Plant drought resistance related protein TaDTGIP1, and coding gene and application thereof

Abstract

The present application belongs to the technical field of plant genetic engineering and genetic improvement, and particularly relates to a plant drought resistance related protein TaDTGIP1, a coding gene thereof and application. The present application clones a plant drought resistance related protein TaDTGIP1 and a coding gene thereof, and finds that the plant drought resistance related protein TaDTGIP1 has a good regulation effect on plant drought resistance. Increasing the use of the plant drought resistance related protein TaDTGIP1 and the coding gene thereof can be used for cultivating drought-resistant plant varieties, and has important significance in breeding and research for improving plant drought resistance.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and genetic improvement technology, specifically relating to a plant drought resistance-related protein TaDTGIP1, its encoding gene, and its applications. Background Technology

[0002] Plants grow and develop in complex and ever-changing environments, often subjected to abiotic stresses. Drought is a major abiotic factor affecting and limiting plant growth and development, and can even lead to plant death, severely impacting agricultural production. Therefore, developing drought-resistant crop varieties has always been one of the main goals of agricultural science and technology research.

[0003] Wheat (Triticum aestivum L.) is the primary source of carbohydrates and protein for humans, and the demand for wheat continues to grow. Therefore, there is an urgent need to find effective breeding methods to increase wheat yield. Although global wheat planting area and yield have increased dramatically with the development and innovation of breeding technologies, abiotic stresses such as drought, high temperatures, and high salinity severely impact wheat production. Among these environmental stressors, drought poses the most serious threat to agricultural production.

[0004] To mitigate or suppress the impact of drought on crop growth, development, and yield, genetic improvement is frequently employed to cultivate drought-resistant varieties. Research indicates that the key to genetic improvement of crop drought resistance lies in the cloning and utilization of superior drought-resistant genes. Therefore, the discovery of drought-resistant genes in wheat is of great significance for breeding drought-resistant wheat varieties and increasing wheat yield. Although some studies have identified genes with drought-resistant functions, such as TaUreG, TaH2A.9, and TaASR1, current technology has not reported any related applications of the TaDTGIP1 gene and its encoded protein in plant drought resistance. Summary of the Invention

[0005] The purpose of this invention is to provide a plant drought resistance-related protein TaDTGIP1, its encoding gene, and its applications. The plant drought resistance-related protein TaDTGIP1 and its encoded nucleic acid molecule can effectively regulate plant drought resistance, providing protein and gene resources for cultivating drought-resistant plants.

[0006] This invention provides a plant drought resistance-related protein, TaDTGIP1, which comprises proteins as shown in 1) or 2):

[0007] 1) Proteins consisting of the amino acid sequence shown in SEQ ID NO:1;

[0008] 2) Proteins derived from 1) whose amino acid sequence in 1) has been substituted, deleted, or added with one or more amino acids and are associated with plant drought resistance.

[0009] The present invention also provides a nucleic acid molecule encoding the plant drought resistance-related protein TaDTGIP1 described in the above technical solution.

[0010] Preferably, the nucleic acid molecule includes the DNA molecule described in any one of the following a) to d);

[0011] a) A DNA molecule as shown in SEQ ID NO:2;

[0012] b) DNA molecules as shown in SEQ ID NO:2, from 80 to 1216 bp;

[0013] c) A DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the DNA sequence defined in a) or b) and encodes the plant drought resistance-related protein TaDTGIP1.

[0014] d) DNA molecules that hybridize under stringent conditions to the DNA sequence defined in 1), 2), or 3) and encode the plant drought-resistant protein TaDTGIP1.

[0015] The present invention also provides an expression cassette, which includes the nucleic acid molecule described in the above technical solution.

[0016] The present invention also provides a recombinant vector, the recombinant vector comprising a base vector and a nucleic acid molecule or expression cassette inserted into the base vector, wherein the nucleic acid molecule is the nucleic acid molecule described in the above technical solution; and the expression cassette is the expression cassette described in the above technical solution.

[0017] The present invention also provides an engineered bacterium, which includes the nucleic acid molecule described in the above technical solution, the expression cassette described in the above technical solution, or the recombinant vector described in the above technical solution.

[0018] This invention also provides the application of the plant drought resistance-related protein TaDTGIP1, the nucleic acid molecule, the expression cassette, the recombinant vector, or the engineered bacteria described in the above-mentioned technical solutions in regulating plant drought resistance and / or cultivating drought-resistant plants.

[0019] Preferably, the regulation of plant drought resistance includes: increasing the content or activity of the plant drought resistance-related protein TaDTGIP1 in the target plant, thereby reducing the plant's drought resistance;

[0020] Alternatively, it can reduce the content or activity of the plant drought-resistant protein TaDTGIP1 in the target plant, thereby improving the plant's drought resistance.

[0021] This invention also provides a method for cultivating drought-resistant transgenic plants, characterized in that the drought-resistant transgenic plants are obtained through steps as described in I) or II):

[0022] I) Reduce the content or activity of TaDTGIP1, a plant drought resistance-related protein, in the target plant;

[0023] II) Inhibit the expression of nucleic acid molecules encoding the plant drought-resistant protein TaDTGIP1 in the target plant;

[0024] The plant drought resistance-related protein TaDTGIP1 is the plant drought resistance-related protein TaDTGIP1 described in the above technical solution or the plant drought resistance-related protein TaDTGIP1 encoded by the nucleic acid molecule described in the above technical solution.

[0025] Preferably, the plant includes wheat.

[0026] Beneficial effects:

[0027] This invention provides a plant drought resistance-related protein, TaDTGIP1, which comprises proteins as shown in 1) or 2): 1) a protein with an amino acid sequence as shown in SEQ ID NO:1; 2) a protein derived from 1) by substitution, deletion, or addition of one or more amino acids in the amino acid sequence of 1) and which is related to plant drought resistance. This invention cloned the plant drought resistance-related protein TaDTGIP1 and its encoding gene, and found that it has a good regulatory role in plant drought resistance, increasing the applicability of TaDTGIP1 and its gene. It can be used to breed drought-resistant plant varieties, which is of great significance in breeding and research to improve plant drought resistance. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0029] Figure 1 This shows the expression pattern of the TaDTGIP1 gene under abiotic stress in Example 1.

[0030] Figure 2 Subcellular localization of the TaDTGIP1-GFP fusion protein in Example 2;

[0031] Figure 3The results of qPT-PCR for the T3 generation transgenic wheat plants in Example 3;

[0032] Figure 4 The phenotype of the T3 generation transgenic wheat plants in Example 3 after drought treatment and rehydration for 3 days;

[0033] Figure 5 The survival rate of T3 generation transgenic wheat plants in Example 3 after drought treatment and rehydration for 3 days is statistically shown.

[0034] Figure 6 The aboveground fresh weight of the T3 generation transgenic wheat plants under normal growth and drought stress in Example 3;

[0035] Figure 7 The aboveground dry weight of the T3 generation transgenic wheat plants under normal growth and drought stress in Example 3;

[0036] Figure 8 The water loss rate of detached leaves of T3 generation transgenic wheat plants in Example 3;

[0037] Figure 9 The stomatal density of leaves in the transgenic wheat under normal growth conditions in Example 3;

[0038] Figure 10 The stomatal aperture of leaves in transgenic wheat under normal growth and drought stress conditions in Example 3;

[0039] Figure 11 The results are infrared thermal imaging of the transgenic wheat plants under normal growth and drought stress conditions in Example 3.

[0040] Figure 12 The leaf temperature of the transgenic wheat plant under normal growth and drought stress conditions in Example 3 is shown. Detailed Implementation

[0041] This invention provides a plant drought resistance-related protein, TaDTGIP1, which comprises proteins as shown in 1) or 2):

[0042] 1) Proteins consisting of the amino acid sequence shown in SEQ ID NO:1;

[0043] 2) Proteins derived from 1) whose amino acid sequence in 1) has been substituted, deleted, or added with one or more amino acids and are associated with plant drought resistance.

[0044] In the present invention, the amino acid sequence shown in SEQ ID NO:1 is: METETERESRKRPRVA AAAAEGPSLVAAAAAGDPSLAAAAEYAAWLEEMAVHEAKAAEFRKNLLANPLTAEQLRERKEAAWEKYNNESWERARDNWIWPFEADTDISCMRFTNENINDPNYPRLVCTMATLQIVSVQVKEITDGLHWPLDVYGFVAVRDVVDRKRIMVFNRERDDYQRITEQDSYLTLTGPTRGVV MTIDPSYLEAKLKVRGATESEDKDLSKFAKTYRLGCYLPIKHTSKLCTLELQHYTVCSSVEATIRVQVIEGQFPRDFRGVLTASTDSESGVMISLLDFSSSDELPVDADGSVKLSRQVVSVKKGGNLKVSIWQHGVGEEEDQEITAASFVAKEAETSTNYMPMKKWKCWMEVSVAWSLFSCW.

[0045] As one embodiment, to facilitate the purification of the protein described in 1), one or more of the following tags can be attached to the amino or carboxyl terminus of the amino acid sequence shown in SEQ ID NO:1: Poly-Arg, Poly-His, FLAG, Strep-tag I, and c-myc. As one embodiment, the Poly-Arg consists of 5-6 amino acid residues, with the amino acid sequence RRRRR (SEQ ID NO:3) or RRRRR R (SEQ ID NO:4). As one embodiment, the Poly-His consists of 2-10 H amino acid residues; as another embodiment, the Poly-His consists of 6 H amino acid residues, with the amino acid sequence HHHHHH (SEQ ID NO:5). As one embodiment, the amino acid sequence of the FLAG is DYKDDDDK (SEQ ID NO:6). As one embodiment, the amino acid sequence of the Strep-tag II is WSHPQFEK (SEQ ID NO:7). As one embodiment, the amino acid sequence of the c-myc is EQKLISEEDL (SEQ ID NO:8).

[0046] As one implementation method, the preparation method of the plant drought resistance-related protein TaDTGIP1 includes artificial synthesis or biological expression of nucleotide molecules encoding the plant drought resistance-related protein TaDTGIP1.

[0047] The present invention also provides a nucleic acid molecule encoding the plant drought resistance-related protein TaDTGIP1 described in the above-described technical solutions. As one embodiment, the nucleic acid molecule includes any one of the following: a) to d); a) a DNA molecule as shown in SEQ ID NO:2; b) a DNA molecule as shown in the 80th to 1216th bp segments of SEQ ID NO:2; c) a DNA molecule encoding the plant drought resistance-related protein TaDTGIP1 that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the DNA sequence defined in a) or b); d) a DNA molecule encoding the plant drought resistance-related protein TaDTGIP1 that hybridizes under stringent conditions to the DNA sequence defined in 1), 2), or 3). In this invention, the DNA molecule shown in SEQ ID NO:2 is the full-length cDNA sequence encoding the plant drought resistance-related protein TaDTGIP1; the DNA molecule shown in SEQ ID NO:2, from 80 to 1216, encodes the coding region sequence of the plant drought resistance-related protein TaDTGIP1.

[0048]

[0049] As one embodiment, the stringent conditions can be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 2×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 1×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.5×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA. Hybridization can be performed in a mixed solution of EDTA, followed by rinsing at 50°C in 0.1×SSC and 0.1% SDS; alternatively, hybridization can be performed at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 65°C in 0.1×SSC and 0.1% SDS; alternatively, hybridization can be performed in a solution of 6×SSC and 0.5% SDS at 65°C, followed by washing once each with 2×SSC and 0.1% SDS and 1×SSC and 0.1% SDS.

[0050] The present invention also provides an expression cassette, which includes the nucleic acid molecule described in the above technical solution.

[0051] This invention also provides a recombinant vector, which includes a base vector and a nucleic acid molecule or expression cassette inserted into the base vector. The nucleic acid molecule is the nucleic acid molecule described in the above-described technical solution; the expression cassette is the expression cassette described in the above-described technical solution. In one embodiment, the base vector includes a plasmid vector; in another embodiment, the plasmid vector can be a binary Agrobacterium vector or a vector suitable for plant microbombardment; in yet another embodiment, the plasmid vector can be, but is not limited to, pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pCAMBIA3301, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb.

[0052] In one embodiment, the recombinant vector may further include the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylated signal and any other DNA fragment involved in mRNA processing or gene expression. In another embodiment, the polyadenylated signal can guide the addition of polyadenylated acid to the 3' end of the mRNA precursor; for example, the untranslated region of the 3' transcription of Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthase) and plant genes (such as the soybean storage protein gene) has similar functions. In another embodiment, when constructing a recombinant vector using the gene, any type of enhanced promoter (such as the cauliflower mosaic virus (CAMV) 35S promoter, the maize ubiquitin promoter), constitutive promoter, or tissue-specific expression promoter (such as a seed-specific expression promoter) can be added before its transcription initiation nucleotide. These can be used alone or in combination with other plant promoters. In one implementation, when constructing a recombinant vector using the gene of the present invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. In another implementation, the translation control signals and start codons are widely available and can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. As one implementation method, to facilitate the identification and screening of transgenic plant cells or plants, the recombinant vector can be processed, such as by adding genes that can be expressed in plants that encode enzymes or luminescent compounds that produce color changes (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinic acid, the hph gene that confers resistance to the antibiotic hygromycin, the dhfr gene that confers resistance to methatrexate, and the EPSPS gene that confers resistance to glyphosate), or chemical reagent resistance marker genes (such as herbicide resistance genes), or mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose.

[0053] In a specific embodiment of the present invention, the base vector of the recombinant vector is pCAMBIA3301, and the recombinant vector is obtained by inserting the DNA molecule shown in SEQ ID NO:2 from 80 to 1216 bp between the Hind III and EcoRI restriction sites.

[0054] This invention also provides an engineered bacterium, which includes the nucleic acid molecule, expression cassette, or recombinant vector described in the above-described technical solutions. In one embodiment, the initial strain of the engineered bacterium is *Agrobacterium*; in another embodiment, the *Agrobacterium* is *Agrobacterium tumefaciens*; in a specific embodiment, the initial strain is *Agrobacterium tumefaciens* EHA105.

[0055] This invention also provides the application of the plant drought resistance-related protein TaDTGIP1, the nucleic acid molecule, the expression cassette, the recombinant vector, or the engineered bacteria described in the above-mentioned technical solutions in regulating plant drought resistance and / or cultivating drought-resistant plants. As one embodiment, regulating plant drought resistance includes: increasing the content or activity of the plant drought resistance-related protein TaDTGIP1 in the target plant to reduce the plant's drought resistance; or, reducing the content or activity of the plant drought resistance-related protein TaDTGIP1 in the target plant to increase the plant's drought resistance.

[0056] In one embodiment, the plant can be a grass (Poaceae); in another embodiment, the grass can be wheat. In a specific embodiment, the present invention introduces a nucleic acid molecule encoding the plant drought-resistance-related protein TaDTGIP1 into wheat, resulting in transgenic wheat with weakened drought resistance. This demonstrates that the plant drought-resistance-related protein TaDTGIP1 is of great significance in breeding and research to provide plant drought resistance. Drought-resistant plants can be bred by knocking out or reducing the nucleic acid molecule encoding TaDTGIP1. In one embodiment, the knockout or reduction method can be gene editing technology.

[0057] This invention also provides a method for cultivating drought-resistant transgenic plants, characterized in that the drought-resistant transgenic plants are obtained through steps as described in I) or II): I) reducing the content or activity of the plant drought-resistant protein TaDTGIP1 in the target plant; II) inhibiting the expression of the nucleic acid molecule encoding the plant drought-resistant protein TaDTGIP1 in the target plant; wherein the plant drought-resistant protein TaDTGIP1 is the plant drought-resistant protein TaDTGIP1 described in the above technical solution or the plant drought-resistant protein TaDTGIP1 encoded by the nucleic acid molecule described in the above technical solution. As one embodiment, the plant can be a grass; as another embodiment, the grass can be wheat.

[0058] As one implementation method, the method for cultivating drought-resistant transgenic plants described in I) or II) is achieved by knocking out or reducing the nucleic acid molecule encoding the plant drought-resistant related protein TaDTGIP1 in the plant; as another implementation method, the knockout or reduction method is not particularly limited, and conventional knockout or reduction techniques in the art, such as gene editing technology, can be used.

[0059] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0061] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0062] The biomaterials used in the following examples are as follows:

[0063] Vector pCAMBIA3301: described in the following literature: Regulatory changes in TaSNAC8-6A area associated with drought tolerance in wheat seedlings. Plant Biotechnol J 2019. Available to the public from Northwest A&F University.

[0064] The vector pTF486 is described in the following literature: ABA-induced sugar transporter TaSTP6 promotes wheat susceptibility to stripe rust. Plant Physiol. 2019, 181(3): 1328-1343, and is available to the public from Northwest A&F University;

[0065] Agrobacterium tumefaciens strain EHA105: described in the following literature: Mei F, Chen B, Du L, Li S, Zhu D, et al. (2022) A gain-of-function allele of a DREB transcription factor gene ameliorates drought tolerance in wheat. Plant Cell 34:4472-4494, available to the public from Northwest A&F University;

[0066] The wheat variety Chinese Spring is documented in the following literature: Regulatory changes in TaSNAC8-6A are associated with drought tolerance in wheat seedlings. Plant Biotechnol J 2019. It is available to the public from Northwest A&F University.

[0067] Wheat variety Fielder: documented in the following literature: Regulatory changes in TaSNAC8-6A areas associated with drought tolerance in wheat seedlings. Plant Biotechnol J 2019. Available to the public from Northwest A&F University.

[0068] Example 1

[0069] Acquisition of the protein TaDTGIP1 and its encoding gene

[0070] I. Cloning of the protein TaDTGIP1 and its encoding gene

[0071] Seeds of the wheat cultivar Chinese Spring were germinated at 25℃ for three days. The germinated seeds were then transferred to nutrient soil or nutrient solution for two weeks of cultivation. The whole plant was then quick-frozen in liquid nitrogen, ground, and total RNA was extracted. Reverse transcription was performed to obtain cDNA. Using this cDNA as a template, PCR amplification was performed with primers 5'-ATGGAGACGGAGACGGAG-3' (SEQ ID NO:9) and 5'-TTACCAACAGCTGAAAAGG-3' (SEQ ID NO:10). The amplification product was subjected to agarose gel electrophoresis, and a 1137bp DNA fragment was isolated, purified, and sequenced. The results showed that the sequence of this DNA fragment was as shown in positions 80 to 1216 of SEQ ID NO:2.

[0072] SEQ ID NO:2 is the full-length cDNA sequence of TaDTGIP1, the protein shown in SEQ ID NO:1, from the wheat cultivar Chinese Spring. Positions 1-79 are the 5' non-coding region, positions 80-1216 are the coding sequence, and positions 1127-1518 are the 3' non-coding region. The gene encoding TaDTGIP1 is named TaDTGIP1.

[0073] II. Expression analysis of TaDTGIP1 gene under drought stress

[0074] Take Chinese Spring wheat cultivar seeds, germinate them at 25℃ for three days, then transfer the germinated seeds to nutrient soil or nutrient solution for two weeks of cultivation, and then perform the following treatments:

[0075] Drought stress was addressed by table drying, where seedlings at the three-leaf stage were placed on a table (temperature 20℃; humidity 50%), and leaves were taken at 0, 1, 3, 6, 12, and 24 hours after treatment and then rapidly frozen in liquid nitrogen. For abscisic acid (ABA) treatment, the roots of seedlings at the three-leaf stage were immersed in 100 μM ABA aqueous solution, and leaves were taken at 0, 1, 3, 6, 12, 24, and 48 hours after treatment and then rapidly frozen in liquid nitrogen.

[0076] The above samples were ground, total RNA was extracted, and reverse transcription was performed to obtain cDNA. Using this cDNA as a template, and 5'-ATGGAAGTCAGTGTCGCCTG-3' (SEQ ID NO:11) and 5'-TCCA TGCAAGCACCGATCAT-3' (SEQ ID NO:12) as primers (to amplify the TaDTGIP1 gene), real-time quantitative PCR (qRT-PCR) was performed. The wheat gene TaActin1 was used as an internal control, and the internal control primers were the same as in step 4 of Example 3. -△△Ct The expression pattern of the TaDTGIP1 gene was analyzed using computational methods, and the results are as follows: Figure 1 As shown.

[0077] Depend on Figure 1 It can be concluded that the TaDTGIP1 gene is upregulated in leaves under drought stress and ABA treatment.

[0078] Example 2

[0079] Subcellular localization of TaDTGIP1-GFP fusion protein

[0080] Using Chinese Spring (CS) cDNA as a template, PCR amplification was performed using 5'-ATGGAGACGGAGACGGAG-3' (SEQ ID NO:9) and 5'-CCAACAGCTGAAAAGG-3' (SEQ ID NO:10) as primers. The target gene was cloned and ligated between the BamHI and Sal I restriction sites of the expression vector pTF486. PEG / Ca 2+ The method of transforming wheat (Chinese Spring) protoplasts was used, with transformation of empty vector pTF486 as a control. The results were observed under a laser confocal microscope. Figure 2 As shown.

[0081] Depend on Figure 2It can be concluded that the green fluorescence in protoplasts transformed with the empty vector pTF486 is distributed throughout the cell, while the green fluorescence in protoplasts transformed with the TaDTGIP1-GFP fusion protein vector is only distributed in the cell nucleus, indicating that the TaDTGIP1 protein is a nuclear localized protein.

[0082] Example 3

[0083] Overexpression of the gene TaDTGIP1 reduces wheat drought resistance

[0084] 1. Construction of recombinant vectors

[0085] The DNA fragment shown at positions 80 to 1216 of SEQ ID NO:2 was cloned into the HindIII and EcoRI restriction sites of pCAMBIA3301 (located downstream of the Ubi promoter), and the recombinant vector pCAMBIA3301-GZ was obtained by sequencing. This recombinant vector expresses the TaDTGIP1 protein shown in SEQ ID NO:1.

[0086] 2. Obtaining recombinant Agrobacterium tumefaciens

[0087] The recombinant vector pCAMBIA3301-GZ was transformed into Agrobacterium tumefaciens strain EHA105 to obtain recombinant Agrobacterium Y containing the recombinant vector pCAMBIA3301-GZ.

[0088] The empty vector pCAMBIA3301 was transformed into Agrobacterium tumefaciens strain EHA105 to obtain recombinant Agrobacterium CK containing the empty vector pCAMBIA3301.

[0089] 3. Obtaining genetically modified wheat

[0090] Recombinant Agrobacterium Y was transformed into the wheat variety Fielder (hereinafter also referred to as wild-type wheat) using Agrobacterium-mediated gene transformation to obtain T0 generation plants, which were then planted in a greenhouse (16h light / 8h darkness). T0 generation plants were identified as positive by PCR, and after self-pollination, T1 generation seeds were obtained. T1 generation plants were also identified as positive by PCR, and after self-pollination, T2 generation seeds were obtained. Simultaneously, positive and negative seedlings were randomly selected and subjected to qRT-PCR detection according to step 4 to determine the expression level of TaDTGIP1 overexpression. T2 generation plants were also identified as positive by PCR, and after self-pollination, T3 generation seeds were obtained.

[0091] The recombinant Agrobacterium CK was transformed into the wheat variety Fielder (hereinafter also referred to as wild-type wheat) using the method described above until the T3 generation pCAMBIA3301 wheat line was obtained.

[0092] T0 generation represents the plants that grow from the current generation after transformation; T1 generation represents the seeds produced by self-pollination of T0 generation and the plants that grow from them; T2 generation represents the seeds produced by self-pollination of T1 generation and the plants that grow from them; T3 generation represents the seeds produced by self-pollination of T2 generation and the plants that grow from them.

[0093] The specific steps of the Agrobacterium-mediated gene transformation method described above are as follows:

[0094] Recombinant Agrobacterium Y was inoculated into YEB liquid medium containing 25 mg / L spectinomycin and cultured at 28°C with shaking until OD reached. 600 The concentration was 0.5. Wheat embryos were placed in 2 mL centrifuge tubes filled with preservation solution and heat-treated at 46℃ for 3 min, followed by centrifugation at 4℃ and 2000 rpm for 10 min. The prepared recombinant Agrobacterium was added to the treated embryos and cultured in the dark at 22℃ for 3 days. The embryos were then transferred to a new culture medium and cultured in the dark at 28℃ for 7-10 days. Screening was performed using different concentrations of glufosinate, and finally, the embryos were transferred to differentiation medium (differentiation medium composition: 1.9 g / L). -1 KNO3, 1.65g L -1 NH4NO3, 170 mg / L -1 KH2PO4, 180.54 mg / L -1 MgSO4, 332.02 mg / L -1 CaCl2, 0.83 g L -1 KI, 6.2 mg / L -1 H3BO3, 16.9 mg / L -1 MnSO4, 8.6 mg / L -1 ZnSO4·7H2O, 0.25 mg / L -1 Na₂MoO₄·2H₂O, 27.8 mg / L -1 FeSO4·7H2O, 37.3 mg / L -1 EDTA, 0.1g / L -1 Myo-inositol, 0.4 mg / L -1 Vitamin B1, 0.5 mg / L -1 Thidiazuron, 200mg L -1 Timentin, 30mg L - 1 Hygromycin (5mg L) -1 Phosphinothricin (pH=5.8) was added to the culture medium, and after differentiation, the samples were transferred to rooting medium (composition: 1.9 g / L). -1 KNO3, 1.65g L -1 NH4NO3, 170 mg / L -1KH2PO4, 180.54 mg / L -1 MgSO4, 332.02 mg / L -1 CaCl2, 0.83 g L -1 KI, 6.2 mg / L -1 H3BO3, 16.9 mg / L -1 MnSO4, 8.6 mg / L -1 ZnSO4·7H2O, 0.25 mg / L - 1 Na₂MoO₄·2H₂O, 27.8 mg / L -1 FeSO4·7H2O, 37.3 mg / L -1 EDTA, 0.1g / L -1 Myo-inositol, 0.4 mg / L - 1 Vitamin B1, 200mg / L -1 Timentin, 15mg L -1 Hygromycin (5mg L) -1 They were cultured on phosphinothricin (pH=5.8) and then transferred to nutrient soil after reaching a certain size.

[0095] 4. qPCR detection of genetically modified wheat

[0096] The wild-type wheat, T3 generation pCAMBIA3301 wheat line, and T3 generation TaDTGIP1 wheat line (OE1-OE9) obtained in step 3 were used to isolate total RNA using the TRIZOL (Biotopped) method. Genomic contamination was then eliminated using the DNAseI (Takara) method, and the concentration was determined using a Nanodrop 1000 (Thermo Scientific product, USA). 5 μg of each RNA was run on 0.8% agarose gel. 1 μg of total RNA was used to synthesize cDNAs using recombinant M-MLV reverse transcriptase with 1 μg of Oligo(dT)23 (Promega) as primers. The TaDTGIP1 cDNA was quantified by qRT-PCR using specific primers F2 and R2, with the wheat TaActin1 gene as an internal control. The internal control primers were a primer pair consisting of FC2 and RC2. A 2... -△△Ct The method was used to calculate the expression level of the target gene, and the results are as follows: Figure 3 As shown, "**" indicates a significant difference (P < 0.01).

[0097] The sequences of the primers mentioned above are as follows:

[0098] F2: 5'-ATGGAAGTCAGTGTCGCCTG-3' (SEQ ID NO: 11);

[0099] R2: 5'-TCCATGCAAGCACCGATCAT-3' (SEQ ID NO: 12);

[0100] FC2: 5'-AAATCTGGCATCACACTTTCTAC-3' (SEQ ID NO: 13);

[0101] RC2: 5'-GTCTCAAACATAATCTGGGTCATC-3' (SEQ ID NO: 14).

[0102] The expression level of the target gene TaDTGIP1 in T3 generation TaDTGIP1 transgenic wheat lines OE1-OE9 was significantly higher than that in wild-type WT. Furthermore, the expression level of TaDTGIP1 was significantly higher in these transgenic wheat lines than in wild-type WT. Figure 3 The results showed that two T3 generation transgenic positive lines with high expression levels were selected for subsequent experiments and named OE8 and OE9, respectively.

[0103] 5. Phenotypic analysis of drought resistance in transgenic wheat

[0104] T3 generation TaDTGIP1 wheat lines (OE8, OE9), wild-type wheat (WT), and T3 generation pCAMBIA3301 wheat lines were transferred to pots containing 250g of nutrient soil. After 21 days of normal growth, a drought treatment (i.e., watering was stopped) was applied. After 25 days, when phenotypic differences became obvious (WT plants showed significant leaf drying, while OE8 and OE9 plants exhibited severe leaf wilting), rehydration was initiated. Three days after rehydration, the survival rate of each line was calculated (plants showing normal growth and harvest were defined as surviving plants, while plants severely affected by drought and unable to grow or harvest were defined as dead plants; the survival rate was the percentage of surviving plants in each line out of the total number of plants). The experiment was repeated three times, with at least 24 plants from each line in each replicate. The average value was used for statistical analysis. The results are shown below. Figure 4 As shown.

[0105] Depend on Figure 4 It can be seen that the leaves of the T3 generation wheat line transformed into TaDTGIP1 showed a higher degree of wilting and drying after rehydration than those of wild-type wheat.

[0106] Three days after rehydration, the survival rates of each plant line were calculated as follows: Figure 5 As shown in the figure, where "**" indicates a significant difference (P < 0.01). It can be seen that the survival rate of the T3 generation transgenic TaDTGIP1 wheat lines after rehydration was 15%–18%, significantly lower than that of wild-type wheat.

[0107] There was no significant difference between the T3 generation pCAMBIA3301 wheat line and the wild-type wheat.

[0108] 6. Fresh and dry weight of aboveground parts of transgenic wheat plants under normal growth and drought stress

[0109] T3 generation wheat lines transgenic to TaDTGIP1 (OE8, OE9), wild-type wheat (WT), and T3 generation wheat lines transgenic to pCAMBIA3301 were transplanted into pots containing 250g of potting soil and 96-well hydroponic boxes (12cm×8cm×11cm) containing 500mL of 1 / 2 Hoagland nutrient solution, respectively. After 21 days of growth under normal conditions, the fresh weight of the above-ground parts of each hydroponic plant was measured. Leaf samples were then dried in an oven (60-80℃) for 2 days before being weighed dry. Soil-grown plants were subjected to a drought treatment (i.e., watering was stopped) after 25 days of growth. When significant phenotypic differences were observed (i.e., WT plant leaves were noticeably withered while OE8 and OE9 plant leaves were severely wilted), they were rehydrated. Three days after rehydration, the fresh weight of the above-ground parts of each plant line was measured, and then dried in an oven for 2 days to obtain the dry weight.

[0110] The results for fresh weight and dry weight are as follows: Figure 6 and Figure 7 As shown, in Figures 6-7 In the figure, "**" indicates a significant difference (P < 0.01), and there are 24 biological replicates in each group.

[0111] It is evident that, under normal growth and drought stress conditions, the fresh weight and dry weight of the T3 generation TaDTGIP1 wheat line were both lower than those of wild-type wheat.

[0112] 7. Water loss rate of detached leaves of transgenic wheat plants

[0113] T3 generation wheat lines transgenic to TaDTGIP1 (OE8, OE9), wild-type wheat (WT), and T3 generation wheat lines transgenic to pCAMBIA3301 were transferred to 96-well hydroponic boxes (12cm×8cm×11cm) containing 500mL of 1 / 2 Hoagland nutrient solution. After 21 days of growth under normal conditions, 2-3 leaf segments from the same part of each plant were cut and placed on filter paper for 13 hours. Weighing was performed every 0.5 hours for the first 6 hours and every 1 hour for the next 6 hours. The experiment was repeated three times, with at least 5 plants from each line in each replicate. The average value was used for statistical analysis. The results are shown below. Figure 8 As shown, "*" and "**" indicate significant differences, P < 0.05 and P < 0.01, respectively.

[0114] Depend on Figure 8 It can be concluded that the water loss rate of detached leaves of T3 generation TaDTGIP1 wheat lines is higher than that of wild-type wheat.

[0115] 8. Stomatal density of leaves in transgenic wheat under normal growth conditions

[0116] Wild-type wheat (WT), T3 generation pCAMBIA3301 wheat lines, and T3 generation TaDTGIP1 wheat lines (OE8 and OE9) were transferred to 96-well hydroponic boxes (12cm×8cm×11cm) containing 500mL of 1 / 2×Hogland nutrient solution. After 21 days of growth under normal conditions, the mesophyll tissue on the surface of the normally growing wheat leaves was scraped off with a scalpel, and the stomatal density was observed under a light microscope. The results are as follows. Figure 9 As shown.

[0117] Depend on Figure 9 It can be concluded that under normal growth conditions, the stomatal density of T3 generation wheat lines transformed into TaDTGIP1 is not significantly different from that of wild-type wheat.

[0118] 9. Observation of stomatal aperture in transgenic wheat under drought stress

[0119] Two leaves (from the same location) of two-week-old wild-type wheat (WT), T3 generation pCAMBIA3301 wheat line, and T3 generation TaDTGIP1 wheat line (OE8, OE9) were sampled and placed in stomatal culture medium [0.05M KNO3, 10mM MES, 50μM CaCl2 (pH=6.15)] under light conditions (300 to 500 μmol·m⁻¹). -2 ·s -1 Cultured at 25℃ for 1.5 hours (normal growth). For drought treatment, placed in stomatal culture medium [0.05M KNO3 / 10mM, MES / 50μM CaCl2 (pH=6.15)] under light conditions (300 to 500 μmol·m⁻¹). -2 ·s -1 The leaves were cultured at 25℃ for 1.5 hours (normal growth), followed by exposure to air for 10 minutes. Subsequently, the samples were incubated in a continuous cycle of darkness, light, and light-to-dark for 30 minutes or 1 hour. Then, the adaxial side of the leaf epidermis was peeled off with a blade (leave upright), and the leaf was placed on a glass slide for observation and photographing of individual stomata under a 20x microscope. Stomatal length and width were measured using ImageJ, and stomatal aperture was calculated. Results are as follows: Figure 10 As shown, "*" and "**" indicate significant differences, P < 0.05 and P < 0.01, respectively.

[0120] Depend on Figure 10As can be seen, under normal growth conditions, the stomatal aperture of leaves in the T3 generation TaDTGIP1 wheat line was significantly higher than that in the wild-type wheat plant. After drought stress, the stomatal aperture of all materials decreased, but the stomatal aperture of the T3 generation TaDTGIP1 wheat line was still significantly higher than that of the wild-type wheat (WT) plant.

[0121] There was no significant difference between the T3 generation pCAMBIA3301 wheat line and wild-type wheat.

[0122] 10. Leaf temperature of transgenic wheat plants under normal growth and drought stress conditions

[0123] T3 generation wheat lines transgenic to TaDTGIP1 (OE8, OE9), wild-type wheat (WT), and T3 generation wheat lines transgenic to pCAMBIA3301 were transferred to pots containing 250g of nutrient soil. After growing under normal conditions for 21 days, measurements were taken using an infrared thermal imaging instrument. Subsequently, a drought treatment was applied (i.e., watering was stopped). After 20-30 days, when phenotypic differences became obvious, i.e., the leaves of WT plants were visibly withered while the leaves of OE8 and OE9 lines were severely wilted, measurements were taken again.

[0124] The results are as follows Figure 11 and Figure 12 ,exist Figure 12 In the figure, "**" indicates a significant difference (P < 0.01).

[0125] Depend on Figure 11 and Figure 12 It can be observed that the leaf temperature of T3 generation TaDTGIP1 transgenic wheat lines was lower than that of wild-type wheat under normal growth and drought stress conditions. Therefore, the TaDTGIP1 gene may play an important role in the stomatal closure process in response to drought.

[0126] The results of the above examples demonstrate that the TaDTGIP1 protein and its encoding gene have the function of regulating plant drought resistance. Overexpression of the TaDTGIP1 encoding gene in plants can weaken plant drought resistance. Therefore, drought resistance in plants can be improved and drought-resistant plants can be cultivated by knocking out or reducing the TaDTGIP1 encoding gene in plants.

[0127] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for breeding transgenic wheat for drought resistance breeding and drought resistance mechanism research, characterized in that, By introducing nucleic acid molecules encoding TaDTGIP1, a plant drought-resistant protein, into recipient wheat using transgenic technology, drought-resistant transgenic wheat was obtained. The amino acid sequence of the plant drought-resistant protein TaDTGIP1 is shown in SEQ ID NO:

1.

2. The application of the method of claim 1 in wheat drought-resistant breeding.

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