Plant drought-resistant related protein TaDTGIP1 as well as coding gene and application thereof

Abstract

The invention belongs to the technical field of plant genetic engineering and genetic improvement, and particularly relates to a plant drought-resistant related protein TaDTGIP1 as well as a coding gene and application thereof. The plant drought-resistant related protein TaDTGIP1 and the coding gene thereof are obtained through cloning, it is found that the plant drought-resistant related protein TaDTGIP1 and the coding gene thereof have a good regulation effect in the aspect of plant drought resistance, the application of the plant drought-resistant related protein TaDTGIP1 and the coding gene thereof is increased, and the plant drought-resistant related protein TaDTGIP1 and the coding gene thereof can be used for cultivating drought-resistant plant varieties and have important significance in breeding and research for improving the plant drought resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering and genetic improvement, and specifically relates to a plant drought resistance-related protein TaDTGIP1 and a coding gene and application thereof. Background Art

[0002] Plants grow and develop in a complex and changing environment and are often subject to adverse stress. Drought is the main adverse factor that affects and restricts plant growth and development, and can even cause plant death, seriously affecting agricultural production. Therefore, cultivating 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 main 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 yields. Although the global wheat planting area and yield have achieved rapid growth with the development and innovation of breeding technology, abiotic stresses such as drought, high temperature, and high salt have seriously affected wheat yields. Among the many environmental stress factors, drought is the most serious threat to agricultural production.

[0004] In order to alleviate or inhibit the impact of drought environment on crop growth, development and yield, genetic improvement is now mostly used to cultivate drought-resistant varieties. And studies have shown that the key to genetic improvement of crop drought tolerance is the cloning and utilization of excellent drought-resistant genes. Therefore, the mining of drought-resistant genes in wheat is of great significance for breeding drought-resistant wheat varieties and increasing wheat yields. Although some genes with drought-resistant functions have been discovered in some studies, such as TaUreG, TaH2A.9 and TaASR1, there are currently no reports on the TaDTGIP1 gene and its encoded protein in plant drought resistance in the existing technology. Summary of the invention

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

[0006] The present invention provides a plant drought resistance-related protein TaDTGIP1, wherein the plant drought resistance-related protein TaDTGIP1 comprises the protein shown in 1) or 2):

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

[0008] 2) A protein derived from 1) in which the amino acid sequence in 1) is substituted, deleted or added with one or more amino acids and which is 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 comprises a DNA molecule as described in any one of a) to d) below;

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

[0012] b) a DNA molecule as shown in bp 80 to 1216 of SEQ ID NO: 2;

[0013] c) a DNA molecule having 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 encoding the plant drought resistance-related protein TaDTGIP1;

[0014] d) A DNA molecule that hybridizes with the DNA sequence defined in 1) or 2) or 3) under stringent conditions and encodes the plant drought resistance-related protein TaDTGIP1.

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

[0016] The present invention also provides a recombinant vector, which includes a basic vector and a nucleic acid molecule or an expression cassette inserted into the basic 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 comprises 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] The present invention also provides the use of the plant drought resistance-related protein TaDTGIP1 described in the above technical solution, or the nucleic acid molecule described in the above technical solution, or the expression cassette described in the above technical solution, or the recombinant vector described in the above technical solution, or the engineering bacteria described in the above technical solution in regulating plant drought resistance and / or cultivating drought-resistant plants.

[0019] Preferably, the regulating plant drought resistance comprises: increasing the content or activity of the plant drought resistance-related protein TaDTGIP1 in the target plant to reduce the drought resistance of the plant;

[0020] Or, the content or activity of the plant drought resistance-related protein TaDTGIP1 in the target plant is reduced to improve the drought resistance of the plant.

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

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

[0023] II) inhibiting the expression of the nucleic acid molecule encoding the plant drought resistance-related 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 comprises wheat.

[0026] Beneficial effects:

[0027] The present invention provides a plant drought resistance-related protein TaDTGIP1, and the plant drought resistance-related protein TaDTGIP1 includes a protein as shown in 1) or 2): 1) a protein composed of an amino acid sequence as shown in SEQ ID NO: 1; 2) a protein derived from 1) in which the amino acid sequence in 1) is substituted, deleted or added with one or more amino acids and is related to plant drought resistance. The present invention cloned the plant drought resistance-related protein TaDTGIP1 and its encoding gene, and found that it has a good regulatory effect on plant drought resistance, increasing the use of the plant drought resistance-related protein TaDTGIP1 and its gene, which can be used to cultivate drought-resistant plant varieties, and has important significance in breeding and research to improve plant drought resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0029] Figure 1 is the expression pattern of the TaDTGIP1 gene under abiotic stress in Example 1;

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

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

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

[0033] Figure 5 The survival rate statistics of the T3 transgenic wheat plants in Example 3 after drought treatment and rehydration for 3 days;

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

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

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

[0037] Fig. 9 is the stomatal density of the leaves of the transgenic wheat under normal growth conditions in Example 3;

[0038] Fig.10 is the stomatal aperture of the leaves of the transgenic wheat under normal growth and drought stress conditions in Example 3;

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

[0040] Fig.12 It is the leaf temperature of the transgenic wheat plants under normal growth and drought stress conditions in Example 3. DETAILED DESCRIPTION

[0041] The present invention provides a plant drought resistance-related protein TaDTGIP1, wherein the plant drought resistance-related protein TaDTGIP1 comprises the protein shown in 1) or 2):

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

[0043] 2) A protein derived from 1) in which the amino acid sequence in 1) is substituted, deleted or added with one or more amino acids and which is associated with plant drought resistance.

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

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

[0046] As an embodiment, the preparation method of the plant drought resistance-related protein TaDTGIP1 includes artificial synthesis or biological expression of a nucleotide molecule 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 technical solution. As an embodiment, the nucleic acid molecule includes a DNA molecule as described in any one of a) to d) below; a) a DNA molecule as shown in SEQ ID NO:2; b) a DNA molecule as shown in 80 to 1216 bp of SEQ ID NO:2; c) a DNA molecule having 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 encoding the plant drought resistance-related protein TaDTGIP1; d) a DNA molecule that hybridizes with the DNA sequence defined in 1) or 2) or 3) under stringent conditions and encodes the plant drought resistance-related protein TaDTGIP1. In the present invention, the DNA molecule shown in SEQ ID NO: 2 is a cDNA sequence encoding the full-length plant drought resistance-related protein TaDTGIP1; the DNA molecule shown in SEQ ID NO: 2, numbers 80 to 1216, encodes the coding region sequence of the plant drought resistance-related protein TaDTGIP1.

[0048]

[0049] As an embodiment, the stringent conditions may be as follows: 50°C, in 7% sodium dodecyl sulfate (SDS), 0.5 M Na 3 PO 4 Hybridize in a mixed solution of 1mM EDTA and rinse in 2×SSC, 0.1% SDS at 50°C; or in 7% SDS, 0.5M Na 3 PO 4 Hybridize in a mixed solution of 1 mM EDTA and rinse in 1×SSC, 0.1% SDS at 50°C; or in 7% SDS, 0.5 M Na 3 PO 4 Hybridize in a mixed solution of 1mM EDTA and rinse in 0.5×SSC, 0.1% SDS at 50°C; or wash in 7% SDS, 0.5M Na 3 PO 4 Hybridize in a mixed solution of 1mM EDTA and rinse in 0.1×SSC, 0.1% SDS at 50°C; or in 7% SDS, 0.5M Na 3 PO 4 Hybridize in a mixed solution of 1mM EDTA and rinse in 0.1×SSC, 0.1% SDS at 65°C; or hybridize in a solution of 6×SSC, 0.5% SDS at 65°C, and then wash the membrane once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS.

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

[0051] The present invention also provides a recombinant vector, the recombinant vector comprising a basic vector and a nucleic acid molecule or an expression cassette inserted into the basic vector, the nucleic acid molecule being the nucleic acid molecule described in the above technical solution; the expression cassette being the expression cassette described in the above technical solution. As an embodiment, the basic vector comprises a plasmid vector; as another embodiment, the plasmid vector may be a binary Agrobacterium vector or a vector that can be used for plant microprojectile bombardment; as another embodiment, the plasmid vector may be but is not limited to pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pCAMBIA3301, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb.

[0052] As an embodiment, the recombinant vector may also contain the 3' non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression; as another embodiment, the polyadenylic acid signal may guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as the Agrobacterium crown gall induction (Ti) plasmid gene (such as the rouge synthase Nos gene) and the 3' non-translated region of the plant gene (such as the soybean storage protein gene) have similar functions. As an embodiment, when the gene is used to construct a recombinant vector, any enhanced promoter (such as the cauliflower mosaic virus (CAMV) 35S promoter, the maize ubiquitin promoter (Ubiquitin)), constitutive promoter or tissue-specific expression promoter (such as the seed-specific expression promoter) may be added before the transcription start nucleotide thereof, and they may be used alone or in combination with other plant promoters. As an embodiment, when using the gene of the present invention to construct a recombinant vector, an enhancer may also be used, including a translation enhancer or a transcription enhancer. These enhancer regions may be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence; as another embodiment, the sources of the translation control signal and the start codon are wide-ranging and may be natural or synthetic. The translation start region may come from a transcription start region or a structural gene. As an embodiment, in order to facilitate the identification and screening of transgenic plant cells or plants, the recombinant vector can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can 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 phosphinothricin, the hph gene that confers resistance to the antibiotic hygromycin, and the dhfr gene that confers resistance to methatrexate, and the EPSPS gene that confers resistance to glyphosate) or chemical 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 by bp 80 to 1216 of SEQ ID NO: 2 between the Hind III and EcoR I restriction sites.

[0054] The present invention also provides an engineered bacterium, the engineered bacterium comprising 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. As an embodiment, the initial strain in the engineered bacterium is Agrobacterium; as another embodiment, the Agrobacterium is Agrobacterium tumefaciens; in a specific embodiment, the initial strain is Agrobacterium tumefaciens EHA105.

[0055] The present invention also provides the use of the plant drought resistance-related protein TaDTGIP1 described in the above technical solution, or the nucleic acid molecule described in the above technical solution, or the expression cassette described in the above technical solution, or the recombinant vector described in the above technical solution, or the engineering bacteria described in the above technical solution in regulating plant drought resistance and / or cultivating drought-resistant plants. As an embodiment, the regulation of plant drought resistance includes: increasing the content or activity of the plant drought resistance-related protein TaDTGIP1 in the target plant, reducing the drought resistance of the plant; or, reducing the content or activity of the plant drought resistance-related protein TaDTGIP1 in the target plant, and increasing the drought resistance of the plant.

[0056] As an embodiment, the plant may be a grass plant; as another embodiment, the grass plant may be wheat. In a specific embodiment, the present invention introduces a nucleic acid molecule encoding the plant drought resistance-related protein TaDTGIP1 into wheat, and the drought resistance of the obtained transgenic wheat is weakened, thereby proving that the plant drought resistance-related protein TaDTGIP1 is of great significance in breeding and research to provide plant drought resistance, and can be used to cultivate drought-resistant plants by knocking out or knocking down the nucleic acid molecule encoding the plant drought resistance-related protein TaDTGIP1; as an embodiment, the knocking out or knocking down method may be gene editing technology.

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

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

[0059] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

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

[0061] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0062] The biological material information in the following examples is as follows:

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

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

[0065] Agrobacterium tumefaciens EHA105 strain: 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 Agriculture and Forestry University;

[0066] Wheat variety Chinese Spring: recorded in the following literature: Regulatory changes in TaSNAC8-6A are associated with drought tolerance in wheat seedlings. Plant Biotechnol J 2019. Available to the public from Northwest Agriculture and Forestry University;

[0067] Wheat variety Fielder: recorded in the following document: Regulatory changes in TaSNAC8-6A areasassociated with drought tolerance in wheat seedlings. Plant Biotechnol J 2019. Available to the public from Northwest Agriculture and Forestry University.

[0068] Example 1

[0069] Obtaining the protein TaDTGIP1 and its encoding gene

[0070] 1. Cloning of protein TaDTGIP1 and its encoding gene

[0071] Seeds of the cultivated wheat variety Chinese Spring were germinated at 25° C. for three days, and then the germinated seeds were transferred to nutrient soil or nutrient solution for culture for two weeks. The whole plant was quickly frozen in liquid nitrogen and ground, and total RNA was extracted. Reverse transcription was performed to obtain cDNA, and PCR amplification was performed using the cDNA as a template and 5'-ATGGAGACGGAGACGGAG-3' (SEQ ID NO: 9) and 5'-TTACCAACAGCTGAAAAGG-3' (SEQ ID NO: 10) as primers. The amplified product was subjected to agarose gel electrophoresis, and a 1137 bp DNA fragment was separated and purified for sequencing. The results showed that the sequence of the 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 encoding the protein TaDTGIP1 shown in SEQ ID NO:1 in the wheat cultivar Chinese Spring, wherein positions 1 to 79 are the 5' non-coding region, positions 80 to 1216 are the coding sequence, and positions 1127 to 1518 are the 3' non-coding region. The gene encoding the protein TaDTGIP1 is named gene TaDTGIP1.

[0073] 2. Expression analysis of TaDTGIP1 gene under drought stress

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

[0075] Drought stress was treated by table drying. The three-leaf seedlings were placed on a table (temperature of 20°C and humidity of 50%), and the leaves treated for 0, 1, 3, 6, 12, and 24 hours were quickly frozen in liquid nitrogen. For abscisic acid (ABA) treatment, the roots of the three-leaf seedlings were immersed in 100 μM ABA aqueous solution, and the leaves treated for 0, 1, 3, 6, 12, 24, and 48 hours were quickly frozen in liquid nitrogen.

[0076] The above samples were ground respectively, total RNA was extracted, reverse transcribed, and cDNA was obtained. The cDNA was used as a template, 5'-ATGGAAGTCAGTGTCGCCTG-3' (SEQ ID NO: 11) and 5'-TCCA TGCAAGCACCGATCAT-3' (SEQ ID NO: 12) were used as primers (to amplify the TaDTGIP1 gene) for real-time fluorescence quantitative PCR (qRT-PCR), and the wheat gene TaActin1 was used as an internal reference. The internal reference primers were the same as those in step 4 of Example 3, and 2 -△△Ct The expression pattern of TaDTGIP1 gene was analyzed by computational method. Figure 1 shown.

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

[0078] Example 2

[0079] Subcellular localization of TaDTGIP1-GFP fusion protein

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

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

[0082] Example 3

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

[0084] 1. Construction of recombinant vector

[0085] The DNA fragment shown at positions 80 to 1216 of SEQ ID NO:2 was cloned between the HindIII and EcoR I restriction sites of pCAMBIA3301 (located downstream of the Ubi promoter), and confirmed by sequencing to obtain a recombinant vector pCAMBIA3301-GZ, which expressed the TaDTGIP1 protein shown in SEQ ID NO:1.

[0086] 2. Obtaining recombinant Agrobacterium tumefaciens

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

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

[0089] 3. Obtaining genetically modified wheat

[0090] The recombinant Agrobacterium Y was transformed into the wheat variety Fielder (hereinafter also referred to as wild-type wheat) by Agrobacterium-mediated gene transformation to obtain T0 plants, which were planted in a greenhouse (16h-light / 8h-dark); T0 plants were identified by PCR to obtain positive plants, and T1 seeds were obtained after self-pollination; T1 plants were identified by PCR to obtain positive plants, and T2 seeds were obtained after self-pollination. At the same time, positive seedlings and negative seedlings were randomly selected for qRT-PCR detection according to the method in step 4 to determine the expression level of overexpressed TaDTGIP1. T2 plants were identified by PCR to obtain positive plants, and T3 seeds were obtained after self-pollination.

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

[0092] The T0 generation refers to the plants grown from the transformation generation; the T1 generation refers to the seeds produced by self-pollination of the T0 generation and the plants grown from them; the T2 generation refers to the seeds produced by self-pollination of the T1 generation and the plants grown from them. The T3 generation refers to the seeds produced by self-pollination of the T2 generation and the plants grown from them.

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

[0094] The recombinant Agrobacterium Y was inoculated into YEB liquid medium containing 25 mg / L spectinomycin and cultured at 28°C with shaking until OD 600 0.5. Place wheat embryos in a 2mL centrifuge tube filled with preservation solution, heat treat at 46°C for 3 minutes, and centrifuge at 4°C and 2000 rpm for 10 minutes. Add the prepared recombinant Agrobacterium to the treated embryos, culture in the dark at 22°C for 3 days, transfer to a new culture medium and culture in the dark at 28°C for 7-10 days. Screen with different concentrations of glufosinate and finally transfer to differentiation medium (the composition of differentiation medium is: 1.9gL -1 KNO 3 , 1.65 g L -1 NH 4 NO 3 , 170mg L -1 KH 2 PO 4 , 180.54mg L -1 MgSO 4 , 332.02mg L -1 CaCl 2 , 0.83 g L -1 KI, 6.2 mg L -1 H 3 BO 3 , 16.9mg L -1 MnSO 4 , 8.6 mg L -1 ZnSO 4 7H 2 O, 0.25 mg L -1 Na 2 MoO 4 ·2H 2 O, 27.8 mg L -1 FeSO 4 7H 2 O, 37.3mg L -1 EDTA, 0.1 g L -1 Myo-inositol, 0.4 mg L -1 Vitamin B1, 0.5mg L -1 Thidiazuron, 200mg L-1 Timentin, 30 mg L - 1 Hygromycin (5mg L -1 phosphinothricin, pH = 5.8) and transferred to rooting medium (rooting medium composition: 1.9 g L -1 KNO 3 , 1.65 g L -1 NH 4 NO 3 , 170mg L -1 KH 2 PO 4 , 180.54mg L -1 MgSO 4 , 332.02mgL -1 CaCl 2 , 0.83 g L -1 KI, 6.2 mg L -1 H 3 BO 3 , 16.9mg L -1 MnSO 4 , 8.6 mg L -1 ZnSO 4 7H 2 O, 0.25 mg L - 1 Na 2 MoO 4 ·2H 2 O, 27.8 mg L -1 FeSO 4 7H 2 O, 37.3mg L -1 EDTA, 0.1 g L -1 Myo-inositol, 0.4 mg L - 1 Vitamin B1, 200mg L -1 Timentin, 15 mg L -1 Hygromycin (5mg L -1 phosphinothricin), pH = 5.8), and when they reach a certain size, they are transferred into nutrient soil.

[0095] 4. qPCR detection of genetically modified wheat

[0096] The wild-type wheat, T3 generation pCAMBIA3301 transgenic wheat lines, and T3 generation TaDTGIP1 transgenic wheat lines (OE1-OE9) obtained in step 3 were used to isolate total RNA using the TRIZOL (Biotopped) method, followed by DNAseⅠ (Takara) method to eliminate genomic contamination, and then the concentration was determined using Nanodrop1000 (Thermo Scientific product, USA), and 5 μg was uniformly taken to run 0.8% agarose gel. 1 μg of total RNA was taken, and cDNAs were synthesized using recombinant M-MLV reverse transcriptase and 1 μg Oligo (dT) 23 (Promega) as primers. The cDNA of the gene TaDTGIP1 was quantified by qRT-PCR using specific primers F2 and R2, and the wheat gene TaActin1 was used as an internal reference, and the internal reference primers were a primer pair consisting of FC2 and RC2, and 2 -△△Ct The expression level of the target gene was calculated by Figure 3 As shown, "**" indicates significant difference P < 0.01.

[0097] The sequences of the above primers 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 the T3 generation TaDTGIP1-transfected wheat lines OE1-OE9 was significantly higher than that in the wild type WT. Figure 3 Based on the results, two T3 transgenic positive lines with higher expression levels were selected for subsequent experiments and named OE8 and OE9 respectively.

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

[0104] The T3 generation transgenic TaDTGIP1 wheat lines (OE8, OE9), wild-type wheat (WT) plants, and T3 generation transgenic pCAMBIA3301 wheat lines were transferred to pots filled with 250 g of nutrient soil. After growing under normal conditions for 21 days, drought treatment (i.e., watering was stopped). After 25 days, when the phenotypic difference was obvious, i.e., the leaves of the WT plants were obviously dried up while the leaves of the OE8 and OE9 lines were severely wilted, the plants were rewatered. After 3 days of rewatering, the survival rate of the plants of each line was counted (the plants that showed normal growth and harvesting were defined as surviving plants, and the plants that showed severe drought damage and could not grow and harvest normally were defined as dead plants; the survival rate was the percentage of the number of surviving plants in each line to the total number of plants). The experiment was repeated 3 times, and the number of plants of each line in each repetition was not less than 24. The average value was taken for statistical analysis, and the results are shown in the figure. Figure 4 shown.

[0105] Depend on Figure 4 It can be seen that the degree of leaf wilting and drying of the T3 generation TaDTGIP1 transgenic wheat line after rehydration is higher than that of the wild-type wheat.

[0106] After 3 days of rehydration, the survival rate of each strain was counted. Figure 5 As shown, "**" indicates significant difference P < 0.01. It can be seen that the survival rate of the T3 generation TaDTGIP1 transgenic wheat lines after rehydration was 15% to 18%, which was significantly lower than that of the wild-type wheat.

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

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

[0109] The T3 transgenic TaDTGIP1 wheat lines (OE8, OE9), wild-type wheat (WT) and T3 transgenic pCAMBIA3301 wheat lines were transplanted into pots filled with 250 g of nutrient soil and 96-well hydroponic boxes (12 cm × 8 cm × 11 cm) filled with 500 mL of 1 / 2 Hoagland nutrient solution, respectively. After the hydroponic plants grew under normal conditions for 21 days, the fresh weight of the aboveground parts of each line was weighed, and then the leaf samples were placed in an oven (60-80°C) and dried for 2 days before weighing the dry weight. After the soil-cultured plants grew under normal conditions for 21 days, drought treatment (i.e., watering was stopped) was performed, and after 25 days of growth, when the phenotypic differences were obvious (i.e., the leaves of the WT plants were obviously dried up, while the leaves of the OE8 and OE9 lines were severely wilted), they were rehydrated. After 3 days of rehydration, the fresh weight of the aboveground parts of the plants of each line was weighed, and then placed in an oven for 2 days to obtain the dry weight.

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

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

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

[0113] The T3 transgenic TaDTGIP1 wheat lines (OE8, OE9), wild-type wheat (WT) plants, and T3 transgenic pCAMBIA3301 wheat lines were transferred to a 96-well hydroponic box (12cm×8cm×11cm) filled with 500mL 1 / 2 Hoagland nutrient solution. After the hydroponic plants grew under normal conditions for 21 days, 2-3 leaves from the same part of each plant were cut and placed on filter paper for 13h. They were weighed every 0.5h for the first 6h and every 1h for the next 6h. The experiment was repeated 3 times, and the number of plants of each line in each repetition was not less than 5. The average value was taken for statistical analysis. The results are shown in the figure. Figure 8 As shown in the figure, "*" and "**" indicate significant differences at 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 transgenic wheat lines is higher than that of wild-type wheat.

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

[0116] The wild-type wheat (WT), T3-generation pCAMBIA3301-transfected wheat lines, and T3-generation TaDTGIP1-transfected wheat lines (OE8, OE9) were transferred to a 96-well hydroponic box (12 cm × 8 cm × 11 cm) filled with 500 mL of 1 / 2× Hoagland nutrient solution. After 21 days of growth under normal conditions, the mesophyll tissue on the surface of the wheat leaves under normal growth was scraped off with a surgical blade, and the stomatal density was observed under a light microscope. The results are as follows: Fig. 9 shown.

[0117] Depend on Fig. 9 It can be concluded that under normal growth conditions, the stomatal density of the T3 generation TaDTGIP1 transgenic wheat lines had no significant difference from that of the wild-type wheat.

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

[0119] The second leaf (same part) of 2-week-old wild-type wheat (WT), T3-generation pCAMBIA3301-transfected wheat lines, and T3-generation TaDTGIP1-transfected wheat lines (OE8 and OE9) were sampled and placed in stomatal culture medium [0.05 M KNO 3 , 10 mM MES, 50 μM CaCl 2 (pH = 6.15), light conditions (300 to 500 μmol·m -2 ·s -1 , 25℃) for 1.5 hours (normal growth). For drought treatment, place the cells in stomatal culture medium [0.05M KNO 3 / 10mM,MES / 50μM CaCl 2 (pH = 6.15)], light conditions (300 to 500 μmol·m -2 ·s -1 , 25℃) for 1.5 hours (normal growth), and then the leaves were exposed to air for 10 minutes. Subsequently, the samples were incubated in continuous darkness, light, and transition from light to dark for 30 minutes or 1 hour. Then, the adaxial side of the leaf epidermis (the front of the leaf) was peeled off with a blade, and the leaves were placed on a slide. Individual stomata were observed and photographed under a 20x microscope. Image J was used to measure the length and width of the stomata, and the stomatal aperture was calculated. The results are shown in Fig.10 As shown in the figure, "*" and "**" indicate significant differences at P < 0.05 and P < 0.01, respectively.

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

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

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

[0123] T3 generation transgenic TaDTGIP1 wheat lines (OE8, OE9), wild-type wheat (WT) plants and T3 generation transgenic pCAMBIA3301 wheat lines were transferred into pots containing 250 g of nutrient soil and grown under normal conditions for 21 days before measurement using an infrared thermal imaging instrument. They were then subjected to drought treatment (i.e., watering was stopped). After 20-30 days, when the phenotypic differences were obvious, i.e., the leaves of the WT plants were obviously dried up while the leaves of the OE8 and OE9 lines were severely wilted, they were measured again.

[0124] The results are as follows Fig.11 and Fig.12 ,exist Fig.12 In the table, “**” indicates significant difference (P < 0.01).

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

[0126] The above results show that TaDTGIP1 protein and its coding gene have the function of regulating plant drought resistance. Overexpression of the coding gene of protein TaDTGIP1 in plants can weaken the drought resistance of plants. Furthermore, the coding gene of protein TaDTGIP1 can be knocked out or knocked down in plants to improve plant drought resistance and cultivate drought-resistant plants.

[0127] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A plant drought resistance-related protein TaDTGIP1, characterized in that: The plant drought resistance-related protein TaDTGIP1 includes the protein shown in 1) or 2): 1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; 2) A protein derived from 1) in which the amino acid sequence in 1) is substituted, deleted or added with one or more amino acids and which is associated with plant drought resistance.

2. A nucleic acid molecule encoding the plant drought resistance-related protein TaDTGIP1 according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule includes the DNA molecule described in any one of a) to d) below; a) a DNA molecule as shown in SEQ ID NO: 2; b) a DNA molecule as shown in bp 80 to 1216 of SEQ ID NO: 2; c) a DNA molecule having 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 encoding the plant drought resistance-related protein TaDTGIP1; d) A DNA molecule that hybridizes with the DNA sequence defined in 1) or 2) or 3) under stringent conditions and encodes the plant drought resistance-related protein TaDTGIP1.

4. An expression cassette, characterized in that The expression cassette comprises the nucleic acid molecule of claim 2 or 3.

5. A recombinant vector, characterized in that: The recombinant vector comprises a basic vector and a nucleic acid molecule or an expression cassette inserted into the basic vector, wherein the nucleic acid molecule is the nucleic acid molecule according to claim 2 or 3; and the expression cassette is the expression cassette according to claim 4.

6. An engineered bacterium, characterized in that: The engineered bacteria comprises the nucleic acid molecule according to claim 2 or 3, the expression cassette according to claim 4, or the recombinant vector according to claim 5.

7. Use of the plant drought resistance-related protein TaDTGIP1 according to claim 1, or the nucleic acid molecule according to claim 2 or 3, or the expression cassette according to claim 4, or the recombinant vector according to claim 5, or the engineered bacteria according to claim 6 in regulating plant drought resistance and / or cultivating drought-resistant plants.

8. The use according to claim 7, characterized in that: The regulating plant drought resistance comprises: increasing the content or activity of the plant drought resistance-related protein TaDTGIP1 in the target plant to reduce the drought resistance of the plant; Or, the content or activity of the plant drought resistance-related protein TaDTGIP1 in the target plant is reduced to improve the drought resistance of the plant.

9. A method for cultivating drought-resistant transgenic plants, characterized in that: The drought-resistant transgenic plant is obtained by the steps described in Ⅰ) or Ⅱ): Ⅰ) reducing the content or activity of plant drought resistance-related protein TaDTGIP1 in target plants; II) inhibiting the expression of the nucleic acid molecule encoding the plant drought resistance-related protein TaDTGIP1 in the target plant; The plant drought resistance-related protein TaDTGIP1 is the plant drought resistance-related protein TaDTGIP1 described in claim 1 or the plant drought resistance-related protein TaDTGIP1 encoded by the nucleic acid molecule described in claim 2 or 3.

10. The use according to claim 7 or 8 or the method according to claim 9, characterized in that: The plants include wheat.

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