CN114591409B - Application of TaDTG6 Protein in Improving Plant Drought Resistance - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of TaDTG6 protein in improving drought resistance of plants. According to the invention, the coding gene of the TaDTG6 protein is overexpressed in the plant, so that the content and activity of the TaDTG6 protein in the plant are improved, and the drought resistance of the plant is remarkably improved. Therefore, the TaDTG6 protein has important significance in the research of improving the drought resistance of plants, and can be used for cultivating drought-resistant plant varieties.

Description

Application of TaDTG6 Protein in Improving Plant Drought Resistance

technical field

The invention belongs to the technical field of plant genetic engineering, and in particular relates to the application of TaDTG6 protein in improving plant drought resistance.

Background technique

Plants grow and develop in a complex and changeable environment, and are often subjected to adversity stress, among which drought is the main stress factor that affects and limits plant growth and development, and even causes 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.

Wheat (Triticum aestivum L.) is the most important carbohydrate and protein source for people all over the world. It is estimated that by 2050, the global wheat output will need to increase by at least 60% to meet the needs of the global population at that time. Therefore, it is urgent to find effective breeding methods to increase wheat yield. Although the global wheat planting area and yield have increased by leaps and bounds, abiotic stresses such as drought, high temperature, and high salinity have seriously affected wheat yield. Among many environmental stress factors, the threat of drought to agricultural production is the most serious worldwide problem. Studies have shown that the key to genetic improvement of crop drought tolerance is the cloning and utilization of excellent drought tolerance genes. Therefore, mining drought-resistant genes in wheat is of great significance for breeding drought-resistant wheat varieties and improving wheat yield.

Contents of the invention

The purpose of the present invention is to provide the application of TaDTG6 protein in improving the drought resistance of plants, and the drought resistance of plants can be improved by increasing the expression level of TaDTG6 protein in plants.

The invention provides the application of TaDTG6 protein in improving the drought resistance of plants;

The amino acid sequence of the TaDTG6 protein is shown in SEQ ID No.1.

Preferably, the nucleotide sequence of the gene encoding the TaDTG6 protein is shown in SEQ ID No.2.

Preferably, the plants include monocots and/or dicots.

Preferably, the monocot includes wheat, and the dicot includes Arabidopsis.

The present invention provides a method for overexpressing exogenous TaDTG6 protein in plants, the method comprising: connecting the gene encoding TaDTG6 protein with an expression vector, transforming Agrobacterium and transforming plants in sequence; the amino acid sequence of the TaDTG6 protein is as follows: Shown in SEQ ID No.1.

Preferably, the expression vector includes a binary Agrobacterium vector or a vector used for plant microprojectile bombardment.

Preferably, the expression vector includes any one of pGKX, pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pCAMBIA3301, pWMB006, pWMB0010, pBI121, pCAMBIA1391-Xa and pCAMBIA1391-Xb.

Preferably, the gene is connected between the BamHI and XhoI restriction sites of the pGKX;

The gene was connected between the HindIII and EcoRI restriction sites of pCAMBIA3301.

Preferably, the Agrobacterium includes Agrobacterium tumefaciens EHA105 or Agrobacterium tumefaciens strain GV3101 containing pSoup plasmid.

The present invention provides a method for cultivating drought-resistant plants. The gene encoding the TaDTG6 protein is overexpressed in the target plant by the method described in the above-mentioned technical solution to obtain the drought-resistant plants.

Beneficial effect:

The invention provides the application of TaDTG6 protein in improving the drought resistance of plants. By overexpressing the coding gene of the TaDTG6 protein in plants, the content and activity of TaDTG6 protein in plants are increased, and the drought resistance of plants is significantly improved. Therefore, the TaDTG6 protein is of great significance in the study of improving the drought resistance of plants, and can be used to breed drought-resistant plant varieties.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following will briefly introduce the drawings required in the embodiments.

Fig. 1 is the expression pattern of TaDTG6 gene under drought stress in embodiment 1;

Fig. 2 is the transcription activation activity analysis of TaDTG6 protein in embodiment 2;

Fig. 3 is the subcellular localization of TaDTG6-GFP fusion protein in embodiment 3;

Fig. 4 is the electrophoresis result of the RT-PCR product of T3 generation transgenic Arabidopsis plant in embodiment 4;

Fig. 5 is the phenotype of T3 generation transgenic Arabidopsis plants in Example 4 after drought treatment and rehydration for 6 days;

Fig. 6 is the statistical result of the survival rate of T3 generation transgenic Arabidopsis plants in Example 4 after drought treatment and rehydration for 6 days;

Fig. 7 is the qPT-PCR result of T3 generation silent wheat line in embodiment 5;

Fig. 8 is the phenotype of T3 generation silenced wheat lines in Example 5 after drought treatment and rehydration for 3 days;

Fig. 9 is the statistical result of the survival rate of T3 generation silent wheat lines in Example 5 after drought treatment and rehydration for 3 days;

Fig. 10 is the qPT-PCR result of T3 generation overexpression wheat line in embodiment 6;

Fig. 11 is the phenotype of the overexpressed wheat line of the T3 generation in Example 6 after drought treatment and rehydration for 3 days;

Fig. 12 is the statistical result of the survival rate of the T3 generation overexpressed wheat lines in Example 6 after drought treatment and rehydration for 3 days;

Figure 13 is the GO analysis of up-regulated expression genes under normal growth conditions of overexpressed wheat in Example 6;

Figure 14 is the GO analysis of down-regulated expression genes under normal growth conditions of overexpressed wheat in Example 6;

Fig. 15 is the GO analysis of the up-regulated expression gene under the drought stress condition of overexpressing wheat in embodiment 6;

Fig. 16 is the GO analysis of down-regulated expression genes under the drought stress condition of overexpressed wheat in Example 6.

Detailed ways

The invention provides an application of TaDTG6 protein in improving plant drought resistance; the amino acid sequence of the TaDTG6 protein is shown in SEQ ID No.1.

The nucleotide sequence of the gene encoding the TaDTG6 protein in the present invention is preferably shown in SEQ ID No.2. The plants of the present invention preferably include monocotyledonous plants and/or dicotyledonous plants, and the monocotyledonous plants preferably include wheat; the dicotyledonous plants preferably include Arabidopsis thaliana. In the embodiment, wheat is only used as a representative of monocotyledonous plants, and Arabidopsis is used as a representative of dicotyledonous plants for illustration, but it cannot be regarded as the entire protection scope of the present invention. The invention achieves the technical effect of improving plant drought resistance by increasing the expression level of TaDTG6 gene encoding TaDTG6 protein in plants, or increasing the content or activity of TaDTG6 protein in plants.

The present invention also provides a method for overexpressing exogenous TaDTG6 protein in plants, the method comprising: connecting the gene encoding the TaDTG6 protein with an expression vector, transforming Agrobacterium and transforming plants in sequence. The plants described in the present invention preferably include dicotyledonous plants and/or monocotyledonous plants, and the monocotyledonous plants and dicotyledonous plants are preferably the same as above, which will not be repeated here.

The invention connects the gene encoding the TaDTG6 protein with the expression vector to obtain the recombinant expression vector. The expression vector of the present invention is preferably selected according to the type of plant, and further preferably includes a binary Agrobacterium vector or a vector for plant microprojectile bombardment, more preferably includes pGKX, pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pCAMBIA3301, pWMB006 , pWMB0010, pBI121, pCAMBIA1391-Xa and pCAMBIA1391-Xb. When the plant is Arabidopsis, the expression vector is preferably pGKX, and when the plant is wheat, the expression vector is preferably pCAMBIA3301. In the present invention, the gene is preferably connected between the BamHI and XhoI restriction sites of pGKX; the gene is connected between the HindIII and EcoRI restriction sites of pCAMBIA3301. In the present invention, there is no special limitation on the connection method between the gene and the expression vector, and conventional operations in the field can be adopted. If there is no special limitation in the present invention, there is no special requirement on the source of the relevant expression vector.

The expression vector of the present invention preferably also includes the 3' untranslated region of the foreign gene, that is, includes the polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal of the present invention can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as: Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopain synthase Nos gene) or plant gene ( Such as soybean storage protein gene), the untranslated region transcribed at the 3' end has similar functions. In the present invention, when constructing the recombinant expression vector, any enhanced promoter, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin promoter of corn, can preferably be added before its transcription start nucleotide (Ubiquitin), constitutive promoter or tissue-specific expression promoter. The enhanced promoter of the present invention is preferably used alone or in combination with other plant promoters. In the present invention, when constructing the recombinant expression vector, an enhancer is preferably used, and the enhancer preferably includes a translation enhancer or a transcription enhancer. The enhancer region of the enhancer in the present invention is preferably the ATG initiation codon or the initiation codon of the adjacent region. The enhancer region of the present invention must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The sources of translational control signals and initiation codons of the enhancers of the present invention are preferably natural or synthetic. The translation initiation region of the enhancer of the present invention is preferably from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the present invention preferably also includes processing the recombinant expression vector used, such as adding enzymes that can express in plants, genes that encode color-changing compounds, luminescent compounds, and markers for antibiotics gene, chemical resistance marker gene, or mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. The gene of the luminescent compound of the present invention is preferably the GUS gene or the luciferase gene; the marker gene of the antibiotic is preferably the nptII gene that confers resistance to kanamycin and related antibiotics, and the gene that confers resistance to the herbicide phosphinothricin. The bar gene that confers resistance to the antibiotic hygromycin, the hph gene that confers resistance to the antibiotic hygromycin, the dhfr gene that confers resistance to methrexate, or the EPSPS gene that confers resistance to glyphosate; the chemical resistance marker gene is preferably herbicide resistance Gene.

To obtain the recombinant expression vector, the present invention preferably transforms the recombinant vector into Agrobacterium to obtain the recombinant Agrobacterium. The type of Agrobacterium described in the present invention is preferably selected according to the plant species, preferably including Agrobacterium tumefaciens EHA105 or Agrobacterium tumefaciens strain GV3101 containing pSoup plasmid, such as when the plant is Arabidopsis, the Agrobacterium preferably It is Agrobacterium tumefaciens strain GV3101 containing pSoup plasmid, and when the plant is wheat, the Agrobacterium is preferably EHA105. In the present invention, the transformation method of the recombinant vector is not specifically limited, and conventional transformation methods in the art can be used.

After obtaining the recombinant Agrobacterium, the present invention transforms the recombinant Agrobacterium into plants, that is, completes the overexpression process. The method of the present invention for transforming the recombinant Agrobacterium into plants preferably includes a flower bud soaking method or an Agrobacterium-mediated gene transformation method. The present invention has no special limitation on the specific operation process of the method, and conventional operations in the field can be used.

The present invention also provides a method for cultivating drought-resistant plants. The gene encoding the TaDTG6 protein is overexpressed in the target plant by using the method described in the above technical solution to obtain the drought-resistant plants. The cultivation method of the present invention is the same as the specific operation process in the method of overexpressing exogenous TaDTG6 protein in plants, and will not be repeated here.

In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

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

Vector pGKX: described in the following documents: Qin F, Sakuma Y, Tran LS, Maruyama K, Kidokoro S, et al. (2008) Arabidopsis DREB2A-interacting proteins function as RINGE3ligases and negatively regulate plant drought stress-responsive geneexpression.Plant Cell 20 :1693-1707. Publicly available from Northwest A&F University;

Vector pCAMBIA3301: described in the following literature: Regulatory changes in TaSNAC8-6Aareassociated with drought tolerance in wheat seedlings. Plant Biotechnol J 2019. Publicly available from Northwest Agriculture and Forestry University.

Vector pTF486: recorded in the following literature: ABA-induced sugar transporterTaSTP6promotes wheat susceptibility to stripe rust.PlantPhysiol.2019,181(3):1328-1343, available to the public from Northwest Agriculture and Forestry University;

Agrobacterium tumefaciens GV3101+pSoup strain: described in the following documents: Scholthof HB, Alvarado VY, Vega-Arreguin JC, Ciomperlik J, Odokonyero D, et al. (2011) Identification of an ARGONAUTE for antiviral RNA silencing in Nicotiana benthamiana.Plant Physiol 156:1548-1555, publicly available from Northwest A&F University;

Agrobacterium tumefaciens EHA105 strain: recorded in the following literature: Regulatory changes in TaSNAC8-6Aare associated with drought tolerance in wheat seedlings.Plant Biotechnol J2019. The public can obtain from Northwest Agriculture and Forestry University;

Arabidopsis thaliana (Columbia ecotype) (col-0; hereinafter referred to as wild-type Arabidopsis): recorded in the following literature: Yamaguchi-Shinozaki K, Shinozaki K (1994) Anovel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 6:251-264. Publicly available from Northwest Agriculture and Forestry University;

Wheat variety Chinese Spring (Chinese Spring): documented in the following literature: Regulatory changes in TaSNAC8-6A are associated with drought tolerance in wheat seedlings. PlantBiotechnol J 2019. Publicly available from Northwest Agriculture and Forestry University;

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. Publicly available from Northwest A&F University.

Example 1 The acquisition of TaDTG6 protein and its coding gene

1. Cloning of protein TaDTG6 and its coding gene

Take the seeds of wheat cultivar Chinese Spring, accelerate germination at 25°C for three days, transfer the germinated seeds to nutrient soil or nutrient solution for two weeks, take the whole plant, freeze it in liquid nitrogen, grind it, extract total RNA, and perform reverse transcription , to obtain cDNA, using the cDNA as a template, SEQ ID No.3: 5'-ATGGAGCGGGCGTACTGCG-3' and SEQ ID No.4: 5'-TCAGGTGCGCGCCATCAGTA-3' as primers for PCR amplification, the amplified product was agar Glycogel electrophoresis, separation and purification of a 642bp DNA fragment and sequencing, the results show that the sequence of the DNA fragment is as shown in the 1-642 positions shown in SEQ ID No.2.

SEQ ID No.2 is the full-length coding sequence encoding the protein TaDTG6 shown in SEQ ID No.1 in the wheat cultivar Chinese Spring, and the gene encoding the TaDTG6 protein is named gene TaDTG6.

2. Expression analysis of TaDTG6 gene under drought stress

Take Chinese Spring seeds of wheat cultivar, after three days of accelerating germination at 25°C, transfer the germinated seeds to nutrient soil or nutrient solution for two weeks, and then carry out the following treatments: the drought stress adopts the method of table top drying treatment, and the three-leaf stage The seedlings are placed on the table (temperature is 20° C.; humidity is 50%), and the seedlings treated for 0, 1, 3, 6, 12, 24, and 48 hours are respectively taken and quick-frozen in liquid nitrogen. Grind the above samples respectively, extract total RNA, perform reverse transcription, and obtain cDNA, using the cDNA as a template, SEQ ID No.5: 5'-CGCAAACTCTCCTTACTACCTCA-3' and SEQ ID No.6: 5'-ATCCACGTCGATGAGCATGC- 3' is a primer (amplification of TaDTG6 gene) for real-time fluorescent quantitative PCR (qRT-PCR) to analyze the expression pattern of TaDTG6 gene.

The results are shown in FIG. 1 , the expression level of TaDTG6 gene was significantly increased during the induction process of wheat seedlings by drought stress, which was an up-regulated expression.

Example 2 Analysis of the transcription activation activity of TaDTG6 protein

Using the cDNA of Chinese Spring (CS) as a template, SEQ ID No. 3: 5'-ATGGAGCGGGCGTACTGCG-3' and SEQ ID No. 4: 5'-TCAGGTGCGCGCCATCAGTA-3' were used as primers for PCR amplification to obtain a 642bp PCR product.

After sequencing, the PCR product has the 1-642th fragment shown in SEQ ID No.2, which is the TaDTG6 gene.

The above PCR product was cloned and ligated into the yeast expression vector pGBKT7 (Clontech, 630489) to obtain a recombinant vector (expressing TaDTG6 protein), and then the recombinant vector was transformed into yeast strain AH109 (Shanghai Sixin Biotechnology Co., Ltd., addgene 0278; containing report genes HIS3 and ADE2), and the transformed empty vector pGBKT7 was used as a control to obtain recombinant yeast strains TaDTG6 and pGBKT7-Control, respectively. The AH109 recombinant yeast strain was spread on the plate of auxotrophic medium, and the transcription activation activity of TaDTG6 protein was analyzed by plaque growth.

The results are shown in Figure 2, the recombinant yeast strain TaDTG6 can grow on the plate of SD/-Trp (single deficiency) medium. On SD/-T-H (two deficiencies), SD/-T-H-A (three deficiencies) auxotrophic medium plates, the yeast strain (pGBKT7-Control) containing the pGBKT7-Control plasmid could not grow normally, while the recombinant yeast strain TaDTG6 could not grow normally. able to grow normally.

The above results show that: TaDTG6 protein has transcriptional activation activity and can act as a transcriptional activator.

Example 3 Subcellular localization of TaDTG6-GFP fusion protein

Using the cDNA of Chinese Spring (CS) as a template, SEQ ID No.7: 5'-ATGGAGCGGGCGTACTGCG-3' and SEQ ID No.8: 5'-GGTGCGCGCCATCAGTAGTT-3' as primers for PCR amplification, the target gene was cloned and Ligated into the expression vector pTF486, transformed into wheat (Chinese Spring) protoplasts, and the transformed empty vector pTF486 was used as a control, respectively observed under a confocal laser microscope.

The results are shown in Figure 3. The green fluorescence in the protoplasts transformed with the empty vector pTF486 is distributed throughout the cell, while the green fluorescence in the protoplasts transformed with the TaDTG6-GFP fusion protein vector is only distributed in the nucleus, indicating that TaDTG6 is a nuclear localization protein.

Example 4 Overexpression of gene TaDTG6 improves drought resistance of Arabidopsis

1. Construction of recombinant vector

The DNA fragment shown in the 1-642 position of SEQ ID No.2 is cloned into the NotI and XhoI restriction sites of the vector pGKX (located downstream of the 35S promoter), and confirmed by sequencing to obtain the recombinant vector pGZ, the The recombinant vector expresses the TaDTG6 protein shown in SEQ ID No.1.

2. Acquisition of recombinant Agrobacterium tumefaciens

Transform the recombinant vector pGZ into the Agrobacterium tumefaciens GV3101+pSoup strain to obtain recombinant Agrobacterium X containing the recombinant vector pGZ;

The empty vector pGKX was transformed into the Agrobacterium tumefaciens GV3101+pSoup strain to obtain the recombinant Agrobacterium Y containing the empty vector pGKX.

3. Obtaining transgenic Arabidopsis

The recombinant Agrobacterium X was transformed into wild-type Arabidopsis thaliana by flower bud soaking method, and the T1 generation seeds were harvested; the T1 generation seeds were screened with MS medium containing 30mg/L kanamycin, and the resistant seedlings were planted and harvested to obtain T2 generation seeds; the T2 generation seeds are screened with MS medium containing 30mg/L kanamycin, and the kanamycin resistance segregation ratio is selected to meet the 3:1 kanamycin resistant seedlings for planting, and the petri dish is randomly selected The resistant seedlings were detected by RT-PCR according to the method in step 4 to determine the Arabidopsis thaliana strain overexpressing TaDTG6 and harvest the seeds to obtain Arabidopsis thaliana seeds overexpressing TaDTG6 in a single copy of the T3 generation. The T3 generation seeds were used to contain 30mg Three Arabidopsis lines that no longer produce kanamycin-resistant T3 homozygous transgene TaDTG6 were screened on MS medium with /L kanamycin, which were named TL1, TL2 and TL3, respectively.

The recombinant Agrobacterium Y was transformed into wild-type Arabidopsis thaliana by the flower bud soaking method, and was screened according to the above method to obtain 3 T3 homozygous Arabidopsis strains transformed into empty vectors that no longer produce kanamycin resistance in the T3 generation , respectively named Y1, Y2 and Y3 (collectively referred to as Y in phenotype experiments).

The T1 generation represents the seeds produced by the transformation generation and the plants grown from it; the T2 generation represents the seeds produced by the selfing of the T1 generation and the plants grown by it; the T3 generation represents the seeds produced by the selfing of the T2 generation and the plants grown by it grown plants.

The concrete steps of above-mentioned flower bud soaking method are as follows:

Take the recombinant Agrobacterium X or Y and inoculate it in LB liquid medium containing 50 mg/L kanamycin and 5 mg/L tetracycline, culture it with shaking at 28°C until the OD600 is 0.8, centrifuge at 25°C, 5000 rpm for 2 minutes, Remove the supernatant, and resuspend the bacterium with a resuspension solution (the solvent is water, the concentrations of the solute sucrose and silwet77 are 50g/L and 0.02% (volume percentage) respectively) to obtain the infection solution. Spot the dye solution on the flower buds and growth points, cover them with a film, keep them moisturized for 2 days, grow under normal conditions, and harvest the seeds.

4. RT-PCR detection of transgenic Arabidopsis

Take the T3 generation homozygous TaDTG6 transgenic Arabidopsis lines (TL1-TL3), the T3 generation homozygous transgenic Arabidopsis lines (Y) plants obtained in step 3, and wild type Arabidopsis (CK) , Total RNA was isolated by TRIZOL (Biotopped) method, followed by DNAse I (Takara) method to eliminate genomic contamination, and then Nanodrop1000 (Thermo Scientificproduct, USA) was used to measure the concentration, and 5 μg was uniformly taken and run on 0.8% agarose gel. Take 1 μg of total RNA, use recombinant M-MLV reverse transcriptase, and use 1 μg Oligo(dT)23 (Promega) as a primer to synthesize cDNAs, and use specific primers F1 and R1 to PCR amplify the cDNA of the gene TaDTG6 to The gene Actin2 in Arabidopsis was used as an internal reference, and the primers were FC1 and RC1. The PCR amplification products were subjected to agarose gel electrophoresis, and the results are shown in Figure 4.

The sequences of the above primers are as follows: and

F1: 5'-ATGGAGCGGGCGTACTGCG-3' (SEQ ID No.9);

R1: 5'-TCAGGTGCGCGCCATCAGTA-3' (SEQ ID No. 10);

FC1: 5'-GGTAACATTGTGCTCAGTGGTGG-3' (SEQ ID No. 11);

RC1: 5'-GCATCAATTCGATCACTCAGAG-3' (SEQ ID No. 12).

The results in Fig. 4 show that the target gene TaDTG6 is not expressed in the wild-type Arabidopsis CK plants; while the expression levels of the target gene TaDTG6 in the transgenic Arabidopsis lines TL1-TL3 are very high.

5. Phenotype analysis of drought resistance of transgenic Arabidopsis

Take wild-type Arabidopsis (CK), Arabidopsis thaliana lines (TL1, TL2, TL3) homozygous transgene TaDTG6 of the T3 generation, and Arabidopsis lines of the T3 generation homozygous transgenic empty vector 7-day-old plants, Transfer to the bowl that 150g nutrient soil is housed, after growing under normal conditions for 32 days, carry out drought treatment (i.e. stop watering), after 14 days, the difference of phenotype is obvious that wild-type CK strain rosette leaves are severely dry and TL1, TL2, When the rosette leaves of TL3 strain were severely wilted, rewater. After 6 days of rehydration, the survival rate of each strain plant was counted (the plants that showed normal growth and harvest were defined as surviving plants, and the plants that showed severe drought damage and could not grow normally and harvest were defined as dead plants; The survival rate is the percentage of the number of surviving plants in the total number of plants in each line). The experiment was repeated 4 times, and the number of plants in each line was not less than 16 in each repetition, and the average value was taken for statistical analysis.

The phenotype observation results are shown in Figure 5. It can be seen that compared with CK, the Arabidopsis lines of the T3 generation homozygous transgene TaDTG6 have a higher plant survival rate after rehydration.

The survival rate was counted, and the results are shown in Figure 6. After rehydration, the plant survival rate of the Arabidopsis line of the T3 homozygous transgene TaDTG6 was 86%-92%, which was significantly higher than that of CK.

There was no significant difference in the results between T3 homozygous transgenic Arabidopsis lines and wild-type Arabidopsis (CK).

The above results indicated that compared with wild-type Arabidopsis or CK, Arabidopsis homozygous transgene TaDTG6 in the T3 generation had improved drought resistance.

The results of Example 4 show that the protein TaDTG6 and its encoding gene have the function of regulating the drought resistance of plants, and overexpressing the encoding gene of protein TaDTG6 in plants can improve the drought resistance of plants.

Example 5 Silence of TaDTG6 gene reduces drought resistance of wheat

1. Construction of recombinant vector

The DNA fragment shown in the 220-517 position of SEQ ID No.2 was cloned after amplification with primers SEQ ID No.13: 5'-AAGCAGAGGCTATGGCTCGGCA-3' and SEQ ID No.14: 5'-TGAGGTAGTAAGGAGAGTTTGCG-3' Between the BamHI and XhoI restriction sites of pWMB006 (located downstream of the Ubi promoter), and confirmed by sequencing, the recombinant vector pWMB006-GZ was obtained, and the recombinant vector expressed the TaDTG6 protein shown in SEQ ID No.1.

2. Acquisition of recombinant Agrobacterium tumefaciens

The recombinant vector pWMB006-GZ was transformed into Agrobacterium tumefaciens EHA105 strain to obtain recombinant Agrobacterium Y containing the recombinant vector pWMB006-GZ.

The empty vector pWMB006 was transformed into the Agrobacterium tumefaciens EHA105 strain to obtain the recombinant Agrobacterium CK containing the empty vector pWMB006.

3. Obtaining genetically modified wheat

The recombinant Agrobacterium Y was transformed into the wheat variety Fielder (hereinafter also referred to as wild-type wheat) by the Agrobacterium-mediated gene transformation method, and the T0 generation plants were obtained, and planted in the greenhouse (16h-light/8h-dark); the T0 generation plants Positive plants were obtained after PCR identification, and T1 generation seeds were obtained after selfing; positive plants were obtained after T1 generation plants were identified by PCR, T2 generation seeds were obtained after selfing, and positive seedlings and negative seedlings were randomly selected according to the method of step 4 qRT-PCR detection was performed to determine the expression level of overexpressed TaDTG6. T2 generation plants were identified by PCR to obtain positive plants, and T3 generation seeds were obtained after selfing.

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 transgenic pWMB006 wheat strain was obtained.

The T0 generation represents the plants grown from the transformed generation; the T1 generation represents the seeds produced by the selfing of the T0 generation and the plants grown from it; the T2 generation represents the seeds produced by the selfing of the T1 generation and the plants grown from it. The T3 generation represents the seeds produced by selfing of the T2 generation and the plants grown from it.

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

Recombinant Agrobacterium Y was inoculated in YEB liquid medium containing 25 mg/L spectinomycin, and cultured with shaking at 28°C until OD600 was 0.5. Place the young wheat embryos in a 2mL centrifuge tube filled with preservation solution, heat-treat at 46°C for 3 minutes, and centrifuge at 4°C at 2000 rpm for 10 minutes. The prepared recombinant Agrobacterium was added to the treated immature embryos, cultured in the dark at 22°C for 3 days, then transferred to a new medium and cultured in the dark at 28°C for 7-10 days. Screened through different concentrations of glufosinate, and finally transferred to the differentiation medium, and then transferred to the rooting medium for cultivation after differentiation, and then moved into the nutrient soil after a certain size.

4. qRT-PCR detection of transgenic wheat

Take the wild-type wheat obtained in step 3, the T3 generation-transferred pWMB006 wheat line, and the T3 generation-transferred pWMB006-GZ wheat line (RI1, RI2, RI3), and use the TRIZOL (Biotopped) method to isolate total RNA, followed by DNAse I ( Takara) method to eliminate the contamination of the genome, and then use Nanodrop1000 (Thermo Scientific product, USA) to measure the concentration, uniformly take 5 micrograms and run 0.8% agarose gel. Take 1 microgram of total RNA, use recombinant M-MLV reverse transcriptase, and use 1 microgram of Oligo(dT)23 (Promega) as a primer to synthesize cDNAs, and use specific primers F2 and R2 to perform qRT-PCR on the cDNA of the gene TaDTG6 For quantification, the wheat gene TaActin1 was used as an internal reference, and the results are shown in Figure 7.

The sequences of the above primers are as follows:

F2: 5'-CGCAAACTCTCCTTACTACCTCA-3' (SEQ ID No. 15);

R2: 5'-ATCCACGTCGATGAGCATGC-3' (SEQ ID No. 16);

FC2: 5'-AAATCTGGCATCACACTTTCTAC-3' (SEQ ID No. 17);

RC2: 5'-GTCTCAAACATAATCTGGGTCATC-3' (SEQ ID No. 18).

The results in Fig. 7 show that the expression level of the target gene TaDTG6 in the T3-transferred pWMB006-GZ wheat line RI1-RI3 is significantly lower than that in the wild-type WT.

5. Phenotypic analysis of drought resistance of transgenic wheat

Take the T3 generation-transferred TaDTG6 wheat lines (RI1, RI2, RI3), wild-type wheat (WT) plants, and the T3 generation-transferred pWMB006 wheat line, and transfer them to a pot containing 250 g of nutrient soil. After growing for 21 days under normal conditions, After 20-30 days of drought treatment (that is, stop watering), the phenotype difference is obvious, that is, the leaves of RI1, RI2, and RI3 strains are obviously dry, and the leaves of WT plants are severely wilted. After 3 days of rehydration, the survival rate of each strain plant was counted (the plants that showed normal growth and harvest were defined as surviving plants, and the plants that showed severe drought damage and could not grow normally and harvest were defined as dead plants; The survival rate is the percentage of the number of surviving plants in the total number of plants in each line). The experiment was repeated 3 times, and the number of plants of each line was not less than 45 in each repetition, and the average value was taken for statistical analysis.

The results are shown in Figure 8. It can be seen that the survival rate of the T3 transgenic pWMB006-GZ wheat line after rehydration is lower than that of the wild-type wheat.

Three days after rehydration, the survival rate of each strain was counted as shown in Figure 9. It can be seen that the survival rate of the T3 generation pWMB006-GZ wheat line after rehydration was 25% to 32%, which was significantly lower than that of the wild type wheat.

There was no significant difference in the results of T3 transgenic pWMB006 wheat line and wild-type wheat.

Example 6 Overexpression of gene TaDTG6 improves drought resistance of wheat

1. Construction of recombinant vector

The DNA fragment shown in the 1-642 position of SEQ ID No.2 is cloned between HindIII and the EcoRI restriction site of pCAMBIA3301 (be positioned at Ubi promotor downstream), and through sequencing confirmation, obtain recombinant vector pCAMBIA3301-GZ, the The recombinant vector expresses the TaDTG6 protein shown in SEQ ID No.1.

2. Acquisition of recombinant Agrobacterium tumefaciens

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

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

3. Obtaining genetically modified wheat

The recombinant Agrobacterium Y was transformed into the wheat variety Fielder (hereinafter also referred to as wild-type wheat) by the Agrobacterium-mediated gene transformation method, and the T0 generation plants were obtained, and planted in the greenhouse (16h-light/8h-dark); the T0 generation plants Positive plants were obtained after PCR identification, and T1 generation seeds were obtained after selfing; positive plants were obtained after T1 generation plants were identified by PCR, T2 generation seeds were obtained after selfing, and positive seedlings and negative seedlings were randomly selected according to the method of step 4 qRT-PCR detection was performed to determine the expression level of overexpressed TaDTG6. T2 generation plants were identified by PCR to obtain positive plants, and T3 generation seeds were obtained after selfing.

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 transgenic pCAMBIA3301 wheat strain was obtained.

The T0 generation represents the plants grown from the transformed generation; the T1 generation represents the seeds produced by the selfing of the T0 generation and the plants grown from it; the T2 generation represents the seeds produced by the selfing of the T1 generation and the plants grown from it. The T3 generation represents the seeds produced by selfing of the T2 generation and the plants grown from it.

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

Recombinant Agrobacterium Y was inoculated in YEB liquid medium containing 25 mg/L spectinomycin, and cultured with shaking at 28°C until OD600 was 0.5. Place the young wheat embryos in a 2mL centrifuge tube filled with preservation solution, heat-treat at 46°C for 3 minutes, and centrifuge at 4°C at 2000 rpm for 10 minutes. The prepared recombinant Agrobacterium was added to the treated immature embryos, cultured in the dark at 22°C for 3 days, then transferred to a new medium and cultured in the dark at 28°C for 7-10 days. Screened through different concentrations of glufosinate, and finally transferred to the differentiation medium, and then transferred to the rooting medium for cultivation after differentiation, and then moved into the nutrient soil after a certain size.

4. qRT-PCR detection of transgenic wheat

Take the wild-type wheat obtained in step 3, the T3-transferred pCAMBIA3301 wheat line, and the T3-transferred TaDTG6 wheat line (OE1-OE3), and use the TRIZOL (Biotopped) method to isolate the total RNA, and then use the DNAse I (Takara) method to eliminate Genome contamination, and then use Nanodrop1000 (Thermo Scientific product, USA) to measure the concentration, uniformly take 5 micrograms and run 0.8% agarose gel. Take 1 microgram of total RNA, use recombinant M-MLV reverse transcriptase, and use 1 microgram of Oligo(dT)23 (Promega) as a primer to synthesize cDNAs, and use specific primers F2 and R2 to perform qRT-PCR on the cDNA of the gene TaDTG6 For quantification, the wheat gene TaActin1 was used as an internal reference, and the results are shown in Figure 10.

The sequences of the above primers are as follows:

F2: 5'-CGCAAACTCTCCTTACTACCTCA-3' (SEQ ID No. 15);

R2: 5'-ATCCACGTCGATGAGCATGC-3' (SEQ ID No. 16);

FC2: 5'-AAATCTGGCATCACACTTTCTAC-3' (SEQ ID No. 17);

RC2: 5'-GTCTCAAACATAATCTGGGTCATC-3' (SEQ ID No. 18).

The results in Figure 10 show that the expression level of the target gene TaDTG6 in the T3-transferred TaDTG6 wheat line OE1-OE3 is significantly higher than that of the wild-type WT.

5. Phenotypic analysis of drought resistance of transgenic wheat

Take the T3 generation of TaDTG6 wheat lines (OE1, OE2, OE3), wild-type wheat (WT) plants and T3 generation of pCAMBIA3301 wheat lines, and transfer them to a pot containing 250g of nutrient soil. After growing for 21 days under normal conditions, After 20-30 days of drought treatment (that is, stop watering), the phenotype difference is obvious, that is, the leaves of the WT plants are obviously dry and the leaves of the OE1, OE2, and OE3 lines are severely wilted. After 3 days of rehydration, the survival rate of each strain plant was counted (the plants that showed normal growth and harvest were defined as surviving plants, and the plants that showed severe drought damage and could not grow normally and harvest were defined as dead plants; The survival rate is the percentage of the number of surviving plants in the total number of plants in each line). The experiment was repeated 3 times, and the number of plants of each line was not less than 45 in each repetition, and the average value was taken for statistical analysis.

The results are shown in Figure 11. It can be seen that the survival rate of T3-transferred TaDTG6 wheat lines after rehydration is higher than that of wild-type wheat.

Three days after rehydration, the survival rate of each line was counted as shown in Figure 12. It can be seen that the survival rate of T3-transferred TaDTG6 wheat lines after rehydration was 77%-85%, which was significantly higher than that of wild-type wheat.

There was no significant difference in the results of T3 transgenic pCAMBIA3301 wheat line and wild-type wheat.

6. Transgenic wheat RNA-seq analysis

Take the 8-day-old plants of the T3 generation-transferred TaDTG6 wheat lines (OE1, OE2) and wild-type (WT) wheat seedlings, and carry out PEG stress treatment for 0 h and 6 h. At least 3 seedlings for each line, use the TRIZOL (Biotopped) method The total RNA was isolated, and then the concentration was measured by Nanodrop1000 (Thermo Scientific product, USA). After passing the test, it was sent to Beijing Boao Biotechnology Co., Ltd. for transcriptome sequencing and data analysis.

The results are shown in Figures 13-16.

The results in Fig. 13-Fig. 16 show that in the T3-transferred TaDTG6 wheat lines under normal growth and drought treatment, genes involved in biological pathways such as water stress response and ABA stress response are generally up-regulated.

The results of Examples 5 and 6 show that the TaDTG6 protein and its coding gene have the function of regulating the drought resistance of plants, and the overexpression of the TaDTG6 protein coding gene in plants can improve the drought resistance of plants.

Although the foregoing embodiment has described the present invention in detail, it is only a part of the embodiments of the present invention, rather than all embodiments, and people can also obtain other embodiments according to the present embodiment without inventive step, these embodiments All belong to the protection scope of the present invention.

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Claims (6)

1. Application of TaDTG6 protein in improving drought resistance of plants;

   the amino acid sequence of the TaDTG6 protein is shown as SEQ ID No. 1;

   the plant is wheat and/or Arabidopsis thaliana.
2. 2. A cultivation method of drought-resistant plants is characterized in that a method of over-expressing exogenous TaDTG6 protein in plants is adopted to over-express a gene encoding the TaDTG6 protein in target plants to obtain the drought-resistant plants;

   the method for over-expressing exogenous TaDTG6 protein in plants comprises the following steps: connecting a gene encoding TaDTG6 protein with an expression vector, and sequentially transforming agrobacterium and transforming plants; the amino acid sequence of the TaDTG6 protein is shown as SEQ ID No. 1;

   the plant is wheat and/or Arabidopsis thaliana.
3. 3. A method of growing according to claim 2, wherein the expression vector comprises a binary agrobacterium vector or a vector for plant microprojectile bombardment.
4. 4. A method of breeding according to claim 3, wherein the expression vector comprises pGKX or pCAMBIA3301.
5. 5. The cultivation method as claimed in claim 4, wherein said gene is ligated between BamHI and XhoI cleavage sites of pGKX;

   alternatively, the gene was ligated between HindIII and EcoRI sites of pCAMBIA3301.
6. 6. The cultivation method as claimed in claim 2, wherein said Agrobacterium comprises Agrobacterium tumefaciens EHA105 or Agrobacterium tumefaciens strain GV3101 containing pSoup plasmid.

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