CN114213515A - Gene OsR498G0917707800.01 and application of protein coded by same in regulation of rice chalkiness - Google Patents

Abstract

The invention discloses a gene OsR498G0917707800.01 and application of a protein coded by the gene in regulation and control of rice chalkiness, and relates to the field of plant genetic engineering. The invention discloses a protein coded by a gene OsR498G0917707800.01, such as 1) or 2) or 3): 1) protein with amino acid sequence shown as SEQ ID No. 2; 2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No.2 and has the same functions as the protein in 1); 3) a fusion protein obtained by connecting a label at the N end and/or the C end of the amino acid sequence of 1) or 2). The invention discovers that the OsR498G0917707800.01 gene in the rice is knocked out, and the rice chalkiness degree can be obviously reduced.

Description

Gene OsR498G0917707800.01 and application of protein coded by same in regulation of rice chalkiness

Technical Field

The invention relates to the field of plant genetic engineering, in particular to a gene OsR498G0917707800.01 and application of protein coded by the gene OsR498G0917707800.01 in regulation and control of rice chalkiness.

Background

Rice (Oryza sativa L.) is native to China and is one of the important food crops in the world. The rice is the most important grain crop in China, the yield of the rice is at the head of the grain crop, and the sowing area of the rice accounts for 1/3 of the total area of the grain crop. In China, besides the improvement of the rice yield, the improvement of the rice quality is also an important target of rice breeding. Chalkiness is one of important characters for measuring the quality of rice, directly influences the appearance quality and commodity circulation of the rice and influences the processing quality of the rice. The chalkiness reduction is one of the main targets of rice quality breeding. The research on the quality gene function is helpful to promote the breeding of new high-quality rice varieties and the industrialization of rice, and further improves the grain production benefit.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a gene for regulating and controlling rice chalkiness and a protein coded by the gene, wherein the gene is named as OsR498G0917707800.01 gene, the coded protein is named as protein OsR498G0917707800.01, the expression level of the protein OsR498G0917707800.01 is reduced or the expression of the protein OsR49G0917707800.01 is blocked, and the rice chalkiness degree can be obviously reduced.

In a first aspect of the present invention, there is provided a use of any one of the proteins 1) to 3) in regulation of rice chalkiness:

1) protein with amino acid sequence shown as SEQ ID No. 2;

2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No.2 and has the same functions as the protein in 1);

3) a fusion protein obtained by connecting a label at the N end and/or the C end of the amino acid sequence of 1) or 2).

In some embodiments of the present invention, in the above-mentioned protein, the amino acid sequence of the protein described in 1) is represented by SEQ ID No.2 and consists of 469 amino acid residues.

In some embodiments of the present invention, the protein may be artificially synthesized, or may be obtained by synthesizing a gene encoding the protein and then performing biological expression.

In some embodiments of the present invention, the tag in 3) of the above proteins refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracing and/or purification of the target protein. The tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In some embodiments of the invention, the regulation of rice chalkiness is a reduction in rice whiteness.

In some embodiments of the invention, the rice is specifically Hunan early indica No. 11, Hunan early indica No. 24, Hunan early indica No.7 or other rice varieties with OsR498G0917707800.01 alleles identical to Hunan early indica No. 11.

In a second aspect of the present invention, there is provided a use of any one of the biomaterials of (1) to (4) for regulating rice chalkiness,

(1) a nucleic acid molecule encoding said protein of the first aspect of the invention;

(2) an expression cassette comprising the nucleic acid molecule of (1);

(3) a recombinant vector comprising the nucleic acid molecule of (1) or the expression cassette of (2);

(4) a recombinant microorganism comprising the nucleic acid molecule of (1) or the expression cassette of (2) or the recombinant vector of (3).

In some embodiments of the present invention, in the above biological material, (1) the nucleic acid molecule may be a DNA, such as a cDNA, a genomic DNA or a recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In some embodiments of the present invention, in the above biological material, the nucleotide sequence of the nucleic acid molecule in (1) is shown as SEQ ID No.1, and the nucleic acid molecule consists of 1410 nucleotides.

In some embodiments of the present invention, the expression cassette in (2) in the above biological material is a DNA molecule capable of expressing the above protein in a host cell, and the DNA molecule may include an enhancer sequence.

In some embodiments of the present invention, in the above biological material, the recombinant vector in (3) comprises a nucleotide sequence shown as SEQ ID No. 1.

In some embodiments of the present invention, in the above biological material, the vector in (3) may be a plasmid, a cosmid, a phage, or a viral vector.

In some embodiments of the present invention, in the above-mentioned biomaterial, the carrier in (3) is

-Blunt Simple Cloning vector。

In some embodiments of the present invention, in the above biological material, the recombinant microorganism in (4) may be yeast, bacteria, algae, or fungi.

In some embodiments of the present invention, in the above-mentioned biological material, the recombinant microorganism described in (4) is Escherichia coli.

In some embodiments of the present invention, in the above-mentioned biological material, the recombinant microorganism in (4) is Escherichia coli DH5 α.

In a third aspect of the invention, there is provided a product for regulating rice chalkiness, the product comprising a protein according to the first aspect of the invention, or a biomaterial according to the second aspect of the invention, or any one of (1) to (4):

(1) a nucleic acid molecule which disrupts the expression level of a gene of the protein of the first aspect of the invention and/or which inhibits the activity of the protein and/or which reduces the content of the protein;

(2) an expression cassette comprising the nucleic acid molecule of (1);

(3) a recombinant vector comprising the nucleic acid molecule of (1) or the expression cassette of (2);

(4) a recombinant microorganism comprising the nucleic acid molecule of (1) or the expression cassette of (2) or the recombinant vector of (3);

wherein the biomaterial comprising a protein according to the first aspect of the invention or a biomaterial according to the second aspect of the invention is used to increase rice chalkiness;

the biomaterial comprising any one of (1) to (4) is used for reducing rice chalkiness.

In some embodiments of the present invention, in the above products, (1) the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In some embodiments of the present invention, in the above product, the recombinant vector in (3) is a recombinant vector having a sequence represented by SEQ ID No.5 or SEQ ID No. 6.

In some embodiments of the present invention, in the above product, the vector in (3) is pYLCISPR/Cas 9P 35s-H plasmid or LB-DNAi plasmid.

In some embodiments of the present invention, in the above product, the recombinant microorganism in (4) may be yeast, bacteria, algae or fungi.

In some embodiments of the present invention, in the above product, the recombinant microorganism in (4) is Escherichia coli.

In some embodiments of the present invention, in the above product, the recombinant microorganism in (4) is Escherichia coli DH5 α.

In some embodiments of the invention, the rice is specifically Hunan early indica No. 11, Hunan early indica No. 24, Hunan early indica No.7 or other rice varieties with OsR498G0917707800.01 alleles identical to Hunan early indica No. 11.

In a fourth aspect of the present invention, there is provided a use of the protein according to the first aspect of the present invention or the biomaterial according to the second aspect of the present invention in any one of (1) to (3), wherein:

(1) the application in rice breeding;

(2) the application in culturing gene knockout rice plants with lowered chalkiness degree;

(3) the application in preparing and cultivating the gene knockout rice plant product with lowered chalkiness degree.

In some embodiments of the invention, the rice is specifically Hunan early indica No. 11, Hunan early indica No. 24, Hunan early indica No.7 or other rice varieties with OsR498G0917707800.01 alleles identical to Hunan early indica No. 11.

In some embodiments of the present invention, among the above-mentioned applications, the application in plant breeding described in (1) may specifically be to cross rice in which the expression of a gene of the protein of the first aspect of the present invention is disrupted and/or the activity of the protein of the first aspect of the present invention is inhibited and/or the content of the protein of the first aspect of the present invention is reduced with other rice to breed rice.

In a fifth aspect of the present invention, there is provided a method for reducing rice chalkiness, which comprises disrupting the expression level of a gene encoding a protein of the first aspect of the present invention in a target rice plant and/or inhibiting the activity of a protein of the first aspect of the present invention in a target rice plant and/or reducing the content of a protein of the first aspect of the present invention in a target rice plant to obtain a knock-out rice plant; the chalkiness degree of the gene knockout rice plant is reduced compared with that of the rice plant.

In some embodiments of the present invention, the method of disrupting the expression level of a gene encoding a protein of the first aspect of the present invention in a target rice plant and/or inhibiting the activity of a protein of the first aspect of the present invention in a target rice plant and/or reducing the content of a protein of the first aspect of the present invention in a target rice plant is performed by knocking out or suppressing or mutating the gene encoding a protein of the first aspect of the present invention in the target rice plant.

In some embodiments of the present invention, the method of disrupting the expression level of a gene encoding a protein of the first aspect of the present invention in a target rice plant and/or inhibiting the activity of a protein of the first aspect of the present invention in a target rice plant and/or reducing the content of a protein of the first aspect of the present invention in a target rice plant is achieved by knocking out the gene encoding the protein of the first aspect of the present invention in the target rice plant by CRISPR/Cas9 technology.

In some embodiments of the invention, the target sequences in the CRISPR/Cas9 technology are shown as SEQ ID No.5 (corresponding to position 118-140 of SEQ ID No.1) and/or SEQ ID No.6 (corresponding to position 371-393 of SEQ ID No. 1).

In some embodiments of the invention, the CRISPR/Cas9 technology comprises any one of (1) - (3) a CRISPR/Cas9 gene-editing recombinant vector:

(1) the recombinant vector pYLCRISPR/Cas9-gRNA1 contains gRNA1 and Cas9 encoding genes, and the gRNA1 sequence is shown as SEQ ID No. 15;

(2) the recombinant vector LB-DNAi/gRNA2 contains gRNA2, and the gRNA2 sequence is shown in SEQ ID No. 16;

(3) the recombinant vector pYLCRISPR/Cas9-OsR498G0917707800.01 contains gRNA1, gRNA2 and Cas9 encoding genes, wherein the sequence of gRNA1 is shown as SEQ ID No.15, and the sequence of gRNA2 is shown as SEQ ID No. 16.

In some embodiments of the invention, the adapter primer pair of gRNA1 is shown as SEQ ID No.7 and SEQ ID No. 8.

In some embodiments of the invention, the adapter primer pair of gRNA2 is shown as SEQ ID No.9 and SEQ ID No. 10.

In some embodiments of the invention, the rice is specifically Hunan early indica No. 11, Hunan early indica No. 24, Hunan early indica No.7 or other rice varieties with OsR498G0917707800.01 alleles identical to Hunan early indica No. 11.

In a sixth aspect of the present invention, there is provided the use of the method of the fifth aspect of the present invention in (1) or (2),

(1) the application in rice breeding;

(2) the application in culturing gene knockout rice plants with lowered chalkiness degree.

In some embodiments of the present invention, among the above-mentioned applications, the application in plant breeding described in (1) may be specifically that the knockout rice plant obtained by the method of the fifth aspect of the present invention is crossed with other rice to breed rice.

In some embodiments of the invention, the rice is specifically Hunan early indica No. 11, Hunan early indica No. 24, Hunan early indica No.7 or other rice varieties with OsR498G0917707800.01 alleles identical to Hunan early indica No. 11.

The invention has the beneficial effects that:

the invention discovers that the rice gene OsR498G0917707800.01 and the coding protein thereof are related to rice chalkiness, and the rice gene OsR498G0917707800.01 and the coding protein thereof are knocked out, so that the rice chalkiness degree can be obviously reduced. Therefore, the OsR498G0917707800.01 gene can be used as an effective target spot of rice genetic breeding, and has good application prospects in the aspects of rice breeding and rice quality improvement.

The invention also provides a method for culturing OsR498G0917707800.01 gene-knocked-out rice plants, which can effectively knock out OsR498G0917707800.01 gene, and compared with wild rice plants, the OsR498G0917707800.01 gene-knocked-out rice plants constructed by the method have the advantages that the chalkiness degree is obviously reduced, and the rice quality is effectively improved.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 is a plasmid map of pYLCRISPR/Cas9-OsR498G0917707800.01 in the example of the present invention.

FIG. 2 shows the result of gene sequence analysis of OsR498G0917707800.01 gene of OsR498G0917707800.01 plant (pYLCIRPR/Cas 9-OsR498G0917707800.01-1) in the example of the present invention.

FIG. 3 is a comparison graph of appearance and shape of an OsR498G0917707800.01 gene knockout plant (pYLCRISPR/Cas9-OsR498G0917707800.01-1) and a wild-type plant (X11) in the embodiment of the invention.

FIG. 4 is a comparison graph of grain phenotype of OsR498G0917707800.01 gene knockout plant (pYLCISPR/Cas 9-OsR498G0917707800.01-1) and wild-type plant (X11) in the embodiment of the invention.

FIG. 5 shows the results of the chalkiness of OsR49G0917707800.01 knock-out plants (pYLCRISPR/Cas 9-OsR49G0917707800.01-1) and wild type plants (X11) in the examples of the present invention.

FIG. 6 shows the results of chalkiness rate of OsR49G0917707800.01 gene knockout plants (pYLCRISPR/Cas 9-OsR49G0917707800.01-1) and wild type plants (X11) in the examples of the present invention.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.

The reagents, methods and equipment used in the following examples are all conventional in the art. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer.

Used in the following examples

Fastpfu DNApolymeras, 10 × Easypfu Buffer and

-Blunt Simple Cloning vector supplied by Beijing Quanjin Biotechnology Ltd; the Gel recovery and purification Kit HiPure Gel Pure DNAMini Kit and HiPure Plasmid Micro Kit are provided by Meiji Biotechnology Co., Ltd, Guangzhou; 2 × Taq mix was supplied by Ranuncut Biotech, Inc. of Boling, Beijing; pYLCRISPR/Cas 9P 35s-H plasmid was supplied by Wuhanbo Biotech, Inc.; LB-DNAi plasmid was supplied by Wuhanbo Biotech, Inc.

Cloning of OsR498G0917707800.01 gene

(1) Extraction of cDNA

First, leaves of 7 d-old rice (X11) seedlings were pulverized with liquid nitrogen, and 100mg of the pulverized leaves was transferred to an RNase-free 2mL centrifuge tube, and RNA was extracted using a Plant tissue RNA extraction Kit HiPure Plant RNA Mini Kit (purchased from Shanghai Mi-Zhi Biotech Co., Ltd.). The purity and integrity of the RNA were checked by 1% agarose gel electrophoresis and the concentration of the RNA was checked by a microplate reader.

② cDNA was synthesized by using reverse transcription kit EasyScript One-Step gDNAremoval and cDNAsynthesis SuperMix (purchased from Beijing Quanyujin Biotechnology Co., Ltd.). The reaction system is shown in Table 1.

Table 1:

the reaction system is subjected to short-term centrifugation in a minitype table centrifugeMixing the above components, placing into PCR instrument, incubating at 42 deg.C for 30min, and heating at 85 deg.C for 5s

RT/RI Enzyme Mix and gDNA Remover inactivation) to obtain cDNA. After the reaction is finished, the mixture is placed into a refrigerator at the temperature of 20 ℃ below zero for storage and standby.

(2) Amplification of OsR498G0917707800.01 Gene

The nucleotide sequence of the OsR498G0917707800.01 gene of the rice is as follows:

5’-ATGAAGAAGACGGTGGTTCTCTACCCCGGCCTCGCCGTCGGCCACCTGAACCCCATGATGGAGCTCGCCGACGTCTTCCTGGACCACGGCTACGCCGTCGCCGTGGCGCTCATCGACCCGTCGGTCATGGAGAACGAAGCCAACCTCGCCGCCGCCGTCGCCCGCGCCGTCTCCTCCAAGAGCTCCACCATCTCCTTCCACACGCTCCCGGGCATCCCGGACCCTCCCTCGCTCGTCTTCAACGATCAGTTCTTCAAGAACTACTTCGACCTCGTGCGACGCCACAACGAGCACCTCCACGACTTCCTCCGCTCCGTGCGGGGCCTCCATGCCGTGGTCATCGACGCATCGTGCGCCCATGCCCATGAAGCCGCGAGGAAGCTGGGAGTCCCTGTCTTGATGTTCTACCCGTCCAACGCCGGCCACCTCGCCGTTAACTTGCAGACTCCTCTGCTTGTTGACGGGTTCAAGAAGCATCTGGGAGGAGATAGTACTAGTCCTGTCGAGTTCTTGGGTGTTCGACCCATGTCGGCTTCTCACTTGGCTGGCCTTTTTGGGCCGATTAGCGAGGTGAACAAGGATTTCGAGGCCATGATTTTTGCCGGTGCGCGCATGAACGCGGAGTTCGACGGAATCCTGATCAACACGTCCGTGTCGCTGGAGGAGCGGGCGCTGCGAGCTCTCGCCGACCCGCGCTGCTGCCCCGACGGCGTGGTAATCCCGCCGGTGTACGCCGTGGGGCCACTGGTCGACAAAGCCGCCGCCGCCGCCGGTGATGAGAGCAGCCGACATCAGTGCCTCGTGTGGCTCGACGGACAACCCGACCGCAGCGTCGTGTTCCTCTGCTTCGGGAGCATCGCCGACGCATGTGAACAGTCCGACCAGCAGCTGAAGGAGATCGCCGCCGGCCTGGACAAGTCCGGCCACCGCTTCCTGTGGGTGGTTCGGGCAACCAGCACCCAACACCTCGACGCGCTCCTACCGGAGGTGTTCTTCGCAAGAACCAGCGGCCGCGGCCTCGTCGTCAACAGCTGGGTGCCCCAGCCGAGCATCCTCCGCCACCGCGCCACCGCCGCGTTCGTGACGCACTGCGGGTGGAACTCGGTGCTAGAGGGGATCACCGCGGGGGTGCCGATGCTCTGCTGGCCGCTGTACGCGGAGCAGAGGATGAACAAGGTGCTCATGGTGGAGGACATGGGCGTCGGCGTGGAGATGGAGGGATGGCTGGAAGGGCTGGTGACCGCCGAGGAGGTGGAGACGAAGGTGAGGCTGGTCATGGAGTCCGAGCATGGAAGGAAGGTTAGAGAGCGTGTCGAGGCGCACAGAGATGGCGTGGCCATGGCCTGGAAAGATGGTGGCTCGTCGCGTGTCGCGTTTGCCCGTCTCATGTCTGAATTGCTCAACGTGTGA-3’(SEQ ID No.1)。

the amino acid sequence of the protein coded by the rice OsR498G0917707800.01 gene (SEQ ID No.1) is as follows:

MKKTVVLYPGLAVGHLNPMMELADVFLDHGYAVAVALIDPSVMENEANLAAAVARAVSSKSSTISFHTLPGIPDPPSLVFNDQFFKNYFDLVRRHNEHLHDFLRSVRGLHAVVIDASCAHAHEAARKLGVPVLMFYPSNAGHLAVNLQTPLLVDGFKKHLGGDSTSPVEFLGVRPMSASHLAGLFGPISEVNKDFEAMIFAGARMNAEFDGILINTSVSLEERALRALADPRCCPDGVVIPPVYAVGPLVDKAAAAAGDESSRHQCLVWLDGQPDRSVVFLCFGSIADACEQSDQQLKEIAAGLDKSGHRFLWVVRATSTQHLDALLPEVFFARTSGRGLVVNSWVPQPSILRHRATAAFVTHCGWNSVLEGITAGVPMLCWPLYAEQRMNKVLMVEDMGVGVEMEGWLEGLVTAEEVETKVRLVMESEHGRKVRERVEAHRDGVAMAWKDGGSSRVAFARLMSELLNV(SEQ ID No.2)。

the sequence of an amplification primer F1/R1 of the OsR498G0917707800.01 gene is as follows:

F1：5’-ATGAAGAAGACGGTGGTTCTC-3’(SEQ ID No.3)；

R1：5’-TCACACGTTGAGCAATTCAGA-3’(SEQ ID No.4)。

taking the cDNA extracted in the step (1) as a template, and adopting a primer F1/R1 and high-fidelity DNA polymerase

Amplification of the target gene was carried out by FastPfu DNA Polymerase. The amplification system is shown in Table 2.

Table 2:

the reaction procedure is as follows: 3min at 95 ℃; 95 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 2min, 35 cycles; 5min at 72 ℃.

And after reaction, obtaining a PCR product.

(3) Recovery and ligation of fragments of interest

And (3) running the PCR product in the step (2) on 1% agarose Gel electrophoresis, cutting a target band with the size of about 1410bp, and recovering by using a Gel recovery and purification Kit HiPure Gel Pure DNA Mini Kit, wherein the operation is strictly performed according to the instruction, so that the recovered product is obtained.

Connecting the recovered product to

-Blunt Simple Cloning vector. Wherein, the reaction system is as follows: 4 μ L of recovered product and 1 μ L

-a Blunt Simple Cloning vector, under the reaction conditions: at 25 ℃ for 15min, the ligation product (

-Blunt Simple Cloning vector/OsR498G0917707800.01)。

(4) Transformation of

mu.L of the ligation product from step (3) and 10. mu.L of LKCM (containing 0.5M KCl, 0.15M CaCl) were added to the centrifuge tubes₂And 0.25M MgCl₂) And 35. mu.L ddH₂And O. Taking 50 mu L of DH5 alpha competent cells out of the centrifuge tube from minus 80 ℃, placing the cells on ice, quickly adding the cells into the centrifuge tube after the cells are completely melted, lightly blowing and uniformly mixing the cells by using a gun head, then sequentially standing the centrifuge tube on the ice for 30min, thermally shocking the centrifuge tube in water bath at 42 ℃ for 30s, standing the centrifuge tube on the ice for 5min, then adding 500 mu L of LB liquid culture medium into the centrifuge tube in a super clean bench, culturing the cells for 1h at 37 ℃ and 180rpm, and then centrifuging the cells for 5min at 6000 Xg. A portion of the supernatant was discarded in a clean bench, 200. mu.L of the supernatant was retained to resuspend the cells, and the cell suspension was then spread evenly on LB solid medium (containing 50mg/L kanamycin) and cultured by inversion at 37 ℃ for 12 hours.

(5) Identification of Positive clones

Gently dipping a single colony on the LB solid medium obtained in the step (4) in a super clean bench by using a tip, and dissolving the single colony in 4 mu L ddH₂And (3) adding 5 mu L of 2 xTaq mix, 0.5 mu L F1(10 mu M) and 0.5 mu L R1(10 mu M) into the O, uniformly mixing, and amplifying according to the reaction program in the step (1) to obtain a PCR product.

The PCR product was run on a 1% agarose gel and a sample with a single band of interest of about 1410bp in size was identified as a positive clone.

(6) Extraction of plasmids

Picking single colonies identified as positive clones in step (5)Dissolved in 20mL of LB liquid medium (containing 50mg/L kanamycin), cultured at 37 ℃ and 180rpm to OD₆₀₀About 0.6. And (3) treating the bacterial liquid by using a HiPure Plasmid Micro Kit to extract plasmids, wherein the operation is strictly carried out according to the instruction.

And sending the extracted plasmid to an organism of the department of Onychidae for sequencing, slightly reversing and uniformly mixing bacterial liquid with correct sequencing and 50% glycerol according to the proportion of 1:1(V/V), and storing in a refrigerator at the temperature of 80 ℃ below zero.

Construction of OsR498G0917707800.01 Gene knockout Rice plants

An OsR498G0917707800.01 gene knockout rice plant is constructed by using a CRISPR/Cas9 system in an early indica type 11 (X11).

1. Construction method

a. Design target

Two specific targets (Target 1 and Target2) were designed by an online design tool (http:// skl. scau. edu. cn/home /) with the following specific sequences:

target 1: 5'-GCTTCGTTCTCCATGACCGACGG-3' (SEQ ID No.5, i.e. corresponding to position 118-140 of SEQ ID No. 1);

target 2: 5'-AGGGACTCCCAGCTTCCTCGCGG-3' (SEQ ID No.6, corresponding to position 371 and 393 of SEQ ID No. 1).

b. Construction of recombinant vector containing gRNA1 and gRNA2

Grnas were designed for two Target sequences (Target 1 and Target2), respectively, and adapter primers for the grnas were synthesized. Wherein, the sequences of gRNA1 aiming at Target 1 and an adapter primer PF2/PR2 thereof are as follows:

gRNA1：5’-TGCAGCTTCGTTCTCCATGACCGAGTTT-3’(SEQ ID No.15)；

PF2：5’-cagtGGTCTCatgcagcttcgttctccatgaccga-3’(SEQ ID No.7)；

PR2：5’-cagtGGTCTCaaaactcggtcatggagaacgaagc-3’(SEQ ID No.8)；

the sequences of gRNA2 and its adapter primer PF3/PR3 for Target2 are as follows:

gRNA2：5’-TGCAAGGGACTCCCAGCTTCCTCGGTTT-3’(SEQ ID No.16)

PF3：5’-cagtGGTCTCatgcaagggactcccagcttcctcg-3’(SEQ ID No.9)；

PR3：5’-cagtGGTCTCaaaaccgaggaagctgggagtccct-3’(SEQ ID No.10)。

PF2 and PR2 were dissolved in ddH, respectively₂O to give a stock solution with a concentration of 10. mu.M, 5. mu.L each was added to 40. mu.L of ddH₂And O, uniformly mixing and reacting, wherein the reaction procedure is as follows: pre-denaturation at 95 deg.C for 10min, denaturation at 55 deg.C for 10min, and annealing at 14 deg.C for 5min to obtain gRNA 1.

The operation steps of PF3 and PR3 are the same as above, and the gRNA2 is obtained.

The annealed primer strands were subjected to enzyme digestion ligation, and the enzyme digestion ligation systems of gRNA1 and gRNA2 are shown in tables 3 and 4, respectively.

Table 3:

components	Volume of
		gRNA1	2μL
pYLCRISPR/Cas9 P35s-H plasmid	1.5μL
		T4 DNA ligase	0.5μL
10×Buffer	1μL
		Eco31I	0.5μL
ddH2O	4.5μL
		Total amount of	10μL

Table 4:

and (3) carrying out enzyme digestion and ligation reaction on the enzyme digestion and ligation systems of the gRNA1 and the gRNA2 at 37 ℃ for 2h to obtain a ligation product 1 and a ligation product 2 respectively. The ligation product 1 and the ligation product 2 were transformed and plasmid-extracted according to the steps (4) and (6) of the above examples, respectively, to obtain pYLCRISPR/Cas9-gRNA1 and LB-DNAi/gRNA2, respectively. pYLCRISP R/Cas9-gRNA1 and LB-DNAi/gRNA2 were sent to the prokaryote for sequencing.

The pYLCRISPR/Cas9-gRNA1 and LB-DNAi/gRNA2 which are sequenced correctly are subjected to enzyme digestion and connected to obtain a recombinant vector pYLCRISPR/Cas9-OsR498G0917707800.01 containing gRNA1 and gRNA 2. The reaction system is shown in Table 5.

Table 5:

components	Volume of
		Sequencing correctly pYLCRISPR/Cas9-gRNA1	1μL
Correctly sequenced LB-DNAi/gRNA2	1.5μL
		LguI	0.5μL
T4 DNAligase	0.5μL
		10×Buffer	1μL
ddH2O	5.5μL
		Total amount of	10μL

The reaction system is subjected to enzyme digestion and ligation reaction for 2h at 37 ℃, and a ligation product (pYLCRISPR/Cas9-OsR498G0917707800.01) is obtained. The plasmid map of pYLCRISPR/Cas9-OsR498G0917707800.01 is shown in FIG. 1.

The ligation products were transformed and positive clone identified according to the steps (4) and (5) of the above examples. Wherein, the primer used for identifying the positive clone is PF4/PR4, and the sequence is as follows:

PF4：5’-GCGATTAAGTTGGGTAACGCCAGGG-3’(SEQ ID No.11)；

PR4：5’-ACCGGTAAGGCGCGCCGTAGT-3’(SEQ ID No.12)；

the reaction procedure is as follows: 3min at 95 ℃; 95 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 2min, 35 cycles; 5min at 72 ℃.

The PCR product was run on a 1% agarose gel and a sample with a single band of interest of about 312bp in size was identified as a positive clone.

Plasmids are extracted according to the step (5) in the embodiment, and the plasmids obtained by extraction are sent to a prokaryote for sequencing.

c. Construction of OsR498G0917707800.01 Gene knockout Rice plants

And c, sending the pYLCRISPR/Cas9-OsR498G0917707800.01 plasmid extracted from the bacterial liquid with correct sequencing in the step b to Wuhanbo remote biotechnology limited to construct and obtain an OsR498G0917707800.01 gene knockout rice plant. The specific construction steps are as follows:

(1) and (3) disinfection: firstly, mechanically shelling seeds (X11), screening high-quality embryo-containing rice grains, placing the selected rice grains in a sterilized triangular flask in an ultra-clean workbench, soaking for 30s in 75% ethanol, and washing with sterile water for 1 time. Soaking in 84 disinfectant for 30min, and cleaning with sterile water for 1min for 3 times; soaking in sterile water for 60 min.

(2) Induction: pouring out sterile water for finally soaking the seeds in the step (1), subpackaging the rice grains on culture dishes containing an induction culture medium by using a spoon, uniformly placing the rice grains on each dish by using tweezers, sealing a flat plate by using a sealing film, culturing for 4-8 days at 28 ℃, separating the rice grains from buds by using the tweezers, and continuously culturing for 2 days after pulling out the rice to obtain the callus.

(3) And (3) transformation: after the pYLCRISPR/Cas9-OsR498G0917707800.01 plasmid is introduced into the agrobacterium EHA105, the recombinant strain EHA105-pYLCRISPR/Cas9-OsR498G0917707800.01 is obtained. Transferring the callus obtained in the step (2) into a sterile triangular flask, and pouring OD₆₀₀Soaking 0.1-0.2 of recombinant strain EHA105-pYLCRISPR/Cas9-OsR498G0917707800.01 bacterial solution for 10 min; transferring the callus onto sterile filter paper board, air drying on a super clean bench for 30min, and shaking every 10min to dry water. Transferring the air-dried callus onto co-culture medium (50-70 pieces/dish), and culturing in the co-culture box at 20 deg.C in dark for 48-72 h. Transferring the callus into a sterile triangular flask, washing with sterile water for 6 times, adding cephalosporin water (1g/L) to soak the callus, and shake-culturing at 30 deg.C and 180rpm for 30 min. Then washing the callus with cephalosporin water (1g/L) for 1 time, transferring to a sterile filter paper board, airing for 2h, and shaking once every 30min to dry the water.

(4) Screening: transferring the dried callus after being washed by the cephalothin water in the step (3) to a screening culture medium (containing 20mg/L of hygromycin) for culturing for 20 days, and transferring the callus to a new screening culture medium (containing 35mg/L of hygromycin); after 30 days of culture, the callus with the resistant callus was selected and transferred to a new selection medium (containing 35mg/L hygromycin) and cultured in the dark at 30 ℃ for 7 days.

(5) Differentiation and rooting: transferring the callus obtained in the step (4) to a sterile paper filtering plate, placing for 24h in the dark, transferring the callus to a differentiation culture medium, culturing for 15d at 25-27 ℃ under illumination, wherein the callus obviously turns green and leaves primordium emerge, transferring the callus to a new differentiation culture medium, and culturing for 10d at 28-30 ℃ to obtain seedlings. Transferring the seedling to rooting culture medium, culturing until the height of seedling is about 10cm, transplanting to large pot, and performing normal field management to obtain T₀And (5) generation of rice.

Identification of OsR498G0917707800.01 Gene-knocked-out Rice plants

C, the T constructed in the step c is₀Selfing the rice generation to obtain T₁Rice generation and T extraction₁The genome DNA of the leaf of the rice generation is amplified by using a primer PF5/PR5, the product is sent to a prokaryote for sequencing, and the sequencing result is compared with the sequence (SEQ ID No.1) of a wild plant before transgenosis. Wherein the sequence of the primer PF5/PR5 is specifically as follows:

PF5：5’-CGAGCTATTCCACCCTCCC-3’(SEQ ID No.13)；

PR5：5’-CCCAAGAACTCGACAGGACTAG-3’(SEQ ID No.14)；

the reaction procedure is as follows: 3min at 95 ℃; 95 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 2min, 35 cycles; 5min at 72 ℃.

The comparison results are shown in fig. 2.

As can be seen from FIG. 2, T is compared with the wild type plant sequence (SEQ ID No.1)₁In the rice generation (marked as pYLRISPR/Cas 9-OsR498G0917707800.01-1), one less nucleotide, namely G, is in the site sequence corresponding to Target 1(SEQ ID No.5), and three less nucleotides, namely GGA, is in the site sequence corresponding to Target 2(SEQ ID No. 6). This indicates that the OsR498G0917707800.01 gene knockout rice plant is successfully constructed.

Chalk character identification of OsR498G0917707800.01 gene knockout rice plant

The correct OsR498G0917707800.01 gene knockout rice plant (T) identified in wild type rice (X11) and the above examples₁Generation of rice, as: pYLCRISPR/Cas 9-OsR49G0917707800.01-1) are respectively cultured to be mature, plant forms in different periods are observed, the seed phenotypes of the X11 and OsR49G0917707800.01 gene knockout rice plants are compared, and the chalkiness and chalkiness of the X11 and OsR49G0917707800.01 gene knockout rice plants are measured according to the national rice standard GB/T1354-2018.

The results of the plant morphology comparison are shown in FIG. 3.

As can be seen from FIG. 3, the OsR498G0917707800.01 gene knockout rice plant has no difference in vegetative growth compared with the wild type rice plant.

Comparison of the grain phenotype is shown in fig. 4, and statistics of chalkiness and chalkiness are shown in fig. 5 and fig. 6, respectively.

As can be seen from FIG. 4, compared with X11, the chalkiness of OsR498G0917707800.01 gene knockout rice plants are obviously reduced. From fig. 5 and fig. 6, it can be seen that the chalkiness rate of the grain of the OsR498G0917707800.01 gene knockout rice plant is not obviously changed compared with that of X11, but the chalkiness degree is greatly reduced from 41.33% to 15.65%.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

Sequence listing

<110> Hunan agriculture university

<120> gene OsR498G0917707800.01 and application of protein coded by same in regulation of rice chalkiness

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1410

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 1

atgaagaaga cggtggttct ctaccccggc ctcgccgtcg gccacctgaa ccccatgatg 60

gagctcgccg acgtcttcct ggaccacggc tacgccgtcg ccgtggcgct catcgacccg 120

tcggtcatgg agaacgaagc caacctcgcc gccgccgtcg cccgcgccgt ctcctccaag 180

agctccacca tctccttcca cacgctcccg ggcatcccgg accctccctc gctcgtcttc 240

aacgatcagt tcttcaagaa ctacttcgac ctcgtgcgac gccacaacga gcacctccac 300

gacttcctcc gctccgtgcg gggcctccat gccgtggtca tcgacgcatc gtgcgcccat 360

gcccatgaag ccgcgaggaa gctgggagtc cctgtcttga tgttctaccc gtccaacgcc 420

ggccacctcg ccgttaactt gcagactcct ctgcttgttg acgggttcaa gaagcatctg 480

ggaggagata gtactagtcc tgtcgagttc ttgggtgttc gacccatgtc ggcttctcac 540

ttggctggcc tttttgggcc gattagcgag gtgaacaagg atttcgaggc catgattttt 600

gccggtgcgc gcatgaacgc ggagttcgac ggaatcctga tcaacacgtc cgtgtcgctg 660

gaggagcggg cgctgcgagc tctcgccgac ccgcgctgct gccccgacgg cgtggtaatc 720

ccgccggtgt acgccgtggg gccactggtc gacaaagccg ccgccgccgc cggtgatgag 780

agcagccgac atcagtgcct cgtgtggctc gacggacaac ccgaccgcag cgtcgtgttc 840

ctctgcttcg ggagcatcgc cgacgcatgt gaacagtccg accagcagct gaaggagatc 900

gccgccggcc tggacaagtc cggccaccgc ttcctgtggg tggttcgggc aaccagcacc 960

caacacctcg acgcgctcct accggaggtg ttcttcgcaa gaaccagcgg ccgcggcctc 1020

gtcgtcaaca gctgggtgcc ccagccgagc atcctccgcc accgcgccac cgccgcgttc 1080

gtgacgcact gcgggtggaa ctcggtgcta gaggggatca ccgcgggggt gccgatgctc 1140

tgctggccgc tgtacgcgga gcagaggatg aacaaggtgc tcatggtgga ggacatgggc 1200

gtcggcgtgg agatggaggg atggctggaa gggctggtga ccgccgagga ggtggagacg 1260

aaggtgaggc tggtcatgga gtccgagcat ggaaggaagg ttagagagcg tgtcgaggcg 1320

cacagagatg gcgtggccat ggcctggaaa gatggtggct cgtcgcgtgt cgcgtttgcc 1380

cgtctcatgt ctgaattgct caacgtgtga 1410

<210> 2

<211> 469

<212> PRT

<213> Rice (Oryza sativa L.)

<400> 2

Met Lys Lys Thr Val Val Leu Tyr Pro Gly Leu Ala Val Gly His Leu

1 5 10 15

Asn Pro Met Met Glu Leu Ala Asp Val Phe Leu Asp His Gly Tyr Ala

20 25 30

Val Ala Val Ala Leu Ile Asp Pro Ser Val Met Glu Asn Glu Ala Asn

35 40 45

Leu Ala Ala Ala Val Ala Arg Ala Val Ser Ser Lys Ser Ser Thr Ile

50 55 60

Ser Phe His Thr Leu Pro Gly Ile Pro Asp Pro Pro Ser Leu Val Phe

65 70 75 80

Asn Asp Gln Phe Phe Lys Asn Tyr Phe Asp Leu Val Arg Arg His Asn

85 90 95

Glu His Leu His Asp Phe Leu Arg Ser Val Arg Gly Leu His Ala Val

100 105 110

Val Ile Asp Ala Ser Cys Ala His Ala His Glu Ala Ala Arg Lys Leu

115 120 125

Gly Val Pro Val Leu Met Phe Tyr Pro Ser Asn Ala Gly His Leu Ala

130 135 140

Val Asn Leu Gln Thr Pro Leu Leu Val Asp Gly Phe Lys Lys His Leu

145 150 155 160

Gly Gly Asp Ser Thr Ser Pro Val Glu Phe Leu Gly Val Arg Pro Met

165 170 175

Ser Ala Ser His Leu Ala Gly Leu Phe Gly Pro Ile Ser Glu Val Asn

180 185 190

Lys Asp Phe Glu Ala Met Ile Phe Ala Gly Ala Arg Met Asn Ala Glu

195 200 205

Phe Asp Gly Ile Leu Ile Asn Thr Ser Val Ser Leu Glu Glu Arg Ala

210 215 220

Leu Arg Ala Leu Ala Asp Pro Arg Cys Cys Pro Asp Gly Val Val Ile

225 230 235 240

Pro Pro Val Tyr Ala Val Gly Pro Leu Val Asp Lys Ala Ala Ala Ala

245 250 255

Ala Gly Asp Glu Ser Ser Arg His Gln Cys Leu Val Trp Leu Asp Gly

260 265 270

Gln Pro Asp Arg Ser Val Val Phe Leu Cys Phe Gly Ser Ile Ala Asp

275 280 285

Ala Cys Glu Gln Ser Asp Gln Gln Leu Lys Glu Ile Ala Ala Gly Leu

290 295 300

Asp Lys Ser Gly His Arg Phe Leu Trp Val Val Arg Ala Thr Ser Thr

305 310 315 320

Gln His Leu Asp Ala Leu Leu Pro Glu Val Phe Phe Ala Arg Thr Ser

325 330 335

Gly Arg Gly Leu Val Val Asn Ser Trp Val Pro Gln Pro Ser Ile Leu

340 345 350

Arg His Arg Ala Thr Ala Ala Phe Val Thr His Cys Gly Trp Asn Ser

355 360 365

Val Leu Glu Gly Ile Thr Ala Gly Val Pro Met Leu Cys Trp Pro Leu

370 375 380

Tyr Ala Glu Gln Arg Met Asn Lys Val Leu Met Val Glu Asp Met Gly

385 390 395 400

Val Gly Val Glu Met Glu Gly Trp Leu Glu Gly Leu Val Thr Ala Glu

405 410 415

Glu Val Glu Thr Lys Val Arg Leu Val Met Glu Ser Glu His Gly Arg

420 425 430

Lys Val Arg Glu Arg Val Glu Ala His Arg Asp Gly Val Ala Met Ala

435 440 445

Trp Lys Asp Gly Gly Ser Ser Arg Val Ala Phe Ala Arg Leu Met Ser

450 455 460

Glu Leu Leu Asn Val

465

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgaagaaga cggtggttct c 21

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tcacacgttg agcaattcag a 21

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gcttcgttct ccatgaccga cgg 23

<210> 6

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

agggactccc agcttcctcg cgg 23

<210> 7

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cagtggtctc atgcagcttc gttctccatg accga 35

<210> 8

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cagtggtctc aaaactcggt catggagaac gaagc 35

<210> 9

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cagtggtctc atgcaaggga ctcccagctt cctcg 35

<210> 10

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cagtggtctc aaaaccgagg aagctgggag tccct 35

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gcgattaagt tgggtaacgc caggg 25

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

accggtaagg cgcgccgtag t 21

<210> 13

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cgagctattc caccctccc 19

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cccaagaact cgacaggact ag 22

<210> 15

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tgcagcttcg ttctccatga ccgagttt 28

<210> 16

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tgcaagggac tcccagcttc ctcggttt 28

Claims (10)

1.1) -3) in regulation of rice chalkiness, characterized in that,

1) protein with amino acid sequence shown as SEQ ID No. 2;

2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No.2 and has the same functions as the protein in 1);

3) a fusion protein obtained by connecting a label at the N end and/or the C end of the amino acid sequence of 1) or 2).

Use of any one of the biomaterials of (1) to (4) for the regulation of rice chalkiness,

(1) a nucleic acid molecule encoding the protein of claim 1;

(2) an expression cassette comprising the nucleic acid molecule of (1);

(3) a recombinant vector comprising the nucleic acid molecule of (1) or the expression cassette of (2);

(4) a recombinant microorganism comprising the nucleic acid molecule of (1) or the expression cassette of (2) or the recombinant vector of (3).

3. The use according to claim 2, wherein the nucleotide sequence of said nucleic acid molecule in (1) is as shown in SEQ ID No. 1.

4. A product for regulating rice chalkiness, which comprises the protein of claim 1, or the biomaterial of claim 2 or 3, or any one of the biomaterials of (1) to (4):

(1) a nucleic acid molecule which disrupts the expression level of a gene of the protein of claim 1 and/or inhibits the activity of the protein and/or reduces the content of the protein;

(2) an expression cassette comprising the nucleic acid molecule of (1);

(3) a recombinant vector comprising the nucleic acid molecule of (1) or the expression cassette of (2);

(4) a recombinant microorganism comprising the nucleic acid molecule of (1) or the expression cassette of (2) or the recombinant vector of (3);

wherein the biomaterial comprising the protein of claim 1 or the biomaterial of claim 2 or 3 is used for improving rice chalkiness;

the biomaterial containing any one of the biomaterials of (1) to (4) is used for reducing rice chalkiness.

5. Use of the protein of claim 1 or the biomaterial of claim 2 or 3 in any one of (1) to (3), wherein:

(1) the application in rice breeding;

(2) the application in culturing gene knockout rice plants with lowered chalkiness degree;

(3) the application in preparing and cultivating the gene knockout rice plant product with lowered chalkiness degree.

6. A method for reducing rice chalkiness, which comprises disrupting the expression level of a gene encoding the protein of claim 1 in a target rice plant and/or inhibiting the activity of the protein in the target rice plant and/or reducing the content of the protein in the target rice plant to obtain a knock-out rice plant; the chalkiness degree of the gene knockout rice plant is reduced compared with that of the rice plant.

7. The method according to claim 6, wherein the method for disrupting the expression level of the gene encoding the protein of claim 1 in the target rice plant and/or inhibiting the activity of the protein of claim 1 in the target rice plant and/or reducing the content of the protein of claim 1 in the target rice plant is achieved by knocking out or suppressing or mutating the gene encoding the protein of claim 1 in the target rice plant.

8. The method according to claim 7, wherein the method for disrupting the expression level of the gene encoding the protein of claim 1 in the rice plant of interest and/or inhibiting the activity of the protein of claim 1 in the rice plant of interest and/or reducing the content of the protein of claim 1 in the rice plant of interest is achieved by knocking out the gene encoding the protein of claim 1 in the rice plant of interest by CRISPR/Cas9 technology.

9. The method according to claim 8, characterized in that the target sequence in the CRISPR/Cas9 technology is SEQ ID No.5 and/or SEQ ID No. 6.

10. Use of the method of any one of claims 6 to 9 in (1) or (2),

(1) the application in rice breeding;

(2) the application in culturing gene knockout rice plants with lowered chalkiness degree.

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