Application of osmorn1 gene in regulating cold tolerance and / or yield of rice

Abstract

The application discloses OsMORN1 Application of a gene in regulating cold tolerance and / or yield of rice. The application proves that a method for knocking out the gene makes the low-temperature germination rate of the mutant at 16 DEG C significantly lower than that of the wild type, and the cold tolerance of the mutant at the seedling stage also presents a downward trend compared with the wild type. This indicates that the gene has a positive regulation effect on regulating the low-temperature germination of rice seeds and the cold tolerance at the seedling stage, which has important research significance for studying the cold tolerance regulation network of rice. Meanwhile, it can be seen that overexpression or up-regulation of the gene can significantly improve the yield of crops including plant height, ear length, grain length, grain width and thousand-grain weight. In general, overexpression or up-regulation of the gene can simultaneously improve the yield and grain size. OsMORN1 OsMORN1 OsMORN1 OsMORN1 ​​​​

Description

Technical Field

[0001] This invention relates to the field of plant gene breeding application technology, and particularly to... OsMORN1 Application of genes in regulating cold tolerance and yield in rice. Background Technology

[0002] Rice, as one of the most important food crops, is the staple food for more than half of the world's population. Originating in tropical and subtropical regions, rice is highly sensitive to low temperatures and chilling injury. Low temperatures limit the expansion of rice cultivation to higher altitudes and latitudes, thus limiting the area under rice cultivation. Frequent low temperatures in rice-growing areas also severely impact rice yield and quality. Rice plants are susceptible to low temperatures throughout their life cycle. The direct impact of chilling injury is a decrease in grain filling rate, leading to a significant reduction in yield and threatening regional food security. Therefore, improving the cold resistance of rice is of great importance.

[0003] In this era of climate change, both biotic and abiotic environmental stresses pose a serious threat to sustainable food production. Other worrying factors include declining arable land supply, soil degradation, and unsustainable farming practices. Therefore, developing high-yielding crop plants with substantial stress resistance is of paramount importance. Consequently, there is an urgent need to breed new, stress-tolerant, and high-yielding rice varieties. Summary of the Invention

[0004] The purpose of this invention is to overcome the current problems related to the cold tolerance and yield of rice, and to provide... OsMORN1 Application of gene-related biomaterials.

[0005] OsMORN1 The gene is located on chromosome 10, Chr10:22316008-22320296, with the following locus number: LOC Os10g41534 (phytozome database), its full-length genome sequence is 4288 bp, including 3 exons, 2 introns, 5′ untranslated region (5′UTR), and 3′ untranslated region (3′UTR), and its full-length cDNA is 1404 bp (SEQ ID NO.1), encoding 468 amino acids (SEQ ID NO.3).

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] OsMORN1 Applications of gene-related biomaterials, including:

[0008] The biomaterial is any one or more of the following substances: A, B, C, D, E, and F:

[0009] A. OsMORN1Full-length genomic DNA of a gene or its homologous nucleic acid molecules;

[0010] B. OsMORN1 Gene cDNA or its homologous nucleic acid molecules;

[0011] C. OsMORN1 Gene-encoded OsMORN1 Protein or its homologous amino acid sequence;

[0012] D. OsMORN1 The promoter of gene expression or its homologous nucleic acid molecules;

[0013] E. Substances that can increase the expression level and / or activity of substances A, B, or D;

[0014] F. Substances that can increase the expression level and / or activity of substance C.

[0015] Furthermore, the nucleotide sequence of the full-length genomic DNA described in A, as shown by the gene locus number in the phytozome database, is... LOC_Os10g41534 sequence is shown.

[0016] Furthermore, the homologous nucleic acid molecules mentioned in A refer to nucleic acid molecules that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with full-length genomic DNA.

[0017] Furthermore, the nucleotide sequence of the cDNA described in B is shown in SEQ ID NO.1.

[0018] Furthermore, the homologous nucleic acid molecules described in B refer to nucleic acid molecules that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the sequence shown in SEQ ID NO.1.

[0019] Furthermore, the amino acid sequence of the OsMORN1 protein described in C is shown in SEQ ID NO.3.

[0020] Furthermore, the homologous amino acid sequence mentioned in C refers to an amino acid sequence that has at least 60%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the sequence shown in SEQ ID NO.3.

[0021] Furthermore, the nucleotide sequence of the promoter described in D is shown in SEQ ID NO.2.

[0022] Furthermore, the homologous nucleic acid molecules described in D refer to nucleic acid molecules that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the sequence shown in SEQ ID NO.2.

[0023] Furthermore, the application is any one or more of the following applications 1), 2), and 3):

[0024] 1) Application of positive regulation of cold tolerance in rice;

[0025] 2) Application of positive regulation of rice yield;

[0026] 3) Application in improving rice grain shape.

[0027] Furthermore, application 1) includes any one or more of the following: applications to improve the germination rate of rice seeds at low temperatures and applications to improve the survival rate of rice seedlings at low temperatures.

[0028] Furthermore, application 2) includes any one or more of the following: applications to improve spikelet fertility in rice, applications to improve grain yield per plant in rice, applications to improve the number of effective panicles in rice, applications to increase the number of filled grains per panicle in rice, applications to increase the seed setting rate in rice, applications to increase plant height in rice, and applications to increase the thousand-grain weight in rice.

[0029] Furthermore, application 3) includes any one or more of the following: applications that increase the grain length of rice and applications that increase the grain width of rice.

[0030] Furthermore, the substance E is selected from: recombinant vectors containing corresponding nucleic acid molecules, expression cassettes, transgenic cell lines, transgenic plant tissues, or recombinant bacteria.

[0031] Furthermore, the recombinant vector used is selected from the pEarleyGate100 plant expression vector containing the CaMV 35S promoter.

[0032] Furthermore, the substance F is selected from: recombinant vectors containing coding genes with corresponding amino acid sequences, expression cassettes, transgenic cell lines, transgenic plant tissues, or recombinant bacteria.

[0033] Furthermore, the rice mentioned includes Nipponbare.

[0034] The present invention has the following advantages and effects compared with the prior art:

[0035] Experiments have shown that, OsMORN1 Knocking out the gene in the wild-type rice variety Nipponbare significantly reduced seed germination rate and seedling survival rate at 16℃ compared to the wild type, and also significantly reduced yield. OsMORN1 Overexpression of the gene in the wild-type cultivar Nipponbare significantly increased yield compared to the wild type. Specifically, overexpressing plants showed significant increases in plant height, number of primary / secondary branches, grain length, grain width, and thousand-grain weight compared to the wild type. This demonstrates... OsMORN1 The potential applications of genes in agricultural production. Attached Figure Description

[0036] Picture 1 for OsMORN1 The relative expression of the gene in its knockout mutants and overexpression lines; among them. ACTIN1 As an internal reference, wild-type OsMORN1 Gene expression levels were compared using a 1:1 ratio; t-tests were used for significance analysis, * P < 0.05, ** P < 0.01.

[0037] Picture 2 Figure 1 shows the statistical results of the germination rate of OsMORN1 knockout mutants under low temperature stress and the results of the study on regulating the cold tolerance of rice seedlings; where (ab) represents the results. osmorn1 The cumulative germination rate of mutant seeds at room temperature and low temperature over time; (c).WT, osmorn1 Morphological phenotype of seedlings after cold treatment; seedlings were grown at 30°C for 3 weeks, then treated at 4°C for 48 h, and then recovered at 30°C for 7 days; plants were photographed after cold treatment and recovery; scale bar = 5 cm; (d). Survival rate was determined based on the mutants and WT shown in (a); numerical values ​​are expressed as mean ± SD of three biological replicates (n = 30 per replicate); t-test was used for significance analysis, * P < 0.05, ** P < 0.01.

[0038] Picture 3 Figure 1 shows the results of the study on the regulatory effect of OsMORN1 on rice seed yield; (a) plant type of mutant and WT plants; BAR = 20 cm; (bf) spikelet fertility (b), grain yield per plant (c), number of effective panicles (d), number of filled grains per panicle (e), and seed setting rate (f); agronomic traits of 30 rice plants of each line were measured; values ​​obtained from 30 biological replicates are expressed as mean ± standard deviation; t-test showed that the asterisks were significantly different from the WT values ​​(*P<0.05, **P<0.01 and ***P<0.001).

[0039] Picture 4Plant and panicle types of OsMORN1 overexpressing plants are shown in the figures. (a) Plant type of WT plants (Nipponbare NIP) and overexpressing plants, BAR = 20 cm; (b) Panicle type of WT plants and overexpressing plants, BAR = 5 cm; (c) Panicle length of WT plants and overexpressing plants; (d) Plant height of WT plants and overexpressing plants. Agronomic traits of 30 rice plants from each line were measured. Values ​​obtained from 30 biological replicates are expressed as mean ± standard deviation. A t-test showed that the values ​​marked with an asterisk (*P<0.05, **P<0.01 and ***P<0.001) were significantly different from the WT values.

[0040] Picture 5 This is a diagram illustrating the regulation of rice yield by OSMORN1; (a) Schematic diagram of grain length in WT plants, mutants, and overexpressing plants, BAR=1 cm; (b) Schematic diagram of grain width in WT plants, mutants, and overexpressing plants, BAR=1 cm; (c) Grain length in WT plants, mutants, and overexpressing plants; (d) Grain width in WT plants, mutants, and overexpressing plants; (e) 1000-grain weight in WT plants, mutants, and overexpressing plants; Values ​​obtained from 30 biological replicates are expressed as mean ± standard deviation; t-test showed that the values ​​marked with asterisks were significantly different from the WT values ​​(*P<0.05, **P<0.01 and ***P<0.001). Detailed Implementation

[0041] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0043] All raw materials and equipment used in this invention are commercially available products that can be directly purchased from the market, and the primer sequences used are synthesized by Shanghai Jierui Biotechnology Co., Ltd.

[0044] OsMORN1 The gene is located on chromosome 10, Chr10:22316008-22320296, with the following locus number: LOC Os10g41534 (Phytozome database), its full-length genome sequence is 4288 bp, including 3 exons, 2 introns, 5′ untranslated region (5′UTR), and 3′ untranslated region (3′UTR). Its full-length cDNA is 1404 bp (SEQ ID NO.1), encoding 468 amino acids (SEQ ID NO.3).

[0045] The following examples use pEarley Gate100 The plasmid was purchased from the Biovector plasmid vector bacterial strain cell protein antibody gene depository center (NTCC).

[0046] Example 1 OsMORN1 Construction of gene knockout and / or overexpression vectors

[0047] OsMORN1 CDS amplification of genes

[0048] Total RNA was extracted from seedlings of cultivated rice Nipponbare using a plant total RNA extraction kit (TIANGEN, DP432). Using the total RNA as a template, cDNA sequences were synthesized using the lnRcute lncRNA cDNA first-strand synthesis kit (TIANGEN, KR202). Using the cDNA as a template and M1-F and M1-R as primers, PCR amplification was performed to obtain the PCR product OsMORN1. The nucleotide sequence of this PCR product is shown in SEQ ID NO.1.

[0049] The M1-F and M1-R sequences are as follows:

[0050] M1-F: 5'-ATGCACCACCACCACCACCTC-3'

[0051] M1-R: 5'-TCACACTACTGACAACGGTAG-3'.

[0052] 2) OsMORN1 Construction of knockout and / or overexpression vectors

[0053] according to OsMORN1 Based on gene structure characteristics, two specific target sites were selected from exon 1 and exon 2. Knockout vectors were constructed using the CRISPR / Cas9 system described in "CRISPR-GE: A convenient software toolkit for CRISPR-based genome editing". OsMORN1 Knockout vector. The primer sequences used are as follows:

[0054] gRT1-MORN1: 5'-CCCCGTCATGACCTCCTCCTGTTTTAGAGCTAGAAAT-3'

[0055] U3T1-MORN1: 5'-AGGAGGAGGTCATGACGGGTGCCACGGATCATCTGC-3'

[0056] gRT2-MORN1: 5'-TCACAGCTATAGGGGACCCAGTTTTAGAGCTAGAAAT-3'.

[0057] Using the above OsMORN1 The full-length CDS sequence of the gene was constructed using the Gateway vector system. OsMORN1 The full-length CDS of the gene is constructed on a substrate containing the CaMV 35S promoter. pEarley Gate100 Then obtain from plant expression vectors OsMORN1 Overexpression vector.

[0058] The obtained OsMORN1 Knockout vectors and / or overexpression vectors were transformed into rice callus tissue using Agrobacterium. Rice callus tissue containing the knockout vector was selectively cultured on Hyg medium; rice callus tissue containing the overexpression vector was selectively cultured on Bar medium. After cultivation, the desired results were obtained. OsMORN1 Mutants and / or overexpressed transgenic plants. The selection medium was changed after 2 weeks, and cultured for another 2-3 weeks. Vigorous, dense callus tissue was selected and transferred to pre-differentiation medium, and cultured in the dark at 26-28℃ for about 1 week. Then, the callus tissue was transferred to differentiation medium and cultured under light at 26-28℃ until regenerated plants differentiated. Total DNA was extracted from the transgenic rice. Using the DNA as a template, specific primers were designed based on the tag gene on the vector and our target gene for PCR amplification. The positive transgenic plants were identified by combining the amplified bands with sequencing. The selected positive transgenic plants are... OsMORN1 Mutants and / or overexpressed transgenic plants.

[0059] 3) OsMORN1 Validation of expression levels in mutants and / or overexpressing plants

[0060] The results obtained in (2) above OsMORN1 Total RNA was extracted from seedlings of mutant and / or overexpressing transgenic plants using a plant total RNA extraction kit (TIANGEN, DP432). Using the total RNA as a template, cDNA sequences were synthesized using the lnRcute lncRNAcDNA first-strand synthesis kit (TIANGEN, KR202). Then, using the obtained cDNA as a template, targeting… OSMORN1 Genes were analyzed using real-time quantitative PCR to detect their presence in various materials. OSMORN1 Gene expression levels at the transcriptional level. The experiment was repeated three times, and the average value was taken. The primer sequences used are as follows:

[0061] OsActin-F: 5'- TGGCATCTCTCAGCACATTCC-3'

[0062] OsActin-R: 5'- TGCACAATGGATGGGTCAGA-3'

[0063] OsMORN1-qPCR-F: 5'-TGGCCACTACCATTTCAGG-3'

[0064] OsMORN1-qPCR-R: 5'-GACCATTGGCAAAACTGTAGACT-3'.

[0065] Example 2: Cold Tolerance Test of Mutants and Wild Types

[0066] 1) Evaluation criteria for seed germination

[0067] The ears of mature plants in the experimental field were cut and dried in a glass greenhouse for a week to obtain the seeds. Seed threshing was then carried out. Seed germination was conducted according to the method described in "Influence of isopropylmalate synthase OsIPMS1 on seedvigour associated with amino acid and energy metabolism in rice". A seed was considered to have germinated when the radicle emerged from the seed coat. A seedling was considered to have survived when the root reached the seed length and the shoot reached half the seed length. Each experiment was repeated at least three times.

[0068] 2) Cold resistance test during the budding stage

[0069] Cold tolerance during the germination stage was assessed using the germination rate of 30 seeds. Homozygous mutant seeds and wild-type seeds were placed separately in moist petri dishes and cultured at 16°C for 13 days. The control group was cultured at 30°C for 10 days during the same period. Germination rates were recorded daily for both treatments and the control group. Each sample was subjected to three biological replicates.

[0070] 3) Cold tolerance test during the seedling stage

[0071] To assess cold tolerance in the seedling stage, 3-week-old mutant and wild-type seedlings were treated at 4°C for 2 days. After recovering at 30°C for 7 days, survival rate was determined by the percentage of surviving seedlings out of the total number of seedlings. Each sample was subjected to 16 biological replicates. All experiments were repeated at least three times.

[0072] Example 3: Measurement and Statistical Analysis of Agronomic Traits

[0073] All plants were planted in the field with a longitudinal spacing of 10-15 cm and a lateral spacing of 13-20 cm. After the seeds matured, phenotypic photos and measurements were taken, as detailed below:

[0074] Analysis of panicles: Thirty individual plants were collected to statistically analyze the yield per plant, number of effective panicles, number of filled grains per panicle, panicle length, primary branches, secondary branches, and seed setting rate.

[0075] Analysis of plant height: 30 individual plants were collected for plant height measurement. The measurement started from the above-ground part of the rice plant. The panicle was held close to a ruler and stretched until the highest point of the panicle was reached. This was the plant height data of that individual plant.

[0076] Biomass analysis: All aboveground tissues from 30 individual plants were collected, dried at 45-50℃, and then weighed and measured in length and / or width.

[0077] All data were analyzed using Graphpad Prism 8 software. Standard deviation is expressed as SD, and t-test was used for analysis of variance.

[0078] Picture 1 for OsMORN1 The relative expression of the gene in its knockout mutants and overexpression lines; among them. ACTIN1 As an internal reference, wild-type OsMORN1 Gene expression levels were compared using a 1:1 ratio; t-tests were used for significance analysis, * P < 0.05, ** P < 0.01.

[0079] Picture 2 Figure 1 shows the statistical results of the germination rate of OsMORN1 knockout mutants under low temperature stress and the results of the study on regulating the cold tolerance of rice seedlings; where (ab) represents the results. osmorn1 The cumulative germination rate of mutant seeds at room temperature and low temperature over time; (c).WT, osmorn1 Morphological phenotype of seedlings after cold treatment; seedlings were grown at 30°C for 3 weeks, then treated at 4°C for 48 h, and then recovered at 30°C for 7 days; plants were photographed after cold treatment and recovery; scale bar = 5 cm; (d). Survival rate was determined based on the mutants and WT shown in (a); numerical values ​​are expressed as mean ± SD of three biological replicates (n = 30 per replicate); t-test was used for significance analysis, * P < 0.05, ** P < 0.01.

[0080] Picture 3Figure 1 shows the results of the study on the regulatory effect of OsMORN1 on rice seed yield; (a) plant type of mutant and WT plants; BAR = 20 cm; (bf) spikelet fertility (b), grain yield per plant (c), number of effective panicles (d), number of filled grains per panicle (e), and seed setting rate (f); agronomic traits of 30 rice plants of each line were measured; values ​​obtained from 30 biological replicates are expressed as mean ± standard deviation; t-test showed that the asterisks were significantly different from the WT values ​​(*P<0.05, **P<0.01 and ***P<0.001).

[0081] Picture 4 Plant and panicle types of OsMORN1 overexpressing plants are shown in the figures. (a) Plant type of WT plants (Nipponbare NIP) and overexpressing plants, BAR = 20 cm; (b) Panicle type of WT plants and overexpressing plants, BAR = 5 cm; (c) Panicle length of WT plants and overexpressing plants; (d) Plant height of WT plants and overexpressing plants. Agronomic traits of 30 rice plants from each line were measured. Values ​​obtained from 30 biological replicates are expressed as mean ± standard deviation. A t-test showed that the values ​​marked with an asterisk (*P<0.05, **P<0.01 and ***P<0.001) were significantly different from the WT values.

[0082] Picture 5 This is a diagram illustrating the regulation of rice yield by OSMORN1; (a) Schematic diagram of grain length in WT plants, mutants, and overexpressing plants, BAR=1 cm; (b) Schematic diagram of grain width in WT plants, mutants, and overexpressing plants, BAR=1 cm; (c) Grain length in WT plants, mutants, and overexpressing plants; (d) Grain width in WT plants, mutants, and overexpressing plants; (e) 1000-grain weight in WT plants, mutants, and overexpressing plants; Values ​​obtained from 30 biological replicates are expressed as mean ± standard deviation; t-test showed that the values ​​marked with asterisks were significantly different from the WT values ​​(*P<0.05, **P<0.01 and ***P<0.001).

[0083] from Picture 2 As can be seen from this, knockout OsMORN1 After gene selection, the germination rate at 16℃ was significantly lower than that of the wild type, and the cold tolerance of the mutant seedlings also showed a decreased trend compared to the wild type. This indicates that... OsMORN1 It has a positive regulatory effect on the low-temperature germination of rice seeds and the cold tolerance of seedlings, which is of great research significance for studying the cold tolerance regulatory network of rice. Meanwhile, from... Picture 3 It can be seen that, compared with the wild type, the mutant showed a significant decrease in grain yield per spikelet, number of effective spikes, number of filled grains per spike, and seed setting rate. Picture 4It can be seen that there is overexpression or upregulation OsMORN1 Genes can significantly improve crop yield, including a significant increase in plant height and ear length compared to the wild type. Simultaneously, from... Picture 5 It can be seen that there is overexpression or upregulation OsMORN1 After gene expression, compared with the wild type, overexpressing plants showed significant increases in seed length, seed width, and thousand-seed weight, while mutants showed no significant differences in seed length, seed width, and thousand-seed weight compared with the wild type. In summary, overexpression or upregulation... OsMORN1 Genes can improve both yield and grain size simultaneously. OsMORN1 Genes have potential applications in agricultural production.

[0084] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Overexpression OsMORN1 The application of genes that increase plant height, panicle length, grain length, and / or grain width in rice is characterized by: The aforementioned OsMORN1 The nucleotide sequence of the gene's cDNA is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that: The overexpression is achieved by containing OsMORN1 The recombinant vector, expression cassette, transgenic cell line, transgenic plant tissue, or recombinant bacteria that contain the nucleotide sequence of the cDNA of the gene are used to achieve this.

3. The application according to claim 1, characterized in that: The aforementioned OsMORN1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

3.

4. The application according to claim 2, characterized in that: The recombinant vector used was selected from the pEarley Gate100 plant expression vector containing the CaMV 35S promoter.

5. The application according to claim 1, characterized in that: The rice variety mentioned includes Nipponbare.

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