Application of MOC1 gene in enhancing low-temperature stress resistance of rice in seedling stage

Abstract

The invention relates to application of an MOC1 gene in enhancing low-temperature stress resistance in a rice seedling stage. Specifically, the rice MOC1 gene participating in positive regulation and control of low-temperature stress resistance is cloned in rice, so that the rice MOC1 gene is overexpressed, and compared with a wild plant, the rice MOC1 gene overexpressed plant shows the low-temperature-resistant characteristic, and the survival rate is remarkably increased. The discovery of the new function of the rice MOC1 gene provides a new gene target and resource for improving the low-temperature stress resistance genetic breeding of rice.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more particularly to... MOC1 Application of genes in enhancing rice seedling resistance to low temperature stress. Background Technology

[0002] As a staple food globally, rice is highly susceptible to the inhibition of growth and development by low-temperature stress. Cold damage causes economic losses of up to tens of billions of dollars worldwide each year, and in China, it also leads to the reduction of millions of tons of rice production (Lou et al., 2007; Zhu et al., 2015; Sun et al., 2022). Therefore, improving the cold resistance of rice seedlings is crucial for ensuring food security and promoting direct seeding cultivation technology.

[0003] Currently, strategies for addressing cold stress in rice mainly fall into two categories: chemical regulation and genetic improvement. Chemical methods, such as pretreatment with melatonin before sowing (Li et al., 2021) or spraying with specific chemical agents (Sun et al., 2020; Back, 2021), can effectively alleviate cold stress and promote germination, but the latter is limited in application due to its high cost. In contrast, genetic improvement, through the introduction of cold-resistant genes, is considered a more fundamental solution. For example, researchers have identified… OsCTB4a , OsMAPK3 , OsCBL7 and OsSAPK6 These genes are functional genes that participate in the cold adaptation process of rice by regulating signaling pathways such as phosphorylation (Zhang et al., 2017; Li et al., 2022; Jia et al., 2022; Guo et al., 2022; Lou et al., 2022).

[0004] However, cold resistance in rice is a complex quantitative trait, finely regulated by a multi-gene network (Chen et al., 2018; Zu et al., 2023; Ding et al., 2023). The number of genes currently cloned and identified is limited, far from sufficient to unravel its complex genetic network. Therefore, discovering new cold resistance-related genes and elucidating their mechanisms of action is crucial for improving cold resistance breeding strategies and overcoming current research bottlenecks.

[0005] MOC1 It encodes a GRAS family protein located in the nucleus that controls axillary meristem formation during both vegetative and reproductive growth stages. MOC1MOC1 is expressed in the epidermis and subcutaneous cells of axillary buds before morphological changes occur, and subsequently throughout the axillary bud and in later leaf axillary primordia. MOC1 plays a crucial role in the formation of leaf axillary meristems and axillary buds, and also promotes the outward growth of axillary buds. Mutant moc1 It has no tillers, only one main stem, and fewer inflorescence axes and spikelets than the wild type. gnp6 and moc1 Alleles, mutants gnp6 The number of tillers decreased, but the number of high-level tillers increased significantly. The panicle length and the number of primary and secondary branches decreased, leading to a reduction in the number of grains per panicle. There were also fewer lateral spikelets on the secondary branches, resulting in a lower grain filling rate. These results indicate that MOC1 / GNP6 regulates tillering and panicle development in rice (Zhang et al. 2021). MOC3 and MOC1 are not only key factors in tiller bud initiation but also regulate tiller bud elongation. MOC3 can directly bind to… FON1 The promoter activates the latter's expression. Although MOC1 cannot directly bind to... FON1 The promoter, but as a co-activator of MOC3, is further activated in the presence of MOC3. FON1 Expression, and FON1 It can positively regulate the elongation of tillers. FON1 Loss-of-function mutants can form normal shoots, but the shoots elongate outwards defectively, leading to a reduction in tiller number (Shao et al. 2019). The tillering regulator MOC1 is prevented from degradation by binding to the DELLA protein SLR1. Gibberellins (GAs) induce SLR1 degradation, leading to stem elongation and MOC1 degradation, thereby reducing tiller number (Liao et al. 2019). Rice ( Oryza sativa L. is an important food crop, and MOC1 is a transcription factor of the rice GRAS family. However, there are no related research reports on its effects on low temperature stress. Summary of the Invention

[0006] Based on existing technologies, this invention has discovered a method for overexpressing [the gene] in rice plants. MOC1 Genes can enhance the resistance of rice plants to low-temperature stress during the seedling stage; therefore, this invention provides, in one aspect, a rice... MOC1 Application of genes in improving rice seedling resistance to low-temperature stress. On the other hand, this invention also provides a method for improving rice seedling resistance to low-temperature stress, comprising: overexpressing rice... MOC1 Genes. Furthermore, this invention also provides rice. MOC1 Application of genes in genetic breeding to improve rice seedling resistance to low temperature stress.

[0007] In this regard, the technical solutions of the present invention include, but are not limited to, the following: In one aspect, the present invention provides rice MOC1The application of the gene in improving rice seedling low-temperature stress resistance and / or rice variety improvement genetic breeding is characterized by overexpression of the rice MOC1 Genes, the rice MOC1 The gene encodes the amino acid sequence shown in SEQ ID NO: 3.

[0008] In another aspect, the present invention provides a rice-containing... MOC1 The application of recombinant gene vectors in genetic breeding for improving rice seedling resistance to low-temperature stress and / or rice variety improvement is characterized by overexpression of the aforementioned rice... MOC1 Genes, the rice MOC1 The gene encodes the amino acid sequence shown in SEQ ID NO: 3.

[0009] In one aspect, the recombinant vector of the present invention comprises an overexpression promoter Ubi.

[0010] Preferably, the recombinant vector of the present invention is a recombinant pCAMBIA1301 vector containing the overexpression promoter Ubi.

[0011] In one aspect, the present invention provides the application of genetically engineered bacteria in improving rice seedling resistance to low-temperature stress and / or in genetic breeding for rice variety improvement, characterized in that the genetically engineered bacteria contains rice... MOC1 Recombinant vectors of the gene, overexpressing the rice MOC1 Genes, the rice MOC1 The gene encodes the amino acid sequence shown in SEQ ID NO: 3. Preferably, the genetically engineered bacteria of the present invention are genetically engineered Escherichia coli or Agrobacterium.

[0012] In one aspect, the genetically engineered bacteria of the present invention are genetically engineered Agrobacterium.

[0013] In one aspect, the genetically engineered bacterium described in this invention is a genetically engineered Agrobacterium GV3101.

[0014] In another aspect, the present invention provides a method for improving the resistance of rice seedlings to low-temperature stress and / or for genetic breeding to improve rice varieties, characterized in that the method comprises: overexpressing rice... MOC1 Genes, the rice MOC1 The gene encodes the amino acid sequence shown in SEQ ID NO: 3.

[0015] In one aspect, the rice variety improvement described in this invention aims to enhance the resistance of rice seedlings to low-temperature stress.

[0016] In another aspect, the present invention provides a method for obtaining rice plants with improved traits, comprising the following processing steps: (1) Infecting rice callus tissue with genetically engineered bacteria; and (2) Infected rice callus tissue was cultured into rice plants; The genetically engineered bacteria contained rice MOC1 Recombinant vectors of the gene, overexpressing the rice MOC1 Genes, the rice MOC1 The gene encodes the amino acid sequence shown in SEQ ID NO: 3.

[0017] In another aspect, the present invention provides the application of rice MOC1 protein in improving rice seedling resistance to low temperature stress and / or in genetic breeding for rice variety improvement.

[0018] In one aspect, the amino acid sequence of the rice MOC1 protein of the present invention is shown in SEQ ID NO: 3.

[0019] In one aspect, the improved trait described in this invention is enhanced resistance to low-temperature stress during the rice seedling stage.

[0020] In one aspect, the present invention provides for constructing overexpression MOC1 The method / steps for gene vectors include: [the following steps are described in the original text, which are not directly related to the gene vector method / steps described in the original text.] MOC1 The nucleotide sequence of the gene coding region was ligated into the vector pCAMBIA1301, with the primers designed as follows: MOC1-ox -F: 5'-ACTAGGGTCTCGCACCATGCTCCGGTCACTCCACT-3'; (SEQ ID NO: 4) MOC1-ox -R: 5'-ACTAGGGTCTCTCGCCCGACGACGACGGCTGCCA-3'; (SEQ ID NO: 5).

[0021] In one aspect, the present invention also provides for constructing overexpression MOC1 The method / steps for genetically engineered bacteria include: overexpressing the strain described in this invention... MOC1 The gene vector was transformed into the Agrobacterium strain.

[0022] In one aspect, the present invention also provides for obtaining overexpression MOC1 The method / steps for producing gene-modified rice plants include: using the overexpression method described in this invention. MOC1 Genetically engineered bacteria infect rice callus tissue, which is then differentiated and rooted.

[0023] In one aspect, the rice of the present invention MOC1 The gene encodes the amino acid sequence shown in SEQ ID NO: 3.

[0024] In one aspect, the rice of the present invention MOC1 The nucleotide sequence of the gene coding region is shown in SEQ ID NO: 1.

[0025] In one aspect, the rice described in this invention is the japonica rice variety Zhonghua 11 ( Rice . Sativa L.spp. Japanese , var. Zhonghua11 ).

[0026] Preferably, the improved low-temperature stress resistance of rice described in this invention is manifested in that, compared to control ordinary rice plants, MOC1 Rice seedlings with overexpressed genes showed significantly higher survival rates after being treated with low temperatures during the seedling stage.

[0027] In one aspect, the low temperature described in this invention is below 12°C. In another aspect, the low temperature described in this invention is below 11°C, below 10°C, below 9°C, below 8°C, below 7°C, below 6°C, below 5°C, or below 4°C. In one aspect, the low temperature described in this invention is 4°C.

[0028] In one aspect, the present invention will MOC1 The gene was introduced as the target gene into the japonica rice variety Zhonghua 11 ( Rice . Sativa L. spp. Japanese , var. Zhonghua11 (obtained from) MOC1 The T0 generation of overexpressing plants were continuously self-crossed to obtain homozygous, highly expressing T2 generation lines, which were named [the gene name is missing here]. MOC1-ox1 and MOC1-ox2 .

[0029] In one aspect, the present invention utilizes CRISPR / Cas9 gene editing technology to modify the rice variety Hua 11 ( Oryza.Sativa L. spp. Japanese , var. Zhonghua11 ) MOC1 Genes were de-functionalized to prepare... MOC1 Plants with missing gene function moc1-1 and moc1-2 .

[0030] In one aspect, in this invention, MOC1 The nucleotide sequence of the protein-coding region of the gene is shown in SEQ ID NO: 1. MOC1 The full-length sequence of the gene is shown in SEQ ID NO: 2. MOC1 The gene encodes a GRAS family transcription factor, composed of 441 amino acids, the amino acid sequence of which is shown in SEQ ID NO.3, specifically: SEQ ID NO: 3: MLRSLHSSSSSDTDNNSGGCKNNGGGGGEAAAAVEGGGDQRAVAAAAPSTRDLLLACADLLQRGDLPAARRAAEIVLAAASPRGDAADRLAYHFARALALRVDAKAGGHG HVVVGGGAARPASSGAYLAFNQIAPFLRFAHLTANQAILEAVDGARRVHILDLDAVHGVQWPPLLQAIAERADPALGPPEVRVTGAGADRDTLLRTGNRLRAFARSIHLPF HFTPLLLSCATTAPHHVAGTSTGAAAAASTAAAATGLEFHPDETLAVNCVMFLHNLAGHDELAAAFLKWVKAMSPAVVTIAEREAGGGGGGGGDHHIDDLPRRVGVAMDHYSA VFEALEATVPPGSRERLAVEQEVLGREIEAAVGPSGGRWWRGIERWGGAARAAGFAARPLSAFAVSQARLLLRLHYPSEGYLVQEARGACFLGWQTRPLLSVSAWQPSSS* Compared with the prior art, the present invention has the following beneficial effects: (1) This invention achieves the effect of overexpressing the gene in ordinary rice plants by overexpressing the gene in ordinary rice plants. MOC1 The gene was discovered to have a novel use in enhancing rice seedling resistance to low-temperature stress, and it provides an important genetic resource for breeding rice varieties resistant to low-temperature stress.

[0031] (2) This invention utilizes transgenic technology to provide a method for breeding rice germplasm resistant to low-temperature stress, and obtains rice germplasm resistant to low-temperature stress. MOC1 Homozygous plants with gene overexpression. Attached Figure Description

[0032] Figure 1 Rice in Example 2 MOC1 Loss-of-function editing target sites and mutation types (AD).

[0033] Figure 2 Rice in Example 3 MOC1 Plants with missing gene function moc1-1 and moc1-2 Survival rates of wild-type rice plants (ZH11) after low-temperature stress; where A is a photograph of plant growth; B is a bar chart of plant survival rates.

[0034] Figure 3 Rice in Example 2 MOC1In gene overexpression materials MOC1 Schematic diagram of gene expression levels (A), rice in Example 3 MOC1 Gene overexpression plants MOC1-ox1 and MOC1-ox2 Photographs (B) and bar charts (C) of plant growth after low-temperature stress on wild-type rice plants (ZH11). Detailed Implementation

[0035] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0036] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0037] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0038] Example 1 Rice MOC1 Gene cloning, construction of gene overexpression engineered bacteria and gene editing engineered bacteria 1. Extraction of total RNA from rice Total RNA was extracted from young rice leaves using the Tiangen Plant total RNA extraction kit according to the instructions. The extracted RNA was then reverse transcribed using the Thermo Fisher reverse transcription kit according to the instructions to obtain cDNA (SEQ ID NO: 1), which was stored at -20℃ for later use.

[0039] 2. MOC1 Construction of gene overexpression engineered bacteria A Design rice MOC1 Primers for specific amplification of gene coding region sequences are as follows: MOC1-ox -F: 5'-ACTAGGGTCTCGCACCATGCTCCGGTCACTCCACT-3'; (SEQ ID NO: 4) MOC1-ox-R: 5'-ACTAGGGTCTCTCGCCCGACGACGACGGCTGCCA-3'; (SEQ ID NO: 5).

[0040] Using the cDNA (complementary DNA) obtained by reverse transcription of total RNA from the leaves of common rice plant 11 as a template, the cDNA was amplified by polymerase chain reaction (PCR). MOC1 The nucleotide sequence of the gene exons was ligated into the vector pCAMBIA1301 containing the overexpression promoter Ubi, and transformed into competent DH5α Escherichia coli by heat shock at 42°C and then plated.

[0041] Successfully transformed monoclonal DH5α Escherichia coli colonies were selected, and the culture solution after shaking was sent to a sequencing company for sequencing. The sequencing results showed that the vector contained... MOC1 The nucleotide sequences of the gene exons were obtained, and plasmids were extracted using a plasmid extraction kit from TransGen Biotech. The plasmids were electroporated into GV3101 Agrobacterium competent cells, incubated at 28°C for two days, and then plasmids were picked for PCR verification, yielding the product containing the overexpression vector pCAMBIA1301- MOC1 Agrobacterium engineered strain A.

[0042] 3. MOC1 Construction of gene-edited engineered bacteria B Based on the genome sequence (SEQ ID NO: 1), specific target sequences were searched online (http: / / crispr.hzau.edu.cn / CRISPR / ), and the target sequences “GGTGGCTGCAAGAACAATGGCGG (SEQ ID NO: 6) and CCGGGGCGTACCTGGCGTTCAAC (SEQ ID NO: 7)” were selected. Complementary primers were synthesized based on these target sequences. Bsa The vector pHun4c12 was digested with restriction endonuclease I, and the linearized vector was obtained by gel extraction. Subsequently, the fusion fragment was ligated into the linearized pHun4c12 vector (Jiang et al., 2019, Mutation of Inositol 1,3,4-trisphosphate 5 / 6-kinase6 Impairs Plant Growth and Phytic Acid Synthesis in Rice. Plants 8(5):114). Enzyme digestion confirmed the correct vector, and further sequencing confirmed that the target sequence had been incorporated into the vector. The correct vector was named pHun4c12- MOC1-1 / 2 Simultaneously, the thermal shock conversion method was used to convert pHun4c12- MOC1-1 / 2When introduced into Agrobacterium strain EHA105, the gene-editing vector pHun4c12- is obtained. MOC1 Agrobacterium engineered strain B was used for subsequent genetic transformation.

[0043] Example 2 Rice MOC1 Obtaining homozygous plants with gene overexpression and homozygous plants with gene loss of function 1. Agrobacterium-mediated genetic transformation of rice Using pCAMBIA1301- MOC1 Agrobacterium engineered strain A overexpression vector and strain containing pHun4c12- MOC1 Gene-editing Agrobacterium B engineered strain B was used to infect rice callus tissue, and after screening on differentiation and rooting media, the corresponding genetically transformed plants were obtained.

[0044] 2. Obtaining homozygous overexpression lines A small amount of leaves and roots from the T0 generation overexpression seedlings were taken, and total RNA was extracted from the plant materials (roots and leaves) using the RNeasy Plant RNA Mini Kit (Qiagen, Hilden, Germany). cDNA was then reverse transcribed using 1 μg of total RNA, oligo-dT18 primers, and the GoScript™ reverse transcription system (Promega). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using a SYBR Green GoTaq qPCRMaster Mix (Promega, WI, USA). Rice OsACTIN Genes were used as internal controls, and 2 were used. -ΔΔCt Method calculation MOC1 Relative expression level.

[0045] Table 1 Primer list for quantitative real-time polymerase chain reaction (qRT-PCR)

[0046] T0 generation transgenic overexpression plants were self-pollinated to obtain T1 generation seeds. From each T0 generation overexpression plant, 6-9 positive plants were taken and self-pollinated again to produce T2 generation seeds, which were then segregated and analyzed. When all T2 generation seedlings produced from the positive T1 generation plants tested positive, the T1 generation plant was considered a homozygous overexpression plant, and thus the T1 generation was obtained. MOC1 Transgenic pure lines with overexpression of the gene are named MOC1-ox1 and MOC1-ox2 Two plants MOC1 See gene expression levels Figure 3 A; otherwise, it is a heterozygous plant.

[0047] 3. Obtaining homozygous plants with gene function loss Young leaves were selected from T0 generation transgenic rice at the 3-4 leaf stage, and genomic DNA was extracted using the CTAB method. Endogenous DNA from the transgenic rice was then amplified using pHun4c12-MOC1 specific primers. MOC1 Gene sequencing was used to verify the PCR products, and the results showed that... MOC1 The gene has undergone single base insertion and deletion, i.e., acquired MOC1 Transgenic pure lines with gene function loss were named as follows: moc1- 1 and moc1-2 (See) Figure 1 ).

[0048] Example 3 MOC1 Experiment on the resistance of rice to low temperature stress by gene overexpression The rice obtained in Example 2 MOC1 Overexpression of T2 generation homozygous lines: MOC1-ox-1 and MOC1-ox-2 , MOC1 Transgenic pure lines with gene function loss: moc1-1 and moc1-2 Compared with ordinary rice plants, low temperature stress was applied.

[0049] To assess the cold resistance of rice plants, rice seedlings soaked for 10 days were transferred to either 4°C (experimental group) or 30°C (control group) for 4 days, followed by a 3-day recovery period at 30°C, and survival rates were analyzed. All treatments included three biological replicates of 30 to 40 plants for survival determination. All tests were conducted in an artificial indoor environment under the following conditions (relative humidity: 65–75%; 16 hours light / 8 hours darkness).

[0050] By overexpressing homozygous lines in the T2 generation MOC1-ox1 and MOC1-ox2 , MOC1 Plants with missing gene function moc1-1 and moc1-2 After cold-treated for 4 days and cultured under normal conditions for 3 days, the survival rate of the 10-day-old plants of the control variety Zhonghua 11 was calculated. MOC1 The survival rate of overexpressing rice was significantly higher than that of the control after low-temperature treatment (see...). Figure 3 B and 3C); and MOC1 Plants with missing gene function moc1-1 and moc1-2 The survival rate of rice after low-temperature treatment was significantly lower than that of the control (see...) Figure 2 (A and 2B).

[0051] The above experiments demonstrate that overexpression MOC1 Genes can enhance rice's resistance to low-temperature stress. MOC1Loss of gene function reduced the resistance of rice to low-temperature stress, indicating that... MOC1 Genes have significant application value in plant genetic engineering for resistance to low temperature stress.

Claims

1. Oryza sativa MOC1 the use of the gene in improving the low temperature stress resistance at the seedling stage of rice and / or genetic breeding of rice varieties, characterized in that, overexpressing the rice MOC1 gene, the rice MOC1 gene encodes an amino acid sequence as set forth in SEQ ID NO:

3.

2. Recombinant vector comprising a rice MOC1 gene for improving the resistance of rice seedlings to low temperature stress and / or improving the genetic breeding of rice varieties, characterized in that, overexpressing said rice MOC1 gene, the rice MOC1 gene encoding an amino acid sequence as set forth in SEQ ID NO:

3.

3. The application of genetically engineered bacteria in improving the low temperature stress resistance of rice seedlings and / or improving the genetic breeding of rice varieties, characterized in that, The genetically engineered bacteria comprise a recombinant vector containing a rice MOC1 gene, overexpression of the rice MOC1 gene, the rice MOC1 gene encodes an amino acid sequence as shown in SEQ ID NO:

3.

4. A method for improving the resistance to low temperature stress at the seedling stage of rice and / or improving the genetic breeding of a rice variety, characterized in that, The method comprises overexpressing a rice MOC1 gene, the rice MOC1 gene encodes an amino acid sequence as shown in SEQ ID NO:

3.

5. A method for obtaining a rice plant with improved traits, characterized in that, comprising the following processing steps: (1) using genetically engineered bacteria to infect rice callus; and (2) culturing the infected rice callus into rice plants; The genetically engineered bacteria comprise a recombinant vector comprising a rice MOC1 gene, wherein the rice MOC1 gene is overexpressed in the rice MOC1 gene encodes an amino acid sequence as shown in SEQ ID NO:

3.

6. Use according to any one of claims 1 to 3 or method according to 4 or 5, characterized in that, The rice MOC1 The nucleotide sequence of the gene coding region is shown as SEQ ID NO:

1.

7. Application of rice MOC1 protein in improving low-temperature stress resistance of rice seedlings and / or genetic breeding of rice varieties.

8. Use according to claim 7, characterized in that, The amino acid sequence of the rice MOC1 protein is shown as SEQ ID NO: 3.

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