Application of rice osrbohI gene in promoting rice growth

Abstract

The application discloses application of a rice OsRbohI gene in promoting growth of rice and relates to the technical field of bioengineering. The application can improve plant height and root length and increase grain size by cloning the rice OsRbohI gene, performing genetic transformation in rice and overexpressing the rice OsRbohI gene; loss of the function of the OsRbohI gene can cause dwarfing of the plant, shortening of the root and reduction in the grain size. Changing the plant type and grain type of rice by means of genetic engineering technology has important research significance for improving crop production. Based on the close relationship between rice and other monocotyledonous plants, the rice OsRbohI gene can regulate growth and development of rice, especially the plant height, root development and grain size. The research result can also be applied to other monocotyledonous plants to cause change in the plant type and grain type.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to the application of the rice OsRbohI gene in promoting rice growth. Background Technology

[0002] Rice is one of the world's most important food crops, providing essential calories for more than half the world's population. Rapid population growth places higher demands on rice production, making the cultivation of high-yield and high-quality rice a top priority for plant breeders. Rice yield depends not only on the number of panicles, grain weight, and the number of grains per panicle, but also on plant height and growth status. Grain shape, as a complex agronomic trait, also plays an indirect role in rice yield. In breeding applications, grain size is the main factor determining grain weight, which is positively correlated with grain length, width, and thickness. Therefore, understanding the genetic and molecular basis of grain size is crucial for rice improvement. In addition, shorter stalks improve lodging resistance, density tolerance, fertilizer tolerance, and yield. Therefore, exploring dwarf rice germplasm resources and applying them to variety improvement is particularly important. Roots play a crucial role in the plant growth cycle, not only absorbing nutrients and water but also helping the plant anchor itself in the soil and influencing its response to the external environment. Therefore, given the potential of root development to affect crop production, optimizing root structure is crucial for promoting the next green revolution. In conclusion, discovering and identifying genes controlling rice growth and development, and using modern biological techniques to improve rice plant architecture and cultivate superior varieties to increase rice yield has both theoretical significance and practical value for rice production.

[0003] Reactive oxygen species (ROS) are a class of single-electron reduction products of oxygen in the body. They are a general term for highly reactive and oxidizing oxygen-containing compounds, including superoxide anion (O2). - Hydrogen peroxide (H2O2), hydroxyl radicals (·OH), and singlet oxygen ( 1 O2, etc. Since the emergence of anaerobic photosynthetic organisms approximately 250 million years ago, ROS has been considered to play a dual role in plant biology. Although ROS has long been considered toxic to cells, a range of evidence demonstrates that ROS, at specific levels, can function as ubiquitous signaling molecules to regulate plant development and stress adaptation. Current research also indicates that the beneficial or harmful effects of ROS on plants are indeed related to specific thresholds in ROS levels. To regulate plant biological processes, changes in ROS levels should not exceed the threshold between redox biological and cytotoxic or inhibitory levels. Therefore, excessively low or high ROS levels can affect normal plant growth, while maintaining ROS levels within an appropriate range can promote plant growth and development.

[0004] The sites and pathways of ROS production are diverse, including chloroplast photosynthesis, the final stage of mitochondrial aerobic respiration (oxidative phosphorylation), peroxisome photorespiration, and the plasma membrane oxidoreductase system. One key component of the plasma membrane oxidoreductase system is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, also known as respiratory burst oxidase homolog (Rboh), which is present in the plasma membrane systems of almost all animals and plants. In 1996, the first plant NADPH oxidase gene was cloned from rice and named RbohA. Subsequently, Rboh genes were discovered in other plants such as Arabidopsis thaliana, tomato, tobacco, and potato. To date, nine and ten Rboh genes have been identified in rice and Arabidopsis thaliana, respectively. Studies have shown that ROS produced by NADPH oxidase encoded by respiratory burst oxidase homologs (Rbohs) play beneficial roles in plant growth and development, such as seed germination, pollen tube development, bud growth, and root development.

[0005] Because the functional domains of the rice OsRbohs family are diverse, they possess different functions and regulatory mechanisms in stress responses and / or normal growth and development. Phylogenetic analysis indicates that OsRbohI is the most recently evolved gene in the rice Rbohs family. Studies have found that the amino acid sequence of the protein OsRbohI is most similar to AtRbohD, with a sequence homology of 66%. AtRbohD is involved in many developmental processes and stress responses, such as stomatal closure, systemic signal transduction, pathogens, damage, and salt stress. However, the function of the OsRbohI gene in regulating growth and development in rice has not been reported.

[0006] Therefore, using modern biotechnology to construct transgenic plants in rice and alter rice plant height, root system, and grain size is of great significance for increasing crop yield. Summary of the Invention

[0007] This invention provides the application of the rice OsRbohI gene in promoting rice growth. The aim is to provide the role of the rice OsRbohI gene in rice growth and development, especially in regulating rice plant height, root system, and grain size. Knocking out this gene in rice can lead to dwarfing of the plant, shorter roots, and smaller grains. Overexpression of this gene can appropriately increase plant height, root growth, and grain size, which plays an important role in improving crop yield.

[0008] This invention provides the application of the rice OsRbohI gene in promoting rice growth. Promoting rice growth can increase rice yield, and / or increase rice plant height, root length, and grain size.

[0009] Preferably, the base sequence of the OsRbohI gene is shown in SEQ ID NO.1.

[0010] This invention also provides a method for regulating rice plant height, root length, or grain size. When it is necessary to increase rice plant height, root length, or grain size, the OsRbohI gene in rice is overexpressed; when it is necessary to decrease rice plant height, root length, or grain size, the OsRbohI gene in rice is silenced or knocked out.

[0011] The present invention also provides a method for increasing crop yield by overexpressing the OsRbohI gene in a monocotyledonous plant.

[0012] Preferably, the crop is rice.

[0013] Preferably, the base sequence of the OsRbohI gene is shown in SEQ ID NO.1.

[0014] The method specifically includes the following steps:

[0015] (1) Construct an OsRbohI gene overexpression vector and insert the OsRbohI gene sequence into the plant expression vector;

[0016] (2) The OsRbohI gene overexpression vector from step (1) was introduced into rice cells for overexpression, and transgenic plants were obtained after culturing.

[0017] Preferably, the plant expression vector is pUN1301.

[0018] In step (2), the OsRbohI gene overexpression vector is transferred into Agrobacterium genetically engineered bacteria and then infects rice cells; the Agrobacterium genetically engineered bacteria is Agrobacterium EHA105 strain.

[0019] Preferably, the cells infected by Agrobacterium are derived from callus induced by rice seeds.

[0020] The rice variety can be Nipponbare, but is not limited to Nipponbare.

[0021] The beneficial effects of this invention are as follows:

[0022] Cloning the rice OsRbohI gene and performing genetic transformation in rice plants revealed that overexpression of the OsRbohI gene increased plant height, root length, and grain size. Constructing a gene knockout vector by designing a knockout target site for the OsRbohI gene and performing genetic transformation in rice showed that loss of OsRbohI gene function led to dwarfing, shorter roots, and smaller grains. Utilizing genetic engineering techniques to alter rice plant architecture and grain shape agronomic traits has significant research implications for improving crop production.

[0023] Based on the close relationship between rice and other monocotyledonous plants, the rice OsRbohI gene in this invention can regulate rice growth and development, particularly its role in regulating plant height, root development, and grain size. This finding can also be applied to other monocotyledonous plants to induce changes in plant architecture and grain shape. Attached Figure Description

[0024] Figure 1 Figure (A) shows the expression of the OsRbohI gene in the overexpression line and the genome sequence editing of the OsRbohI gene in the knockout line; where **p < 0.01.

[0025] Figure 2 Comparison of seedling height and root appearance of wild-type (NIP), rice overexpression and knockout mutant transgenic lines (OE-OsRbohI-1, OE-OsRbohI-2, Osrbohi-1, Osrbohi-2) (A), and comparison of seedling height (B) and root appearance (C) (bar = 2 cm; n = 30); where *p < 0.05, **p < 0.01.

[0026] Figure 3 Comparison of mature plant height (A) and data (B) between wild-type (NIP) and transgenic rice lines (OE-OsRbohI-1, OE-OsRbohI-2, Osrbohi-1, Osrbohi-2); where **p<0.01.

[0027] Figure 4 Comparison of grain size (A) and grain length (B), grain width (C), and grain thickness (D) between wild-type (NIP) and transgenic rice lines (OE-OsRbohI-1, OE-OsRbohI-2, Osrbohi-1, Osrbohi-2) (bar = 1 cm; n = 30); where *p < 0.05, **p < 0.01. Detailed Implementation

[0028] Example 1: Construction of rice OsRbohI gene vector and rice genetic transformation (1) Construction of overexpression vector

[0029] ① Based on the sequence of gene OsRbohI (Os11g0537400, base sequence as shown in SEQ ID NO.1, amino acid sequence of the encoded protein as shown in SEQ ID NO.2) in Primer 5, specific primers were designed, and BamHI and KpnI restriction enzyme sites were added to the 5' end of the primers, respectively, as follows:

[0030] Primer 1:

[0031] Primer 2:

[0032] ② PCR amplification using full-length cDNA as a template:

[0033] High-fidelity enzymes are used HS DNA Polymerase (TaKaRa Code: DR010A) system:

[0034]

[0035] The PCR cycling conditions were as follows: 98℃ pre-denaturation for 5 minutes; 98℃ denaturation for 10 seconds, 55℃ annealing for 15 seconds, 72℃ extension for 90 seconds, for 30 cycles; and finally, 72℃ extension for 10 minutes to end the reaction.

[0036] ③ Recover the PCR product and digest plasmid pUN1301 with BamHI and KpnI restriction enzymes. The system is as follows: 10×K Buffer 2.5 μL, BamHI 1 μL, KpnI 1 μL, plasmid 10 μL, ddH2O 35.5 μL.

[0037] Mix briefly twice by vortexing, centrifuge slightly, and incubate in a 37°C water bath for 2 hours.

[0038] ④ Double enzyme digestion ligation:

[0039] The ligation reaction was performed using Taraka's T4 ligase, and the system is as follows:

[0040]

[0041] The ligation reaction conditions were: 10℃ for 3 minutes, 6℃ for 6 seconds (increasing by 0.2℃ per cycle), 16℃ for 3 minutes, 18℃ for 1 minute, for a total of 19 cycles; 65℃ for 15 minutes, and stored at 12℃.

[0042] The ligation product was transformed into E. coli DH5α, positive clones were selected for PCR verification and sequenced, and plasmids were extracted for later use.

[0043] (2) Knockout vector construction

[0044] 1) Based on the website "http: / / skl.scau.edu.cn / targetdesign / ", a specific knockout target for the OsRbohI gene was designed, with the following sequence:

[0045] Knockout target: GACCATGTGACCCCACCTGC.

[0046] 2) The pYLCRISPR / Cas9Pubi-H vector was digested using the BsaI restriction enzyme from NEB:

[0047] The system is as follows:

[0048]

[0049]

[0050] The enzyme digestion conditions were: 37℃ for 30 minutes.

[0051] 3) Target knockout synthesis:

[0052] The synthesized primer target sequence is as follows:

[0053] OsRbohI-aFP:

[0054] OsRbohI-aRP:

[0055] The primer annealing system is as follows:

[0056] OsRbohI-aFP 9μL,

[0057] OsRbohI-aR 9μL,

[0058] Add ddH2O to a final volume of 20 μL.

[0059] Annealing conditions: 95℃ for 5 minutes, then remove and place at room temperature.

[0060] 4) Ligation of the knockout target site with the enzyme digestion vector:

[0061] The ligation reaction was performed using Taraka's T4 ligase, and the system is as follows:

[0062]

[0063] The ligation reaction conditions were: 10℃ for 3 minutes, 6℃ for 6 seconds (increasing by 0.2℃ per cycle), 16℃ for 3 minutes, 18℃ for 1 minute, for a total of 19 cycles; 65℃ for 15 minutes, and stored at 12℃.

[0064] The ligation product was transformed into E. coli DH5α, positive clones were selected for PCR verification and sequenced, and plasmids were extracted for later use.

[0065] (3) Take out EHA105 competent cells from the -80℃ freezer and transform Agrobacterium using liquid nitrogen freeze-thaw method.

[0066] The competent cells were thawed on ice. 5 μL of the target plasmid extracted in step (1) or (2) was added to the competent cells. After mixing, the cells were placed on ice for about 30 min. Then, all the mixture in the centrifuge tube was placed in liquid nitrogen for 1 minute to freeze. After that, the cells were placed in a 37°C water bath for 2 minutes to thaw. Then, 1 mL of antibiotic-free LB liquid nutrient medium was added to resuspend the cells. The cells were then placed on a shaker at 28°C and 200 rpm for 2 hours to recover. After that, the cells were spread on YEP plates containing the corresponding antibiotics (rifampicin 50 mg / L, kanamycin sulfate 50 mg / L) and incubated upside down at 28°C for 2 days until a single colony grew.

[0067] Single clones of Agrobacterium were selected from the grown cells, and positive single clones were identified by colony PCR. These clones were then used to transform rice seed-induced callus using a rapid genetic transformation method.

[0068] Hygromycin was used to screen resistant callus and induce differentiation into seedlings. RNA was extracted from the leaves of the differentiated seedlings and subjected to quantitative real-time PCR to determine gene expression levels. DNA was extracted from the leaves of the differentiated seedlings, and the target genome sequence was amplified and sequenced to identify target mutations. The primers used are as follows:

[0069] qRT-OsRbohI-F:

[0070] qRT-OsRbohI-R:

[0071] qRT-OsActin-F:

[0072] qRT-OsActin-R:

[0073] OsRbohICAS-F:

[0074] OsRbohICAS-R:

[0075] Rice OsRbohI gene overexpression mutants OE-1 and OE-2, and rice OsRbohI gene knockout mutants KO-1 and KO-2 were obtained.

[0076] Example 2: Gene expression in rice OsRbohI gene overexpression lines and gene editing in knockout lines

[0077] To detect gene expression in transgenic rice lines of the OsRbohI gene, total RNA was extracted from the leaves of positive seedlings and subjected to quantitative real-time PCR to determine gene expression levels. DNA was extracted from the leaves of edited seedlings, and the amplified fragments were sent to Hangzhou Shangya Company for sequencing.

[0078] like Figure 1 As shown, sequencing results revealed that the gene expression levels of both identified overexpression lines were significantly increased, and both DNA single strands of the sequenced gene-edited lines underwent mutations.

[0079] Example 3: Observation of the traits of transgenic rice lines

[0080] To investigate the role of OsRbohI in rice growth and development, we observed transgenic overexpression lines (OE-1 and OE-2) and OsRbohI knockout mutants (KO-1 and KO-2). We found that compared with the wild type, the plant height at the seedling and maturity stages was significantly higher. Figure 2 and Figure 3 and root length ( Figure 2 Significant changes were observed in all gene expression levels. Specifically, compared to gene knockout mutants (KO-1 and KO-2), overexpression (OE-1 and OE-2) transgenic lines promoted stem growth and root formation in the early seedling stage. Furthermore, OE-1 and OE-2 showed greater plant height at maturity than the wild type, while KO-1 and KO-2 showed shorter plant heights. Additionally, compared to the wild type, the overexpression transgenic lines exhibited significantly different grain sizes. Figure 4 Significant changes occurred. Specifically, compared to the wild type, the grain traits (length, width, and thickness) of OE-1 and OE-2 were significantly increased, while the grain length and width of the KO-1 and KO-2 mutants were slightly decreased, with no significant change in thickness. This indicates that overexpression of the OsRbohI gene can promote the growth of the aboveground parts and roots, and increase grain size.

Claims

1. Overexpression in rice OsRbohI The application of genes in promoting rice growth, the aforementioned OsRbohI The gene's base sequence is shown in SEQ ID NO.1, and the promotion of rice growth is manifested in increased rice plant height, root growth, and larger grains.

2. A method for regulating rice plant height, root length, or grain shape, characterized in that, When it is necessary to increase the height of rice plants, the length of the root system, or the size of the grains, the rice... OsRbohI Gene overexpression; when it is necessary to reduce rice plant height, shorten root system, or shrink grain size, the gene is overexpressed in rice. OsRbohI Gene silencing or knockout; The OsRbohI The base sequence of the gene is shown in SEQ ID NO.

1.

3. A method for promoting rice growth, characterized in that, Overexpression in rice OsRbohI Genes, the ones mentioned OsRbohI The gene's base sequence is shown in SEQ ID NO.1, and the promotion of rice growth is manifested in increased rice plant height, root growth, and larger grains.

4. The method as described in claim 3, characterized in that, Includes the following steps: (1) Constructing overexpression OsRbohI Gene vector, will OsRbohI Gene sequences are inserted into plant expression vectors; (2) Overexpress the steps in (1) OsRbohI Gene vectors were introduced into rice cells for overexpression, and transgenic plants were obtained after culturing.

5. The method as described in claim 4, characterized in that, The plant expression vector is pUN1301.

6. The method as described in claim 4, characterized in that, In step (2), the overexpression of step (1) will be performed. OsRbohI The method of introducing gene vectors into rice cells involves overexpressing... OsRbohI After the gene vector was transferred into the Agrobacterium genetically engineered strain, it infected rice cells; the Agrobacterium genetically engineered strain was Agrobacterium EHA105.

7. The method as described in claim 6, characterized in that, The cells infected by Agrobacterium were derived from callus induced by rice seeds.

Images