Application of miR9772 and precursor sequence thereof in regulation and control of drought resistance and salt resistance of wheat

Abstract

The invention relates to the technical field of gene engineering, in particular to application of miR9772 and a precursor sequence thereof in regulation and control of drought resistance and salt resistance of wheat. The application of the wheat miR9772, the precursor sequence of the wheat miR9772, the biological material containing the encoding gene of the wheat miR9772 or the biological material containing the encoding gene of the precursor sequence of the wheat miR9772 in any one of the following items: a, regulating and controlling the drought resistance of the wheat; b, regulating the salt stress resistance of wheat; c, breeding drought-resistant wheat; d, breeding salt-tolerant wheat; and e, improving drought resistance and salt resistance of wheat germplasm resources. According to the invention, the wheat-specific miR9772 and the precursor thereof are characterized for the first time to regulate the drought resistance and salt resistance of wheat, and a new gene resource is provided for improving the stress resistance of crops.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to the application of miR9772 and its precursor sequence in regulating drought resistance and salt tolerance of wheat. Background Art

[0002] As one of the major food crops in the world, wheat (Triticum aestivum L.) covers one-fifth of the world's crop planting area, and more than 40% of the global population uses it as the main food source. However, due to the continuous deterioration of the global climate, environmental stresses such as drought, salinity, temperature changes, and heavy metal pollution have significantly affected the yield and quality of wheat. Therefore, developing wheat varieties with stress resistance and stable yields has become a key goal in current wheat breeding work. In-depth research and understanding of the key genes and their molecular mechanisms involved in coping with abiotic stresses are of crucial significance for ensuring global food security. Drought and salinity stresses are the main abiotic factors restricting crop growth and yield formation. In order for plants to maintain normal growth and development under these adverse conditions, they must sense and respond to various disturbances or stresses by adjusting their own cellular and physiological states. Mining and applying drought- and salt-tolerant genes in wheat and its relatives to breed new drought- and salt-tolerant wheat varieties is the most direct and effective strategy to cope with drought and saline-alkali hazards.

[0003] MicroRNA (miRNA) is a class of non-coding RNA molecules composed of 21-22 nucleotides. They act as regulators at the post-transcriptional level. By binding to the mRNA of target genes and under the action of the RISC (RNA-induced silencing) complex, they regulate gene expression by degrading mRNA or inhibiting its translation. This process is involved in various biological processes such as the growth and development of organisms, morphological construction, and response to adversity. In the field of plants, in recent years, significant progress has been made in the discovery of plant salt-tolerant miRNAs and the study of their regulatory mechanisms. Numerous studies have revealed the key regulatory role of miRNA in the process of plant perception, response, and adaptation to salt stress. Multiple miRNA families related to plant salt stress signal response have been identified and reported, such as miR156, miR159, miR166, miR167, miR172, miR319, and miR393, etc., which are highly conserved in species such as Arabidopsis thaliana, rice, and maize. Nevertheless, the research on the function of miRNAs in Triticeae plants under salt stress is still in its infancy. Therefore, exploring and applying miRNAs related to drought resistance and salt tolerance in Triticeae plants is of great significance for enriching wheat stress-resistant gene resources and promoting wheat stress-resistant breeding. Summary of the Invention

[0004] The object of the present invention is to provide the application of miR9772 and its precursor sequence in regulating the drought resistance and salt tolerance of wheat.

[0005] In order to achieve the above object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the application of wheat miR9772, the precursor sequence of wheat miR9772, the biological material containing the coding gene of wheat miR9772 or the biological material containing the coding gene of the precursor sequence of wheat miR9772 in any one of the following:

[0007] a. For regulating the drought resistance ability of wheat;

[0008] b. For regulating the salt stress tolerance ability of wheat;

[0009] c. For breeding drought-resistant wheat;

[0010] d. For breeding salt-tolerant wheat;

[0011] e. For improving the drought resistance and salt tolerance of wheat germplasm resources;

[0012] The nucleotide sequence of the said miR9772 is shown as SEQ ID NO.1; the precursor sequence of the said miR9772 is shown as SEQ ID NO.2 to NO.10.

[0013] Preferably, the said biological material includes any one of the following:

[0014] d1. A DNA molecule encoding the coding gene of the said wheat miR9772 or a DNA molecule encoding the coding gene of the precursor sequence of wheat miR9772;

[0015] d2. An expression cassette containing the DNA molecule described in d1;

[0016] d3. A recombinant vector containing the DNA molecule described in d1 or a recombinant vector containing the expression cassette described in d2;

[0017] d4. A recombinant microorganism containing the DNA molecule described in d1 or a recombinant microorganism containing the expression cassette described in d2, or a recombinant microorganism containing the recombinant vector described in d3;

[0018] d5. A transgenic wheat cell line containing the DNA molecule described in d1, or a transgenic wheat cell line containing the expression cassette described in d2, or a transgenic wheat cell line containing the recombinant vector described in d3;

[0019] d6. A transgenic wheat tissue or organ containing the DNA molecule described in d1, or a transgenic wheat tissue or organ containing the expression cassette described in d2, or a transgenic wheat tissue or organ containing the recombinant vector described in d3.

[0020] Preferably, the recombinant vector is an overexpression vector or a silencing vector.

[0021] The present invention also provides a method for improving the drought resistance and salt tolerance of wheat, by reducing the expression level or content of the wheat miR9772 in the target wheat plants to improve the drought resistance and salt tolerance of wheat.

[0022] Preferably, specifically, an STTM silencing vector is constructed by the short tandem target technology and introduced into the target wheat plants to achieve the reduction of the expression level or content of wheat miR9772.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The miR9772 or its precursor sequence disclosed in the present invention is derived from Triticeae plants, including wild emmer wheat, durum wheat, Aegilops tauschii and common wheat; the mature sequence of miR9772 is as shown in SEQ ID NO.1, and its precursor sequence is as shown in SEQ ID NOs.2 - NO.10. The result of multiple sequence alignment analysis shows that miR9772 is highly conserved in Triticeae crops and encodes the same mature sequence. The present invention constructs the miR9772 precursor coding sequence derived from wild emmer wheat into an overexpression vector for the first time, and constructs a miR9772-STTM silencing vector at the same time. The two vectors are introduced into wheat Fielder by genetic transformation to construct miR9772 overexpression lines and silencing lines; the identification results of the drought resistance and salt tolerance of each line show that miR9772 negatively regulates the drought resistance and salt tolerance of wheat. The present invention characterizes for the first time the function that the Triticeae-specific miR9772 and its precursor can regulate the drought resistance and salt tolerance of wheat, providing new gene resources for improving the stress resistance of crops.

[0025] The present invention verifies the function of the Triticeae-specific miR9772 in wheat for the first time. The research results reveal that overexpressing the miR9772 precursor sequence in wheat Fielder increases the expression level of Ttu-miR9772 and reduces the drought resistance and salt tolerance of transgenic overexpression plants; silencing miR9772 by the STTM technology reduces the expression level of miR9772 and improves the drought resistance and salt tolerance of transgenic silencing plants. It shows that the negative regulator miR9772 can be used as a gene editing target to improve the drought resistance and salt tolerance of wheat by gene editing breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the cloning electrophoresis map of the Td-mR9772 precursor.

[0027] Figure 2Analysis of wheat miR9772 and its precursor sequence. Among them, (A) is the secondary structure analysis of the pre-Td-miR9772 precursor of wild emmer wheat; (B) is the multiple sequence alignment analysis of the wheat miR9772 precursor sequence. The mature sequence of miR9772 is within the red box. In the sequence names, Tu represents Triticum urartu, Tt represents Triticum durum, Td represents wild emmer wheat, Ta represents common wheat, and Ae represents Aegilops tauschii.

[0028] Figure 3 Analysis of the expression characteristics of Td-miR9772 under salt stress. Among them, (A) is the analysis of the expression level of the salt-tolerant line A5 of wild emmer wheat; (B) is the analysis of the expression level of the salt-sensitive line C2 of wild emmer wheat.

[0029] Figure 4 Schematic diagram of the miR9772 expression vector structure and analysis of the expression level of transgenic lines. Among them, (A) is the structure of the Td-miR9772-OE overexpression vector; (B) is the structure of the miR9772-STTM silencing vector; (C) is the analysis result of the expression of the T3 generation of the Td-miR9772 overexpression line; (D) is the analysis result of the expression of the T3 generation of the miR9772 silencing line.

[0030] Figure 5 Analysis results of the phenotypes of miR9772 transgenic lines under salt stress. Among them, (A) is the phenotypic morphology of each line after salt stress treatment; (B) is the statistical analysis result of the survival rate after salt treatment.

[0031] Figure 6 Analysis results of the phenotypes of miR9772 overexpression lines under drought stress. Among them, (A) are the morphological diagrams of each line before drought stress and 3 days after rehydration after stress treatment, and (B) is the statistical analysis result of the survival rate 3 days after rehydration.

[0032] Figure 7 Analysis results of the phenotypes of miR9772 silencing lines under drought stress. Among them, (A) are the morphological diagrams of each line before drought stress and 3 days after rehydration after stress treatment, and (B) is the statistical analysis result of the survival rate 3 days after rehydration. Detailed implementation manners

[0033] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Experimental materials: The salt-tolerant line A5 and salt-sensitive line C2 of wild emmer wheat, and common wheat Fielder; preserved in the College of Agronomy, Northwest A&F University.

[0035] The sequences involved in the present invention are as follows:

[0036]

[0037]

[0038]

[0039] Example 1 Amplification of pre-Td-miR9772 precursor sequence

[0040] 1. Select plump and uniform-sized seeds of the salt-tolerant line A5 of wild emmer wheat. After disinfection and sterilization, sow them on filter paper, treat them at 4°C for 3 days until they show white tips, and then transfer the germinated seeds to nutrient soil and culture them at 22°C for two weeks to obtain seedlings.

[0041] 2. Take the leaves of wheat seedlings and extract DNA using the CTAB method. Using the obtained genomic DNA as a template, perform PCR amplification with the primer pair consisting of pre-Td-miR9772-F (5’-AGGAAATCCTATCCTTGTACTGC-3’; SEQ ID NO.11) and pre-Td-miR9772-R (5’-TTGGATTATTTTCGGTGTGCAGG-3’; SEQ ID NO.12) to obtain the PCR amplification product of the Td-miR9772 precursor sequence.

[0042] Table 1 PCR amplification system

[0043]

[0044] 3. The PCR reaction program is: pre-denaturation at 95°C for 30 sec; denaturation at 95°C for 5 sec, annealing at 55°C for 10 sec, extension at 72°C for 1 min, with 35 cycles; extension at 72°C for 10 min.

[0045] 4. Detect the PCR product by 1% agarose gel electrophoresis ( Figure 1 ).

[0046] 5. Ligate the PCR amplification product to a cloning vector to obtain the pMD18-Td-miR9772 recombinant cloning vector, transform it into Escherichia coli, and pick monoclonal colonies for sequencing.

[0047] The experimental results show that the Td-miR9772 precursor sequence of the PCR amplification product is consistent with the reference sequence Td-miR9772-5B (as shown in SEQ ID NO.2). Fold the secondary structure of the Td-miR9772 precursor sequence using the RNAfold online software (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi) Figure 2In (A), its precursor sequence folds into a stable secondary stem-loop structure, which conforms to the structural characteristics of miRNA precursors.

[0048] Example 2 Identification and Sequence Analysis of miR9772

[0049] The mature sequence of miR9772 is 21 bp in length and is specifically shown as SEQ ID NO.1. Further, using the keyword "miR9772" to search the miRbase database (https: / / www.mirbase.org / ) and the Rfam database (https: / / rfam.org / ), it is found that miR9772 is only found in wheat crops, including common wheat (Triticum aestivum L.), durum wheat (Triticum turgidum L. var. durum), Triticum urartu Thumanjan ex Gandilyan, wild emmer wheat (Triticum turgidum L. var. dicoccoides), and Aegilops tauschii Coss., indicating that miR9772 is a wheat-specific miRNA. Sequence analysis shows that the sequence region encoded by miR9772 is located on chromosome 5; multiple sequence alignment analysis shows that the precursor sequences of wheat miR9772 are highly conserved and encode the exactly same mature sequence of miR9772 Figure 2 In (B); the precursor sequences are shown as SEQ ID NO.2 to NO.10.

[0050] Example 3 Analysis of the Expression Characteristics of Td-miR9772 under Salt Stress Conditions

[0051] 1. Select 50 seeds each of the salt-tolerant line A5 and the salt-sensitive line C2 of wild emmer wheat that are plump and of the same size. Disinfect them with sodium hypochlorite for 10 min, wash them 8 times with distilled water, then place them in a petri dish filled with ceramsite, add 5 mL of water, take them out after placing them at 4°C for 3 days, and place them in a culture room for cultivation. When the seedling height grows to 5 cm, transplant them into a hydroponic box.

[0052] 2. When the seedlings have two leaves and one heart, treat them with 150 mM NaCl, and take their leaves at 0, 0.5, 3, 8, and 27 h respectively for RNA extraction. Use the Trizol method to extract the total RNA of the leaves, and then use the miRNA cDNA first-strand synthesis kit to synthesize cDNA.

[0053] 3. Using the reverse-transcribed cDNA as a template and Td-U6 as an internal reference, design a specific upstream primer (qmiR9772-F) according to the mature sequence of miR9772, and use the primers provided with the reagent as the downstream primer for fluorescence quantitative PCR.

[0054] The upstream primer for detecting the expression level of Td-miR9772 is 5'-TGAGATGAGATTACCCCATAC-3'; SEQ ID NO.13. The primer pair for detecting the expression level of Td-U6 is 5'-GGGGACATCCGATAAAAT-3'; SEQ ID NO.14 and 5'-TTTGTGCGTGTCATCCTT-3'; SEQ ID NO.15.

[0055] Fluorescent quantitative PCR system:

[0056] 1) Preparation of reaction system

[0057] Refer to the TB Premix Ex Taq TM II (Tli RNaseH Plus) instruction manual for the preparation method of the Applied Biosystems 7500 / 7500Fast Real-Time PCR System. First, prepare the PCR reaction solution on ice according to the amount of reaction number + 2, and then aliquot it into each PCR tube after the first step of the reaction.

[0058] Table 2 Fluorescent quantitative PCR reaction system

[0059]

[0060]

[0061] 2) Real-Time PCR reaction program

[0062] The Real-Time PCR reaction program performs PCR amplification according to the standard two-step method program in the instruction manual. The specific reaction program is as follows:

[0063] Table 3 Reaction program

[0064]

[0065] The detection results are shown in Figure 3 . The results show that the expression of Td-miR9772 is induced by salt stress, down-regulated in the salt-tolerant line A5, and up-regulated in the salt-sensitive line C2, suggesting that it may be a negative regulator of salt stress.

[0066] Example 4 Construction of overexpression and silencing vectors of Td-miR9772 and Agrobacterium transformation

[0067] (1) Construction of the overexpression vector of Td-miR9772

[0068] Digest the CUB expression vector with two enzymes, Spe I and Sac I. The digestion system is as follows: 1 μg of CUB plasmid, 1 μl each of Spe I and Sac I enzymes, 3 μl of 10×Quick buffer, and make up to 30 μl with ddH 2 O; After mixing the reagents, place them in a PCR instrument and digest at 37 °C for 3 h, and inactivate the enzymes at 85 °C for 15 min to obtain a linearized vector. According to the designed sequence of the Td-miR9972 precursor and the flanking sequences of the vector digestion sites, seamless cloning primers Td-miR9772-F (5’-TGAAAAAGAGGGGGATTACCCGGGACCCTAATTCCCAAAACGAAGC-3’; SEQ ID NO.16) and Td-miR9772-R (5’-GAACGATCGGGGAAATTCGAGCTCAAGGTTTCCAAGAAGATGACAAC-3’; SEQ ID NO.17) were designed and synthesized. Use the seamless cloning primers to PCR amplify the target fragment from the extracted plasmid of the cloning vector pMD-Td-miR9772, cut and recover the gel by agarose gel electrophoresis, and ligate the gel recovery product with the linearized vector. The ligation system is as follows: 1 μl of the gel recovery target fragment, 2 μl of the linearized vector, and 3 μl of DNA assembly mix. After mixing, place it in a PCR instrument and ligate at 50 °C for 30 min. After ligation, transform Escherichia coli, pick monoclonal colonies and perform colony PCR with the universal primers pUbi-F (5’-GGCATATGCAGCA TCTATTC-3’; SEQ ID NO.18) and M13-R (5’-CAGGAAACAGCTATGAC-3’; SEQ ID NO.19). After colony PCR identification, send the bacterial solution with the correct band size to the company for sequencing to determine whether the sequence is correct. Subsequently, expand and culture the correct bacterial solution and extract the plasmid to construct the pUbi::Ttu-miR9772::Nos overexpression vector Figure 4 in (A)]. The PCR system and procedure are as follows:

[0069] Table 4 PCR identification reaction system

[0070]

[0071] PCR reaction procedure: Pre-denature at 95 °C for 30 sec; Denature at 95 °C for 5 sec, anneal at 55 °C for 30 sec, extend at 72 °C for 1 min, cycle 35 times; Extend at 72 °C for 10 min.

[0072] (2) Construction of the miR9772 silencing vector

[0073] To further verify the function of miR9772, based on the identical sequences of miR9772 in Triticeae crops, a silencing vector of miR9772 was constructed and transferred into wheat Fielder to silence miR9772 in the recipient wheat and reduce its expression level. According to the structure of the STTM silencing vector, oligonucleotide fragments were ligated according to the STTM structure through a 48-bp linker sequence linker-STTM to obtain the STTM9772 sequence, and SpeⅠ and SacⅠ restriction enzyme sites were added to both ends of the STTM9772 sequence. The Ttu-STTM9772 sequence with restriction enzyme sites was synthesized onto the intermediate vector pUC57, and seamless cloning primers were designed simultaneously; the STTM9772 structure was amplified by PCR using the seamless cloning primers STTM9772-F (5’-CTTTGAAAAAGAGGGGGATTACCCGGGGTATGGGGTACTAATCTC ATC-3’; SEQ ID NO.20) and STTM9772-R (5’-GAACGATCGGGGAAATTCGAGCTCTG AGATGAGATTAGTACCCCATAC-3’; SEQ ID NO.21) and recovered. The vector was linearized, ligated, transformed, and verified as described above to construct the pUbi::STTM9772::Nos silencing vector Figure 4 in (B).

[0074] Table 5 PCR amplification system

[0075]

[0076]

[0077] PCR reaction program: pre-denaturation at 95℃ for 30 sec; denaturation at 95℃ for 5 sec, annealing at 58℃ for 30 sec, extension at 72℃ for 1 min, 35 cycles; extension at 72℃ for 10 min.

[0078] (3) Transformation of Agrobacterium with miR9772 overexpression and STTM9772 silencing vectors

[0079] Add 2 μg of the pUbi::pre-Td-miR9772::Nos recombinant plasmid and 2 μg of the pUbi::STTM9772::Nos recombinant plasmid to 100 μL of Agrobacterium tumefaciens GV3101 competent cells respectively, mix gently, and place on ice for 30 minutes; quickly freeze in liquid nitrogen for 10 minutes, and then heat shock in a 37°C water bath for 5 minutes; add 0.6 mL of antibiotic-free liquid LB medium, culture at 28°C with low-speed oscillation at 180 revolutions per minute for 3 hours; then centrifuge at 4000 revolutions per minute to remove 0.6 mL of the supernatant, and suspend the remaining 100 μL of bacteria with a pipette and evenly spread them on an LB plate medium containing 50 μg / mL rifampicin and 50 μg / mL kanamycin, and incubate in the dark at 28°C for 48 h; pick the transformed colonies and culture them in a liquid LB medium containing 50 μg / mL rifampicin and 50 μg / mL kanamycin and perform PCR amplification detection to obtain Agrobacterium strains containing the pUbi::pre-Td-miR9772::Nos recombinant vector and the pUbi::STTM9772::Nos recombinant vector respectively.

[0080] Example 5 Establishment and Screening of miR9772 Transgenic Wheat Plants

[0081] Transform the Agrobacterium containing the overexpression of Td-miR9772 and the silencing recombinant vector of STTM9772 into the wheat variety Fielder (hereinafter also referred to as wild-type wheat) by Agrobacterium-mediated gene transformation method to obtain T0 generation plants, and plant them in a phytotron (16 h - light / 8 h - dark); take about 0.1 g of transgenic wheat leaves of 1-month-old T0 generation, extract DNA using the CTAB method and perform PCR identification using the universal primers pUbi-F (5’-GGCATATGCAGCATCTATTC-3’; Sequence 18) and M13-R (5’-CAGGAAACAGCTATGAC-3’; Sequence 19) to obtain positive plants, and self-cross to obtain T1 generation seeds; harvest the seeds of T1 generation positive seedlings individually, dry them in an oven at 37°C for generation multiplication, and the seeds of each individual plant are a plant line until the T 3 generation homozygous plant line is screened.

[0082] Table 6 PCR Amplification System

[0083]

[0084]

[0085] PCR reaction procedure: Pre-denature at 95°C for 30 sec; denature at 95°C for 5 sec, anneal at 58°C for 30 sec, extend at 72°C for 1 min, cycle 35 times; extend at 72°C for 10 min.

[0086] Furthermore, leaves of positive individual plants of the T3 generation were taken and Total RNA was extracted, reverse transcribed into cDNA using a miRNA cDNA first-strand synthesis kit, and then the expression level of Ttu-miR9772 in transgenic plants was verified using the SYBR fluorescent quantitative PCR method. The upstream primer used to detect the Ttu-miR9772 gene was qmiR9772-F5'-TGAGATGAGATTACCCCATAC-3'; sequence 13, and the lower primer was the primer provided by the miRNA cDNA first-strand synthesis kit; wheat TaU6 was used as an internal reference, and the primer pair was TaU6-F (5'-GGGGACATCCGATAAAAT-3' sequence 14) and TaU6-R (5'-TTTGTGCGTGTCATCCTT-3'; sequence 15).

[0087] Table 7 Fluorescence quantitative PCR reaction system

[0088]

[0089] Table 8 Fluorescence quantitative PCR reaction program

[0090]

[0091] The relative expression results of miR9772 gene are shown in Figure 6 The results showed that the overexpression lines [ Figure 4 The expression level of miR9772 in the STTM silenced line [ Figure 4 The expression level of miR9772 in (D)] was significantly lower than that in the wild type.

[0092] Example 6 Analysis of salt tolerance phenotype of miR9772 transgenic wheat

[0093] The seeds of T3 transgenic wheat overexpressing lines (OE9, OE28), miR9772 silencing lines (STTM13, STTM26) and wild-type wheat (WT) were sterilized and sown on culture dishes with double-layer filter paper. They were treated at 4°C for 3 days and then transferred to room temperature for germination for 3 days. The seedlings with consistent germination were transplanted into hydroponic boxes and cultured with 1 / 2 Hoagland nutrient solution for 10 days. Then, they were treated with nutrient solution containing 200 mM NaCl. The mortality rate was calculated after 30 days of stress treatment.

[0094] Results Figure 5: After 30 days of salt stress treatment, it was found that OE9772 was severely dried and died, with a survival rate of 20.83%. The Ttu-miR9772-silenced transgenic plants (STTM9772) grew relatively well, with a survival rate of 89.58%. The survival rate of the wild type was between the two, at 47.92%. The results showed that the gene silencing conferred better salt tolerance. Ttu-miR9772 played a negative regulatory role under salt stress.

[0095] Example 7 Analysis of the drought-resistant phenotype of miR9772 transgenic wheat

[0096] 1. Analysis of the drought-resistant phenotype of miR9772 overexpression lines

[0097] Seeds of the T3 generation miR9772 overexpression lines (OE9, OE28) and wild-type wheat (WT) plants were sown on a petri dish lined with double-layer filter paper, kept with sufficient moisture. After the seeds germinated and sprouted, they were transferred to pots filled with 250 g of nutrient soil and grown to the three-leaf stage under normal conditions. After thoroughly watering the substrate, watering was stopped for drought treatment. After 14 days, when the phenotypic differences were obvious, i.e., the leaves of the OE9 and OE28 lines were significantly dried and the leaves of the WT plants were severely wilted, rehydration was carried out. Three days after rehydration, the survival rates of the plants in each line were counted (plants that could grow normally and set seeds were defined as surviving plants, and plants that were severely damaged by drought and could not grow and set seeds 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 set with 3 replicates, and the average value was taken for statistical analysis. The statistical results are as Figure 6 shown. It can be seen that the survival rate of the T3 generation miR9772 overexpression lines after rehydration was lower than that of wild-type wheat.

[0098] 2. Analysis of the drought resistance of miR9772-silenced lines

[0099] Seeds of the T3 generation miR9772-silenced lines (STTM13, STTM26) and wild-type wheat (WT) plants were sown on a petri dish lined with double-layer filter paper, kept with sufficient moisture. After the seeds germinated and sprouted, they were transferred to pots filled with 250 g of nutrient soil and grown to the three-leaf stage under normal conditions. After thoroughly watering the substrate, watering was stopped for drought treatment. After 14 days, when the phenotypic differences were obvious, i.e., the leaves of the WT line were significantly dried and the leaves of the STTM13 and STTM26 plants were severely wilted, rehydration was carried out. Three days after rehydration, the survival rates of the plants in each line were counted. The experiment was set with 3 replicates, and the average value was taken for statistical analysis. The statistical results are as Figure 7 shown. It can be seen that the survival rate of the T3 generation miR9772-silenced lines after rehydration was higher than that of wild-type wheat. The above is only the preferred embodiment of the present invention.

Claims

1. Use of wheat miR9772, a precursor sequence of wheat miR9772, a biological material containing a gene encoding wheat miR9772, or a biological material containing a gene encoding a precursor sequence of wheat miR9772 in any of the following: a. Used to regulate wheat drought resistance; b. Used to regulate wheat's ability to tolerate salt stress; c. Used for breeding drought-resistant wheat; d. Used for breeding salt-tolerant wheat; e. Used to improve the drought resistance and salt tolerance of wheat germplasm resources; The nucleotide sequence of miR9772 is shown in SEQ ID NO.1; the precursor sequence of miR9772 is shown in SEQ ID NO.2 to NO.

10.

2. The use according to claim 1, characterized in that: The biological material includes any of the following: d1, a DNA molecule encoding the gene encoding wheat miR9772 according to claim 1 or a DNA molecule encoding the gene of the precursor sequence of wheat miR9772; d2, an expression cassette containing the DNA molecule described in d1; d3, a recombinant vector containing the DNA molecule described in d1 or a recombinant vector containing the expression cassette described in d2; d4, a recombinant microorganism containing the DNA molecule described in d1, or a recombinant microorganism containing the expression cassette described in d2, or a recombinant microorganism containing the recombinant vector described in d3; d5, a transgenic wheat cell line containing the DNA molecule described in d1, or a transgenic wheat cell line containing the expression cassette described in d2, or a transgenic wheat cell line containing the recombinant vector described in d3; d6. Transgenic wheat tissues and organs containing the DNA molecule described in d1, or transgenic wheat tissues and organs containing the expression cassette described in d2, or transgenic wheat tissues and organs containing the recombinant vector described in d3.

3. The use according to claim 2, characterized in that: The recombinant vector is an overexpression vector or a silencing vector.

4. A method for improving drought resistance and salt tolerance of wheat, characterized in that: The expression level or content of the wheat miR9772 of claim 1 is reduced in the target wheat plant to improve the drought resistance and salt tolerance of wheat.

5. The method according to claim 4, characterized in that Specifically, the STTM silencing vector is constructed through short tandem target technology and introduced into the target wheat plants to achieve a reduction in the expression level or content of wheat miR9772.

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