CN116574755A - Application of PtTINY gene in regulation and control of plant salt stress tolerance - Google Patents

Abstract

The invention discloses an application of PtTINY gene in regulating plant salt stress tolerance, wherein the PtTINY gene has a gene sequence number of XM_002298200.4 in NCBI. According to the invention, the PtTINY over-expression vector pCAMBIA2300-PtTINY is constructed, and the agrobacterium inflorescence infection method is utilized to transform the wild type Arabidopsis thaliana (Clo-0, WT) to obtain an over-expression plant, and an analysis result shows that under salt stress, compared with the wild type Arabidopsis thaliana seedling salt stress tolerance is reduced after over-expression, so that gene resources are provided for crop salt tolerance molecular breeding.

Description

Application of PtTINY gene in regulation and control of plant salt stress tolerance

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of PtTINY genes in regulating plant salt stress tolerance, in particular to application of PtTINY genes in reducing plant salt stress tolerance.

Background

Soil salinization is formed by the accumulation of large amounts of soluble salts in the soil. Soil salinization has become a global problem today, and about 8% of the world is threatened by salinization. China is a country with serious soil salinization, and the salinized soil area accounts for 1/10 of the global salinized soil area. In recent years, under the influence of natural and human factors and other conditions, the salinized area of soil is gradually enlarged, the normal growth and development of plants are seriously influenced, the survival and development of agriculture and animal husbandry and human beings are endangered, and a plurality of economic problems are caused.

The harm of salt stress to plants is mainly three aspects, the first aspect is osmotic stress, the plants grow in a high-concentration ion environment, and the high osmotic pressure causes the plants to lose water, so that leaf pores are closed, photosynthesis is limited, and normal growth metabolism of the plants is affected. The second aspect is ion poisoning, which results in Na due to plant transpiration ⁺ And Cl ^- Excessive accumulation in plant body, excessive Na ⁺ Inhibit the activity of enzymes in plants andand the metabolism of plants is influenced, osmotic pressure balance in the plants is destroyed, competitive inhibition effect is generated on other ions, and the death of the plants is finally caused. In addition to the two hazards above, there is also an indirect oxidative stress effect. Due to metabolic disturbance in the plant body caused by salt stress, the redox system of the plant is damaged, active oxygen and other substances generated in the metabolic process accumulate in the plant body, the photosynthesis of the plant is affected, and finally the plant growth is inhibited.

The adaptation of plants to the environment makes them resistant to salt stress to a certain extent, and in order to alleviate the damage of soil salinization, plants evolve a series of salt tolerance mechanisms including osmotic regulation, metabolic regulation, salt stress sensing and salt tolerance and salt rejection mechanisms. The poplar tree grows rapidly, the wood is grown rapidly, the application is wide, the poplar tree is not only used as a protective forest and an urban greening tree species in northwest areas, but also the wood is used as industrial wood, and the poplar tree has great economic, ecological and social benefits. The research on the salt tolerance of poplar has important significance for the development and utilization of saline-alkali soil in China.

Disclosure of Invention

The invention aims to provide application of PtTINY genes in reducing salt stress tolerance of plants, wherein the PtTINY genes negatively regulate the salt stress tolerance of the plants.

In order to achieve the above purpose, the technical scheme adopted by the invention is summarized as follows:

the PtTINY gene adopted by the invention has a gene Sequence number (Sequence ID) of XM_002298200.4 in NCBI, a messenger RNA (mRNA) Sequence length of the PtTINY gene of 1214bp, and a coding Sequence length of the GhGPX5 gene of 702bp, which comprises 233 amino acids.

The invention also constructs a series of plant expression vectors, recombinant vectors or transgenic plant lines containing the genes, and the functions of host cells containing the vectors in reducing the salt stress tolerance of plants also fall into the protection scope of the invention.

The function of the gene protected by the invention not only comprises the PtTINY gene, but also comprises the function of homologous genes with higher homology (the homology is up to 99%) with the PtTINY gene in the aspect of salt stress resistance.

The PtTINY gene disclosed by the invention has biological functions in plant salt stress tolerance, and is specifically expressed in the following steps: under salt stress, the root length and fresh weight and germination rate of PtTINY gene over-expression strain are lower than those of wild type.

According to the functions, a plant resistant to salt stress can be obtained in a transgenic mode, specifically, a target plant can be transformed by constructing a gene editing vector, and the transgenic plant is obtained, wherein the salt stress resistance of the plant is higher than that of the target plant.

Specifically, the PtTINY gene can be introduced into the plant of interest specifically by the recombinant expression vector. In the method, the recombinant expression vector may be used to transform plant cells or tissues by using conventional biological methods such as Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, electric conduction, agrobacterium mediation, etc., and the transformed plant tissues are cultivated into plants.

In order to improve the excellent properties of plants, in the practical application process, the expression of PtTINY genes in plants can be regulated by a method of over-expressing, silencing or directionally mutating PtTINY genes. Regulating the gene expression level comprises regulating the PtTINY expression by utilizing a DNA homologous recombination technology and an agrobacterium-mediated transformation system to obtain a transgenic plant line.

The invention also provides a novel plant breeding method, which is (1) or (2) or (3):

(1) By reducing the activity of PtTINY protein in the target plant, obtaining a plant with stronger salt stress tolerance than the target plant;

(2) Obtaining a plant with salt stress tolerance lower than that of the target plant by promoting the expression of PtTINY gene in the target plant;

(3) By inhibiting the expression of PtTINY gene in the target plant, a plant having higher salt stress tolerance than the target plant is obtained.

The "promotion of expression of PtTINY gene in a plant of interest" may be achieved as follows (1) or (2) or (3):

(1) Introducing PtTINY gene into target plant;

(2) Introducing strong promoters and/or enhancers;

(3) Other methods are common in the art.

The "means for suppressing expression of PtTINY gene in a target plant" may be a means for knocking out, silencing or targeted mutation of PtTINY gene, and the means for knocking out PtTINY gene may be a means for gene editing.

Wherein the target plant is Arabidopsis thaliana, poplar, and especially mountain new poplar.

Genes of interest, also known as target genes, are used in genetic engineering design and manipulation to recombine genes, alter receptor cell traits and obtain desired expression products. May be of the organism itself or from a different organism.

In the present invention, the plant suitable for the present invention is not particularly limited as long as it is suitable for performing a gene transformation operation such as various crops, flower plants, forestry plants, or the like. The plant may be, for example (without limitation): dicotyledonous, monocotyledonous or gymnosperm plants.

As a preferred mode, the "plant" includes, but is not limited to: poplar, arabidopsis thaliana, and particularly mountain new poplar (Populus davidiana ×Populus boleana), are all suitable genes having this gene or a gene homologous thereto.

As used herein, the term "plant" includes whole plants, parent and progeny plants thereof, and various parts of plants, including seeds, fruits, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, in which the gene or nucleic acid of interest is found. Reference herein to "plant" also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the foregoing comprises the gene/nucleic acid of interest.

The present invention includes any plant cell, or any plant obtained or obtainable by a method therein, as well as all plant parts and propagules thereof. The present patent also encompasses transfected cells, tissues, organs or whole plants obtained by any of the foregoing methods. The only requirement is that the sub-representations exhibit the same genotypic or phenotypic characteristics, and that the progeny obtained using the methods of this patent have the same characteristics.

The invention also extends to harvestable parts of a plant as described above, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. And further to other derivatives of the plants after harvest, such as dry granules or powders, oils, fats and fatty acids, starches or proteins. The invention also relates to a food or food additive obtained from the relevant plant.

The invention has the advantages that:

(1) The invention innovatively clones an AP2/ERF (APETALA 2/ETHYLENE RESPONSIVE FACTOR) transcription factor family gene PtTINY responding to adversity stress in mountain new poplar (Populus davidiana ×Populus boleana). The PtTINY over-expression vector pCAMBIA2300-PtTINY is constructed, and an agrobacterium inflorescence infection method is used for transforming wild type Arabidopsis thaliana (Clo-0, WT) to obtain an over-expression plant, and analysis results show that under salt stress, compared with the wild type PtTINY, the salt tolerance is reduced, that is, the PtTINY gene negatively regulates the tolerance of Arabidopsis thaliana to the salt stress. Therefore, the PtTINY gene plays an important role in regulating and controlling plant salt stress, and has important significance for cultivating poplar varieties capable of resisting external stress conditions.

(2) The plant with salt tolerance can be obtained by a transgenic mode, specifically, a PtTINY gene editing vector can be constructed to transform a target plant, so that the transgenic plant is obtained, the salt tolerance of the plant is higher than that of the target plant, and a new way is provided for plant salt tolerance breeding.

Drawings

FIG. 1 is a phylogenetic tree and amino acid sequence alignment of mountain poplar, arabidopsis thaliana, brassica napus, soybean, maize and rice; FIG. 1A shows that mountain poplar TINY is closer to dicotyledon Arabidopsis thaliana and Brassica napus TINY relatives;

FIG. 1B shows that the amino acid AP2 domain sequence encoded by TINY is highly conserved among different species;

FIG. 2 is a PtTINY expression level analysis; FIG. 2A shows that PtTINY is expressed at the highest level in the root; FIG. 2B shows that 200mM NaCl induces PtTINY expression;

FIG. 3 is a PtTINY overexpression vector construction and heterologous overexpression Arabidopsis expression level analysis; FIG. 3A is a schematic diagram of an over-expression vector structure; FIG. 3B shows PtTINY gene expression levels of different strains;

FIG. 4 is a phenotypic comparison of Arabidopsis wild-type and PtTINY heterologous overexpression lines under salt stress.

FIG. 5 shows the germination percentage comparison of Arabidopsis wild-type and PtTINY heterologous overexpression lines under salt stress.

Detailed Description

The present invention will be described in detail with reference to specific examples. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. The test methods in the following examples are conventional methods unless otherwise specified. The reagents and materials employed, unless otherwise indicated, are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botanicals, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination, and bioinformatics, which will be apparent to one of skill in the art. These techniques are fully explained in the published literature, and the methods of DNA extraction, phylogenetic tree construction, gene editing method, gene editing vector construction, gene editing plant acquisition, etc. used in the present invention can be realized by the methods disclosed in the prior art except the methods used in the examples described below.

The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" as used herein are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-or double-stranded structures. Such nucleic acids or polynucleotides include, but are not limited to, gene coding sequences, antisense sequences, and regulatory sequences of non-coding regions. These terms include a gene. "Gene" or "gene sequence" is used broadly to refer to a functional DNA nucleic acid sequence. Thus, a gene may include introns and exons in genomic sequences, and/or coding sequences in cDNA, and/or cDNA and regulatory sequences thereof. In particular embodiments, for example in relation to isolated nucleic acid sequences, it is preferred that they are cDNA.

Biological material

The arabidopsis Col-0 seeds are preserved in a laboratory;

the overexpression vector pSuper-1300-GFP is stored in a laboratory;

coli DH 5. Alpha. And Agrobacterium GV3101 were kept in the laboratory;

primer synthesis and sequencing were performed by the Optimago company.

Experimental reagent

RNA extraction kits, reverse transcription kits, and fluorescent quantification kits were purchased from nuuzan biotechnology limited;

common reagents such as NaCl are purchased from Shanghai Co;

hygromycin is purchased from soribao biosystems;

MS media was purchased from beijing cool pacing technologies limited;

various endonucleases were purchased from tabacco qingjun biotechnology limited;

one-step cloning enzyme was purchased from nuuzan biotechnology limited;

plasmid miniprep and gel recovery kits were purchased from beijing tiangen biotechnology limited.

Experimental equipment

PCR apparatus was purchased from Bio-rad company;

the refrigerated centrifuge is purchased from Eppendorf corporation;

quantitative PCR instrument was purchased from Bio-rad company;

the autoclave MLS-3750 was purchased from Sanyang, japan;

nucleic acid detector Nanodrop 2000C was purchased from Thermo Scientific company;

the room temperature centrifuge was purchased from Thermo Scientific company.

EXAMPLE 1 cloning of PtTINY Gene and amino acid sequence

Extracting RNA of a new poplar growing 15 days, taking cDNA obtained by reverse transcription reaction as a template, designing a specific Primer by using a gene sequence obtained from NCBI database through Primer premier5.0, cloning a coding sequence of PtTINY gene, converting a nucleic acid sequence into a protein sequence through a transfer (https:// www.expasy.org/resources/on-line tool), downloading Arabidopsis, cabbage type rape, soybean, corn and rice TINY protein sequences at NCBI, further constructing a system evolution tree of different species of TINY by using MEGA software, and comparing the different species of TINY protein sequences by using DNAMAN software.

The result shows that the coding sequence of the PtTINY gene of the populus tremulosa contains 702bp base. The encoded protein comprises 233 amino acids. And mountain poplar is most closely related to dicotyledonous plants Arabidopsis thaliana and Brassica napus (FIG. 1A), the AP2 domains of different species TINY protein sequences are highly conserved (FIG. 1B).

EXAMPLE 2 analysis of expression Pattern of PtTINY Gene

Selecting wild mountain new Yang Tupei seedlings which grow well and have consistent sizes and have the growth time of 25-30d, sampling five tissue parts of a sample leaf, a stem, a root, a xylem and a phloem respectively, rapidly placing the sample into liquid nitrogen, extracting RNA, and carrying out reverse transcription to obtain cDNA.

Wild mountain new poplar tissue culture seedlings are randomly divided into 6 groups of 3 plants. An aqueous solution containing 200mM NaCl was prepared, and seedlings were transferred to an aqueous solution containing salt for stress treatment, and then sampled at 0h, 1h, 3h, 6h, 12h, 24h,6 time points of the treatment, respectively. The whole poplar sample is quickly placed in liquid nitrogen, RNA is extracted, and cDNA is obtained after reverse transcription.

qRT-PCR primers were designed using Primer3 software (http:// frodo. Wi. Mit. Edu /), and Primer specificity was verified by NCBI-Primer blast (https:// www.ncbi.nlm.nih.gov/tools/Primer-blast/index. Cgi), ptACTIN was used as an internal reference gene for qRT-PCR analysis. RT-qPCR experiments Using CFX96 real-time fluorescent quantitative PCR detection System (Bio-Rad, hercules, calif., U.S.A.) and SYBRPremix Ex Taq reagents ^TM The cassette (Takara) was used. Three biological replicates and three technical replicates were performed for each sample. The amplification parameters were as follows: the cycle was repeated 40 times at 95℃for 30 seconds, 95℃for 5 seconds, and 60℃for 30 seconds.

The results indicated that PtTINY was expressed in all tissues tested, but the highest levels were expressed in xylem (FIG. 2A). Salt stress treatment induced expression of ptiny, with expression levels up-regulated about 40-fold at 12 hours post-treatment and about 10-fold at 24 hours (fig. 2B).

Example 3 overexpression of Arabidopsis thaliana verifies the function of PtTINY gene in Arabidopsis thaliana salt stress tolerance

In order to further analyze the function of PtTINY, the inventors constructed PtTINY overexpression vectors p35S-PtTINY-GFP, respectively, to obtain overexpression Arabidopsis plants. The specific process is briefly described as follows:

first, a primer with a restriction enzyme BamHI cleavage site was designed, the sequence being as follows:

1300-PtTINY-F：5′-GAGCTCGGTACCCGGGGATCCATGACGAAAGAAGAAAGCAGTAACAG-3′；

1300-PtTINY-R：5′-CAGGTCGACTCTAGAGGATCCTTAATTCAACTCAGATCCTCCAAAGT-3′；

secondly, performing PCR amplification by using the cDNA sample prepared in the example 1 as a template, and purifying and recovering an amplification product;

thirdly, single enzyme digestion is carried out on the 1300-GFP vector by adopting BamHI, and enzyme digestion products are purified;

fourthly, carrying out homologous recombination connection on the PCR amplification product and the vector after enzyme digestion to construct a 1300-PtTINY over-expression vector;

fifthly, converting the connection product into escherichia coli DH5 alpha by adopting a heat shock conversion method, and carrying out K ⁺ Screening (kanamycin, 50 mug/mL) resistance, selecting positive colonies for PCR detection, amplifying and sequencing correct colonies identified by the PCR detection, and extracting plasmids from bacterial liquid with correct sequencing for later use;

sixthly, the extracted plasmid is transformed into an agrobacterium competent cell GV3101, and the plasmid is preserved at the temperature of minus 80 ℃ for standby;

seventh, transforming wild type Arabidopsis thaliana (Clo-0, WT) by Agrobacterium inflorescence infection, screening the harvested seeds on a 1/2MS medium containing hygromycin, harvesting seeds from a single plant of a potential transgenic plant, and screening on a medium containing hygromycin again until T ₃ Obtaining potential homozygous transgenic plants through generation screening;

analysis of the expression levels of PtTINY in potentially transgenic plants, respectively, using qRT-PCR, revealed that PtTINY expression could not be detected in wild type plants, and PtTINY expression was detected in heterologous over-expression plants (FIG. 3B).

In order to analyze whether PtTINY participates in regulating and controlling the growth process of salt stress inhibition plants, WT and over-expressed PtTINY Arabidopsis seeds are respectively sown on a 1/2MS culture medium containing 0mM,50mM,150mM,200mM NaCl, are treated at a low temperature of 4 ℃ for 3 days, are vertically placed in a greenhouse (21 ℃) with 12h illumination/12 h darkness, and are observed for growth conditions of the Arabidopsis plants.

The results showed that after 7 days of growth on NaCl-free (0 mM) medium, the fresh weight of the overexpressed strain was significantly lower than that of the wild-type (FIGS. 4A, F), representing about 2.71mg of the wild-type fresh weight, and less than 1.87mg of the overexpressed strain. The NaCl treatment with different concentrations obviously inhibits the growth of plants, and the effect of inhibiting the growth of plants is more obvious along with the increase of the NaCl concentration in the 1/2MS culture medium. After 7 days of growth on 1/2MS medium containing 50mM, the fresh weight of the wild-type plants was about 2.46mg, whereas the fresh weight of the overexpressed lines was less than 1.33mg, and the fresh weight of the overexpressed lines was significantly lower than that of the wild-type plants (FIGS. 4B, F). After 7 days of growth on 1/2MS medium containing 150mM, the fresh weight of the wild-type plants was about 0.88mg, while the fresh weight of the overexpressed lines was less than 0.42mg, and the fresh weight of the overexpressed lines was significantly lower than that of the wild-type plants (FIGS. 4C, F). After 7 days of growth on 1/2MS medium containing 200mM, the fresh weight of the wild-type plants was about 0.08mg, while the fresh weight of the overexpressed lines was about 0mg, which was significantly lower than that of the wild-type plants (FIGS. 4D, F). The inhibition rate results of salt stress on the fresh weight of the plants show that the inhibition rate of the over-expressed strain is significantly higher for different concentrations of NaCl than for the wild type strain (FIG. 4H).

After 7 days of growth on NaCl-free (0 mM) medium, the overexpressing strain root length was significantly lower than the wild-type (FIGS. 4A, E), representing about 3.06cm in length for the wild-type root and less than 2.40cm in length for the overexpressing strain root (FIGS. 4A, E). The high-concentration NaCl treatment obviously inhibits the growth of plant roots, and the effect of inhibiting the growth of plant roots is more obvious along with the increase of the NaCl concentration in the 1/2MS culture medium. After 7 days of growth on 1/2MS medium containing 50mM, the wild type plants had a root length of about 3.16cm, whereas the overexpressing lines had a root length of less than 2.13cm, the overexpressing lines had a root length significantly less than the wild type (FIGS. 4B, E). After 7 days of growth on 1/2MS medium containing 150mM, the wild type plants had a root length of about 1.41cm, whereas the overexpressing lines had a root length of less than 0.84cm, the overexpressing lines had a root length significantly less than the wild type (FIGS. 4C, E). After 7 days of growth on 1/2MS medium containing 200mM, the wild type plants had a root length of about 0.20cm, whereas the overexpressing lines had a root length of 0cm, which was significantly lower than the wild type (FIGS. 4D, E). The inhibition rate results of salt stress on plant root length show that the inhibition rate of the over-expressed strain is significantly higher for different concentrations of NaCl than for the wild type strain (FIG. 4G).

To analyze whether PtTINY is involved in salt stress response during germination of Arabidopsis thaliana, we sown wild-type, overexpressed PtTINY Arabidopsis seeds on 1/2MS medium containing 0mM and 200mM NaCl, treated at low temperature of 4℃for 3d, then vertically placed in a 12h light/12 h dark greenhouse (21 ℃) and observed for Arabidopsis thaliana germination. The results showed that after 10 days of growth on NaCl-free (0 mM) medium, the wild-type germination rate was significantly higher than that of the transgenic plants (FIGS. 5A, B), which was shown to be about 98.32% for the wild-type, and about 75.58% for the overexpressing strain. After 10 days of growth on 200mM NaCl-containing medium, the wild-type germination rate was significantly higher than that of transgenic plants (FIGS. 5A, B), which was shown to be about 92.76% for wild-type, while the germination rate was lower than 40.33% for overexpressing lines (FIGS. 5A, B). The results of salt stress on the germination inhibition rate of arabidopsis thaliana showed that the germination inhibition rate of 200mM NaCl on the overexpressed strain was significantly higher than that of the wild-type (fig. 5C).

In conclusion, the salt stress tolerance of the heterologous over-expressed arabidopsis thaliana is reduced, which indicates that the PtTINY gene negatively regulates the tolerance of the arabidopsis thaliana to the salt stress. Therefore, the PtTINY gene plays an important role in regulating and controlling plant salt stress, and has important significance for cultivating poplar varieties capable of resisting external stress conditions.

The above-mentioned embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and other embodiments can be easily made by those skilled in the art through substitution or modification according to the technical disclosure in the present specification, so that all changes and modifications made in the principle of the present invention shall be included in the scope of the present invention.

Claims (8)

1. The application of PtTINY gene in regulating plant salt stress tolerance, which is characterized in that the PtTINY gene can reduce plant salt stress tolerance, and the PtTINY gene has a gene sequence number XM_002298200.4 in NCBI.
2. 2. Use according to claim 1, characterized in that transgenic plants with reduced salt stress tolerance are obtained by constructing a ptiny overexpression vector.
3. 3. The use according to claim 1 or 2, wherein the plant is arabidopsis thaliana, poplar.
4. 4. The use according to claim 3, wherein the poplar is aspen.
5. 5. The use according to claim 1, wherein the salt stress tolerance is manifested as: under salt stress, the root length inhibition rate, the fresh weight inhibition rate and the germination inhibition rate of the PtTINY over-expression strain are all higher than those of a wild type.
6. 6. A plant breeding method characterized in that the method is (1) or (2) below:

   (1) Obtaining a plant with salt stress tolerance lower than that of the target plant by increasing the activity of PtTINY protein in the target plant;

   (2) Obtaining a plant with salt stress tolerance lower than that of the target plant by promoting the expression of PtTINY gene in the target plant;

   the PtTINY gene has a gene sequence number of XM_002298200.4 in NCBI.
7. 7. The method of plant breeding according to claim 6, wherein the plant of interest is Arabidopsis thaliana, poplar.
8. 8. The plant breeding method according to claim 7, wherein the poplar is aspen.