CN113527451A - Wheat heat stress related protein TaANK and coding gene and application thereof - Google Patents

Abstract

The invention discloses a wheat heat stress related protein TaANK, and a coding gene and application thereof. The tolerance of the overexpression transgenic wheat obtained by introducing the TaANK gene into the wheat to heat stress is higher than that of a receptor Fielder, and the tolerance of the TaANK transgenic wheat interfered by RNAi to heat stress is lower than that of the receptor Fielder. The protein and the gene of the invention can play an important role in cultivating heat stress resistant plants.

Description

Wheat heat stress related protein TaANK and coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, and in particular relates to a wheat heat stress related protein TaANK, and a coding gene and application thereof.

Background

Abiotic stresses, such as drought, high temperature and saline environmental factors, severely affect wheat growth and yield. Analyzing the response and signal transduction mechanism of wheat under adversity stress, improving the stress tolerance of wheat, and becoming one of the important tasks of wheat genetic research and wheat variety improvement.

Plants produce a series of responses under stress, accompanied by many physiological, biochemical and developmental changes. The reaction mechanism of the plant to the stress is determined, and scientific data is provided for the research and application of the stress-resistant gene engineering. At present, the research on plant stress resistance has been advanced to the cellular and molecular level, and combined with the research on genetics and genetic engineering, the research on improving the growth characteristics of plants by biotechnology is aimed at improving the adaptability of plants to stress.

Under the adverse conditions of environmental stresses such as drought, high salinity, low temperature and the like, the plant can be correspondingly adjusted on the molecular, cellular and overall levels so as to reduce the damage caused by the environment to the maximum extent and survive. Many genes are induced to express by stress, and the products of the genes not only can be directly involved in the stress response of plants, but also can regulate the expression of other related genes or be involved in signal transduction pathways, so that the plants can avoid or reduce damage, and the resistance to the stress environment is enhanced. It has now been found that plant response stress signaling networks are dominated by the phytohormone signaling pathway, the liposome signaling pathway, the MAPK signaling pathway, the ROS signaling pathway, and the stomata signaling pathway. These signal network systems intimately link the hormonal regulation, metabolism, energy supply and growth and development of plants. This shows that the plant adaptation to stress depends not only on the expression of the stress tolerance related genes, but also on the comprehensive regulation of various signal pathways induced by stress induction. Stress-related gene products can be divided into two broad categories: the products coded by the first gene comprise gene products directly participating in plant stress response, such as ion channel protein, aquaporin, osmotic regulatory factor (sucrose, proline, betaine and the like) synthetase and the like; the second class of genes encodes products including protein factors involved in stress-related signaling and regulation of gene expression, such as protein kinases, transcription factors, and the like.

Disclosure of Invention

The invention aims to provide a wheat heat stress related protein TaANK, and a coding gene and application thereof.

In a first aspect, the invention protects the use of TaANK protein or its related biomaterials in the regulation of plant heat stress resistance; the related biological material is a nucleic acid molecule capable of expressing the TaANK protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule or a substance inhibiting the expression of the nucleic acid molecule;

the TaANK protein is any one of the following proteins:

(A1) a protein having an amino acid sequence of SEQ ID No. 2;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.2 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

The TaANK protein is derived from Triticum aestivum L.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

In the above protein, the tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, etc.

In the above biological material, the nucleic acid molecule is a DNA molecule as described in any one of:

(B1) DNA molecule shown in SEQ ID No. 1;

(B2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes said TaANK protein;

(B3) a DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the DNA sequence defined in (B1) or (B2) and encoding the TaANK protein.

The stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In the above biological material, the recombinant vector may be specifically a recombinant vector obtained by inserting a DNA fragment shown in SEQ ID No.1 between BamHI cleavage sites of a pWMB110 vector.

In the biological material, the recombinant bacteria can be obtained by introducing the recombinant vector into an agrobacterium strain. The agrobacterium strain may specifically be agrobacterium strain EHA 105.

In the above biological material, none of the transgenic plant cell lines comprises propagation material.

In the above biological material, the substance inhibiting the expression of the nucleic acid molecule may specifically be an RNAi interference vector. The RNAi interference vector can be specifically an RNAi interference vector obtained by connecting SEQ ID No.3 with a pWMB110 vector digested by SmaI and SacI.

The application may be embodied as: the activity and/or expression level of the TaANK protein or the nucleic acid molecule capable of expressing the TaANK protein in the plant is increased, and the heat stress resistance of the plant is increased; the activity and/or expression level of the TaANK protein or the nucleic acid molecule capable of expressing the TaANK protein in the plant is reduced, and the heat stress resistance of the plant is reduced. The increased heat stress resistance may in particular be embodied by an increased survival rate under heat stress conditions and/or an increased proline content and/or a decreased MDA content. The reduced heat stress resistance may in particular be embodied by a reduced survival rate under heat stress conditions and/or a reduced proline content and/or an increased MDA content. The heat stress condition may be specifically 42 ℃.

In a second aspect, the present invention provides a method for increasing heat stress resistance in a plant, comprising the step of increasing the expression level and/or activity of a TaANK protein in a recipient plant.

The TaANK protein is as shown before. The increased heat stress resistance may in particular be embodied by an increased survival rate under heat stress conditions and/or an increased proline content and/or a decreased MDA content.

In a third aspect, the present invention provides a method of growing a transgenic plant, comprising the steps of: introducing a nucleic acid molecule capable of expressing a TaANK protein into a recipient plant to obtain a transgenic plant with increased expression level of the TaANK protein; the transgenic plant has increased heat stress resistance as compared to the recipient plant.

The "introduction into a recipient plant of a nucleic acid molecule capable of expressing a TaANK protein" is effected by introducing into said recipient plant an expression vector comprising a gene encoding said TaANK protein.

The coding gene of the TaANK protein is any one of the following DNA molecules:

(B1) DNA molecule shown in SEQ ID No. 1;

(B2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes said TaANK protein;

(B3) a DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the DNA sequence defined in (B1) or (B2) and encoding the TaANK protein.

The expression vector can be a recombinant vector obtained by inserting the DNA fragment shown in SEQ ID No.1 into the BamHI enzyme cutting sites of the pWMB110 vector.

In a fourth aspect, the invention provides a TaANK protein or a biological material related thereto as described in any of the preceding paragraphs, or a method as described in any of the preceding paragraphs, for use in plant breeding. The breeding is aimed at breeding plants with high heat stress resistance.

In a fifth aspect, the invention features method a or method B.

Method A is a method for reducing heat stress resistance in a plant, comprising the step of reducing the expression level and/or activity of a TaANK protein in a recipient plant.

The method B is a method for cultivating transgenic plants, and comprises the following steps: inhibiting or silencing expression of a nucleic acid molecule encoding a TaANK protein in a receptor plant to obtain a transgenic plant with reduced expression level of the TaANK protein; the transgenic plant has reduced heat stress resistance as compared to the recipient plant.

The TaANK protein is as described hereinbefore.

Said "inhibiting or silencing the expression of a nucleic acid molecule encoding a TaANK protein in a recipient plant" may be effected by introducing into the recipient plant an RNAi interference vector. The RNAi interference vector can be specifically an RNAi interference vector obtained by connecting SEQ ID No.3 with a pWMB110 vector digested by SmaI and SacI.

Any of the plants described above is a dicot or monocot;

further, the monocotyledon is a gramineous plant;

further, the gramineous plant is wheat.

The wheat can be wheat variety Fielder.

Experiments prove that the resistance of the transgenic wheat obtained by introducing the TaANK gene into the wheat to the heat stress is higher than that of a receptor wheat Fielder, and the tolerance of RNAi (ribonucleic acid interference) to the heat stress of the wheat is lower than that of the receptor wheat Fielder. The protein and the gene provided by the invention provide a basis for human control of expression of stress resistance and stress tolerance related genes, and play an important role in cultivating plants with enhanced stress resistance and stress tolerance.

Drawings

FIG. 1 is a schematic diagram of the construction of an overexpression and RNAi interference TaANK vector.

FIG. 2 shows PCR positive detection of wheat over-expressed TaANK.

FIG. 3 shows qRT-PCR expression detection of wheat over-expressed TaANK.

FIG. 4 shows the positive PCR assay of TaANK-RNAi wheat.

FIG. 5 shows the detection of the expression level of TaANK-RNAi wheat qRT-PCR.

FIG. 6 shows the phenotypic identification of heat stress of transgenic TaANK wheat.

FIG. 7 is the determination of the physiological index of transgenic TaANK wheat under heat stress.

FIG. 8 is the TaANK subcellular localization.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples,% is by mass unless otherwise specified.

The wheat variety Xiaobaimai (Triticum aestivum L.) in the following examples is described in the literature "Sun Hao et al, screening of transcription factor interacting protein of wheat TaDREB6, Chinese agricultural science, 2011,44 (22): 4740-; the public is also available from the national germplasm resources pool (accession number ZM 242).

The pWMB110 vector, GFP vector pJIT16318 and wheat variety Fielder in the following examples are described in "Xiao-Yu Cui, Yuan Gao, Jun Guo, Tai-Fei Yu, Wei-Jun Zheng, Yong-Wei Liu, Jun Chen, Zhu-Shi Xu and You-Zhi Ma. BES/BZR Transcription Factor TaBZR2 positional Regulation draught Responses by Activation of TaGSTT 11.plant physiology.2019, 605" 605, which was publicly available from the research of crop science of the national academy of sciences, and which was used only for repeating experiments related to the present invention and was not used for other purposes.

Example 1 cloning of TaANK

Treatment of plant material

The whole young plant of Triticum aestivum cv. Xiiaobaimai in trefoil stage, which grows in water culture for about 10 days, is quickly frozen in liquid nitrogen and stored at-80 deg.c for further use.

Second, extraction of Total RNA

Extracting the total RNA of the leaves of the treated wheat seedlings obtained in the first step by using a Trizol method (Tianggen).

Third, obtaining cDNA

The first strand cDNA synthesis is performed using reverse transcriptase XL (AMV). The ds cDNA was synthesized by SMART method and the PCR product was detected by 1.0% agarose gel electrophoresis. And (3) amplifying a wheat cDNA library by designing a proper primer sequence to obtain SEQ ID No. 1. SEQ ID No.1 can also be obtained by artificial synthesis.

The specific primers for amplification are:

TaANK-F2:5’-TGTCACATCTGAAGCGTTGG-3’；

TaANK-R5:5’-TAGTAGTGCCAGTTTCCAAT-3’。

the gene shown in SEQ ID No.1 is named as TaANK gene, the open reading frame of the gene is nucleotides 1 to 642 from the 5' end of the SEQ ID No.1, the protein coded by the gene is named as TaANK protein, the amino acid sequence of the protein is SEQ ID No.2, and the protein consists of 213 amino acid residues.

Example 2 acquisition of transgenic TaANK wheat and stress tolerance analysis thereof

Obtaining of TaANK-transgenic wheat

1. Construction of overexpression TaANK recombinant vector

(1) Amplification of the TaANK Gene

And (3) taking a TaANK fragment which is amplified and recovered from wheat as a template, and adopting primers of TaANK-110F and TaANK-110R for amplification to obtain a PCR product. The primer sequences are as follows, BamHI enzyme digestion recognition sites are respectively introduced into the ends of the primers and are marked by underlines:

TaANK-110F：5'-CGACTCTAGAGGATCCATGTATGCGGACCAGATC-3'；

TaANK-110R：5'-GGGTACCCGGGGATCCCTACGCTCTGGATTCCAA-3'。

and (3) carrying out 1.2% agarose gel electrophoresis on the PCR product, sequencing the PCR product, and indicating that the PCR product has SEQ ID No. 1.

The PCR product was recovered and purified by using the Agarose Gel DNA Purification Kit Ver.2.0(TaKaRa Co., Code No.: DV 807A).

(2) Digesting the pWMB110 vector by using a restriction enzyme BamHI, and recovering a vector skeleton; connecting the enzyme digestion product with a carrier framework to obtain a connection product;

(3) and (3) thermally shocking the ligation product obtained in the step (2) to transform a TOP10 strain (Tiangen, CB104-03), culturing overnight at 37 ℃, and picking positive clones to extract plasmids for sequencing.

Sequencing results show that the plasmid is a vector obtained by inserting the DNA fragment shown in SEQ ID No.1 into BamHI enzyme cutting sites of a pWMB110 vector, the plasmid is named as pWMB110-TaANK, and the information of important elements of the pWMB110-TaANK is shown in figure 1A.

2. Construction of RNAi interference TaANK recombinant vector

(1) The specific operation of constructing the TaANK RNAi interference vector is as follows: artificially synthesizing a 445-639 th nucleotide sequence and a reverse complementary sequence thereof in SEQ ID No.1, taking a corn alcohol dehydrogenase (Adh) gene sequence with 146bp in the middle as an intron, adding a SmaI enzyme cutting site at the 5 'end and adding a SacI enzyme cutting site at the 3' end, wherein the fragment synthesis is finished by Beijing Okkomy Biotech Limited, and the specific synthetic sequence fragment is shown as SEQ ID No. 3.

(2) The pWMB110 vector was digested with SmaI and SacI, and the digested product was recovered using the Agarose Gel DNAPURATION Kit Ver.2.0(TaKaRa Co., Code No.: DV 807A). And connecting the artificially synthesized fragment SEQ ID No.3 in the step (1) with a pWMB110 vector digested by SmaI and SacI to construct a TaANK-pWMB110-RNAi interference vector, wherein the information of important elements of the TaANK-pWMB110-RNAi interference vector is shown in figure 1B.

3. Construction of recombinant bacterium

(1) The recombinant plasmids pWMB110-TaANK and the TaANK-pWMB110-RNAi obtained in the steps 1 and 2 are respectively transformed into agrobacterium EHA105 (purchased from Beijing Beerty Biotech company) to obtain recombinant agrobacterium.

(2) The plasmid of the recombinant agrobacterium is extracted and sequenced, and the result shows that both the pWMB110-TaANK and the TaANK-pWMB110-RNAi recombinant bacteria are positive recombinant agrobacterium and are respectively named as EHA105/pWMB110-TaAN and EHA105/TaANK-pWMB 110-RNAi.

4. Taank transgenic wheat acquisition and detection

(1) Respectively inoculating recombinant agrobacterium tumefaciens EHA105/pWMB110-TaANK and EHA105/TaANK-pWMB110-RNAi into YEP liquid culture medium, and culturing at 28 ℃ and 200rpm for about 30 hours;

(2) transferring the bacterial liquid obtained in the step (1) into YEP liquid culture medium (containing 50 mu g/ml rifampicin and cana), and culturing at 28 ℃ and 200rpm for about 14 hours (bacterial liquid OD600 reaches 1.5-3.0);

(3) collecting the thallus obtained in step (2), centrifuging at 4 deg.C for 10min at 4000g, and diluting with 10% sucrose (containing 0.02% silwet) to OD600 of about 1.0;

(4) in a clean bench, picking agrobacterium tumefaciens bacterial colony with a 1ml syringe needle, injecting wheat variety Fielder embryo, culturing the injected embryo under aseptic and high humidity condition until the tissue culture wheat seedling grows out, and continuously culturing the wheat seedling until T is harvested₀Over-expression and RNAi of the surrogate TaANK interfered with wheat seeds.

Respectively combine T with₀Overexpression of TaANK, RNAi (ribonucleic acid interference) for interfering sowing and selfing of wheat seeds until T is obtained₃Wheat is used as substitute.

(5) And (3) utilizing qRT-PCR, PCR and detecting TaANK overexpression and TaANKRNAi interference wheat positive plants.

The specific operation of qRT-PCR detection of TaANK overexpression and TaANKRNAi interference wheat expression quantity is as follows, TaANK overexpression is respectively extracted, TaANKRNAi interferes with different wheat strains and receptor wheat leaf RNA, reverse transcription is carried out to obtain cDNA, and qRT-PCR detection is carried out by taking the following sequences as primers:

qRT-TaANK-F2：5'-CTGATGATAAATGGAAGC-3'；

qRT-TaANK-R2：5'-CGCCTGGAAGGTAATAGAA-3'。

the detection results of the over-expression strains are shown in FIG. 3, and the results show that the expression quantity of the over-expression strains is obviously improved compared with that of Fielder, and the expression quantities of No.3 and No. 4 are relatively consistent, and are named as OE-3 and OE-4 for subsequent experimental analysis. The results of the detection of the RNAi interference lines are shown in FIG. 5, and show that the expression levels of RNAi-4 and RNAi-6 of the interference lines are obviously reduced relative to Fielder, and are similar, and are named as RNAi-4 and RNAi-6 for subsequent experimental analysis.

For PCR detection, DNA of different wheat strains and receptor wheat leaves interfered by TaANK overexpression and TaANKRNAi are respectively extracted.

For the PCR detection of TaANK overexpression plants, DNA is used as a template, the following sequence is used as a primer for detection, and pWMB110-TaANK plasmid is used as a positive control.

110-JC-F：5'-CCCTgTTgTTTggTgTTACTTCTg-3'；

qRT-TaANK-R2：5'-CGCCTGGAAGGTAATAGAA-3'。

For the PCR detection of TaANK interfering plants, DNA is used as a template, the following sequence is used as a primer for detection, and a TaANK-pWMB110-RNAi interfering vector is used as a positive control.

110-JC-F：5'-CCCTgTTgTTTggTgTTACTTCTg-3'；

RNAi-JianR:5'-CCAAGGTATCTAATCAGCCATC-3'。

As shown in FIGS. 2 and 4, specific bands were detected in both positive lines, and no specific band was detected in the recipient Fidler, indicating that both lines were positive.

Second, obtaining empty carrier wheat

Transforming wheat variety Fielder by adopting pWMB110 vector to replace pWMB110-TaANK and TaANK-pWMB110-RNAi interference vector to obtain T₃Empty vector wheat (pWMB 110).

Stress tolerance analysis of TaANK-transgenic wheat

1.T numbered OE-3 and OE-4, respectively₃TaANKRNAi interfering seeds with RNAi-4 and RNAi-6 numbers, and T₃The empty-transferred carrier wheat (pWMB110) and Fielder seeds grow normally for 5 days in a water culture tray, and each line is taken24 seeds are subjected to heat stress treatment in a constant temperature incubator at 42 ℃, the growth state of the seedlings is observed at different time including 6 hours, 8 hours, 10 hours and 12 hours respectively, photographing is carried out, sampling is carried out when the seeds are subjected to heat treatment for 6 hours, and related physiological indexes (proline content and MDA content) are determined.

The method for measuring the proline content comprises the following steps: proline content was determined using the Proline (PRO) content test kit (Comin, Code No.: PRO-2-Y) from Suzhou Keming Biotechnology, Inc. Weighing about 0.1g of tissue, adding 1mL of extracting solution, and performing ice bath homogenization; then oscillating and extracting for 10min at 90 ℃; 10000g, centrifuging for 10min at 25 ℃, taking supernatant, cooling and then testing. Preheating the spectrophotometer for more than 30min, adjusting the wavelength to 520nm, and adjusting the distilled water to zero. Placing 0.5ml sample, 0.5ml reagent I and 0.5ml reagent II in a test tube with a cover, placing in a boiling water bath, and keeping the temperature for 30min, and shaking once every 10 min. After cooling, adding 1ml of reagent III into the test tube, oscillating for 30s, and standing for a moment to transfer the pigment into the reagent III; sucking 0.8ml-1ml of the upper layer solution into a 1ml glass cuvette, carrying out color comparison at the wavelength of 520nm, recording the absorbance A, and calculating the proline content.

And (3) MDA content determination: MDA content determination was performed using a Malondialdehyde (MDA) test kit (Comin, Code No.: MDA-2-Y) from Suzhou Keming Biotechnology, Inc. About 0.1g of the tissue was weighed, and 1ml of the extract was added to conduct ice bath homogenization. Centrifuging at 8000g and 4 deg.C for 10min, collecting supernatant, and testing on ice. 0.6ml of the first reagent is sucked into a 1.5ml centrifuge tube, then 0.2ml of the sample is added, and the mixture is mixed evenly. Preserving heat in water bath at 95 ℃ for 30min, placing in an ice bath for cooling, and centrifuging for 10min at 10000g and 25 ℃. The supernatant was pipetted into a 200. mu.l glass cuvette and the absorbances at 532nm and 600nm were determined and recorded as A532 and A600 and the MDA content was calculated.

The phenotypic observations are shown in figure 6. The results show that T is numbered OE-3 and OE-4₃After the wheat with over-expressed TaANK is treated at 42 ℃, the growth vigor of the wheat is better than that of Fielder, and TaANKRNAi of RNAi-4 and RNAi-6 interferes with wheat seedlings to show leaf wilting and partially die under the heat treatment condition.

The stress-related physiological indices are shown in fig. 7. The results show that the wheat over-expressing TaANKThe survival rate and proline content after heat treatment were significantly higher than Fielder, while the MDA content was significantly lower than Fielder. TaANKRNAi interferes with the survival rate and proline content of wheat to be significantly lower than Fielder, while the MDA content is significantly higher than Fielder. Under normal conditions, TaANK is over-expressed, TaANKRNAi interference and Fielder wheat seedlings have no obvious difference in phenotype and physiological indexes. Receptors Fielder and T₃The wheat (pWMB110) with the empty vector has no significant difference in each item of data.

The results show that the heat resistance of the transgenic wheat can be obviously improved by over-expressing TaANK in the wheat.

Example 3 analysis of TaANK subcellular localization

First, TaANK-GFP vector construction

Designing a primer with a BamHI enzyme cutting site, amplifying a linear fragment with a joint by using a TaANK-pWMB110 vector as a template, simultaneously cutting the GFP vector by using BamHI enzyme, and recovering a framework. And cloning the TaANK to a GFP vector by using Infusion enzyme to obtain the TaANK-GFP recombinant vector. The specific primers are as follows:

TaANK-GFP-F：5’-TATCTCTAGAGGATCCATGTATGCGGACCAGATC-3’；

TaANK-GFP-R：5’-TGCTCACCATGGATCCCGCTCTGGATTCCAAGGC-3’。

preparation of wheat protoplast

1. 15mL of the enzymatic hydrolysate was prepared and placed in a small beaker.

2. 10 wheat Fielder seedlings (growing for about 1 week) with good growth are taken, the part above the stem is cut into thin strips, and the thin strips are placed in the enzymolysis liquid.

3. The small beaker is wrapped with tinfoil paper, vacuumized for 15min, and then placed on a shaker at 25 ℃ and 50rpm in the dark for about 5h until the leaf cells are completely lysed.

4. Filtering the lysate with a sieve (100 meshes), removing impurities, subpackaging the filtered filtrate (the gun tip is cut off by about 3 cm) in a 2.0ml centrifuge tube, centrifuging at 4 ℃, setting the acceleration to be 2 at 100g for 2 min.

5. The supernatant was discarded, the protoplasts were gently mixed with a pre-cooled W5 solution, centrifuged at 4 ℃ for 100g for 1min, and the acceleration was set to 2.

6. The supernatant was discarded, and the protoplasts were gently mixed with a pre-cooled W5 solution and allowed to stand on ice for 30 min.

7. Centrifuge at room temperature, centrifuge at 100g for 1min, set acceleration to 2, discard supernatant. Each tube of pellet was gently suspended with 0.5ml of MMg solution (this step and the following procedure was carried out at 23 ℃).

8. Mu.g of plasmid was taken and 100. mu.l of protoplast was added. After gentle mixing, 110. mu.l of PEG/Ca solution was added and gently mixed. The mixture was placed in a 25 ℃ heating block and transformed for 25min in the dark. The plasmid used is a recombinant plasmid TaANK-GFP, which expresses a fusion protein of TaANK and GFP, and is started by a 35S strong promoter and terminated by an NOS strong terminator.

9. A440. mu. L W5 solution was added to stop the reaction. Centrifugation was carried out at 23 ℃ and 100g for 1min, with the acceleration being 2.

10. The supernatant was discarded, 1mL of W5 solution was added, mixed gently and mixed, and cultured in a 23 ℃ incubator for 10-16h in the dark.

11. The green fluorescence was observed under a confocal microscope.

TABLE 1 enzymatic hydrolysate

Note: mixing the above materials in water, cooling to room temperature, adding CaCl₂Is prepared by reacting CaCl₂The concentration in the enzymatic hydrolysate was 1M.

TABLE 2W 5 solution

Note: the pH was adjusted to 5.8 with KOH.

TABLE 3 PEG4000 solution

TABLE 4 MMg solution

Note: the pH was adjusted to 5.6 with KOH.

The positioning results are shown in fig. 8. The results showed that TaANK-GFP is located in the nucleus.

Sequence listing

<110> institute of crop science of Chinese academy of agricultural sciences

<120> wheat heat stress related protein TaANK, and coding gene and application thereof

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gacctcgccc ccgaccgcgg cggcgcggac gccgccggcc gggctcgtaa cgcgctgtcc 120

aagaggcaac gacaaactga tgataaatgg aagcatgatc tttaccggga tgatgaatca 180

gcttcaagat cagttgatcc acgggatttg cgattcaagc ttcagaggaa aagctctcag 240

caagggtttg ctggccaaaa gggttccgag gtacgtgatc tacgtgagat gttatctgga 300

acaatgcacg cacaaccatc aaatgttgat cctcaaaaga ggaaaccggt gtcagaggtt 360

gtgaaagtca caaggcgtga aaatgttgat ggaaggcctg tacgcctaag taagaaagta 420

tcaaaaccat caacatccaa gaagacttct cagccaaagg ctgaaagtcc actcgacagt 480

tttctgaaat cactaggact tgagaagtat tctattacct tccaggcgga ggaggttgac 540

atggcggccc tacgtcacat gactgaaagc gatctcaagg cgttgggcat cccgatgggt 600

cctaggaaga agattaccct tgccttggaa tccagagcgt ag 642

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35 40 45

Lys Trp Lys His Asp Leu Tyr Arg Asp Asp Glu Ser Ala Ser Arg Ser

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Val Asp Pro Arg Asp Leu Arg Phe Lys Leu Gln Arg Lys Ser Ser Gln

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Val Asp Gly Arg Pro Val Arg Leu Ser Lys Lys Val Ser Lys Pro Ser

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Thr Ser Lys Lys Thr Ser Gln Pro Lys Ala Glu Ser Pro Leu Asp Ser

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Phe Leu Lys Ser Leu Gly Leu Glu Lys Tyr Ser Ile Thr Phe Gln Ala

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Glu Glu Val Asp Met Ala Ala Leu Arg His Met Thr Glu Ser Asp Leu

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Lys Ala Leu Gly Ile Pro Met Gly Pro Arg Lys Lys Ile Thr Leu Ala

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Leu Glu Ser Arg Ala

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cacatgactg aaagcgatct caaggcgttg ggcatcccga tgggtcctag gaagaagatt 180

acccttgcct tggaatccag agcggatccg atcgaaaaac gggagtctgc ccctaagaca 240

gataagccgc caagaaggcg caagtcaacc gcgagttgtt gtatcatatc tactgacaaa 300

gatcacaaat gggatggctg attagatacc ttggcctccc agatcgattc cgctctggat 360

tccaaggcaa gggtaatctt cttcctagga cccatcggga tgcccaacgc cttgagatcg 420

ctttcagtca tgtgacgtag ggccgccatg tcaacctcct ccgcctggaa ggtaatagaa 480

tacttctcaa gtcctagtga tttcagaaaa ctgtcgagtg gactttcagc ctttggctga 540

gaagtgagct cg 552

Claims (10)

1. The application of TaANK protein or related biological materials thereof in regulating and controlling plant heat stress resistance;

   the related biological material is a nucleic acid molecule capable of expressing the TaANK protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule or a substance inhibiting the expression of the nucleic acid molecule;

   the TaANK protein is any one of the following proteins:

   (A1) a protein having an amino acid sequence of SEQ ID No. 2;

   (A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.2 and has the same function;

   (A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

   (A4) a fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
2. 2. The use of claim 1, wherein:

   the activity and/or expression level of the TaANK protein or the nucleic acid molecule capable of expressing the TaANK protein in the plant is increased, and the heat stress resistance of the plant is increased;

   the activity and/or expression level of the TaANK protein or the nucleic acid molecule capable of expressing the TaANK protein in the plant is reduced, and the heat stress resistance of the plant is reduced.
3. 3. A method for increasing heat stress resistance in a plant, comprising the step of increasing the expression level and/or activity of a TaANK protein in a recipient plant.
4. 4. A method of breeding a transgenic plant comprising the steps of: introducing a nucleic acid molecule capable of expressing a TaANK protein into a recipient plant to obtain a transgenic plant with increased expression level of the TaANK protein; the transgenic plant has increased heat stress resistance as compared to the recipient plant.
5. 5. The method of claim 4, wherein: the "introduction into a recipient plant of a nucleic acid molecule capable of expressing a TaANK protein" is effected by introducing into said recipient plant an expression vector comprising a gene encoding said TaANK protein.
6. 6. The method of claim 5, wherein:

   the coding gene of the TaANK protein is any one of the following DNA molecules:

   (B1) DNA molecule shown in SEQ ID No. 1;

   (B2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes said TaANK protein;

   (B3) a DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the DNA sequence defined in (B1) or (B2) and encoding the TaANK protein.
7. 7. A TaANK protein or a biological material related thereto as claimed in claim 1, or a method as claimed in any one of claims 3 to 6 for use in plant breeding.
8. 8. The use of claim 7, wherein: the breeding is aimed at breeding plants with high heat stress resistance.
9. 9. Method A or method B;

   method A is a method for reducing heat stress resistance in a plant, comprising the step of increasing the expression level and/or reducing the expression level of a TaANK protein in a recipient plant;

   the method B is a method for cultivating transgenic plants, and comprises the following steps: inhibiting or silencing expression of a nucleic acid molecule encoding a TaANK protein in a receptor plant to obtain a transgenic plant with reduced expression level of the TaANK protein; the transgenic plant has reduced heat stress resistance as compared to the recipient plant;

   the TaANK protein is any one of the following proteins:

   (A1) a protein having an amino acid sequence of SEQ ID No. 2;

   (A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.2 and has the same function;

   (A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

   (A4) a fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
10. 10. Use or method according to any of claims 1-9, wherein: the plant is a dicotyledonous plant or a monocotyledonous plant;

    further, the monocotyledon is a gramineous plant;

    further, the gramineous plant is wheat.

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