Application of rice OsCSN5 protein

Abstract

The invention provides an application of a rice OsCSN5 protein, relates to the technical field of crossing of biotechnology and plant breeding, and particularly provides an application of the rice OsCSN5 protein in a method for controlling the length of a main root of rice and the sensitivity of auxin of a rice root system. The invention further provides application of the rice OsCSN5 gene encoding the rice OsCSN5 protein in a method for controlling the length of a main root of rice and the sensitivity of auxin in a rice root system, the amino acid sequence of the rice OsCSN5 protein is shown as SEQ ID No.2, and the nucleotide sequence of the rice OsCSN5 gene is shown as SEQ ID No.1. The regulation and control effect of the OsCSN5 gene on the growth of the rice root system is analyzed for the first time, and a theoretical foundation is laid for further genetic improvement of rice root system molecules.

Description

Technical Field

[0001] The present invention relates to the cross-technical field of biotechnology and plant breeding, and in particular, to an application of a rice OsCSN5 protein, and more particularly, to an application of a rice OsCSN5 protein and a gene encoding the rice OsCSN5 protein in rice breeding. Background Art

[0002] Rice is one of the most common cultivated crops in the world, providing food for more than half of the world's population. Rice relies on its roots to anchor to the soil, absorb water and nutrients, and interact with the environment. The development and function of the root system directly affect the yield and quality of rice. However, there are still many unknowns about the molecular mechanisms of rice root development and regulation, which limits the molecular genetic improvement of rice roots. In view of this, discovering and cloning more root length-related genes and analyzing their genetic mechanisms are of great practical significance for rice genetic breeding. At present, more than 50 genes related to rice root growth and development have been identified. The functions of these genes involve multiple processes such as cell wall synthesis, hormone signal transduction, fatty acid metabolism, and DNA replication, which shows the complexity of rice root growth regulation. Discovering and cloning more root length-related genes will help to explain the molecular mechanisms of rice root development and regulation as soon as possible. Summary of the invention

[0003] The purpose of the present invention is to develop a method for regulating the development of rice root system.

[0004] To achieve the above objectives, the first aspect of the present invention provides an application of rice OsCSN5 protein, which controls the length of rice taproot and the sensitivity of rice root auxin by regulating the expression level of rice OsCSN5 protein in rice and / or regulating the activity of rice OsCSN5 protein, wherein the amino acid sequence of rice OsCSN5 protein is shown in SEQ ID No.2.

[0005] Preferably, the nucleotide sequence of the rice OsCSN5 gene encoding the rice OsCSN5 protein is shown in SEQ ID No.1.

[0006] CSN5 is a subunit of the COP9 signaling complex and has the activity of removing ubiquitin-like molecules from CUL1, thereby affecting the function of ubiquitin ligase E3. In Arabidopsis, csn5 mutants exhibit auxin signaling pathway defects and growth retardation phenotypes. The present invention achieves the purpose of regulating the length of the rice taproot and the auxin sensitivity of the rice root system for the first time by regulating the expression level and / or activity of the rice OsCSN5 protein.

[0007] Preferably, the nucleotide sequence of the rice OsCSN5 gene encoding the rice OsCSN5 protein is shown in SEQ ID No.1.

[0008] The second aspect of the present invention provides a plasmid for regulating the length of rice taproot and the auxin sensitivity of rice root system, wherein the plasmid comprises the nucleotide sequence shown in SEQ ID No.1.

[0009] A third aspect of the present invention provides a host cell for regulating the length of rice taproot and the auxin sensitivity of rice root system. The host cell is Agrobacterium, comprising the plasmid according to claim 3.

[0010] A fourth aspect of the present invention provides a method for regulating the length of the main root of rice, wherein the gene shown in SEQ ID No.1 or the protein shown in SEQ ID No.2 or the aforementioned plasmid or the aforementioned host cell is transferred into rice cells, and transgenic plants are obtained by tissue culture, thereby achieving the purpose of regulating the length of the main root of rice.

[0011] The fifth aspect of the present invention provides a method for regulating the sensitivity of rice root auxin, wherein the gene shown in SEQ ID No.1 or the protein shown in SEQ ID No.2 or the aforementioned plasmid or the aforementioned host cell is transferred into rice cells, and transgenic plants are obtained by tissue culture, thereby achieving the purpose of regulating the sensitivity of rice root auxin.

[0012] The present invention has the following beneficial effects: 1. In the present invention, the regulatory effect of the OsCSN5 gene on rice root growth was analyzed for the first time through the reverse genetics method, laying a theoretical foundation for further molecular genetic improvement of rice roots; 2. The present invention provides a new protein for controlling the growth of rice roots, and provides a gene encoding the protein, which affects the growth of rice roots; and also provides an expression vector containing the knockout gene; 3. The present invention provides an application of the aforementioned expression vector in regulating the development of rice root system. The expression vector containing a partial sequence of the rice root development regulating gene OsCSN5 is transferred into rice to regulate the root length of the rice root system;

[0013] 4. The present invention also provides an application of the above expression vector in regulating the sensitivity of rice to auxin. The expression vector containing the partial sequence of the rice root development regulating gene OsCSN5 is transferred into rice to regulate the sensitivity of rice root to auxin. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The identification result of rice mutants in Example 4 of the specific embodiment of the present invention; Figure 2 This is a comparative diagram of root phenotypes and a statistical diagram of taproot length of wild-type rice Zhonghua 11 and rice mutants after 7 days of normal hydroponics in Example 5 of the specific embodiment of the present invention; Figure 3 This is a comparative diagram of root phenotypes and a statistical diagram of root relative growth of wild-type rice Zhonghua 11 and rice mutants after normal hydroponics and auxin treatment in Example 5 of the specific implementation mode of the present invention. DETAILED DESCRIPTION

[0015] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation method and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the scope of protection of the claims of the present invention.

[0016] It should be noted that the endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0017] Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventionally understood meanings are defined herein for the purpose of clarification or ease of reference, and such definitions herein should not be construed as indicating significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out in accordance with the protocols and parameters given by the manufacturer.

[0018] The purpose of the specific embodiment of the present invention is to provide a protein and nucleic acid that can regulate the length of the main root of rice and the sensitivity of auxin. To achieve the above purpose, the amino acid sequence of the rice OsCSN5 protein for controlling the length of the main root of rice and the sensitivity of the auxin of the rice root system provided by the specific embodiment of the present invention is shown in SEQ ID No. 2, wherein SEQ The sequence of IDNo.2 is: MEPTSSAAMARQTWELENNIPAAASDPDALDAIYRYDEAAQARVQQEKPWANDPHPFRRAKISALALLKMVVHARAGGTIEVMGLMQGKCEGDAIVVMDAAFALPVEGTETRVNAQADAYEYMVEYSTINKQAGRLENVVGWYHSHPGYGCWLSGIDVSTQMLNQQFQEPFLAVVI DPTRTVSAGKVEIGAFRTYPKDYKPPDEPVSEYQTIPLNKIEDFGVHCKQYYALDITYFKSSLDSHLLDLLWNKYWVNTLSSSPLLGNRDYVAGQIFDLADKLEQAEGQLAHSRYGMLMPSQRKKEQEESPLAKVTRDSSKITAEQVHGLMSQVIKDILFNSVHPSNKASTSAPDSSGPEPMVEA.

[0019]

[0020] More specifically, the specific embodiments of the present invention also provide a plasmid comprising the nucleotide sequence shown in SEQ ID No. 1 for regulating the length of the main root of rice and the sensitivity of the root system to auxin of rice.

[0021] More specifically, the specific embodiments of the present invention also provide a host cell comprising the aforementioned plasmid for regulating the length of rice taproot and the auxin sensitivity of rice root system.

[0022] More specifically, the specific embodiments of the present invention also provide a method for regulating the length of the rice taproot by transferring the aforementioned rice OsCSN5 gene or rice OsCSN5 protein or plasmid or host cell into rice cells to obtain transgenic plants through tissue culture.

[0023] More specifically, the specific embodiments of the present invention also provide a method for regulating the auxin sensitivity of rice roots by transferring the aforementioned rice OsCSN5 gene or rice OsCSN5 protein or plasmid or host cell into rice cells to obtain transgenic plants through tissue culture.

[0024] The technical solution of the present invention is further described below through specific embodiments.

[0025] Example 1 Construction of OsCSN5 knockout plasmid Oligo sequence CCACCAGATGAACCTGTGTCAGA (SEQID No. 3) was generated based on the sequence information of rice OsCSN5 gene; Dissolve the synthesized Oligo in water to 10 μM, mix according to the reaction system shown in Table 1, heat at 95℃ for 3 min, and then slowly decrease to 20℃ at about 0.2℃ / s. Construct the Oligo dimer into the CRISPR / Cas vector: Mix the components on ice according to the reaction system shown in Table 2, and react at room temperature (20℃) for 1h after mixing. Transform Escherichia coli: Take 5 μL of the reaction solution and add it to at least 50 μL of competent cells, mix well and let it stand in an ice bath for 30 min; gently take it out, heat shock at 42℃ for 60 s, and immediately put it on ice for 2min; add 500 μL SOB / LB, and culture at 37℃ 200 rpm for 1 h; take an appropriate amount of bacterial solution and spread it on the LB plate containing kanamycin, and culture it upside down at 37℃ overnight. Obtain the OsCSN5 knockout plasmid.

[0026]

[0027]

[0028] Example 2 Preparation of Agrobacterium containing OsCSN5 knockout plasmid Transform Agrobacterium using chemical method. Take a tube of Agrobacterium competent cells from -80℃ freezer and place it on ice. After it is partially thawed, immediately insert it into ice. Add 1 μL of pre-thawed OsCSN5 knockout plasmid, mix gently, and place it on ice for 5 min, liquid nitrogen for 5 min, 37℃ water bath for 5 min, and ice bath for 5 min. Add 700 μL of YEP liquid culture medium to the clean bench, and then place it on a shaker at 28℃ for 2-3 h. Collect the cells by centrifugation at 6000 rpm for 1 min, discard 700 μL of supernatant, gently blow the remaining culture medium with a pipette, resuspend the cells and apply to the plate, invert the plate and place it in a constant temperature culture at 28℃ for 2-3 days, pick a single clone, and obtain Agrobacterium containing OsCSN5 knockout plasmid.

[0029] Example 3 Rice genetic transformation Five hundred seeds of wild-type rice Zhonghua 11 were used to prepare rice callus.

[0030] Seed induction: Wash with alcohol for 30 s, 30% sodium hypochlorite solution for 15 min, and sterile water 5 times to remove excess water, and place in induction medium for culture.

[0031] Callus subculture: Use sterile tweezers to remove the young shoots and seeds, retain the mature callus tissue, and place it on the culture medium for further cultivation.

[0032] Agrobacterium activation: Use a 200 μL pipette tip to dip into the Agrobacterium culture solution, inoculate on a YEP plate, and culture in the dark at 28°C for 2 days.

[0033] Agrobacterium infection: Use the infection solution to wash the carrier on the plate into a suspension, soak the callus tissue with the suspension for 15 minutes, then transfer the callus to sterile filter paper to absorb excess liquid, and place it at 25℃ for 3 days in the dark.

[0034] Screening culture: Wash the callus after dark culture with water containing carbenicillin, dry the surface moisture with the air of the clean bench, and then place it on the screening culture medium for screening culture.

[0035] Differentiation and regeneration: The positive particles grown on the screening medium are transferred to the differentiation medium for regeneration.

[0036] Seedling rooting: Seedlings grown on differentiation medium take root in rooting tubes and undergo root strengthening culture. Rice mutants with knockout of rice OsCSN5 gene are obtained.

[0037] Example 4 Identification of rice mutants DNA extraction: Take an appropriate amount of rice leaves and put them into a 1.5 mL centrifuge tube with steel beads added. After grinding with a grinder for 60 s, add 300 μL TPS extract; incubate at 75°C for 20 min (mix once during the period); centrifuge at 12000 rpm for 10 min, take the supernatant and transfer it to a new 1.5 mL centrifuge tube; add 200 μL isopropanol, mix well, and centrifuge at 12000 rpm for 10 min; discard the supernatant, add 200 μL 70% ethanol, and centrifuge at 12000 rpm for 5 min; discard the supernatant, turn the paper towel upside down, transfer to an ultra-clear table, and blow until the ethanol evaporates.

[0038] The knockout site was amplified using a high-fidelity enzyme, upstream primer GGCACTATCGAGGTCATGGG (SEQ ID No. 4) and downstream primer AGGAGAGGTGATGAAGATAACG (SEQ ID No. 5). 3 μL of the PCR stock solution was taken to detect the target band in 1% agarose gel. After the size was consistent with the expected result, sequencing was performed. The sequencing results were compared and analyzed. The mutation site information is shown in Figure 1 shown.

[0039] Example 5 Phenotypic analysis of rice mutants Rice mutants and wild-type Zhonghua 11 (WT) grown in an artificial climate chamber for 7 days and in good growth condition were neatly arranged on a black curtain. The plant phenotypes were photographed with a digital camera. Ten Oscsn5 and wild-type Zhonghua 11 plants were randomly selected to measure and record the taproot length. The results are as follows: Figure 2 As shown, Figure 2 A in the figure is a comparison of root phenotypes after 7 days of normal hydroponics. Figure 2 B in the figure is a statistical chart of the main root length after 7 days of normal hydroponic culture, where WT is the wild type, and strains Oscsn5-1 and Oscsn5-2 are mutant strains.

[0040] The seeds of wild-type rice Zhonghua 11 and rice mutant strains were sown in normal culture medium and 0.1 μM 2,4-D culture medium, respectively. After 7 days of treatment, photos were taken with a digital camera. The taproot length was counted. Ten plants of each variety were randomly recorded for each treatment. The results are shown in Figure 3 As shown, Figure 3 A in the figure is a comparison of root phenotypes after control culture and auxin treatment. Figure 3 B in the figure is a statistical diagram of relative root growth after auxin treatment, where WT is the wild type, strains Oscsn5-1 and Oscsn5-2 are mutant strains, and Mock is treated with normal culture medium.

[0041] Depend on Figure 2 and Figure 3It can be seen that after knocking out the rice Oscsn5 gene, the length of the rice taproot and the sensitivity of the rice root system to auxin changed, proving the feasibility of the rice Oscsn5 gene in regulating the length of the rice taproot and the sensitivity of the rice root system to auxin.

[0042] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. An application of rice OsCSN5 protein, characterized in that: The length of the rice taproot and the auxin sensitivity of the rice root system are controlled by regulating the expression amount of the rice OsCSN5 protein in rice and / or regulating the activity of the rice OsCSN5 protein, wherein the amino acid sequence of the rice OsCSN5 protein is shown in SEQ ID No.

2.

2. The use according to claim 1, characterized in that The nucleotide sequence of the rice OsCSN5 gene encoding the rice OsCSN5 protein is shown in SEQ ID No.

1.

3. A plasmid for regulating the length of rice taproot and the sensitivity of rice root auxin, characterized in that: The plasmid includes the nucleotide sequence shown in SEQ ID No.

1.

4. A host cell for regulating the length of rice taproot and the sensitivity of rice root auxin, characterized in that: The host cell is Agrobacterium, comprising the plasmid according to claim 3.

5. A method for regulating the length of a rice taproot, characterized in that: The gene shown in SEQ ID No. 1 or the protein shown in SEQ ID No. 2 or the plasmid described in claim 3 or the host cell described in claim 4 is transferred into rice cells, and transgenic plants are obtained by tissue culture, so as to achieve the purpose of regulating the length of the main root of rice.

6. A method for regulating the auxin sensitivity of rice roots, characterized in that: The gene shown in SEQ ID No. 1 or the protein shown in SEQ ID No. 2 or the plasmid described in claim 3 or the host cell described in claim 4 is transferred into rice cells, and transgenic plants are obtained by tissue culture, so as to achieve the purpose of regulating the auxin sensitivity of rice roots.

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