, and reaches a peak value after 200mM salt stress treatment for 6 hours. When the treatment time was prolonged to 12 hours, the amount of VLNHX D expressed was decreased. These results indicate that salt stress induces VLNHX D expression level changes.

The expression pattern of VLNHX D on upland cotton leaves at different concentrations of NaCl treatment was further analyzed and cotton seedlings at the two-leaf one-heart stage were treated with 0, 50, 100, 150 and 200mM NaCl. Since VLNHX D had the highest expression level at 6h, VLNHX D expression level in 6h of leaf treated with NaCl at different concentrations was analyzed. As shown in FIG. 5, it was revealed from the fluorescent quantitative results that as the NaCl concentration was increased, the VLNHX D expression level in the leaf was also increased. This further verifies that salt stress can induce and regulate VLNHX D expression levels.

2. Yeast functional complementation experiment

1. Constructing a yeast expression vector:

(1) PCR amplification and gel recovery were performed using a plasmid containing VLNHX D E.coli as a template, and BamHI and SacI cleavage site sequences (VLNHX D-Y-F/R) were included on the primers, respectively.

(2) Yeast expression vector pYES2 plasmid was linearized with BamHI and SacI restriction enzymes and then gel recovered.

(3) And (3) connecting the VLNHX D gel recovery product containing the enzyme cutting site with the carrier recovery product in the step (2) by utilizing homologous recombinase, and constructing a carrier, wherein the LB culture medium resistance is Amp. The yeast expression vector plasmid pYES2-VLNHX D was obtained.

2. Preparation of yeast competence: coolaber Yeast kit (SK 2401-200T) was used.

3. Yeast plasmid transformation: yeast expression vectors pYES2-VLNHX D and pYES2 (empty, control) were transferred into the prepared yeast competence, respectively.

4. Positive strain verification: the monoclonal strain was selected and cultured overnight in YNB+Ade+Try liquid medium at 30℃and 200rpm. And (3) performing PCR by taking the saccharomycete liquid as a template, and performing positive strain verification. Since the yeast contains cell walls, the pre-denaturation time is prolonged to 5min.

5. Yeast functional complementation assay: yeast salt stress function screening was performed with different APG media. Five nutrients of Ade, ura, try, leu and His were added on the basis of APG medium, and three culture conditions of 0 (control), 40mM NaCl and 50mM NaCl were set.

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

(2) The bacterial liquid was cultured at 30℃in a 200rpm incubator until OD ₆₀₀ =1.2.

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

(4) The diluted four gradient bacteria were spotted at 8. Mu.L each on APG medium containing 0, 40mM NaCl and 50mM NaCl.

(5) The rows were observed by incubating for 5 days at 30℃upside down and photographed with a camera.

Transferring the pYES2-VLNHX3D yeast fusion expression vector into a yeast mutant AXT3, taking transgenic yeast of the empty vector pYES2 as a negative control, taking the positive control as wild type yeast W303, and carrying out salt tolerance analysis on the pYES2-VLNHX3D transgenic yeast. The results are shown in FIG. 6: in APG medium without NaCl, wild type yeast W303, empty vector pYES2 transgenic yeast and pYES2-VLNHX3D transgenic yeast can grow normally, and the growth conditions are basically consistent. In APG medium containing 40mM NaCl, W303 was diluted 2000-fold and still grew normally. Since the mutant AXT3 lacks the endogenous Na ⁺ transporter and AXT3 is inhibited from growing when being poisoned by Na ⁺, the yeast transformed with empty pYES2 is inhibited from growing in a medium of 40mM NaCl and can not grow basically. Growth of pYES2-VLNHX3D transgenic yeast was slightly inhibited after 2000-fold dilution compared to wild-type yeast W303. When the NaCl concentration was increased to 50mM, the growth of W303 was not significantly inhibited, while the growth of both pYES2 and pYES2-VLNHX D showed different degrees of inhibition. Compared with the negative control of the pYES2 transgenic yeast, the growth of the pYES2-VLNHX D yeast is slightly inhibited, the growth condition is better, a small amount of bacterial plaque is still visible after 200 times and 2000 times dilution, and the pYES2 transgenic yeast after 200 times dilution almost cannot grow. These experimental results show that pYES2-VLNHX D not only can partially restore Na ⁺ transport function of yeast mutants, but also the transgenic yeast shows higher salt tolerance.

3. Virus-induced VLNHX D silencing in cotton

1. Construction of a VIGS recombinant expression vector: see in particular the above steps; the cleavage sites are EcoRI and XhoI. Finally, the TRV VLNHX D VIGS vector is obtained.

(1) The VIGS vector TRV VLNHX3D transformed with Agrobacterium (GV 3101),

(2) Positive clone detection

(3) And (5) performing PCR verification by taking the bacterial liquid as a PCR template.

(4) And (3) adding 50% glycerol with the same volume for preserving bacteria after amplifying and culturing bacterial liquid with the correct sequence, and storing at-80 ℃.

2. And planting upland cotton TM-1 under the water planting condition. After the two cotyledons of cotton are fully developed, VIGS bacterial liquid injection is performed.

3. Preparing a heavy suspension: to 500mL of deionized water, 5mL of MES solution, 1mL of AS solution and 5mL of MgSO ₄ solution were added, respectively, and mixed well (AS prepared).

4. And (5) resuspension of bacterial liquid:

(1) 40mL of the overnight cultured Agrobacterium solution containing the VIGS vector was taken and centrifuged at 5000rpm for 5min in a 50mL clean centrifuge tube, and the cells were collected.

(2) The cells were suspended with the formulated resuspension and OD ₆₀₀ = 1.2 was adjusted.

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

5. Cotton injection:

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

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

(3) After infection is completed, the growth is carried out for 24 hours in dark, and then the growth is carried out under normal conditions.

6. Detection of gene silencing efficiency: and 10 days after bacterial liquid injection, the true leaves of the TRV/CLA (positive control) plants show albino characters. Randomly taking the roots, stems and leaves of 3 target gene silencing plants and injecting empty plants to carry out RNA extraction and reverse transcription. The silencing efficiency of the gene is quantitatively detected by using fluorescence.

To further verify that VLNHX D has the function of enhancing salt tolerance, the effect of VLNHX D under cotton salt stress was analyzed using VIGS technology. Virus Induced GENE SILENCING (VIGS) techniques can utilize viruses or bacterial fluids containing gene fragments of interest to infect plants, induce endogenous gene silencing in the plants to cause corresponding physiological and morphological changes, and then study gene function through these changes. The constructed silencing expression vectors TRV: VLNHX3D, TRV:00 (negative control) and TRV:CLA (positive control) are respectively mixed with auxiliary plasmids in equal volume and then injected to the back of cotyledons of cotton seedlings after 10 days of germination. The true leaves of the positive control showed a whitening trait after 12 days (fig. 7).

The efficiency of silencing VLNHX D in TRV VLNHX D plants was examined after the appearance of a albino phenotype in the positive control. RNA from the roots, stems and leaves of TRV VLNHX D and TRV 00 cotton plants were extracted, respectively, and then detected for VLNHX D expression levels using fluorescent quantitative techniques. The results showed that VLNHX D expression levels in roots, stems and leaves of TRV VLNHX D were significantly lower than those of TRV 00 plants (FIG. 7). To ensure the specificity of the gene silencing effect, the corresponding expression level of VLNHX D homologous gene GhNHX3A was also examined. The results in FIG. 8 show that there was no significant difference in the expression levels of GhNHX3A in the leaves, stems and roots of TRV VLNHX D and TRV 00 plants. This suggests that VLNHX D is efficiently and specifically silenced in TRV VLNHX3D silenced cotton plants.

7. Salt treatment of silent plants: after the VIGS injection, when cotton seedlings were grown to two leaves and one heart, 200mM NaCl treatment was performed on plants of TRV 00 (empty) and the silenced gene of interest, respectively. The corresponding mass of NaCl was added to the Hoagland nutrient solution, while the control treatment was carried out with the Hoagland nutrient solution without NaCl. Phenotype observations and photographs of the gene-silenced plants and control plants were performed 10 days after treatment.

As a control group containing 0mM NaCl, 200mM NaCl was applied to the treatment group, and salt tolerance of cotton seedlings was observed after 10 days of treatment. FIG. 9 shows that under normal growth conditions, TRV VLNHX3D silenced plants and TRV 00 grow in agreement; TRV VLNHX D leaves clearly wilted in the yellow and plant height shorter than TRV 00 after 200mM NaCl salt stress treatment. Indicating that plants have increased sensitivity to salt stress when VLNHX D expression is silenced.

8. Ion content determination: taking 9 treated 10 days TRV:00 and TRV: VLNHX D plants (the treated group and the control group are all sampled), separating roots, stems and leaves, marking, deactivating enzyme in an oven at 105deg.C for 5min, oven drying at 75deg.C to constant weight, and grinding into powder.

Sodium ion and potassium ion content determination: the measurement was performed using an atomic absorption method. And measuring the content of sodium ions and potassium ions.

Taking the dried sample powder and sieving the powder with a 100-mesh nylon sieve. Weighing 0.1g of the sieved plant powder, drying the second true leaves, stems and roots of the TRV 00 and TRV GhNHX3D plants at 90 ℃, and grinding into powder. A0.05 g sample of the powder was dissolved in 5mL of concentrated HNO ₃ (nitrification). Dilute with deionized water 12 times and centrifuge. The supernatant was collected and the sodium and potassium ion contents were determined by atomic absorption.

Salt tolerance of plants is related to intracellular ion content. Too high a concentration of ions in the cells can cause toxicity to the cells, thereby reducing the salt tolerance of the plant. The present study measured the Na ⁺ and K ⁺ content in the roots, stems and leaves of TRV VLNHX D and TRV 00 plants and calculated the Na ⁺/K⁺ ratio, further explored the salt tolerance mechanism of VLNHX D. The results in FIG. 10 show that the same levels of Na ⁺ and K ⁺ levels and Na ⁺/K⁺ ratios were shown in both the roots, stems and leaves of the TRV VLNHX D and TRV 00 plants in the control group. After 200mM NaCl treatment, the Na ⁺ content in the roots, stems and leaves of the TRV VLNHX D plant is obviously higher than that of TRV 00; there was no difference in the content of K ⁺ in the TRV VLNHX D roots and stems, but the content of K ⁺ in the TRV VLNHX3D roots was significantly lower than TRV 00.

Compared with the TRV:00 plant, the Na ⁺/K⁺ ratio of roots, stems and leaves of the TRV: VLNHX3D silent plant is obviously increased after salt stress. It is shown that the TRV VLNHX D plant accumulates more Na ⁺ in vivo after being subjected to salt stress, especially the content of K ⁺ in roots is obviously reduced, which results in the TRV VLNHX D cotton plant having higher Na ⁺/K⁺ and increased sensitivity to salt stress.

While particular embodiments of the present invention have been illustrated and described, it will be appreciated 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 agricultural university

Application of <120> gene VLNHX D in regulating Na+ and/or K+ concentration of 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. The application of gene VLNHX D in regulating the concentration of plant cells Na ⁺ and K ⁺, wherein the gene VLNHX D is a nucleotide sequence shown in SEQ ID NO. 1, and the gene VLNHX D has the effect of improving the salt tolerance of plants; compared with a wild plant, silencing Na ⁺ accumulation amount in roots, stems and leaves of the gene VLNHX D plant is increased, K ⁺ accumulation amount in the stems and leaves is unchanged, K ⁺ content in the roots is reduced, and salt tolerance of cotton is improved by regulating steady-state balance of Na+ and K+ in cells;

the protein expressed by the gene VLNHX D is positioned in a vacuole membrane, and the plant is upland cotton.

2. Application of gene VLNHX D in cultivation or detection of salt tolerance transgenic plants, wherein the gene VLNHX D is a nucleotide sequence shown in SEQ ID NO. 1, and the plants are upland cotton; detecting that the gene VLNHX is over-expressed or transferred in 3D, and the salt tolerance of the plant is increased;

the gene VLNHX D was not detected or silenced, and the salt tolerance of the plant was weakened; the protein expressed by the gene VLNHX D is located in the vacuolar membrane.

3. The method for detecting the salt tolerance of the plant is characterized by detecting the existence or the expression condition of a gene VLNHX D of a sample to be detected and judging that the plant containing the gene VLNHX D has higher salt tolerance than a plant with the gene VLNHX D silenced;

the gene VLNHX D is a nucleotide sequence shown in SEQ ID NO. 1;

The plant is upland cotton.

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

5. The method for detecting salt tolerance of plants according to claim 4, wherein the primer pair has a nucleic acid sequence as set forth in SEQ ID NO:2 and SEQ ID NO:3 or SEQ ID NO:4 and SEQ ID NO: shown at 5.

6. The method for detecting salt tolerance of plants according to any one of claims 3 to 5, wherein the sample to be detected comprises a material suitable for tissue culture of sexually reproducing, asexually reproducing or viable cells.

7. The method for detecting salt tolerance of plants according to claim 6, wherein the sample to be detected comprises any one of the following materials: leaves, roots, stems, radicle, embryo, seeds.

8. A method for increasing salt tolerance in a plant, comprising preparing a transgenic plant comprising or overexpressing gene VLNHX D;

the gene VLNHX D is a nucleotide sequence shown in SEQ ID NO. 1;

The plant is upland cotton.