Rice drought-resistant gene and its application in plant drought-resistant breeding

Abstract

The application discloses an application of a drought-resistant gene of rice in regulating drought resistance of plants and / or cultivating drought-resistant plant varieties, and belongs to the technical field of genetic engineering. The nucleotide sequence of the drought-resistant gene is shown in SEQ ID NO. 1, or is completely complementary to the sequence shown in SEQ ID NO. 1, or is a nucleotide sequence for coding the amino acid sequence shown in SEQ ID NO. 2. The drought resistance of plants can be improved by overexpressing the drought-resistant gene, and the application can be applied to plant genetic engineering breeding, and provides a new method for creating or improving new plant drought-resistant germplasm.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a rice drought-resistant gene and application thereof in plant drought-resistant breeding. BACKGROUND

[0002] Drought is the most common abiotic stress, which is extremely harmful to the normal growth and development of plants and can cause wilting of leaves and even death of the whole plant. In the current agricultural production field, as a key component of food crops, the growth status of Gramineae is closely related to the stability of food supply. Common Gramineae food crops such as wheat, rice, corn, millet and broomcorn millet are not only an important source of human staple food, but also occupy an important position in feed and industrial raw materials. Under the current threat of drought, cultivating excellent drought-resistant crop varieties is an important way to solve the problem of drought resistance. However, the genetic regulation network of plant drought resistance is complex, and the molecular regulation mechanisms of plants with different genetic backgrounds are quite different. Normal growth and development of Gramineae food crops requires consumption of a large amount of fresh water, and drought and water shortage is one of the main limiting factors of food production. It is still a technical problem to be solved to mine new rice drought-resistant genes and develop new methods for cultivating drought-resistant plant varieties. SUMMARY

[0003] The purpose of the present application is to provide a rice drought-resistant gene and application thereof in regulating plant drought resistance and / or cultivating drought-resistant plant varieties.

[0004] The present application verifies the regulation effect of the rice drought-resistant gene on the drought resistance of rice through transgenic experiments and phenotype analysis. The nucleotide sequence of the drought-resistant gene is shown in SEQ ID NO. 1, or is a nucleotide sequence completely complementary to the sequence shown in SEQ ID NO. 1, or is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2. The gene is expected to be applied to the genetic engineering breeding of plants and provide a theoretical basis for the creation or improvement of new plant drought-resistant germplasm. Considering the degeneracy of codons, modification of the bases of the above-mentioned nucleotide sequences without changing the amino acid sequence also falls within the protection scope of the present application.

[0005] In one aspect, the present application provides a rice drought-resistant protein, characterized in that the amino acid sequence of the drought-resistant protein is shown in SEQ ID NO. 2.

[0006] In another aspect, the present application provides a rice drought-resistant gene, and the sequence of the drought-resistant gene is a nucleotide sequence shown in SEQ ID NO. 1, or a nucleotide sequence completely complementary to the sequence shown in SEQ ID NO. 1, or a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2.

[0007] In another aspect, the present application provides an expression cassette, a recombinant vector, a recombinant cell, a transgenic plant tissue or a transgenic plant containing the aforementioned drought-resistant gene.

[0008] In another aspect, the present application provides the aforementioned protein or gene for use in regulating drought resistance of a plant.

[0009] Further, the regulation of drought resistance of a plant is to increase the drought stress tolerance of a plant by increasing the expression of the aforementioned protein or gene, or to decrease the drought stress tolerance of a plant by decreasing the expression of the aforementioned protein or gene.

[0010] In another aspect, the present application provides the aforementioned protein or gene for use in breeding drought-resistant plant varieties or improving drought-resistant germplasm resources of a plant.

[0011] Further, a plant variety with high tolerance to drought stress is obtained by increasing the expression of the aforementioned protein or gene, or a plant variety sensitive to drought stress is obtained by decreasing the expression of the aforementioned protein or gene.

[0012] Further, in the aforementioned use, the drought-resistant gene is introduced into a plant cell, tissue or organ, and the transformed plant cell, tissue or organ is cultivated into a plant to obtain a transgenic plant with improved drought resistance.

[0013] In another aspect, the present application also provides a method for improving drought resistance of a plant, characterized in that it comprises the following steps:

[0014] Increasing the expression of the aforementioned drought-resistant protein or gene in a plant.

[0015] Preferably, the expression of the aforementioned drought-resistant protein or gene in a plant is increased by introducing the aforementioned drought-resistant gene into the plant.

[0016] Further, in any of the aforementioned uses or methods, the plant is a monocotyledon.

[0017] Further, the plant is a plant of the family Poaceae.

[0018] Further, the plant is a plant of the genus Oryza.

[0019] Further, the plant is rice.

[0020] Compared with the prior art, the present application has the following advantages:

[0021] 1) The present application provides a new method for regulating drought resistance of a plant and / or breeding drought-resistant plant varieties, i.e. regulating drought resistance of a plant and / or obtaining drought-resistant plant varieties by overexpressing a drought-resistant gene of rice or a protein encoded thereby.

[0022] 2) The new method for regulating drought resistance of plants and / or breeding drought-resistant plant varieties provided by the present application can be used for plants with different genetic backgrounds. BRIEF DESCRIPTION OF DRAWINGS

[0023] The rice drought-resistant gene and its application in plant drought-resistant breeding and its beneficial effects will be described in detail below in combination with the drawings and specific embodiments.

[0024] Figure 1 The figure shows the expression frame schematic diagram of the overexpression vector of the rice drought-resistant gene.

[0025] Figure 2 The figure shows the drought-resistant gene expression analysis in transgenic rice; wherein, Nip represents Nipponbare; OE#7 and OE#8 represent drought-resistant gene overexpression plants.

[0026] Figure 3 The figure shows the PEG simulated drought treatment phenotype of transgenic plants and Nip plants; wherein, Nip represents Nipponbare; OE#7 and OE#8 represent drought-resistant gene overexpression plants; A figure is the growth phenotype of rice seedlings treated with 20% PEG6000 for 7 days, and then rehydrated and grown for 5 days; B figure is the survival rate of rice seedlings treated with 20% PEG6000 for 7 days, and then rehydrated and grown for 5 days.

[0027] Figure 4 The figure shows the soil drought treatment phenotype of transgenic plants and Nip plants; wherein, Nip represents Nipponbare; OE#7 and OE#8 represent drought-resistant gene overexpression plants; A figure is the phenotype of rice seedlings treated with drought for 12 days until all leaves are rolled, and then rehydrated and grown for 10 days; B figure is the survival rate of rice seedlings treated with drought for 12 days until all leaves are rolled, and then rehydrated and grown for 10 days. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be described in detail below, obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0029] Unless otherwise defined, all technical and scientific terms used in the present application have the same meanings as commonly understood by those skilled in the art to which the present application belongs.

[0030] The nucleotide sequence of the rice drought-resistant gene in the embodiments is shown in SEQ ID NO. 1:

[0031] ATGGCCGGCCTGAGTGTCCTTCTTGAGACACACAAGAACGATCACCACCCCAACATGAGGCCTCCCCAAATCATCAGCAAGGCCACCCTCCATAGCCACCCAGAGACGATGTCCTCCTCCTCTCCGGCGACTGCGACGACGGCGACGATGAGCTCCTTCTTGCAGCGCTGCTTCCTCTGCCGCAGGGAGCTCGCCGACGGCAAGGACATCTACATGTACAGAGGGGACAGAGCGTTCTGCAGCGTGGATTGCCGGTGCAAGCAGATCTTCATGGACGAGGACGCCGCCGCCGCCGGCGGCAACTGCGCCGCGGTGCGCGCCGGCCGGCGGCGGGCGGCCGTACCGCGCGAGCAAACCGGCGCCGGTGGCTTCGCGTACTGA.

[0032] The amino acid sequence of the drought resistance protein of rice is shown as SEQ ID NO. 2:

[0033] MAGLSVLLETHKNDHHPNMRPPQIISKATLHSHPETMSSSSPATATTATMSSFLQRCFLCRRELADGKDIYMYRGDRAFCSVDCRCKQIFMDEDAAAAGGNCAAVRAGRRRAAVPREQTGAGGFAY.

[0034] Example 1

[0035] Construction of the overexpression vector of the drought resistance gene of rice

[0036] Take 0.5 g of rice seedlings germinated and grown on moist filter paper for 10 days, extract total RNA using a plant RNA extraction kit (Magen Company), and then amplify the target gene. The specific experiment is as follows:

[0037] Take 1 μg high-quality (OD 260 / OD 280 : 1.8-2.0; OD 260 / OD 230The first-strand cDNA was obtained by reverse transcription (Novagen reverse transcription kit) of the RNA. The cDNA was used as a template for PCR amplification using KOD FX high-fidelity enzyme (ToYoBo). The reaction system was as follows: 2x PCR buffer 10 μL, 2 mM dNTPs 2 μL, F primer (5'-GATTAACAGGGATCCCCCATGGCCGGCCTGAGTGTCCTT-3') 0.5 μL, R primer (5'-GAGACTAGTGGTACCCCCGTACGCGAAGCCACCGGCGC-3') 0.5 μL, cDNA template 1 μL, KOD FX (1 U / μl) 0.4 μL, and water to 20 μL. The reaction conditions were as follows: 98 °C for 5 min; 98 °C for 15 sec, 56 °C for 30 sec, 68 °C for 30 sec, 35 cycles; 68 °C for 5 min. After the reaction, the PCR product was recovered by a gel recovery kit (Magen).

[0038] The pCAMBIA1300-GFP vector (modified from pCAMBIA1300 vector, containing the Ubiquitin 10 promoter and the GFP tag) was linearized by the restriction enzyme Sma I (NEB). The enzyme cutting system was as follows: 2 μg of vector, 2 μL of Sma I endonuclease, 10x rCutSmart buffer 10 μL, and water to 100 μL. The enzyme cutting conditions were as follows: 25 °C, 2 hours. After the vector enzyme cutting product and the PCR product were purified and recovered, the DNA concentration was determined by NanoDrop 2000, and recombination was performed using a recombination kit (Novagen). The recombination system was as follows: target fragment: 14 ng, vector fragment: 130 ng, 5x buffer 2 μL, Exnase II 1 μL, and water to 10 μL. The recombination conditions were as follows: 37 °C, 30 min. The entire product was added to 100 μL of E. coli DH5α competent cells, and the transformation product was plated on LB solid medium (containing kanamycin resistance, and the concentration of kanamycin was 50 mg / L). The culture was incubated at 37 °C overnight, and 4 single colonies were selected for colony PCR identification. Two positive clones were selected for sequencing, and a positive clone containing the drought-resistant gene sequence was obtained. Finally, an overexpression vector containing the drought-resistant target gene was obtained. Figure 1 ).

[0039] Example 2

[0040] Obtaining and identification of drought-resistant rice

[0041] The overexpression vector constructed in Example 1 was introduced into japonica rice variety Nipponbare by Agrobacterium EHA105-mediated genetic transformation method. The T0 generation transgenic seedlings were obtained by selection culture, differentiation, rooting, and seedling culture. The experimental method was referred to the reference "Study on Agrobacterium-mediated high-efficiency rice genetic transformation system", Zheng Jie, Hunan Agricultural Science, 2008, No. 2.

[0042] PCR identification was performed on all transgenic seedlings, and the GFP vector fragment was amplified. Ten positive transgenic seedlings were propagated to obtain T1 and T2 generations. The T2 generation seeds were germinated using a medium containing hygromycin. If the seeds can grow normally, it is proved that the strain is a homozygous strain. Two transgenic strains (OE#7, OE#8) were selected for qRT-PCR detection, and subsequent experiments and analysis were performed.

[0043] The overexpression effect of the drought-resistant gene in the transgenic rice was identified by qRT-PCR as follows:

[0044] 1. The total RNA of the rice leaf at the four-leaf stage was extracted by the plant RNA extraction kit (Magen Company). 1 μg of high-quality (OD 260 / OD 280 : 1.8-2.0; OD 260 / OD 230 ≈ 2.0) RNA was reverse transcribed (Novozyme reverse transcription kit) to obtain the first strand cDNA.

[0045] 2. The cDNA in step 1 was used as a template to detect the expression of the drought-resistant gene by qF (CTTGAGACACACAAGAACGATC) and qR (CCCTCTGTACATGTAGATGTCC) primers. The expression of the rice housekeeping gene UBQ gene was detected by OsUBQ-qF (ACCACTTCGACCGCCACTACT) and OsUBQ-qR (ACGCCTAAGCCTGCTGGTT) primers as an internal reference. The quantitative PCR reagent was SYBR Premix Ex Taq™ (TAKARA Company), and the quantitative PCR instrument was CFX 96 (Bio RAD Company). The reaction system was as follows: 2×PCR buffer 5 μL, qF primer 0.4 μL, qR primer 0.4 μL, cDNA template 1 μL, sterilized water 3.2 μL, and the total volume of the reaction system was 10 μL. The reaction program was as follows: 95℃ for 30 sec; 95℃ for 5 sec, 68℃ for 30 sec, and 45 cycles.

[0046] The results are shown in Table 1. Figure 2As shown, the expression of drought-resistant genes in the selected two strains was greatly improved, and the two overexpression strains were selected for subsequent experiments.

[0047] Example 3

[0048] Phenotype analysis of drought-resistant gene overexpression rice under PEG simulated drought treatment

[0049] The homozygous overexpression plants and wild type Nip seeds were broken dormancy and germinated for 2 days. Then the uniform seedlings were selected and sowed in 96-well plates for further growth. After 2 weeks of growth, the rice seedlings were treated with 20% PEG6000 for about 7 days. Then the PEG6000 solution was washed away and the plants were watered for about 5 days for phenotype observation and photo taking.

[0050] The phenotype results are shown in Figure 3 A. The results show that the drought-resistant gene overexpression rice OE#7 and OE#8 are more drought-resistant than the wild type Nip. The survival rate of the drought-resistant gene overexpression rice OE#7 and OE#8 is significantly higher than that of the wild type Nip (see Figure 3 B).

[0051] Example 4

[0052] Phenotype analysis of drought-resistant gene overexpression rice under soil drought treatment

[0053] The homozygous overexpression plants and wild type Nip seeds were broken dormancy and germinated for 2 days. Then the uniform seedlings were selected and sowed in plant pots filled with soil for further growth. After 3 weeks of growth, watering was stopped and the plants were droughted for about 12 days until all the leaves were rolled. Then the plants were watered for about 10 days for phenotype observation and photo taking.

[0054] The phenotype results are shown in Figure 4 A. The results show that the drought-resistant gene overexpression rice OE#7 and OE#8 are more drought-resistant than the wild type Nip. The survival rate of the drought-resistant gene overexpression rice OE#7 and OE#8 is significantly higher than that of the wild type Nip (see Figure 4 B).

[0055] >SEQ ID NO. 1

[0056] ATGGCCGGCCTGAGTGTCCTTCTTGAGACACACAAGAACGATCACCACCCCAACATGAGGCCTCCCCAAATCATCAGCAAGGCCACCCTCCATAGCCACCCAGAGACGATGTCCTCCTCCTCTCCGGCGACTGCGACGACGGCGACGATGAGCTCCTTCTTGCAGCGCTGCTTCCTCTGCCGCAGGGAGCTCGCCGACGGCAAGGACATCTACATGTACAGAGGGGACAGAGCGTTCTGCAGCGTGGATTGCCGGTGCAAGCAGATCTTCATGGACGAGGACGCCGCCGCCGCCGGCGGCAACTGCGCCGCGGTGCGCGCCGGCCGGCGGCGGGCGGCCGTACCGCGCGAGCAAACCGGCGCCGGTGGCTTCGCGTACTGA.

[0057] >SEQ ID NO. 2

[0058] MAGLSVLLETHKNDHHPNMRPPQIISKATLHSHPETMSSSSPATATTATMSSFLQRCFLCRRELADGKDIYMYRGDRAFCSVDCRCKQIFMDEDAAAAGGNCAAVRAGRRRAAVPREQTGAGGFAY.

[0059] The above description of disclosed embodiments enables one of ordinary skill in the art to make and use the application. Various modifications to the above-described embodiments will be apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of a drought resistance protein or a gene encoding said drought resistance protein in modulating drought resistance in a plant, characterized in that, The amino acid sequence of the drought-resistant protein is shown as SEQ ID NO. 2, the drought-resistant protein is overexpressed to improve the drought resistance of the plant, and the plant is rice.

2. Use of a drought resistance protein or a gene encoding said drought resistance protein for breeding a drought resistant plant variety or for improving a plant's drought resistance germplasm, characterized in that, The amino acid sequence of the drought-resistant protein is shown as SEQ ID NO. 2, the drought-resistant protein is overexpressed to improve the drought resistance of the plant, and the plant is rice.

3. Use according to any one of claims 1-2, characterized in that, The gene encoding the drought-resistant protein is introduced into a plant cell, tissue or organ by constructing an overexpression vector of the gene, and the transformed plant cell, tissue or organ is cultivated into a plant to obtain a transgenic plant with improved drought resistance.

4. A method for improving drought tolerance in plants, characterized by, The method comprises the following steps: The drought-resistant protein with the amino acid sequence shown as SEQ ID NO. 2 or the gene encoding the drought-resistant protein is overexpressed in a plant, and the plant is rice.

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