CN113527451B - 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 to heat stress of the over-expressed transgenic wheat obtained by introducing the TaANK gene into wheat is higher than that of the receptor Fielder, and the tolerance to heat stress of the RNAi interference TaANK transgenic wheat is lower than that of the receptor Fielder. The protein and the gene of the invention 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, in particular to a wheat heat stress related protein TaANK, and a coding gene and application thereof.

Background

Abiotic stresses, such as drought, high temperature and salt-water environmental factors, seriously affect wheat growth and yield. Analyzing response and signal transmission mechanism of wheat under adversity stress, improving stress tolerance of wheat, becoming one of important tasks of wheat genetic research and wheat variety improvement.

Plants produce a series of responses under stress, accompanied by a number of physiological, biochemical and developmental changes. The reaction mechanism of plants to stress is clarified, and scientific arguments are provided for stress resistance genetic engineering research and application. At present, plant stress resistance research is gradually advanced to cellular and molecular levels, and is combined with genetic and genetic engineering research to explore biotechnology for improving plant growth characteristics, so as to improve the adaptation capability of plants to stress.

Under adverse conditions of drought, high salt, low temperature and other environmental stresses, the plants can make corresponding adjustments on molecular, cellular and overall levels so as to reduce the damage caused by the environment to the greatest extent and survive. Many genes are expressed by stress induction, and the products of the genes can directly participate in the stress response of plants, and can regulate the expression of other related genes or participate in signal transduction pathways, so that the plants are prevented or reduced from being damaged, and the resistance to the stress environment is enhanced. It has now been found that plant response stress signaling networks are primarily plant hormone signaling pathways, liposome signaling pathways, MAPK signaling pathways, ROS signaling pathways, and stomatal signaling pathways. These signal network systems closely link the hormonal regulation, metabolism, energy supply, and growth and development of plants. This demonstrates that adaptation of plants to stress depends not only on the expression of stress-tolerance related genes, but also on the integrated regulation of various signaling pathways triggered by stress induction. Gene products related to stress can be divided into two main classes: the products coded by the first type of genes comprise gene products directly involved in plant stress response, such as ion channel proteins, aquaporins, osmotic adjusting factors (sucrose, proline, betaine and the like) synthetases and the like; the products encoded by the second class of genes include 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 provides the use of a tank protein or related biological material thereof for modulating 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 with an amino acid sequence of SEQ ID No. 2;

(A2) A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for 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 with the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

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

The tank protein is derived from wheat genus wheat (Triticum aestivum l.).

The protein can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing.

Among the above proteins, the tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, a SUMO tag, or the like.

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

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

(B2) A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (B1) and which encodes said tank 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 tank protein.

The stringent conditions may be as follows: 50℃in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA in a mixed solution at 65deg.CRinsing in 0.1 XSSC, 0.1% SDS; the method can also be as follows: hybridization was performed in a solution of 6 XSSC, 0.5% SDS at 65℃and then washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

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

In the above biological material, the recombinant bacterium may be obtained by introducing the above recombinant vector into an agrobacterium strain. The agrobacterium strain may specifically be agrobacterium strain EHA105.

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

In the above biological material, the substance that inhibits 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 which is digested by SmaI and SacI.

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

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

The tank protein is as previously described. The increased heat stress resistance may be embodied as an increased survival rate and/or an increased proline content and/or a decreased MDA content under heat stress conditions.

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 tank protein into a recipient plant to obtain a transgenic plant with increased expression of the tank protein; the transgenic plant has increased heat stress resistance as compared to the recipient plant.

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

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

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

(B2) A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (B1) and which encodes said tank 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 tank protein.

The expression vector can 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 a fourth aspect, the invention provides the use of a tank protein as defined in any preceding claim, or a related biological material thereof, or a method as defined in any preceding claim, in plant breeding. The purpose of the breeding is to breed plants with high heat stress resistance.

In a fifth aspect, the present invention protects either method a or method B.

The method A is a method for reducing the heat stress resistance of a plant, and comprises the step of reducing the expression amount 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 tank protein in a recipient plant, resulting in a transgenic plant having reduced expression of the tank protein; the transgenic plant has reduced resistance to thermal stress as compared to the recipient plant.

The tank protein is as described hereinbefore.

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

Any of the plants described above is a dicotyledonous plant or a monocotyledonous plant;

further, the monocotyledonous plant is a plant of the Gramineae family;

still further, the gramineous plant is wheat.

The wheat may specifically be a wheat variety Fielder.

Experiments of the invention prove that the transgenic wheat obtained by introducing the TaANK gene into wheat has higher resistance to the heat stress than a receptor wheat field, and the tolerance of RNAi interference wheat to the heat stress is lower than the receptor wheat field. The protein and the gene provided by the invention provide a foundation for controlling the 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 overexpression and RNAi interference TaANK vector construction.

FIG. 2 shows PCR positive detection of overexpressed TaANK wheat.

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

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

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

FIG. 6 is a representation of the heat stress phenotype identification of transgenic TaANK wheat.

FIG. 7 is a thermal stress physiological index assay of transgenic TaANK wheat.

FIG. 8 shows TaANK subcellular localization.

Detailed Description

The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged. In the following examples, the percentages are by mass unless otherwise indicated.

Common wheat (Triticum aestivum l.) variety small Bai Mai in the following examples is described in literature "Sun Haitao et al, screening of wheat tadeb 6 transcription factor interacting protein, chinese agricultural science, 2011,44 (22): 4740-4747, "the public is available from the national academy of agricultural sciences for crop science, and the biomaterial is used only for repeated experiments related to the invention, and is not used as other uses; the public is also available from the national germplasm resources (number ZM 242).

The pWMB110 vector, GFP vector pJIT16318 and wheat variety Fielder described in the examples below are described in the literature "Xiao-Yu Cui, yuan Gao, jun Guo, tai-Fei Yu, wei-Jun Zheng, yong-Wei Liu, jun Chen, zhao-Shi Xu and Young Ma. BES/BZR Transcription Factor TaBZR2 Positively Regulates Drought Responses by Activation of TaGST11.Plant Physics, 180:605-620", which is available from national academy of agricultural sciences for use only in experiments related to duplicate inventions, and not as other uses.

Example 1 cloning of TaANK

1. Treatment of plant material

The whole seedling in three leaf stage of small Bai Mai (Triticum aestivum cv. Xiaobaimai) grown in water for about 10 days is quick frozen with liquid nitrogen and stored at-80 ℃ for later use.

2. Extraction of Total RNA

Total RNA from leaves of the treated small Bai Mai seedlings obtained in step one was extracted by Trizol method (TianGen).

3. cDNA acquisition

Reverse transcriptase XL (AMV) is used for first strand cDNA synthesis. The ds cDNA was synthesized by SMART method and the PCR products were detected by 1.0% agarose gel electrophoresis. The SEQ ID No.1 was obtained by designing a suitable primer sequence for amplification in a wheat cDNA library. SEQ ID No.1 may also be obtained synthetically.

The specific amplified primers 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 is 1 st-642 th nucleotide from the 5' end of SEQ ID No.1, the protein encoded 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 obtaining of transgenic tank wheat and stress tolerance analysis

1. Obtaining of TaANK-transformed wheat

1. Construction of an overexpressed tank recombinant vector

(1) Amplification of the tank Gene

And (3) taking the fragment containing the TaANK amplified and recovered from the wheat as a template, and amplifying by adopting primers TaANK-110F and TaANK-110R to obtain a PCR product. The primer sequences were as follows, the ends of the primers were respectively introduced with BamHI cleavage recognition sites and underlined:

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

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

the PCR product was subjected to 1.2% agarose gel electrophoresis and sequenced, and the result showed that the PCR product had SEQ ID No.1.

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

(2) Cutting pWMB110 carrier by restriction enzyme BamHI to recover carrier skeleton; connecting the enzyme digestion product with a carrier framework to obtain a connection product;

(3) The ligation product obtained in the step (2) was heat-shock transformed into TOP10 strain (Tiangen, CB 104-03), cultured overnight at 37℃and positive clone extraction plasmids were picked up for sequencing.

Sequencing results show that the plasmid is a vector obtained by inserting a DNA fragment shown in SEQ ID No.1 between BamHI cleavage sites of a pWMB110 vector, and the plasmid is named pWMB110-TaANK, and important element information of the pWMB110-TaANK is shown in FIG. 1A.

2. Construction of RNAi interference TaANK recombinant vector

(1) The specific operation of the construction of the TaANK RNAi interference vector is as follows: the 445 th to 639 th nucleotide sequence and the reverse complementary sequence in SEQ ID No.1 are synthesized artificially, a 146bp corn alcohol dehydrogenase (Adh) gene sequence in the middle is taken as an intron, a SmaI enzyme cutting site is added at the 5 'end, a SacI enzyme cutting site is added at the 3' end, wherein the fragment synthesis is completed by Beijing Oriental biosciences, and a 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 by using Agarose Gel DNAPurification Kit Ver.2.0 (TaKaRa Co., code No.: DV 807A). And the artificially synthesized fragment SEQ ID No.3 in the step (1) is connected with a pWMB110 vector which is digested by SmaI and SacI to construct a TaANK-pWMB110-RNAi interference vector, and the important element information of the TaANK-pWMB110-RNAi interference vector is shown in the figure 1B.

3. Construction of recombinant bacteria

(1) And (3) respectively transforming the recombinant plasmids pWMB110-TaANK and TaANK-pWMB110-RNAi obtained in the step (1) and (2) into agrobacterium EHA105 (purchased from Beijing Bayer Di Biotechnology Co.) to obtain recombinant agrobacterium.

(2) The plasmids of the extracted recombinant agrobacterium were sent to sequencing, and the results show that 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-pWMB110-RNAi.

4. Acquisition and detection of tank-transformed wheat

(1) Recombinant agrobacterium EHA105/pWMB110-TaANK and EHA105/TaANK-pWMB110-RNAi are respectively inoculated into YEP liquid culture medium and cultured at 28 ℃ and 200rpm for about 30 hours;

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

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

(4) Picking agrobacterium colony in a super clean workbench by using a 1ml syringe needle, injecting wheat variety field embryo, culturing the injected embryo under aseptic and high humidity condition until the tissue cultured wheat seedling grows out, and continuously culturing the wheat seedling until T is harvested ₀ Generation tank overexpression and RNAi interfere with wheat seeds.

Respectively T ₀ Over-expression of the generation TAANK, RNAi interferes with wheat seed sowing and selfing until T is obtained ₃ Replacing wheat.

(5) Using qRT-PCR, both TaANK overexpression and TaANKRNAI interference with wheat positive plants were PCR and detected.

The specific operation of detecting the TaANK overexpression and the TaANKRNAI interference wheat expression quantity by qRT-PCR comprises the following steps of respectively extracting the TaANK overexpression, the TaANKRNAI interference different wheat strains and acceptor wheat leaf RNA, carrying out reverse transcription to cDNA, and carrying out qRT-PCR detection by taking the following sequences as primers:

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

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

the results of the detection of the over-expressed strain are shown in FIG. 3, and the results show that the expression level of the over-expressed strain is obviously improved compared with that of the Fielder, and the expression levels of #3 and #4 are relatively consistent, and are named OE-3 and OE-4 for subsequent experimental analysis. The detection of RNAi interfering strains is shown in FIG. 5, and the results show that the expression levels of RNAi-4 and RNAi-6 of the interfering strains are significantly reduced relative to those of Fielder, and the expression levels are similar, and are designated RNAi-4 and RNAi-6 hereinafter for subsequent experimental analysis.

For PCR assays, taANK overexpression and TaANKRNAI interference were extracted separately from different wheat lines and acceptor wheat leaf DNA.

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

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

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

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

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

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

As a result, as shown in FIGS. 2 and 4, a specific band was detected in each of the positive lines, and no specific band was detected in the receptor Fidler, indicating that the positive lines were all obtained.

2. Obtaining of wheat with empty vector

The pWMB110 carrier is adopted to replace pWMB110-TaANK and TaANK-pWMB110-RNAi interference carrier, and the wheat variety Fielder is transformed to obtain T ₃ Empty vector wheat (pWMB 110) was transformed.

3. Stress tolerance analysis of tank-transformed wheat

1.T numbered OE-3 and OE-4, respectively ₃ Taankrenai-interfering seeds, T, from generation over-expressed Taank wheat seeds, numbered RNAi-4 and RNAi-6 ₃ The empty vector wheat (pWMB 110) and Fielder seeds were grown normally in a hydroponic dish for 5 days, 24 seeds were taken per strain, and then heat stress treatment was performed in a 42℃incubator, the seedling growth state was observed at different times including 6 hours, 8 hours, 10 hours and 12 hours, photographing was performed, sampling was performed at 6 hours of heat treatment, and the relevant physiological indexes (proline content, MDA content) were measured.

The method for measuring the content of the proline comprises the following steps: proline content was determined using a Suzhou Ming Biotechnology Co., ltd. Proline (PRO) content test box (Comin Co., code No.: PRO-2-Y). Weighing about 0.1g of tissue, adding 1mL of the extracting solution, and carrying out ice bath homogenization; then shaking and extracting for 10min at 90 ℃;10000g, centrifuging at 25deg.C for 10min, collecting supernatant, cooling, and testing. Preheating the spectrophotometer for more than 30min, adjusting the wavelength to 520nm, and zeroing the distilled water. Taking 0.5ml sample and 0.5ml reagent one and 0.5ml reagent two in a capped test tube, placing in a boiling water bath, preserving the temperature for 30min, and oscillating every 10min. After cooling, adding 1ml of a reagent III into the test tube, oscillating for 30s, standing for a moment, and transferring the pigment into the reagent III; sucking 0.8ml-1ml of the upper layer solution into a 1ml glass cuvette, comparing colors at 520nm wavelength, recording absorbance A, and calculating proline content.

MDA content measurement: MDA content was determined using a Suzhou Ming Biotechnology Co., ltd. Malondialdehyde (MDA) test box (Comin Co., code No.: MDA-2-Y). About 0.1g of tissue was weighed, and 1ml of the extract was added thereto to perform ice bath homogenization. And (5) centrifuging at the temperature of 8000g for 10min at 4 ℃, taking supernatant, and placing on ice for testing. Draw 0.6ml of reagent one into a 1.5ml centrifuge tube, add 0.2ml of sample, mix well. Preserving heat in 95 ℃ water bath for 30min, cooling in ice bath, 10000g, centrifuging at 25 ℃ for 10min. The supernatant was aspirated into 200. Mu.l glass cuvettes, absorbance at 532nm and 600nm was measured, recorded as A532 and A600 and MDA content was calculated.

Phenotypic observations are shown in figure 6. The results show T numbered OE-3 and OE-4 ₃ After the wheat with the over-expression of the tankr is treated under the condition of 42 ℃, the growth vigor is superior to that of a Fielder, and the tankrnai of RNAi-4 and RNAi-6 interferes with wheat seedlings to show leaf wilting and partial death under the condition of heat treatment.

The stress-related physiological index is shown in fig. 7. The results show that both the survival rate and the proline content of the overexpressed tank wheat after heat treatment were significantly higher than Fielder, while the MDA content was significantly lower than Fielder. Tankrnai interferes with the survival rate and proline content of wheat significantly below Fielder, while MDA content is significantly above Fielder. Under normal conditions, the TaANKRNAI interference and Fielder wheat seedlings have no obvious difference in phenotype and physiological indexes. Receptor Fielder and T ₃ The empty vector wheat (pWMB 110) was transformed without significant differences in the various data.

The above results indicate that overexpression of tank in wheat can significantly improve the heat resistance of transgenic wheat.

Example 3 TaANK subcellular localization analysis

1. TaANK-GFP vector construction

The primers with BamHI cleavage sites were designed, and the adapter-containing linear fragment was amplified using the TaANK-pWMB110 vector as a template, while the GFP vector was cleaved with BamHI enzyme, and the backbone was recovered. And cloning the TaANK onto the GFP vector by using an infion 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’。

2. preparation of wheat protoplast

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

2. 10 wheat field seedlings (growing for about 1 week) with good growth vigor are taken, the parts above the stems are cut into thin strips, and the thin strips are placed in enzymolysis liquid.

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

4. The lysate was filtered through a sieve (100 mesh) to remove impurities, and the filtered filtrate (gun tip cut off about 3 cm) was dispensed into 2.0ml centrifuge tubes, centrifuged at 4 ℃,100g,2min and acceleration set at 2.

5. The supernatant was discarded, protoplasts were gently mixed with a pre-chilled W5 solution, centrifuged at 4℃and 100g,1min at an acceleration of 2.

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

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

8. Mu.g of plasmid was added to 100. Mu.l of protoplasts. After gentle mixing, 110. Mu.l of PEG/Ca solution was added and gently mixed. Placing in a heating block at 25 ℃ and converting for 25min in a dark place. Wherein the plasmid is recombinant plasmid TaANK-GFP, which expresses fusion protein of TaANK and GFP, and is started by 35S strong promoter and terminated by NOS strong terminator.

9. A440. Mu. L W5 solution was added to terminate the reaction. Centrifuge at 23℃and 100g for 1min, acceleration was set at 2.

10. The supernatant was discarded, 1mL of the W5 solution was added and gently mixed, and the mixture was placed in an incubator at 23℃for 10-16 hours in the absence of light.

11. Green fluorescence was observed under a confocal microscope.

TABLE 1 enzymatic hydrolysate

Note that: mixing above materials in water, cooling to room temperature, adding CaCl ₂ CaCl is caused to be ₂ The concentration in the enzymatic hydrolysate was 1M.

TABLE 2 W5 solution

Note that: the pH was adjusted to 5.8 with KOH.

TABLE 3 PEG4000 solutions

TABLE 4 MMg solution

Note that: the pH was adjusted to 5.6 with KOH.

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

Sequence listing

<110> institute of crop science at national academy of agricultural sciences

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

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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 (7)

1. Application of TaANK protein or related biological materials 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 with an amino acid sequence of SEQ ID No. 2;

   (A2) A fusion protein obtained by ligating protein tags to the N-terminal and/or C-terminal of the protein defined in (A1);

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

   the expression level of the tank protein or a nucleic acid molecule capable of expressing the tank protein in the plant is reduced, and the plant heat stress resistance is reduced;

   the plant is wheat.
2. 2. A method for increasing heat stress resistance in a plant, comprising the step of increasing the expression level of a tank protein in a recipient plant;

   the TaANK protein is any one of the following proteins:

   (A1) A protein with an amino acid sequence of SEQ ID No. 2;

   (A2) A fusion protein obtained by ligating protein tags to the N-terminal and/or C-terminal of the protein defined in (A1);

   the plant is wheat.
3. 3. A method of growing a transgenic plant comprising the steps of: introducing a nucleic acid molecule capable of expressing a tank protein into a recipient plant to obtain a transgenic plant with increased expression of the tank protein; the transgenic plant has increased heat stress resistance as compared to the recipient plant;

   the TaANK protein is any one of the following proteins:

   (A1) A protein with an amino acid sequence of SEQ ID No. 2;

   (A2) A fusion protein obtained by ligating protein tags to the N-terminal and/or C-terminal of the protein defined in (A1);

   the plant is wheat.
4. 4. A method as claimed in claim 3, wherein: the "introduction of a nucleic acid molecule capable of expressing a tank protein into a recipient plant" is achieved by introducing into the recipient plant an expression vector comprising a gene encoding the tank protein.
5. 5. The method of claim 4, wherein: the coding gene of the TaANK protein is a DNA molecule shown as SEQ ID No.1.
6. 6. Use of the tank protein of claim 1 or its related biological material or the method of any of claims 2 to 5 in plant breeding; the purpose of the breeding is to breed plants with high heat stress resistance; the plant is wheat.
7. 7. Method a or method B;

   the method A is a method for reducing the heat stress resistance of a plant, and comprises the step of reducing the expression level of a TaANK protein in a receptor 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 tank protein in a recipient plant, resulting in a transgenic plant having reduced expression of the tank 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 with an amino acid sequence of SEQ ID No. 2;

   (A2) A fusion protein obtained by ligating protein tags to the N-terminal and/or C-terminal of the protein defined in (A1);

   the plant is wheat.

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