Application of GSP5 protein or coding gene thereof in negative regulation of plant drought resistance

Abstract

The invention relates to the technical field of plant breeding, in particular to application of a GSP5 protein or a coding gene thereof in negative regulation of plant drought resistance. The research finds that the GSP5 protein can negatively regulate the drought resistance of the plant, and the drought resistance of the plant can be effectively improved by knocking out the encoding gene of the GSP5 protein in the plant. The GSP5 protein as well as the coding gene and the application thereof provided by the invention expand an effective solution for researching a rice drought stress response molecular mechanism and cultivating a new drought-tolerant rice variety through a gene editing method, and have important value in the field of plant breeding.

Description

Technical Field

[0001] The invention relates to the technical field of plant breeding, and in particular to the application of GSP5 protein or its encoding gene in negatively regulating plant drought resistance. Background Art

[0002] As one of the important food crops, increasing rice yield is crucial to ensuring food security. However, existing high-yield rice varieties rely on a large amount of irrigation water, and even moderate water deficits can cause serious reductions in rice yields. Cultivating new rice varieties that are water-saving and drought-resistant without or with less reduction in yield can effectively solve the problem of agricultural water resource shortage. However, due to the complexity of stress-related traits and the diversity of field environments, it is relatively difficult to breed high-yield and stable-yield varieties through traditional breeding approaches. With the rise of molecular design breeding, it is possible to aggregate multiple trait genes in a single variety. However, high and stable yields are often interrelated. Currently, the identified and available stress-resistant genes are still limited, and the genetic balance mechanism for high and stable yields is still unclear, which seriously restricts the application of molecular design strategies in high and stable yield breeding.

[0003] Drought stress has a significant inhibitory effect on plant growth and production. In order to cope with drought stress, plants have developed complex drought avoidance, drought tolerance and drought resistance adaptation mechanisms, using a variety of strategies to respond. For example, shortening the life cycle or accelerating flowering to avoid drought, resisting drought by optimizing water use (such as developing deep and thick root systems, closing stomata and increasing the thickness of leaf cuticles), and increasing cell osmotic pressure or increasing antioxidant capacity to cope with drought. In general, the response process of plants to drought stress involves the perception, conduction and amplification of stress signals, followed by changes at the morphological, physiological and molecular levels. Transcription factors play an indispensable role in known drought resistance pathways.

[0004] However, most of the genes currently used to improve drought resistance are achieved through heterologous expression or overexpression, which is not conducive to their application in molecular polymerization breeding. With the rise of molecular design breeding and the rapid development of gene editing technology, there is an urgent need to identify more candidate genes for improving rice drought tolerance. In particular, negative regulatory factors for drought resistance can be processed by gene editing methods on endogenous genes of plants. Although this processing process depends on the introduction of exogenous DNA, it can be quickly separated in the subsequent hybridization process, thereby producing plant individuals that do not carry exogenous DNA fragments at all, which can be directly used in crops, greatly shortening the breeding period. Therefore, finding and cloning negative regulatory genes for drought resistance has important guiding significance for achieving the breeding goal of high and stable yields. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides the use of GSP5 protein or its encoding gene in negatively regulating plant drought resistance.

[0006] In a first aspect, the present invention provides the use of GSP5 protein, or a gene encoding it, or a biological material comprising a gene encoding it in regulating plant drought resistance; the regulation is negative regulation.

[0007] The present invention further provides the use of GSP5 protein, or its encoding gene, or biological materials containing its encoding gene in cultivating plants with high drought resistance.

[0008] The present invention further provides the use of GSP5 protein, or its encoding gene, or biological materials containing its encoding gene in improving plant drought-resistant germplasm resources.

[0009] Furthermore, by reducing the expression level of the GSP5 protein in the plant, the drought resistance of the plant is improved.

[0010] Furthermore, the GSP5 protein comprises any one of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO.1; (2) An amino acid sequence of a protein having the same function as the amino acid sequence shown in SEQ ID NO.1 obtained by replacing, inserting or deleting one or more amino acids.

[0011] Preferably, the similarity between the above amino acid sequence (2) and the amino acid sequence (1) is 99%, 98%, 97%, 95%, 90%, 85% or 80% or more.

[0012] Furthermore, the gene encoding the GSP5 protein includes any one of the following nucleotide sequences: (1) the nucleotide sequence shown in SEQ ID NO.2; (2) A nucleotide sequence encoding a protein with the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.2; (3) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO. 2 under stringent conditions.

[0013] Preferably, the similarity between the above nucleotide sequence (2) and the nucleotide sequence (1) is 99%, 98%, 97%, 95%, 90%, 85% or 80% or more.

[0014] Furthermore, the plant is a monocotyledonous plant or a dicotyledonous plant, preferably rice.

[0015] In a second aspect, the present invention provides a nucleic acid comprising a nucleotide sequence as shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5.

[0016] Among them, SEQ ID NO.3 and SEQ ID NO.4 are the coding gene sequences of the mutated GSP5 protein. SEQ ID NO.5 is the sequence of the gRNA involved in the present invention.

[0017] The present invention further provides a biological material comprising the nucleic acid.

[0018] Furthermore, the biological material is an expression cassette, a vector or a transgenic cell.

[0019] The transgenic cells described in the present invention do not include transgenic cells that have the ability to independently develop into a complete individual, that is, do not include plant varieties.

[0020] In a third aspect, the present invention provides a method for cultivating transgenic plants, comprising: Regulating the expression level of GSP5 protein in the plant; The amino acid sequence comprises any of the following: (1) the amino acid sequence shown in SEQ ID NO.1; (2) an amino acid sequence having the same functional protein obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown in SEQ ID NO.1; Preferably, the expression level of the GSP5 protein in the plant is regulated by any one of the following methods: Transgenic, hybrid, backcross, selfing or asexual reproduction; The transgenic preferably includes: Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation or Agrobacterium-mediated one or more methods. For example, RNA interference is achieved by any of the above methods to achieve the purpose of reducing the expression level of GSP5 protein. Or point mutation is performed based on CRISPR / Cas9 technology to achieve the purpose of reducing the expression level of GSP5 protein.

[0021] Furthermore, the CRISPR / Cas9 technology is based on the gRNA shown in SEQ ID NO.5.

[0022] Furthermore, the CRISPR / Cas9 technology is based on the pBGK032 vector. The vector pBGK032 is disclosed in Yuming Lu, Xiao Ye, Renming Guo, Jing Huang, Wei Wang, Jiuyou Tang, Longtao Tan, Jiankang Zhu, Chengcai Chu, Yangwen Qian, Molecular Plant, 2017, DOI: 10.1016 / j.molp.2017.06.007.

[0023] The present invention has the following beneficial effects: The present invention has found that GSP protein is involved in the regulation of drought stress in plants, and rice strains in which the coding gene of GSP protein is knocked out show obvious improvement in drought resistance. The GSP protein and its coding gene and application provided by the present invention can be used to cultivate highly drought-resistant plants and improve drought-resistant germplasm resources of plants, and have important application value in the field of plant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 It is a comparison diagram of gene sequences and expression levels of wild-type rice, and GSP5-1 and GSP5-2 mutant strains provided in Example 1 of the present invention; wherein A is a gene sequence comparison diagram, and B is an expression level comparison diagram.

[0026] Figure 2 This is a comparison diagram of the drought resistance effects of the wild-type rice provided in Example 1 of the present invention, and the GSP5-1 and GSP5-2 mutant strains; wherein A is a growth status comparison, Control is a normal culture without stopping watering, and B is a watering-stopped treatment.

[0027] Figure 3 It is a comparison chart of leaf water loss rate of wild-type rice provided in Example 1 of the present invention, and GSP5-1 and GSP5-2 mutant strains.

[0028] Figure 4 It is a comparison diagram of the wild-type rice provided in Example 1 of the present invention, and the OE1 and OE2 overexpression strains; wherein A is a plant comparison, B is a survival rate and expression level comparison, and C is a leaf water loss rate comparison. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0031] The experimental materials and reagents involved in the following examples, unless otherwise specified, can be obtained from commercial sources, for example:

[0032] The present invention provides a method for improving drought resistance of rice, comprising the following process: 1. Gene Editing of GSP5 Target Sites

[0033] The 5'-CCGGCGGGAAGGACCAAGTTCAG-3' sequence on the GSP5 gene (SEQ ID NO.2) was selected as the target site (where the underline is the PAM sequence that conforms to NGG), sgRNA was synthesized, introduced into the CRISPR / Cas9 knockout vector pBGK032 containing the Cas9 enzyme expression frame, transformed into Agrobacterium, infected the mature embryo callus of rice Zhonghua 11, and obtained the transgenic strain after regeneration. For the transformation method, see Nishimura, A., Aichi, I., and Matsuoka, M., Nature protocols, 2006, 1, 2796-2802.

[0034] 2. Identification of homozygous mutants with loss of function of GSP5 gene

[0035] The total DNA extracted from leaves of T0 transgenic plants was used as a template for PCR amplification using the following primer pair: HPT-F: 5'-TAGGAGGGCGTGGATATGTC-3' (SEQ ID NO. 6); HPT-R: 5'-TACACAGCCATCGGTCCAGA-3' (SEQ ID NO. 7).

[0036] T0 positive transgenic plants were screened (the size of the PCR amplification product of the positive plants was 845 bp). The total DNA of the leaves of the positive plants was used as a template, and the following primers were designed at both ends of the GSP5 target site: GSP5-F:5'-GCTCAGCTCAAGCTCGCCGG-3' (SEQ ID NO.8); GSP5-R: 5'-ACGCGGAGTCGGCGAAGTTG-3' (SEQ ID NO. 9).

[0037] After amplification, sequencing was performed to screen mutant strains. The T0 generation was self-pollinated to obtain the T1 and T2 generations. The T2 generation plants were screened again to obtain two independent strains that were transgenic negative (T-DNA insertion and separation) but homozygous mutants at the GSP5 target site, named GSP5-1 and GSP5-2. GSP5-1 had a nucleotide base inserted at the target sequence, resulting in a frameshift starting at position G21 of the encoded protein, and GSP5-2 had a nucleotide base deleted at the same position, also resulting in a frameshift starting at position G21 of the encoded protein (e.g. Figure 1 shown).

[0038] 3. Drought resistance experiment of transgenic lines GSP5-1 and GSP5-2

[0039] After the seeds of GSP5-1, GSP5-2 and wild-type control Zhonghua 11 were soaked in the dark at 37°C for germination, they were sown in black pots filled with a 1:1 mixture of vermiculite and nutrient soil, and grown in a light incubator for 7 days at 30°C, 12h light / 12h dark. After one week, excess water was removed from the outside of the pots and watering was stopped for 9 days, followed by watering and normal culture for 7 days. At this time, the wild type was almost completely dead, while most of the mutant strains of GSP5-1 and GSP5-2 were still alive.

[0040] The results are as follows Figure 2 As shown: Statistics show that the survival rate of the wild type is less than 70%, while the survival rate of GSP5-1 and GSP5-2 is about 90%, which illustrates that the loss of GSP5 gene function can improve rice drought resistance.

[0041] 4. Experiment on water loss rate of detached leaves of transgenic lines GSP5-1 and GSP5-2

[0042] After the seeds of GSP5-1, GSP5-2 and wild-type control Zhonghua 11 were soaked in the dark at 37°C for germination, they were sown in black small pots filled with a 1:1 mixture of vermiculite and nutrient soil, and cultured in a light incubator for 7 days at 30°C, 12h light / 12h dark. After one week, about 6 complete leaves were taken from each number and placed at room temperature, and the weight of the leaves was weighed every hour. After obtaining 4 hours of data, the water loss rate of the leaves was calculated.

[0043] The results are as follows Figure 3As shown: Statistical data show that compared with the wild-type leaf weight, the leaf weight of the GSP5-1 and GSP5-2 mutant lines decreased more slowly and the leaf water loss rate was lower, indicating that the loss of GSP5 gene function can improve the drought resistance of rice ( Figure 3 ).

[0044] 5. Drought resistance experiment of overexpression lines OE1 and OE2 and water loss rate experiment of detached leaves

[0045] First, a recombinant vector pCAMBIA1300-35S-FLAG-GSP5 was constructed. The vector was obtained by integrating the GSP5 gene sequence (SEQ ID NO.2) by homologous recombination on the basis of the pCAMBIA1300-35S-FLAG vector. The pCAMBIA1300-35S-FLAG vector is a widely used vector in the art and can be purchased commercially or obtained by connecting a FLAG tag to a pCAMBIA1300-35S vector. The recombinant vector pCAMBIA1300-35S-FLAG-GSP5 was transformed into Zhonghua 11 plants by the same transformation method as step 1 to obtain the GSP5 gene overexpression strains OE1 and OE2 in the Zhonghua 11 background. By repeating the same detached leaf water loss rate experiment as step 4, the following was obtained: Figure 4 Results shown: Statistical data showed that leaf water loss rates of overexpressing lines OE1 and OE2 were significantly increased compared with the leaf weight of the wild type.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of GSP5 protein, or its encoding gene, or biological materials containing its encoding gene in regulating plant drought resistance; the regulation is negative regulation.

2. Use of GSP5 protein, or its encoding gene, or biological materials containing its encoding gene in cultivating plants with high drought resistance.

3. Application of GSP5 protein, or its encoding gene, or biological materials containing its encoding gene in improving plant drought-resistant germplasm resources.

4. The use according to any one of claims 1 to 3, characterized in that: By reducing the expression level of the GSP5 protein in the plant, the drought resistance of the plant is improved.

5. The use according to any one of claims 1 to 4, characterized in that: The GSP5 protein comprises any one of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO.1; (2) An amino acid sequence of a protein having the same function as the amino acid sequence shown in SEQ ID NO.1 obtained by replacing, inserting or deleting one or more amino acids.

6. The use according to any one of claims 1 to 5, characterized in that: The coding gene of the GSP5 protein includes any one of the following nucleotide sequences: (1) the nucleotide sequence shown in SEQ ID NO.2; (2) A nucleotide sequence encoding a protein with the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.2; (3) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO. 2 under stringent conditions.

7. The use according to any one of claims 1 to 6, characterized in that: The plant is a monocotyledonous plant or a dicotyledonous plant, preferably rice.

8. The use according to any one of claims 1 to 7, characterized in that: The biological material is an expression cassette, a vector or a transgenic cell.

9. A nucleic acid, characterized in that The nucleic acid comprises a nucleotide sequence as shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.

5.

10. A method for cultivating transgenic plants, characterized in that: include: Regulating the expression level of GSP5 protein in the plant; The amino acid sequence comprises any of the following: (1) the amino acid sequence shown in SEQ ID NO.1; (2) an amino acid sequence having the same functional protein obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown in SEQ ID NO.1; Preferably, the expression level of the GSP5 protein in the plant is regulated by any one of the following methods: Transgenic, hybrid, backcross, selfing or asexual reproduction; The transgenic method preferably includes one or more methods selected from the group consisting of Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation or Agrobacterium-mediated.

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