CN119331898A - Application of Arabidopsis atpB gene in improving plant stress resistance - Google Patents

Abstract

The invention relates to application of an arabidopsis atpB gene in improving plant stress resistance, belonging to the technical field of plant genetic engineering breeding. In order to provide more stress-resistant genes which can be applied to transgenic technology for improving plant stress resistance, the invention provides application of an arabidopsis atpB gene in improving plant stress resistance, in particular application of the arabidopsis atpB gene in improving plant drought resistance and/or salt resistance, and the method comprises the steps of constructing a plant overexpression vector containing the arabidopsis atpB gene and transforming the plant overexpression vector into plants to cultivate drought-resistant and/or salt-resistant transgenic plants. The invention proves that the arabidopsis atpB gene can respond to drought and salt stress, and the seedling stage phenotype of an arabidopsis atpB over-expressed plant after drought and salt stress treatment is observed, and the result shows that the arabidopsis atpB gene can obviously improve the drought resistance and salt resistance of transgenic plants and is an excellent gene for plant drought resistance and/or salt resistance gene engineering breeding.

Description

Application of arabidopsis atpB gene in improving plant stress resistance

Technical Field

The invention belongs to the technical field of plant genetic engineering breeding, and particularly relates to application of an arabidopsis atpB gene in improving plant stress resistance.

Background

Plant stress resistance refers to the resistance, adaptation and tolerance that plants exhibit when subjected to various environmental factors that are detrimental to their growth and survival. This ability allows plants to maintain their growth and reproduction in stress. In agriculture and forestry production, the yield and quality of crops can be improved by utilizing the stress resistance of plants. By breeding the crop variety with excellent stress resistance, the influence of stress on the growth of the crop can be reduced, and the stress resistance and adaptability of the crop can be improved.

Genetic improvement is a common method for improving stress resistance of plants, and comprises the steps of screening stress-resistant varieties for cross breeding and introducing stress resistance genes into plants by utilizing a genetic engineering technology, so that crops have stronger stress resistance. For example, the saline-alkali tolerance of crops can be obviously improved by introducing the saline-alkali tolerance genes into the crops through a transgenic technology. The stress-resistant gene may be derived from a natural stress-resistant plant or microorganism, or may be a gene of a protein which is produced by a plant in stress and acts as a cell-protecting agent. The number of stress-resistant genes in plants is numerous and the distribution is wide, which makes the screening work of the stress-resistant genes extremely complex. Different stress-resistant genes may have different functions and mechanisms of action, which also increase the difficulty of screening and identifying stress-resistant genes.

Chloroplast ATP synthase is closely related to energy metabolism of plants and plays an important role in interaction of plants with the external environment. In the energy metabolism process of plants, the a subunit-atpA and the β subunit-atpB of the ATP synthase play a critical role, and the atpB plays a central role in regulating the activity of the ATP synthase and the interaction with reaction substrates. Earlier studies found that the Arabidopsis atpB gene has the ability to resist Pseudomonas syringae-Pst.DC3000. However, the antibacterial function and the stress-resistant function of the plants are relatively independent in biology, and are different adaptation mechanisms respectively developed for different external pressures and challenges, and the action objects and principles of the plants are different. Therefore, other functions of the gene cannot be deduced according to the antibacterial function of the Arabidopsis atpB gene, and the stress resistance function of the Arabidopsis atpB gene has not been reported yet.

Disclosure of Invention

In order to provide more stress-resistant genes which can be applied to transgenic technology for improving plant stress resistance, the invention provides application of the arabidopsis atpB gene in improving plant stress resistance.

The technical scheme of the invention is as follows:

Use of the arabidopsis atpB gene for increasing stress resistance in plants.

Further, the nucleotide sequence of the Arabidopsis atpB gene is shown as SEQ ID No. 1.

Further, the application is the application of the Arabidopsis atpB gene in improving drought tolerance and/or salt tolerance of plants.

Further, the application comprises the steps of constructing a plant over-expression vector containing an arabidopsis atpB gene, transforming the constructed plant over-expression vector into a plant, and culturing to obtain a drought-tolerant and/or salt-tolerant transgenic plant.

Further, the plant is a woody plant or a herbaceous plant.

Further, the woody plant is Arabidopsis thaliana.

A plant over-expression vector comprising an arabidopsis atpB gene.

A recombinant genetically engineered bacterium containing the plant over-expression vector.

Further, the plant over-expression vector containing the Arabidopsis atpB gene is constructed by introducing E.coli or Agrobacterium.

The invention has the beneficial effects that:

The invention proves that the arabidopsis atpB gene can respond to drought and salt stress, and the seedling stage phenotype of Col-0, atpB-OE, atpB and atpBpro:atpB plants after drought and salt stress treatment is observed, and compared with Col-0 and atpBpro:atpB, the atpB-OE and the atpB have higher germination rate, green leaf rate and root elongation phenotype. The detection of the seedling stage phenotype and related physiological indexes shows that the atpB-OE improves the active oxygen scavenging capacity of plants, and accumulates higher chlorophyll content and lower MDA content to resist drought and salt stress. The arabidopsis atpB gene can obviously improve drought tolerance and salt tolerance of transgenic plants, and is an excellent gene for plant drought tolerance and/or salt tolerance gene engineering breeding.

Drawings

FIG. 1 is a graph showing comparison of the expression of Arabidopsis atpB gene of example 2 at different drought stress times;

FIG. 2 is a graph showing comparison of the expression of Arabidopsis atpB gene of example 2 at different salt stress times;

FIG. 3 is a comparison of seed germination phenotypes of Col-0, atpB-OE, atpB and atpBpro of example 3, atpB plants on different mannitol concentration media;

FIGS. 4 and 5 are graphs comparing germination rates and green leaf rates of, respectively, col-0, atpB-OE, atpB and atpBpro in example 3, atpB plants grown for 10d on different mannitol concentration media;

FIG. 6 is a photograph of root growth phenotypes of Col-0, atpB-OE, atpB and atpBpro of example 4, atpB plants on different mannitol concentrations of culture medium;

FIG. 7 is a graph comparing root growth coefficients of Col-0, atpB-OE, atpB and atpBpro of example 4, for atpB plants on different mannitol concentrations of culture medium;

FIG. 8 is a photograph showing phenotypes of Col-0, atpB-OE, atpB and atpBpro of example 5, before and after drought treatment, showing values corresponding to each plant as soil moisture content values;

FIG. 9 is a graph showing the comparison of the chlorophyll a content, chlorophyll b content and total chlorophyll content of the Col-0, atpB-OE, atpB and atpBpro plants before and after drought treatment in example 5;

FIG. 10 is a graph comparing MDA content of Col-0, atpB-OE, atpB and atpBpro of example 5, before and after drought treatment;

FIG. 11 is a comparison of seed germination phenotypes of Col-0, atpB-OE, atpB and atpBpro of example 6, atpB plants on different NaCl concentration media;

FIGS. 12 and 13 are graphs comparing germination rates and green leaf rates of, respectively, col-0, atpB-OE, atpB and atpBpro in example 6, atpB plants grown for 10d on different NaCl concentration media;

FIG. 14 is a photograph of root growth phenotypes of Col-0, atpB-OE, atpB and atpBpro of example 7, atpB plants on different NaCl concentration media;

FIG. 15 is a graph comparing root growth coefficients of Col-0, atpB-OE, atpB and atpBpro of example 7, for atpB plants on different NaCl concentrations;

FIG. 16 is a photograph of phenotypes of Col-0, atpB-OE, atpB and atpBpro of example 8, of atpB plants before and after salt stress treatment;

FIG. 17 is a representative photograph of Col-0, atpB-OE, atpB and atpBpro in example 8, NBT, DAB and trypan blue staining of atpB plants before and after salt stress treatment;

FIG. 18 is a graph showing the comparison of the chlorophyll a content, chlorophyll b content and total chlorophyll content of Col-0, atpB-OE, atpB and atpBpro of example 8, before and after salt stress treatment;

FIG. 19 is a graph comparing MDA content of Col-0, atpB-OE, atpB and atpBpro:atpB plants before and after salt stress treatment in example 8.

Detailed Description

The following embodiments are used for further illustrating the technical scheme of the present invention, but not limited thereto, and all modifications and equivalents of the technical scheme of the present invention are included in the scope of the present invention without departing from the spirit and scope of the technical scheme of the present invention. The process equipment or apparatus not specifically mentioned in the following examples are all conventional equipment or apparatus in the art, and the raw materials and the like used in the examples of the present invention are commercially available unless otherwise specified, and the technical means used in the examples of the present invention are all conventional means well known to those skilled in the art.

Example 1

This example provides a method for preparing an atpB-OE with an overexpression of the atpB gene and a method for preparing a replacement plant atpBpro of an atpB gene-mutated Arabidopsis atpB.

Plant material used in this example:

Wild type Arabidopsis Col-0 is Columbia background type Arabidopsis thaliana (Arabidopsis ihaliana), arabidopsis thaliana atpB gene T-DNA insertion mutant number SALK 004946C, and the ABRC website purchased Arabidopsis thaliana mutant seeds, culture conditions were 23℃for 16h light, 8h dark.

The strains and vectors used in this example:

(1) Coli DH 5. Alpha. And Agrobacterium GV3101 (pSoup-P19) were purchased from Shanghai Weidi Biometrics.

(2) The vector pSuper1300-Myc is prepared by adding a 35S promoter before the 5 'end of a polyclonal site sequence of a commercial pCAMBIA1300 vector, and adding a Myc tag after the 3' end of the polyclonal site sequence.

1. The construction method of the Arabidopsis atpB gene overexpression vector and the recombinant genetic engineering bacteria containing the Arabidopsis atpB gene comprises the following steps:

1. the extraction method of the plant RNA comprises the following steps:

And (3) putting the obtained arabidopsis leaves (0.5-1 g) into a 1.5mL microcentrifuge tube without RNase, putting small steel balls into the microcentrifuge tube, quick-freezing with liquid nitrogen, and grinding and crushing with a tissue crusher. Adding 1mL TransZol UP into a centrifugal tube after grinding the plant sample, and vibrating for 5min at room temperature after vortex forming homogenate by a homogenate instrument. Then 200 mu L of pre-chilled chloroform is added, after vortex mixing, the mixture is incubated for 3min at room temperature, 10,000Xg is centrifuged for 15min at 2-8 ℃. After centrifugation, the upper colorless water phase-shifted to a new 1.5mL microcentrifuge tube, 500mL of pre-cooled isopropanol was added, and the mixture was left standing for 10min after inversion and mixing. Centrifuging at 2-8 ℃ for 10min at 10,000Xg, and removing the supernatant. 1mL of 75% ethanol (750. Mu.L of absolute ethanol+250. Mu LDEPC water) was added, vortexed and centrifuged at 7,500 Xg for 5min at 2-8 ℃. Removing the supernatant, airing the precipitate at room temperature, adding 50-100 mu LRNADissolving Solution, incubating at 55-60 ℃ for 10min, and preserving at-70 ℃.

2. Reverse transcription into cDNA

The total plant RNA was reverse transcribed into cDNA using SPARKSCRIPTIIALL-in-one RT SuperMix for qPCR kit, all on ice and using enzyme-free microcentrifuge tubes and tips, specifically:

Sequentially adding the components in table 1 into a centrifuge tube, uniformly mixing, centrifuging for a short time, and storing the product to-20 ℃ after 50 ℃,15min, 80 ℃ and 5 sec.

TABLE 1

3. Gene cloning

The test Arabidopsis thaliana gene was cloned using a wild type Arabidopsis thaliana (Co 1-0) cDNA as a template according to the PCR reaction system and the reaction procedure shown in tables 2 and 3.

TABLE 2

TABLE 3 Table 3

The primer sequences used in this example are as follows:

Forward Primer:TGCTCTAGAATGAGAACAAATCCTACTACTT

Reserve Primer:CGGGGTACCTTTCTTCAATTTACTCTCCATTT

The sequence of the Arabidopsis atpB gene obtained in this example is as follows:

sequence number ATCG00480.1

CDS sequence (1497)

ATGAGAACAAATCCTACTACTTCAAATCCAGAGGTTTCGATACGTGAAAAAAAAAACCTGGGACGTATCGCCCAAATCATTGGTCCGGTACTGGATGTAGCCTTCCCCCCGGGCAAAATGCCTAATATTTACAATGCTCTGGTGGTTAAGGGTCGAGATACTCTTGGTCAAGAAATTAATGTGACTTGTGAAGTACAGCAATTATTAGGAAATAATCGAGTTAGAGCTGTAGCTATGAGTGCGACAGAGGGTTTAAAGAGAGGAATGGACGTGGTTGATATGGGAAATCCTCTAAGTGTTCCAGTCGGCGGAGCGACTCTAGGACGAATTTTCAACGTGCTTGGGGAACCCGTTGATAATTTAGGTCCTGTCGATACTCGCACAACATCTCCTATCCATAAATCCGCGCCTGCTTTTATAGAATTAGATACAAAATTATCGATTTTTGAAACAGGAATTAAAGTAGTAGATCTTTTGGCCCCTTATCGTCGTGGGGGAAAAATTGGACTATTCGGTGGGGCTGGCGTGGGTAAAACAGTACTAATTATGGAATTGATCAACAACATTGCTAAAGCTCATGGTGGTGTATCCGTATTTGGTGGAGTAGGCGAACGGACTCGTGAAGGAAATGATCTTTACATGGAAATGAAAGAATCTGGAGTCATTAATGAACAAAATCTTGCGGAATCCAAAGTGGCCCTAGTTTATGGTCAGATGAATGAACCGCCAGGAGCTCGTATGAGAGTTGGTCTGACTGCCTTAACTATGGCAGAATATTTCCGAGATGTTAATGAGCAAGACGTACTTCTATTTATCGACAATATCTTCCGTTTCGTACAAGCAGGATCCGAGGTATCCGCCTTATTGGGTAGAATGCCTTCTGCTGTGGGTTATCAACCCACCCTTAGTACCGAAATGGGTACTTTACAAGAAAGAATTACTTCTACGAAAAAGGGGTCCATAACCTCTATTCAAGCAGTTTATGTACCTGCAGATGATTTGACTGACCCCGCACCTGCCACCACATTTGCACATTTAGATGCGACTACCGTACTATCAAGAGGATTAGCTGCCAAAGGTATCTATCCAGCGGTAGATCCTTTAGATTCAACGTCAACTATGCTACAACCTCGAATCGTTGGCGAGGAACATTATGAAACTGCGCAACAAGTAAAACAAACTTTACAACGTTACAAGGAGCTTCAGGACATTATAGCTATCCTGGGGTTGGATGAATTATCCGAAGAGGATCGCTTAACCGTAGCAAGAGCACGAAAAATTGAGCGTTTCTTATCACAACCCTTTTTCGTAGCAGAAGTATTTACAGGTTCTCCGGGAAAATATGTTGGTCTAGCGGAAACAATTAGAGGGTTTAATTTGATCCTTTCCGGAGAATTTGATTCTCTTCCCGAACAGGCCTTTTACTTAGTGGGTAACATCGATGAAGCTACTGCGAAGGCTACGAACTTAGAAATGGAGAGTAAATTGAAGAAATGA.

Recovery (purification) of PCR product gel

The test uses a kit for century Gel Extraction Kit, specifically:

Cutting out the DNA strip, placing the DNA strip into a microcentrifuge tube, weighing, adding an equal volume of Buffer PG and 50 ℃ water bath sol, gently reversing the microcentrifuge tube upside down every 2-3 min, cooling to room temperature, loading the microcentrifuge tube into a column, balancing the column, adding 200 mu LBuffer PS,13,000rpm and 1min into an adsorption column, and discarding waste liquid. The solution obtained in the step 2 was put into an adsorption column, allowed to stand at room temperature for 2min,13,000rpm,1min, the waste liquid was poured off, 450. Mu. LBuffer PW (containing absolute ethanol) was added, and 13,000rpm,1min, the waste liquid was poured off. 13,000rpm, air-separating for 1min, pouring out the waste liquid, repeating the steps and centrifuging once. Placing the adsorption column into a new 1.5ml microcentrifuge tube, suspending and dropwise adding 50-100 mu L of ddH₂ O (preheated in a 50 ℃ water bath) to the middle position of the adsorption film, and standing for 2min at room temperature. 12,000rpm,2min, at-20 ℃.

5. Double enzyme cutting

The target gene purification product is subjected to double enzyme digestion, the components in table 4 are sequentially added into a microcentrifuge tube, after being gently mixed, the mixture is incubated for 30min at 37 ℃, 10X DNALoading Buffer is added, agarose gel electrophoresis is performed again, and DNA gel recovery is performed.

TABLE 4 Table 4

The carrier double enzyme digestion reaction system is shown in Table 5, after gentle mixing, the reaction is terminated by incubation at 37℃for 30min and inactivation at 65℃for 20min.

TABLE 5

6. Recombinant plasmid ligation

The recombinant plasmid ligation system shown in Table 6 was used at 16℃for 1 to 3 hours or 4℃overnight.

TABLE 6

7. Transformation of E.coli by heat shock method

Taking DH5 alpha out from-80 deg.C, melting on ice, sucking out 10. Mu.L of the ligation product, adding into the competence, mixing gently, and freezing for 25min. And taking out the mixture after heat shock for 45s in a 42 ℃ water bath, and carrying out ice bath for 2min. 700. Mu.L of LB medium without antibiotics was added thereto, and the culture was performed at 37℃and 200rpm for 1 hour. Centrifuge at 6,000rpm for 1min. Pouring out the supernatant, remaining 100 mu L of bacterial liquid, blowing and uniformly mixing, and then coating on a corresponding culture medium, and culturing for 12-24 hours at 37 ℃.

2. Transformation method of Arabidopsis thaliana of this example

1. Plasmid small scale, the test uses a full gold EasyPure HiPure PLASMIDMINIPREP KIT kit, and detailed steps are shown in the specification.

2. Freeze thawing process of transforming agrobacterium

Taking out GV3101 (pSoup-P19) from the ultra-low temperature refrigerator, melting at room temperature, inserting into ice, and adding 0.01-1 mu gpSuper1300-atpB-MYC plasmid into melted Agrobacterium GV3101 (pSoup-P19) competence. Sequentially standing on ice for 5min, liquid nitrogen for 5min, water-bath at 37deg.C for 5min, and ice-bath for 5min, adding 700 μl of non-resistant LB culture medium into the centrifuge tube, culturing at 28deg.C and 200rpm for 2h, centrifuging the centrifuge tube at 6,000rpm for 1min, and collecting thallus. Approximately 100. Mu.L of supernatant was retained and the resuspended pellet was gently swirled using a micropipette. Subsequently, the bacterial liquid was uniformly spread on the surface of the LB medium containing the antibiotic. Culturing in 28 deg.c incubator for 2-3 d, picking single colony from the plate into LB liquid culture medium with Rif and Kan resistance, culturing in 28 deg.c shaking table at 200rpm for 2-3 d, and taking 1. Mu.L of bacteria liquid for PCR identification. And uniformly mixing the bacterial liquid which is successfully identified with glycerol, and storing the mixture in a refrigerator at the temperature of-80 ℃.

3. Planting of Arabidopsis thaliana

A clean 1.5mL microcentrifuge tube was selected and the seeds to be sterilized were placed in advance in the 1.5mL microcentrifuge tube. In a sterile operation table, 900 mu L of sterile water, 100 mu L of sodium hypochlorite and 1 mu LTween-80 are sequentially added into a centrifuge tube, and the centrifuge tube is shaken for 7-8 min. And in the sterile operation table, the sterilized seeds are washed for 5-6 times by sterile water until the foam disappears. The sterilized seeds were plated on 1/2MS medium with an inoculating loop and cultured in a light incubator for 1 week. And planting arabidopsis, namely uniformly mixing seedling raising soil and vermiculite in a ratio of 1:1, watering until the soil is completely wet, transplanting the arabidopsis seedlings growing to four leaves on a culture medium into the soil, performing film-covered culture for 2d in an arabidopsis climate chamber (22 ℃,16h illumination/8 h darkness), and continuing to perform culture after film uncovering.

4. Transformation of Arabidopsis thaliana

The inflorescence dip-dyeing method is adopted:

pSuper1300-atpB-MYC positive colonies were picked and propagated in 50mL LB liquid medium of 50mg/LKan and 25 mg/LRif. Culturing overnight at 28 ℃ until OD₆₀₀ = 1.8-2.0, centrifuging at 4,000rpm for 10min, and collecting bacteria. The cells were resuspended in sucrose solution (per 100mL of distilled water +5g sucrose +25. Mu. L Sliweet-77), and the bacterial solution was diluted to OD₆₀₀ =0.6 to 0.8. The buds of the arabidopsis plants are carefully placed in bacterial liquid and soaked for about 20s by both hands. After infection, bagging and culturing in dark for 24h, and punching holes on the bag after 24h. After 2d, the bag is removed and the cells are normally cultured in an Arabidopsis climate chamber. After one week, the infection was performed in the same manner.

3. Preparation method of the Hui-patch plant atpBpro of the atpB Gene mutant Arabidopsis atpB of this example

The inflorescence dip-dyeing method is used for converting pSuper1300-atpB-Myc into atpB to obtain T₀ generation atpB seed, then T₀ generation seeds are screened in a screening culture medium (1/2MS+50 mg/L hygromycin), DNA level identification is carried out on T₁ generation positive plants obtained through screening by using hygromycin primers, then total RNA of leaf blades of the atpB and 8 positive plants is extracted by a TRIzol method, the RNA concentration of each sample is adjusted to be consistent, corresponding cDNA is obtained through reverse transcription and used as a template, transcriptional level identification is carried out, the expression level of the atpB gene in the positive plants is detected, after single plant collection of transgenic plants with high expression level is carried out, T₂ generation seeds are sown in the screening culture medium, then DNA level detection is carried out on T₂ plants, the single plant seeds are collected positively, 30 positive plant seeds with the same number are inoculated in the screening culture medium, the cotyledons are green and all surviving transgenic homozygous plants, and two independent homozygous transgenic positive plants atpBpro are selected for the test of the atpB plants.

Example 2

This example demonstrates that the Arabidopsis atpB gene is capable of responding to drought and salt stress.

In this example, wild type Arabidopsis Col-0 with consistent growth in soil was selected and transferred to a nutrient solution containing 1/2Hoagland for 2d, and then transferred to a 1/2Hoagland+200mM mannitol nutrient solution and a 1/2Hoagland+125mM NaCl nutrient solution, respectively, for continuous culture, and sampling was performed at 0h, 1h, 3h, 6h, 9h, 12h and 24 h.

Extracting Arabidopsis RNA by TRIzol method, obtaining cDNA by reverse transcription, and detecting the expression condition of Arabidopsis atpB genes under drought and salt stress at different times by RT-qPCR. The results are shown in FIGS. 1 and 2, where the Arabidopsis atpB gene expression has increased, indicating its ability to respond to drought and salt stress.

Example 3

This example examined wild type Arabidopsis Col-0, attB gene overexpression Arabidopsis attB-OE, attB gene mutation Arabidopsis attb and a restorer plant atpBpro of attB gene mutation Arabidopsis attb the seedling stage phenotype of attB under drought stress.

Col-0, atpB-OE, atpB and atpBpro atpB plants were inoculated onto 1/2MS, 1/2MS+150mM mannitol and 1/2MS+175mM mannitol, respectively, germination tests were performed, and four lines of seed germination were recorded by photographing at 3d, 7d and 11d, and germination rates and green leaf rates of each line grown for 10d on 1/2MS plus mannitol medium at different concentrations were investigated and counted.

Four strain seeds germinate as shown in FIGS. 3-5, when sown on 1/2MS medium for 6d, four strain seeds all germinated, and the germination rates and green leaf rates of atpB-OE and atpB were not significantly different from those of Col-0 and atpBpro:atpB.

After sowing on 1/2MS+150mM mannitol medium, it was found that at 5d, the atpB germination rate had reached 95%, the atpB-OE germination rate was 81%, the Col-0 germination rate was 92%, atpBpro:90%, the atpB green leaf rate was 80%, the atpB-OE green leaf rate was 70%, and the Col-0 green leaf rate was 57%, atpBpro:56%. The cotyledon growth as a whole exhibited a more luxuriant state than that of Col-0 and atpBpro:atpB.

After sowing on 1/2MS+175mM mannitol medium, it was found that at 8d, atpB-OE and atpB all germinated, col-0 and atpBpro that the atpB germination rate was 94%, the atpB green leaf rate was 56%, the atpB-OE green leaf rate was 42%, and the Col-0 green leaf rate was 35%, atpBpro that the atpB green leaf rate was 37%, and the green leaf rate of atpB had a significant difference from that of Col-0, and that the Col-0 cotyledon grown as a whole showed wilting.

The above results demonstrate that atpB and atpB-OE increase the tolerance of plants to drought stress.

Example 4

This example examined wild type Arabidopsis Col-0, attB gene overexpression Arabidopsis attB-OE, attB gene mutation Arabidopsis attb and a complement plant atpBpro of attB gene mutation Arabidopsis attb root elongation phenotype under drought stress.

Col-0, atpB-OE, atpB and atpBpro atpB plants were inoculated onto 1/2MS medium, respectively, and a part of plants of one week old and consistent germination were inoculated onto 1/2MS+175mM mannitol medium for root length detection test, daily observation and photographing recording.

As shown in FIG. 6, the root growth vigor of the four lines was substantially uniform in the 1/2MS medium containing no mannitol, and the root growth was inhibited in the 1/2MS medium containing 175mM mannitol, compared with atpB-OE and atpB, col-0 and atpBpro: the root length of atpB was short.

As shown in FIG. 7, by counting the elongation of the main roots, the elongation rate of the atpB-OE roots was significantly lower than that of Col-0 and atpBpro:atpB, and the elongation rate of the atpB roots was significantly higher than that of Col-0 and atpBpro:atpB, as the treatment days increased, compared with that of Col-0 and atpBpro:atpB, which generally indicated that mutation of the atpB gene increased drought stress tolerance of Arabidopsis thaliana.

Example 5

This example examined wild type Arabidopsis Col-0, attB gene overexpression Arabidopsis attB-OE, attB gene mutation Arabidopsis attb and a complement plant atpBpro of attB gene mutation Arabidopsis attb phenotype and physiological index detection under drought stress.

Col-0, atpB-OE, atpB and atpBpro:atpB plants grown normally at 22℃for 4 weeks were subjected to drought water-break treatment, and seedling morphology changes during stress were observed and recorded by photographing.

The chlorophyll content determination method in the invention comprises the following steps:

(1) The method comprises the following steps of weighing 0.2g of fresh plant leaves under weak light, wiping the surfaces of the fresh plant leaves, and shearing the fresh plant leaves;

(2) Placing into a microcentrifuge tube, vibrating and crushing, adding a small amount of 95% ethanol, washing to a 50mL centrifuge tube in batches, fixing the volume to 50mL, and uniformly mixing;

(3) 95% ethanol is used as a blank, 1mL of the extracting solution is absorbed, and absorbance is measured at the wavelengths of 470nm, 665nm and 649 nm;

Ca=13.95A₆₆₅－6.88A₆₄₉

Cb=24.96A₆₄₉－7.32A₆₆₅

Total chlorophyll concentration = ca+cb

Chlorophyll content= (chlorophyll concentration x extract volume x dilution factor)/fresh sample weight

As shown in FIG. 8, at 20d, compared with the control group, col-0 and atpBpro, the atpB withered, the leaves were subjected to large-area yellowing lodging, the atpB-OE was also the same, and the atpB plants were still strong, and the leaves were lighter in green loss.

Chlorophyll content was closely related to photosynthesis, and thus, the chlorophyll a, chlorophyll b, and total chlorophyll content of the attB plants were measured for Col-0, attB-OE, attB, and atpBpro subjected to drought water-break treatments of 0d and 20 d.

As shown in FIG. 9, it was found by statistical analysis that compared to Col-0 and atpBpro:atpB, the total chlorophyll content in chlorophyll a, chlorophyll b and chlorophyll b was higher than that of Col-0 and atpBpro:atpB, and atpB was more significant up to 1.6 times that of Col-0. Indicating that atpb has greater resistance to drought stress.

The MDA of atpB plants was measured simultaneously for Col-0, atpB-OE, atpB and atpBpro for drought water-break treatments 0d and 20 d.

As shown in FIG. 10, the MDA content of atpB-OE was slightly lower than Col-0 and the MDA content of atpB was significantly lower than Col-0 and atpBpro:atpB 4.7 times lower than Col-0, as compared to Col-0.

The results show that atpB and atpB-OE reduce the oxidative damage degree of Arabidopsis thaliana to drought stress and improve the tolerance of plants to drought stress.

Example 6

This example examined wild type Arabidopsis Col-0, attB gene overexpression Arabidopsis attB-OE, attB gene mutation Arabidopsis attb and a restorer plant atpBpro of attB gene mutation Arabidopsis attb the seedling stage phenotype of attB under salt stress.

Col-0, atpB-OE, atpB and atpBpro atpB plants were inoculated onto 1/2MS, 1/2MS+100mM NaCl and 1/2MS+125mM NaCl medium, respectively, germination tests were performed, and four lines of seed germination were recorded by photographing at 3d, 7d and 11d, and germination rates and green leaf rates of the respective lines grown on 1/2MS plus NaCl medium at different concentrations were investigated and counted.

Four strain seeds germinated as shown in FIGS. 11-13, when sown on 1/2MS medium for 6d, the four strain seeds all germinated, and the germination rates and green leaf rates of atpB-OE and atpB were not significantly different from those of Col-0 and atpBpro:atpB.

After sowing on 1/2MS+100mM NaCl medium, it was found that at 5d, the atpB germination rate was 97%, the atpB-OE germination rate was 94%, the Col-0 germination rate was 81%, atpBpro:84%, and at 7d, the atpB green leaf rate was 95%, the Col-0 and atpBpro:81%, and the atpB-OE green leaf rate was only 61%. atpB showed a more luxuriant state than Col-0 cotyledon growth overall, while atpB-OE showed a more wilting.

After sowing on the 1/2MS+125mM NaCl medium, the overall germination rate of the four strains is about 10% lower than that of the four strains on the 1/2MS+100mM NaCl medium, and at 6d, the green leaf rate of atpb is 8%, and the green leaf rates of the other three strains are all 0, which indicates that the atpb mutant leads to the increased tolerance of Arabidopsis after NaCl treatment.

Example 7

This example examined wild type Arabidopsis Col-0, overexpression of the atpB gene Arabidopsis atpB-OE, mutant Arabidopsis atpB of the atpB gene and a complement plant atpBpro of the mutant Arabidopsis atpB of the atpB gene the root elongation phenotype of the atpB under salt stress.

Col-0, atpB-OE, atpB and atpBpro atpB plants were inoculated onto 1/2MS medium, respectively, and a part of plants of one week old and consistent germination were selected and inoculated onto 1/2MS+100mM NaCl medium for root length detection test, daily observation and photographing recording.

As shown in FIG. 14, the root growth vigor of the four lines was substantially uniform in the 1/2MS medium containing no NaCl, and in the 1/2MS medium containing 100mM NaCl, col-0 and atpBpro: the root length of atpB was shorter and the root growth was suppressed as compared with the atpB-OE and atpB.

As shown in FIG. 15, by counting the elongation of the main roots, the length elongation rate of the atpB-OE roots was slightly higher than that of Col-0 and atpBpro:atpB, and the length elongation rate of the atpB roots was significantly higher than that of Col-0 and atpBpro:atpB, as compared with Col-0 and atpBpro:atpB, with the increase of the treatment days, it was generally demonstrated that both the atpB-OE and the atpB were capable of improving the salt stress tolerance of plants.

Example 8

This example examined wild type Arabidopsis Col-0, attB gene overexpression Arabidopsis attB-OE, attB gene mutation Arabidopsis attb and a complement plant atpBpro of attB gene mutation Arabidopsis attb phenotype and physiological index detection under salt stress.

Col-0, atpB-OE, atpB and atpBpro, atpB potting seedlings were watered at 22℃for 4 weeks and photographed to record the morphological differences of the four lines after five days.

As shown in FIG. 16, at 5d, col-0 and atpBpro, attB changed to yellow and wilt, plants lodged, attB-OE, and attB plants remained robust and leaf green was lighter compared to control group 0 d.

To verify whether atpB-OE and atpB scavenged hydrogen peroxide (H₂O₂) and superoxide anions (O₂ -.) in vivo via the ROS pathway, untreated and treated Arabidopsis thaliana were stained with tetrazolium chloride (NBT), DAB, and trypan blue to observe H₂O₂ accumulation and severity of cell death in leaf cells.

The method for dyeing tetrazolium chloride (NBT) comprises the following steps:

Preparing Nitro Blue Tetrazolium (NBT) staining solution, washing fresh leaves with distilled water, soaking in the staining solution after absorbing surface moisture, preserving at room temperature in dark for more than 8 hours, decolorizing the leaves by soaking in absolute ethyl alcohol, and observing and photographing the staining result by using a microscope (IMAGEVIEW).

The DAB dyeing method in the invention comprises the following steps:

Preparing 1mg/mL DAB (50 mM Tris-AcOH, pH 5.0) solution, cutting off leaves, placing into a centrifuge tube, adding DAB solution until the DAB solution is over a sample, soaking for 8-12 h at room temperature in dark, pouring out the staining solution, placing into a decolorizing solution with the volume ratio of acetic acid to glycerol to ethanol of 1:1:3, and decolorizing in water bath until the leaves are green and disappear. The leaves after 60% glycerol fixation and decolorization were observed under a microscope and photographed, and cells accumulated with hydrogen peroxide were stained as brown spots.

The trypan blue dyeing method comprises the following steps:

Preparing trypan blue dye liquor, wherein the trypan blue dye liquor needs to be prepared in situ. Cutting off the blades, placing into a centrifuge tube, adding trypan blue dye solution until the trypan blue dye solution is over the sample, boiling in boiling water for 2min, and standing at room temperature overnight. The staining solution was poured out, and the leaves were fixed with 60% glycerol after decolorization with 2.5g/mL of chloral hydrate, and the dead cells were stained blue under a microscope.

The MDA content determination method in the invention comprises the following steps:

Weighing 0.2g of plant tissue, shearing, placing into a centrifuge tube, quick freezing by liquid nitrogen, grinding a sample by a tissue vibration breaker, adding 1.6mL of 10% TCA (1 g of powder and 10mL of deionized water) into the centrifuge tube, vortex vibrating for 1min, 12,000rpm,10min, sucking 700 mu L of supernatant, adding 700 mu L of 0.67% TBA (0.67 g of thiobarbituric acid and 1mol/L of NaOH for dissolution, and 10% TCA for constant volume to 100 mL), uniformly mixing, bathing in boiling water for 15min, taking 700 mu L of deionized water and 700 mu L of 0.67% TBA as blank control, and measuring absorbance at wavelengths of 450nm, 532nm and 600nm after cooling.

MDA concentration C (umol/L) =6.45 (A)₅₃₂-A₆₀₀)-0.56A₄₅₀

MDA content (umol/g Fw) =C×V/W

V is the volume of the extracting solution, 1.6mL W is the fresh weight of the sample, 0.2g

As shown in FIG. 17, there are fewer dark blue and dark brown regions of atpB than Col-0 and atpBpro:atpb, indicating that fewer dead cells and ROS are accumulated, there is greater reactive oxygen species scavenging ability, atpB-OE contains more dark blue and dark brown regions, there is less antioxidant capacity, indicating that atpB mutants have reduced levels of oxidative damage in Arabidopsis and improved plant tolerance to salts.

The chlorophyll a, chlorophyll b and total chlorophyll content of the atpB plants were measured for Col-0, atpB-OE, atpB and atpBpro treated with NaCl stress.

As shown in FIG. 18, it was found by statistical analysis that compared to Col-0 and atpBpro:atpB, the total chlorophyll content in chlorophyll a, chlorophyll b and chlorophyll b was higher than that of Col-0 and atpBpro:atpB, and atpB was more significant up to 1.4 times that of Col-0. The atpb mutant significantly improved the tolerance of arabidopsis after NaCl treatment.

The MDA of atpB plants was measured simultaneously for Col-0, atpB-OE, atpB and atpBpro treated with NaCl stress for 0d and 5 d.

As shown in FIG. 19, the MDA content of atpB-OE was slightly lower than that of Col-0 and atpBpro compared with Col-0 and atpBpro:atpB, and the MDA content of atpB was significantly lower than that of Col-0 and 1.8 times lower than that of Col-0.

The above results demonstrate that both overexpression and mutants of atpB enhance salt tolerance in arabidopsis thaliana, but that atpB mutants are more resistant to salt stress.

Both atpB mutant and atpB-OE were resistant to stress reasons:

the reason why the atpB-OE resists salt and drought is that the plant resistance to adversity stress is positively regulated by the atpB gene, and the higher the expression quantity of the atpB, the stronger the plant resistance.

The salt resistance and drought resistance of the atpB mutant are due to tight coupling between the synthesis of several subunits of ATP synthase, and the phenomenon that the expression level of atpB is increased in atpA mutant strain and the expression level of atpA in the atpB mutant is increased is detected through the earlier post-injection transcription level of PstDC 000. Second, the yeast, LCI and BiFC assays demonstrate that the atpA and atpB proteins have an interactive relationship in vivo and in vitro. Again, atpa atpb double mutants do not have the ability to resist adversity stress. It was therefore concluded that atpA and atpB are in a complementary relationship, that either atpA or atpB alone is deleted and that another gene would produce a compensating effect, thereby increasing drought and salt resistance in plants, and that the mutants would also have the ability to resist drought and salt stress.

Claims (9)

Translated fromChinese

1.拟南芥atpB基因在提高植物抗逆性中的应用。1. Application of Arabidopsis atpB gene in improving plant stress resistance.2.如权利要求1所述拟南芥atpB基因在提高植物抗逆性中的应用，其特征在于，所述拟南芥atpB基因的核苷酸序列如SEQ ID No:1所示。2. The use of the Arabidopsis thaliana atpB gene in improving plant stress resistance as claimed in claim 1, characterized in that the nucleotide sequence of the Arabidopsis thaliana atpB gene is shown in SEQ ID No: 1.3.如权利要求1或2所述拟南芥atpB基因在提高植物抗逆性中的应用，其特征在于，所述应用为拟南芥atpB基因在提高植物耐旱性和/或耐盐性中的应用。3. The use of the Arabidopsis thaliana atpB gene in improving plant stress resistance as claimed in claim 1 or 2, characterized in that the use is the use of the Arabidopsis thaliana atpB gene in improving plant drought tolerance and/or salt tolerance.4.如权利要求3所述拟南芥atpB基因在提高植物抗逆性中的应用，其特征在于，所述应用包括构建含有拟南芥atpB基因的植物过表达载体，将所构建的植物过表达载体转化到植物中，培育得到耐旱和/或耐盐转基因植物。4. The use of the Arabidopsis thaliana atpB gene in improving plant stress resistance as claimed in claim 3, characterized in that the use comprises constructing a plant overexpression vector containing the Arabidopsis thaliana atpB gene, transforming the constructed plant overexpression vector into plants, and cultivating drought-resistant and/or salt-resistant transgenic plants.5.如权利要求4所述拟南芥atpB基因在提高植物抗逆性中的应用，其特征在于，所述植物为木本植物或草本植物。5. The use of Arabidopsis thaliana atpB gene in improving plant stress resistance as claimed in claim 4, characterized in that the plant is a woody plant or a herbaceous plant.6.如权利要求5所述拟南芥atpB基因在提高植物抗逆性中的应用，其特征在于，所述草本植物为拟南芥。6 . The use of the Arabidopsis thaliana atpB gene in improving plant stress resistance as claimed in claim 5 , wherein the herbaceous plant is Arabidopsis thaliana .7.一种含有拟南芥atpB基因的植物过表达载体。7. A plant overexpression vector containing the Arabidopsis thaliana atpB gene.8.一种含有权利要求7所述植物过表达载体的重组基因工程菌。8. A recombinant genetically engineered bacterium containing the plant overexpression vector according to claim 7.9.如权利要求8所述重组基因工程菌，其特征在于，将含有拟南芥atpB基因的植物过表达载体导入大肠杆菌或农杆菌构建而成。9. The recombinant genetically engineered bacterium according to claim 8, characterized in that it is constructed by introducing a plant overexpression vector containing the Arabidopsis thaliana atpB gene into Escherichia coli or Agrobacterium.