Application of oslac4 gene in improving rice yield

Abstract

The application discloses application of an OsLAC4 gene in improving rice yield. Researches of the application show that knocking out the OsLAC4 gene of rice can obviously increase the grain and ear type of the rice, and the thousand-grain weight is also obviously increased. It is shown that the OsLAC4 gene is a negative regulation gene of the rice yield, and the effect of the OsLAC4 gene on improving the rice yield is obviously superior to that of an OsLAC20 gene, and high-yield rice can be bred by knocking out the OsLAC4 gene, thereby providing an efficient method for breeding high-yield rice.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering, and more particularly to application of OsLAC4 gene and the encoded protein thereof in improving rice yield. BACKGROUND

[0002] With the rapid growth of global population and the reduction of arable land on earth, food security has become increasingly important. Therefore, cultivating high-yield and high-resistance varieties has become an important issue and research direction for crop geneticists.

[0003] Rice belongs to monocotyledonous plants of Poaceae and is one of the main food crops. The yield of rice is mainly affected by factors such as tiller number, grain number per panicle, and thousand-grain weight, and environment. Therefore, finding genes with normal seed setting rate and increased yield has important guiding significance for production. At present, many yield-related genes have been identified. For example, IPA1 is a transcription factor downstream of miR156, which positively regulates rice tillering, effective grain number, and grain type, and is an ideal rice plant type. DEP1 is a key gene that controls panicle type and grain number per panicle, and plants are erect, and grain number per panicle is increased, thereby significantly improving rice yield. GS3 is a major QTL that controls rice grain length and weight, but has little effect on rice grain width and thickness.

[0004] Chinese Patent with publication number CN111534537B discloses application of OsLAC20 gene in increasing seed setting rate and improving rice yield, and Chinese Patent with publication number CN106480084B discloses application of OsLAC13 and miR397a / b in cultivating high-seed-setting-rate or high-yield rice, but the yield-increasing effect still needs to be improved. At present, application of OsLAC4 gene in improving rice yield has not been reported. SUMMARY

[0005] The present application aims to overcome the above-mentioned defects and deficiencies in the prior art, and provides application of OsLAC4 gene or OsLAC4 protein in significantly improving rice yield.

[0006] The above-mentioned object of the present application is achieved by the following technical solution:

[0007] The sequence of OsLAC4 gene is as follows: rice genome database (http: / / rice.plantbiology.msu.edu / index.shtml), the gene number of OsLAC4 is LOC_Os01g62480, the nucleotide sequence thereof is shown as SEQ ID NO: 1, and the amino acid sequence encoded thereby is shown as SEQ ID NO: 2.

[0008] The present application obtains a rice mutant plant with knocked-out OsLAC4 gene by constructing a knock-out vector of rice OsLAC4 gene and transforming the rice plant, and the results show that the ear type and grain of the rice mutant plant are significantly increased, and the thousand-grain weight is also obviously increased, indicating that the OsLAC4 gene is a negative regulatory gene of rice yield, and its effect on improving rice yield is significantly better than that of OsLAC20 gene, and the OsLAC4 gene can be used for cultivating high-yield rice by knocking out the OsLAC4 gene.

[0009] Therefore, the present application provides the following uses of the OsLAC4 gene and the encoded protein thereof:

[0010] The application of the OsLAC4 gene shown in SEQ ID NO: 1 or the OsLAC4 protein shown in SEQ ID NO: 2 in regulating the yield of rice.

[0011] Specifically, the expression level of the OsLAC4 gene or the OsLAC4 protein in rice is regulated, and then the yield of rice is regulated.

[0012] The application of the OsLAC4 gene shown in SEQ ID NO: 1 or the OsLAC4 protein shown in SEQ ID NO: 2 in improving the yield of rice.

[0013] The application of the OsLAC4 gene shown in SEQ ID NO: 1 or the OsLAC4 protein shown in SEQ ID NO: 2 in creating high-yield rice varieties.

[0014] Specifically, the expression of the OsLAC4 gene or the OsLAC4 protein is inhibited, so as to improve the yield of rice.

[0015] Preferably, a stable genetically OsLAC4 gene knockout mutant rice is constructed.

[0016] Specifically, the stable genetically OsLAC4 gene knockout mutant rice is constructed, including the following steps:

[0017] S1. Constructing an OsLAC4 gene knockout plasmid;

[0018] S2. Transforming the plasmid of step S1 into rice callus;

[0019] S3. Screening and differentiation of the transgenic rice positive callus to obtain a stable genetically OsLAC4 gene knockout mutant plant.

[0020] Preferably, the knock-out vector of the OsLAC4 gene is pYLCRISPRCas9-OsLAC4.

[0021] Further preferably, the sgRNA expression cassette construction sequence of the knockout vector target site of the OsLAC4 gene is as follows:

[0022] P1: 5'- gccgCCTAGTGATGCCCTGAGCT-3';

[0023] 5'- aaacAGCTCAGGGCATCACTAGG-3';

[0024] P2: 5'- gttgGCACAACATCTCGTTGCAC-3';

[0025] 5'- aaacGTGCAACGAGATGTTGTGC-3'.

[0026] The application also provides application of the knockout vector or plasmid of the OsLAC4 gene in improving rice yield or in creating a high-yield rice variety.

[0027] Specifically, the high yield refers to larger ear type, more grains per ear or effective grains, larger grains and heavier thousand-grain weight.

[0028] Compared with the prior art, the application has the following beneficial effects:

[0029] The application discloses application of an OsLAC4 gene in improving rice yield. Researches show that rice plants with the OsLAC4 gene knocked out have more primary branches, larger ear types, and obviously increased length, width and thickness of grains, and obviously increased thousand-grain weight, indicating that the OsLAC4 gene is a negative regulation gene of rice yield, and the effect of the OsLAC4 gene on improving rice yield is obviously better than that of an OsLAC20 gene. Therefore, negative regulation of the OsLAC4 gene can be used for cultivating high-yield rice, and a simple and efficient method for cultivating high-yield rice is provided. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Genotype detection of the OsLAC4 mutant strain knocked out by the CRISPR-Cas9 technology.

[0031] Figure 2 Comparison of the wild type and the mutant strain, wherein the left side is the wild type, and the right side is the mutant strain knocked out by the CRISPR-Cas9 technology.

[0032] Figure 3 Comparison of the ear types of the wild type and the mutant strain, wherein the left side is the wild type, and the right side is the mutant strain knocked out by the CRISPR-Cas9 technology. B is a statistical diagram of the primary branches of the ear types of the wild type and the mutant strain.

[0033] Figure 4Figure 1. Comparison of seed length, width and thickness between wild type and CRISPR-Cas9 knockout OsLAC4 mutant. The upper row is wild type, and the lower row is CRISPR-Cas9 knockout OsLAC4 mutant. A is the statistical chart of seed type between wild type and CRISPR-Cas9 knockout OsLAC4 mutant.

[0034] Figure 5 Figure 2. Comparison of 1000-grain weight between wild type and CRISPR-Cas9 knockout OsLAC4 mutant. DETAILED DESCRIPTION

[0035] The present application is further illustrated by the following description in connection with the drawings and specific examples. The examples do not, however, limit the present application in any form. Unless otherwise specified, the reagents, methods and devices employed in the present application are conventional in the art.

[0036] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0037] Example 1 Construction of pYLCRISPR / Cas9-OsLAC4 knockout plasmid

[0038] 1. Design of target sites for OsLAC4 knockout plasmid

[0039] Two target sites were designed on OsLAC4 using online software CRISPR-P (http: / / skl.scau.edu.cn / targetdesign / ).

[0040] The sgRNA expression cassettes for the two target sites were constructed by primers. The primer sequences are shown as follows:

[0041] P1: 5'-gccgCCTAGTGATGCCCTGAGCT-3';

[0042] 5'-aaacAGCTCAGGGCATCACTAGG-3';

[0043] P2: 5'-gttgGCACAACATCTCGTTGCAC-3';

[0044] 5'-aaacGTGCAACGAGATGTTGTGC-3'.

[0045] 2. The expression cassettes were connected into pYLCRISPR / Cas9 vector with the backbone of binary vector pCAMBIA-1300 and transformed into E. coli to complete the construction of OsLAC4 knockout plasmid.

[0046] Example 2 OsLAC4 knock-out plasmid transformation of rice callus to obtain knock-out rice mutant strain

[0047] 1. Agrobacterium tumefaciens transformation with knock-out plasmid

[0048] The above constructed expression plasmid was transformed into Agrobacterium tumefaciens (commercially available), using a conventional method in the art, and the Agrobacterium tumefaciens strain transformed with the OsLAC4 knock-out plasmid was streaked on YEP medium (10 g yeast extract, 10 g peptone, 5 g NaCl, 15 g agar) containing rifampicin, kanamycin and hygromycin, and incubated at 28°C in the dark for 2-3 days. Single colonies were picked and plated on YEP medium containing the same antibiotics, and incubated at 28°C in the dark for 2 days. An appropriate amount of the bacterial mass was suspended in liquid co-culture medium containing 100 μM acetosyringone, and diluted to an OD 550 of about 0.3, and incubated at 28°C on a shaker (140 rpm) for 40 minutes to prepare Agrobacterium tumefaciens inoculation solution, which can be used for infection.

[0049] The mature seeds of rice were peeled, soaked in 75% alcohol for 1 minute, washed with sterile water several times, treated with 1% sodium hypochlorite twice for 20 minutes each time, shaken several times during the treatment, washed with sterile water several times, and then dried with sterile filter paper. The peeled seeds were inoculated on induction medium (N6 macro, B5 micro, B5 organic, iron salt, 2 mg / L 2,4-D, 30 g / L sucrose, 500 mg / L glutamine, 500 mg / L proline, 3.0 g / L hydrolyzed casein, 3.0 g / L phytagel) to induce embryogenic callus. The induced embryogenic callus was inoculated on fresh induction medium, and the callus in good growth condition was picked every 2-3 weeks and subcultured on fresh induction medium.

[0050] The callus in good growth condition, which was pale yellow, granular and compact in structure, was picked and dried in a sterile triangular flask. The callus was then infected with the prepared Agrobacterium tumefaciens inoculation solution for 20 minutes, with appropriate shaking. After infection, the callus was placed in a culture dish with sterile filter paper and air-dried for 1-2 hours. The callus was then inoculated on co-culture medium with a layer of filter paper, air-dried for about half an hour, and incubated at 26°C in the dark for 2-3 days.

[0051] 2. Screening and differentiation of positive callus of transgenic rice

[0052] The callus after co-cultivation is taken out and placed in a sterile culture dish with three layers of filter paper, dried for about 1 day, and then inoculated on the screening medium and cultured in the dark at 26°C for 14-21 days. Screening is performed twice. The resistant callus with good growth state is inoculated on the pre-differentiation medium and cultured in the light at 26°C. After 21 days, the resistant callus with good growth state and green spots is inoculated on the differentiation medium to make the callus regenerate. When the seedlings differentiated from the resistant callus grow to 4-6 cm, they are transferred to the rooting medium and cultured in the light at 26°C. When the seedlings grow to 10-12 cm with wide leaves, dark green color and healthy root system, the medium and the callus attached to the base are washed away, and the seedlings are planted in pots outdoors to obtain transgenic rice.

[0053] The screening medium has the following formulation: N6 medium in large amount, MS medium in small amount, B5 medium in small amount, 1 g / L hydrolyzed casein, 1 g / L proline, 2 mg / L (2,4-D), 30 g / L sucrose, 50 mg / L hygromycin, 500 mg / L cefotaxime, and 4 g / L plant gel, and the final pH of the screening medium is 5.8.

[0054] The pre-differentiation medium has the following formulation: MS medium, 1 g / L hydrolyzed casein, 20 g / L sucrose, 1 mg / L (2,4-D), 500 mg / L cefotaxime, 50 mg / L hygromycin, and 4 g / L plant gel, and the final pH of the pre-differentiation medium is 5.8.

[0055] The differentiation medium has the following formulation: MS medium, 2 mg / L (6-BA), 0.5 mg / L naphthalene acetic acid, 1 mg / L kinetin, 30 g / L sucrose, 3% sorbitol, and 4 g / L plant gel, and the final pH of the differentiation medium is 5.8.

[0056] The rooting medium has the following formulation: 1 / 2MS medium.

[0057] The formulations of the MS medium, 1 / 2MS medium, and N6 medium used in the example are conventional formulations in the art. The reagents involved in the formulations of the above-mentioned media are commercially available.

[0058] 3. Identification of CRISPR / Cas9 mutant strains

[0059] According to the sequence of genomic DNA in the MSU library, the test primers were designed at the position of about 100-200 bp upstream and downstream of the knockout target. The DNA in the leaves or other tissues of the mutant strain was extracted as a template, and the DNA fragment was obtained by high-fidelity enzyme PCR amplification with outer primers, and then the product was gel electrophoresis and the gel was cut and recovered, and the designed inner primers were sent to the company for sequencing. The sequencing results were analyzed by the website to analyze the knockout effect: http: / / dsdecode.scgene.com / home / . The genotype detection results of the OsLAC4 mutant strain knocked out by CRISPR-Cas9 technology are shown in Figure 1 , which indicates that the OsLAC4 gene is a frameshift mutation.

[0060] Example 3 Rice planting and phenotype analysis of OsLAC4 mutant strain

[0061] The CRISPER-Cas9 knockout mutant strain uses a mutant strain in which OsLAC4 on both chromosomes is inactivated. The rice seeds are germinated with water, and after one week of planting on a flat plate, they are transferred to the field and grown to grain maturity. Among them, the seed size and thousand-grain weight are compared using completely mature and dried seeds. All statistical data are the average of more than 30 rice seedlings.

[0062] Figure 2 For the comparison of wild type and mutant whole plant, among them, the left is wild type, and the right is the mutant strain knocked out by CRISPR-Cas9 technology, which shows that the mutant strain knocked out by CRISPR-Cas9 technology is taller than the WT plant.

[0063] Figure 3 For the comparison of wild type and mutant ear size, among them, the left of A figure is wild type, and the right is the mutant strain knocked out by CRISPR-Cas9 technology, which shows that the mutant strain knocked out by OsLAC4 has much larger ear type than WT. B figure is a statistical diagram of the first branch of wild type and mutant. The gray column represents wild type, and the orange column represents the mutant strain knocked out by CRISPR-Cas9 technology. It shows that the first branch of the mutant strain knocked out by OsLAC4 is more than WT, and the ear type is large.

[0064] Figure 4Figure A is a comparison of seed length, width and thickness between wild type and CRISPR-Cas9 knockout OsLAC4 mutant strains. The upper row is wild type, and the lower row is the CRISPR-Cas9 knockout OsLAC4 mutant strain, indicating that the seed length, width and thickness of the knockout OsLAC4 mutant strain are much larger than that of the WT. Figure B is a statistical chart of seed length, width and thickness of wild type and CRISPR-Cas9 knockout OsLAC4 mutant strains. The gray column represents wild type, and the orange column represents the CRISPR-Cas9 knockout OsLAC4 mutant strain. It is shown that the grain type of the knockout OsLAC4 is indeed increased.

[0065] Figure 5 Figure C is a statistical comparison of the thousand seed weight of wild type and CRISPR-Cas9 knockout OsLAC4 mutant strains. The gray column represents wild type, and the orange column represents the CRISPR-Cas9 knockout OsLAC4 mutant strain. It is shown that the thousand seed weight of the CRISPR-Cas9 knockout OsLAC4 mutant strain is significantly increased compared with WT. The thousand seed weight of the OsLAC4 mutant strain is significantly higher than that of WT by more than 15%, while the thousand seed weight of the OsLAC20 mutant strain is only about 2.3% higher than that of WT. It is shown that the thousand seed weight of the CRISPR-Cas9 knockout OsLAC4 mutant strain is significantly increased compared with WT and the OsLAC20 mutant strain.

[0066] The above Figures 2 to 5 The results show that knocking out the rice OsLAC4 gene can increase the growth rate of rice, significantly improve the yield of rice, increase the size of the grain and the thousand seed weight, and the improvement effect is significantly better than that of the OsLAC20 gene reported in the prior art.

Claims

1. Use of the gene represented by SEQ ID NO: 1 in increasing yield of rice, characterized in that, OsLAC4 Construction of stably inherited OsLAC4 gene knockout mutant rice. ​ 2. As shown in SEQ ID NO: 1 OsLAC4 The application of genes in the creation of high-yield rice varieties is characterized by, Construction of stably inherited OsLAC4 gene knockout mutant rice.

3. Use according to claim 1 or 2, characterized in that, The constructed stable genetic OsLAC4 A gene knockout mutant rice, comprising the steps of: S1. Construction OsLAC4 Gene knockout plasmid; S2. Transform the plasmid of step S1 into rice callus; S3. Screening and differentiation of transgenic rice positive calli to obtain stably inherited OsLAC4 knockout mutant plants.

4. Use according to claim 3, characterized in that, The OsLAC4 The gene knockout plasmid is pYLCRISPR / Cas9- OsLAC4 The construction method is to connect the sgRNA expression cassette of the gene into the pYLCRISPR / Cas9 vector with the backbone of the binary vector pCAMBIA-1300. OsLAC4 The construction method is to connect the sgRNA expression cassette of the gene into the pYLCRISPR / Cas9 vector with the backbone of the binary vector pCAMBIA-1300.

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