CN117417950A - Rice tillering regulatory gene, mutant, preparation method and application thereof - Google Patents

Abstract

The invention discloses a rice tillering regulating gene, a mutant, a preparation method and application thereof, wherein the nucleotide sequence of the rice tillering regulating gene is shown as SEQ ID NO.1 or SEQ ID NO. 2. The invention uses gene editing technology to specifically knock outOsCKX3Genes by reduction ofOsCKX3Expression orOsCKX3Complete loss of function and increaseThe tillering number of the rice, the spike number of the rice and the yield of the rice are improved.

Description

Rice tillering regulatory gene, mutant, preparation method and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a rice tillering regulatory gene, a mutant, a preparation method and application thereof.

Background

With the increasing population of the world, the demand of people for grains is also increasing, and rice is taken as a main grain crop, and is a staple food which is relied on by nearly half of the population of the world. The yield and quality of rice are also becoming more and more of a focus, and the yield of rice is mainly determined by tillers, ear grains and grain weight. Therefore, research on the control gene related to the tiller number of the rice plays a vital role in improving the yield of the rice.

Cytokinins are plant-specific hormones, and are important signal molecules throughout the life of plants, involved in the important processes of plant growth and development. Among plants, cytokinins naturally occurring in several plants mainly include isopentenyl adenine (IP), trans-zeatin (tZ), cis-zeatin (cZ) and Dihydrozeatin (DHZ). Cytokinin is involved in plant embryo development, root growth, senescence delay, stimulation of plant top dominance and the like.

Cytokinin oxidase/dehydrogenase (cytokinin oxidase/dehydrogenase, CKX) is an important cytokinin degrading enzyme in plant cells, and the degradation process is irreversible. Cytokinin oxidase CKX inactivates cytokinins by degrading the unsaturated isoprene of the cytokinin CK side chain to adenine and the corresponding aldehyde. Currently 7 AtCKXs (AtCKX 1-7) are identified in Arabidopsis thaliana. CKX enzymes consist of a FAD domain and a domain of a substrate. Although FAD and substrate domains are conserved among various CKX enzymes, sequences outside of these conserved regions show a strong sequence difference. The biochemical properties and subcellular localization of the CKX enzyme in arabidopsis are also different.

Based on data analysis, 11 are currently identified in rice genomesOsCKXMember 7 family members have been reported, and studies have found that knockouts or decreasesOsCKX2The expression level of (2) can cause cytokinin to accumulate in the rice apical meristem and increase flowersThe number of the powder tubes is obviously increased, and the tillering number and the ear grain number are obviously increased compared with the wild type, so that the yield of the rice is obviously increased.OsCKX3Gene reports are involved in the regulation of the angle of the local branch, and further studies have found that,OsCKX4is involved in the development of adventitious roots of rice,OsCKX4mainly expressed in leaves and roots, preferentially expressed in adventitious root primordia, over-expression and RNAi can promote the formation of adventitious roots, in addition, a single-impurity experiment of yeast shows that,OsCKX4can be directly combined by OsARF25 and cytokinin response factors OsRR2 and OsRR3, which shows thatOsCKX4Can regulate the development of adventitious roots of rice through cytokinin and auxin pathways. There have been reports on the use of the above-mentioned materials,OsCKX9takes part in the degradation process of activating strigolactone driving cytokinin,OsCKX9as a major strigolactone response gene. In addition, rice cytokinin oxidase coding gene by site-directed mutagenesisOsCKX11Analysis of the mutant phenotype changes shows that the functional deficiency of the gene can lead to late senescence of rice leaves.

The rice tillering is an important index for improving the yield of rice, and the research of improving the quantity of rice tillers by a gene editing technology to further achieve the yield increasing effect is obviously insufficient at the present stage.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, provides a rice tillering regulation gene, a mutant, a preparation method and application thereof, and uses a gene editing technology to specifically knock outOsCKX3 gene by makingOsCKX3The complete loss of function increases the tillering number of rice, increases the grain number of rice ears and increases the yield of rice.

The invention provides the following technical scheme:

in a first aspect, a rice tillering control gene is provided, and the nucleotide sequence of the gene is shown as SEQ ID NO.1 or SEQ ID NO. 2.

In a second aspect, a rice tillering control mutant is provided, comprising a gene having a nucleotide sequence as shown in SEQ ID NO.1 or SEQ ID NO. 2.

In a third aspect, there is provided a rice tillering control mutationA method of making a body comprising: for rice geneOsCKX3The nucleotide sequence of (2) is subjected to targeted editing to obtain a mutant with a gene with the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.

Further, the method comprises the following steps:

at the position ofOsCKX3Selecting target spots on genes and designing primers;

connecting the primer with the sgRNA expression cassette, and then amplifying to obtain a target gRNA expression cassette;

connecting the target gRNA expression cassette with pYLCRISPR/Cas9-MH (B) to obtainOsCKX3A gene knockout vector;

will beOsCKX3Transforming agrobacterium with the gene knockout vector, infecting mature rice callus, and then carrying out resistance screening to obtain resistant callus;

differentiation of the resistant callus into seedlings is carried out to obtain rice seedlings of the rice tillering regulation mutant.

Further, inOsCKX3Selecting a target point on the gene, and designing a primer according to the promoter;

the promoters are U6a and U6b;

the primer is OsCKX3-T1-F with a nucleotide sequence shown as SEQ ID NO.3, osCKX3-T1-R with a nucleotide sequence shown as SEQ ID NO.4, osCKX3-T2-F with a nucleotide sequence shown as SEQ ID NO.5 and OsCKX3-T2-R with a nucleotide sequence shown as SEQ ID NO. 6.

Further, the method for ligating primers to the sgRNA expression cassette comprises:

and taking plasmid pYLgRNA-OsU a/LacZ and pYLgRNA-OsU b for enzyme digestion, and connecting the digested plasmid with a corresponding joint of the primer.

Further, the method for amplifying the sgRNA expression cassette after connecting the primers comprises the following steps:

pre-denaturing at 98 ℃ for 5min, denaturing at 98 ℃ for 30s, annealing at 55 ℃ for 15s, extending at 58 ℃ for 30s, circulating for 30 times, extending at 68 ℃ for 5min to obtain amplified PCR products;

and separating the amplified PCR product by agarose gel electrophoresis, and then performing gel cutting recovery by using a gel recovery kit to obtain a recovery fragment, namely the target gRNA expression cassette.

Further, the method of ligating the target gRNA expression cassette to the pYLCRISPR/Cas9-MH (B) comprises:

BsaI endonuclease and T4 ligase are selected for edge ligation, and the target gRNA expression cassette is ligated to the kanamycin-resistant expression vector pYLCRISPR/Cas9-MH (B).

Further, the method also comprises the steps of screening and identifying T0 generation transgenic plants of rice seedlings of the rice tillering regulation mutant:

extracting DNA of the T0 generation transgenic plant, and identifying CAS9 to obtain the T0 generation transgenic plant seed containing CAS 9;

breeding T0 generation transgenic plant seeds containing CAS9 in the ground, harvesting T1 generation for genotyping, designing primers at 500bp upstream and downstream of the target point, and amplifying the target sequence;

and (3) after amplification, agarose gel electrophoresis is carried out, correct colony amplification culture of the strip is verified, plasmids are extracted, and the mutant genotype is obtained after sequencing.

In a fourth aspect, there is provided an application of the rice tillering control gene of the first aspect, or the rice tillering control mutant of the second aspect, or the mutant prepared by the method of the third aspect, in controlling rice tillering.

Compared with the prior art, the invention has the beneficial effects that:

(1) The invention provides a rice tillering regulating gene which can increase the tillering number of rice, increase the grain number of rice ears and increase the yield of rice when being applied to rice planting;

(2) The invention also provides a rice tillering regulation mutant and a preparation method thereof, and the rice tillering regulation mutant is specifically knocked out by using a gene editing technologyOsCKX3Gene, obtaining multi-tiller rice genetic transformation homozygous line with genetic background of Japanese sunny, by makingOsCKX3The complete loss of function further increases rice spike grains, and further, the biological function of increasing the tiller number of rice based on the OsCKX3 loss of function can improve the existing rice variety, increase the rice spike grain number and provide a new theoretical basis for rice high-product seed breeding through means such as gene editing, RNA interference, T-DNA insertion, genetic transformation, molecular auxiliary breeding and the like.

Drawings

FIG. 1 is a map of expression vector pYLCRISPR/Cas9-MH (B) in an embodiment of the invention;

FIG. 2 is a schematic illustration of an embodiment of the present inventionOsCKX3Schematic representation of gene structure map and mutation type annotation;

FIG. 3 is a schematic illustration of an embodiment of the present inventionOsCKX3A gene knockout homozygous mutant expression profile;

FIG. 4 is a schematic illustration of an embodiment of the present inventionOsCKX3Phenotype photograph of homozygous mutant with knockout in the field;

FIG. 5 is a schematic illustration of an embodiment of the present inventionOsCKX3A high statistical histogram of the homozygous mutant knocked out and the wild type Japanese single plant of the control;

FIG. 6 is a schematic illustration of an embodiment of the present inventionOsCKX3A statistical histogram of the tillering numbers of the homozygous mutant knocked out and the wild type Japanese individual strain of the control;

FIG. 7 is a schematic illustration of an embodiment of the inventionOsCKX3A statistical histogram of the yield of the homozygous mutant knocked out and the wild type Japanese single plant of the control;

FIG. 8 is a schematic illustration of an embodiment of the inventionOsCKX3Single plant spike pattern diagram of gene knockout homozygotic mutant and control wild type Japanese sunny;

FIG. 9 is a schematic illustration of an embodiment of the inventionOsCKX3The homozygous mutant is knocked out and compared with wild type Japanese sunny;

FIG. 10 is a schematic illustration of an embodiment of the inventionOsCKX3And (3) a second-level branch count chart of the homozygous mutant with the wild type Japanese sunny comparison.

Detailed Description

The invention is further described below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.

Example 1

The embodiment provides a rice tillering regulating gene, and the nucleotide sequence of the gene is shown as SEQ ID NO.1 or SEQ ID NO. 2.

SEQ ID NO.1：

ATGGAGGTTGCCATGGTCTGCACAAGAGTTAACCTGCTCATCCTCATCCTTTCTCTCTGCTCCCCATACAAGTTTATACAGAGCCCCATGGACTTTGGCCCTTTGAACCTCCTTCCCACCACCACCACTGCATCCAGTGACTTTGGTAGAATCCTCTTCCACTCCCCATCTGCAGTTCTAAAGCCCCAAGCTCCAAGGGATATATCCCTGCTTCTCAGCTTCCTCTCTGCCTCACCTCTTGGCAAGGTGACGGTGGCAGCCAGGGGAGCAGGTCACTCCATCCATGGACAGGCACAGGCCCTTGATGGCATTGTAGTGGAGATGAGCTCCTTGCCTTCTGAGATTGAATTCTACAGAAGAGGAGAAGGAGATGTTTCTTATGCTGATGTGGGTGGTGGAATCATGTGGATTGAGCTTCTGGAGCAGAGCTTGAAGCTTGGGCTGGCTCCAAACTGATTACCTCTATCTCACCATTGGTGGAACCTTGTCCAATGCTGGCATCAGTGGGCAGACATTCAAGCATGGACCTCAGATTAGCAATGTTCTACAGCTGGAAGTAGTCACAGGTGAGAGATGCATAATGAAACATTAAAAAATATTGATAAGGTCTTATATTTGCCCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTTCTCTCAACGTCTTTCTTGCCCAACGTCTTTTAACTGATGGGCAGTAGATTTTCTGTTCCTCTTTGCTTGACAGGCAGGGGTGAGATTGTGACCTGTTCACCCACCAAGGATGCAGAGCTATTCAATGCTGTTCTTGGAGGCCTTGGGCAGTTCGGCATCATTACTAGGGCAAGGATCCTGCTGCAGGAAGCTCCACAGAAAGTATGCAGCTTTAGAACTGTCCTTTGCATATTAGTATATTATTATATGATCTTAACCATTATTTCTGCATGTCATGTTTCTGACACGCATATATATAATTAAACAAATCTGGAATTTCATTTTAGGTGAAGTGGGTTAGAGCCTTCTATGATGATTTCGCCACCTTCACCAAAGACCAGGAGTTGCTGGTGTCAATGCCGGTTTTGGTGGACTATGTGGAAGGTTTCATCGTGTTGAATGAGCAATCCCTTCACAGCTCCTCCATTGCCTTCCCTACAAATGTTGACTTCAACCCAGATTTTGGCACCAAGAACAACCCTAAGATCTACTACTGCATAGAGTTTGCTGTCCACGATTACCAGAACAAGAACATCAATGTGGAACAGGTAAGCTCCAAAATTTACTCTGCTTTTTGTGAACTTATCAAGTCATGGTAATGATCAAACAAGTCTTCATCTATTTCAGGTCGTGGAAGTCATCTCAAGGCAGATGAGCCACATAGCATCCCACTTGTATAGCGTGGAGGTGTCCTACTTTGATTTTCTCAACAGGGTTAGGATGGAGGAGATGAGCCTTAGGAACAGTGGGCTCTGGGAGGTGCACCATCCATGGTTGAACATGTTCGTGCCAAGCGCTGGGATTAGTGACTTCAGGGACCTGCTCATGGACAGCATCTCACCGGATAACTTCGAGGGCCTCATCCTCATCTACCCACTCCTCAGACACAAGTAAGCAGTACTGCCCACTAATGCTAAATAGGCTTCACATTATGATATGCCAACATGAGGGCACAGCAATGCGTGGAAGCATTATTTGTGAGAAAAAGGTTATCTATTGGTTATTTAATTGATCTCTCTTTCATGATCAGCAACATTAGCTGCCAAGCAGTCCTAGTAGGACAAGTACTTGTCTGCATTATCCAACGTGGTGCCCCTTTGTGTTATAGTATTAGTCCCTTTTGGCATTAATATGTTGGTCCATTTTTTTTCAATTTTACAACTGCCCAGTGTAGAAGATATTTACATGCCTGATTAGGTTAATATTTCAGTGTGGGGCGTCTTTTTGCTCTTCTTTGGAAACATATGTTGCTCTTTTCATAGCATTTATGATTTGAGTGTGTTCTCATCGATTCTGTAAATGTTAATGTCATCTATTTATGATTTGGTAGCATCCAGCTATTTATGATATCAGGAAGAGAGCCTTTCTTTTTAATAAATGATGTTTGATTTATCCTTCTGCCTAGTCCCACTTAGGTTCTAAAGCCCTAGTATCATTACTTCATCAGTATTCTCTTGTTGGTCCCGCTTAGGTCTGTTCTTGTGGTAAGAAGTAAAGCTTCTGCTTTTCACATATTAAGAGGGCTGGGGCCTGAGGGGGATCCATTCTTGCCTAGGCTTTCAGCGCGGCAGATAAGTTTCAGGCTTGCTTCATCCAAACGTTGGACACAAGAATGGGAATATGCGATAAGTTAGGCAGTTAGTTTGTCTAATGCTGTGGCAGCTTATTTTTGTTGGAAAGTTACATGTTTGCATAAAATAATTTTTTGGTCGAAAGTCCACACCTACATGGCTACAGCGACTTTATTTGTAGACTGTTGCTTGATTACGAATTGTATCATGTTTTATGGGTCCCTCAAAATAGAATGCCAGTTATCATCAGCATATTATGATTCAACTGAAGCAATAGCTTAATCAGTGGCATCCTACTTTTCAGAGTAGTTTGGTCACCTTTGTTCAGTAGCAACAGTGAATATAGAAACAGAAGTTGCTTGATCTCTCTTACATTTTAGTTTTTTGACTTCAATGAAAAGCATACCGATACTTTGGCTTGTTTGTGCTTAGATAAATATGCTTATTCCAGCCCATGATAAATAATTATCTAGGTCAAATGGAACTAATGTTGACTATCGGCAAACTATTTGTTGTGATGTTGACAAGGCACAAATTCTGTGCGGTGTTAAAAATCAAACAAGTTTTATGATCTCATTGACCTCAGATGAAAGAATTTGTAATGTGATCCTCCTTCCAAGAAGTTTATACAAGTTTATCTGCAAGGGGGGTTTAAAACTACTCTTGGTGGCAGGCAGAGTCAGCTCTAATCTTGTTGAACCAATGAACCTTAACAAAAGATTCATGTTCACTGTACTGAAATAATTTAACAGCACCAATGTGGACTTCACTCCCCCAAACACTAAAACAAACCTCTCTTGAGTCAGAATACACTGAACTAGTACTGAATGTCAGCTATATTATCTTGTACTGCTCATTTTTTTTTCATTTCTAATAATTCTGATACTCTGTTAGATGCTGGGATATCATCTAATTGTTGCACTATTTATGCCTCTCACTGACAGGTGGGACACCAACACGTCGGTCGTGTTACCGGACTCCGGGTCGACGGACCAGGTGATGTACGCGGTGGGCATCCTCCGGTCGGCGAACCCCGACGACGGGTGCTCCCACCACTGCCTCCAGGAGCTCCTCCTCCGCCACCGCCGCCTCGCCGGCGCCGCCGCGTCGGGCCTCGGCGCGAAGCAGTACCTCGCCCACCACCCGACCCCGGCCGGCTGGCGCCGCCACTTCGGCCGGCGGTGGGAGCGGTTCGCCGATCGCAAGGCCCGGTTCGACCCGAGGTGCATCCTCGGGCCAGGCCAGGGTATATTCCCCAGGGACAGCAGCAGCAGCAATGGCGCATTTGCAAGCTATAGCTGA。

SEQ ID NO.2：

ATGGAGGTTGCCATGGTCTGCACAAGAGTTAACCTGCTCATCCTCATCCTTTCTCTCTGCTCCCCATACAAGTTTATACAGAGCCCCATGGACTTTGGCCCTTTGAACCTCCTTCCCACCACCACCACTGCATCCAGTGACTTTGGTAGAATCCTCTTCCACTCCCCATCTGCAGTTCTAAAGCCCCAAGCTCCAAGGGATATATCCCTGCTTCTCAGCTTCCTCTCTGCCTCACCTCTTGGCAAGGTGACGGTGGCAGCCAGGGGAGCAGGTCACTCCATCCATGGACAGGCACAGGCCCTTGATGGCATTGTAGTGGAGATGAGCTCCTTGCCTTCTGAGATTGAATTCTACAGAAGAGGAGAAGGAGATGTTTCTTATGCTGATGTGGGTGGTGGAATCATGTGGATTGAGCTTCTGGAGCAGAGCTTGAAGCTTGGACTGATTACCTCTATCTCACCATTGGTGGAACCTTGTCCAATGCTGGCATCAGTGGGCAGACATTCAAGCATGGACCTCAGATTAGCAATGTTCTACAGCTGGAAGTAGTCACAGGTGAGAGATGCATAATGAAACATTAAAAAATATTGATAAGGTCTTATATTTGCCCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTTCTCTCAACGTCTTTCTTGCCCAACGTCTTTTAACTGATGGGCAGTAGATTTTCTGTTCCTCTTTGCTTGACAGGCAGGGGTGAGATTGTGACCTGTTCACCCACCAAGGATGCAGAGCTATTCAATGCTGTTCTTGGAGGCCTTGGGCAGTTCGGCATCATTACTAGGGCAAGGATCCTGCTGCAGGAAGCTCCACAGAAAGTATGCAGCTTTAGAACTGTCCTTTGCATATTAGTATATTATTATATGATCTTAACCATTATTTCTGCATGTCATGTTTCTGACACGCATATATATAATTAAACAAATCTGGAATTTCATTTTAGGTGAAGTGGGTTAGAGCCTTCTATGATGATTTCGCCACCTTCACCAAAGACCAGGAGTTGCTGGTGTCAATGCCGGTTTTGGTGGACTATGTGGAAGGTTTCATCGTGTTGAATGAGCAATCCCTTCACAGCTCCTCCATTGCCTTCCCTACAAATGTTGACTTCAACCCAGATTTTGGCACCAAGAACAACCCTAAGATCTACTACTGCATAGAGTTTGCTGTCCACGATTACCAGAACAAGAACATCAATGTGGAACAGGTAAGCTCCAAAATTTACTCTGCTTTTTGTGAACTTATCAAGTCATGGTAATGATCAAACAAGTCTTCATCTATTTCAGGTCGTGGAAGTCATCTCAAGGCAGATGAGCCACATAGCATCCCACTTGTATAGCGTGGAGGTGTCCTACTTTGATTTTCTCAACAGGGTTAGGATGGAGGAGATGAGCCTTAGGAACAGTGGGCTCTGGGAGGTGCACCATCCATGGTTGAACATGTTCGTGCCAAGCGCTGGGATTAGTGACTTCAGGGACCTGCTCATGGACAGCATCTCACCGGATAACTTCGAGGGCCTCATCCTCATCTACCCACTCCTCAGACACAAGTAAGCAGTACTGCCCACTAATGCTAAATAGGCTTCACATTATGATATGCCAACATGAGGGCACAGCAATGCGTGGAAGCATTATTTGTGAGAAAAAGGTTATCTATTGGTTATTTAATTGATCTCTCTTTCATGATCAGCAACATTAGCTGCCAAGCAGTCCTAGTAGGACAAGTACTTGTCTGCATTATCCAACGTGGTGCCCCTTTGTGTTATAGTATTAGTCCCTTTTGGCATTAATATGTTGGTCCATTTTTTTTCAATTTTACAACTGCCCAGTGTAGAAGATATTTACATGCCTGATTAGGTTAATATTTCAGTGTGGGGCGTCTTTTTGCTCTTCTTTGGAAACATATGTTGCTCTTTTCATAGCATTTATGATTTGAGTGTGTTCTCATCGATTCTGTAAATGTTAATGTCATCTATTTATGATTTGGTAGCATCCAGCTATTTATGATATCAGGAAGAGAGCCTTTCTTTTTAATAAATGATGTTTGATTTATCCTTCTGCCTAGTCCCACTTAGGTTCTAAAGCCCTAGTATCATTACTTCATCAGTATTCTCTTGTTGGTCCCGCTTAGGTCTGTTCTTGTGGTAAGAAGTAAAGCTTCTGCTTTTCACATATTAAGAGGGCTGGGGCCTGAGGGGGATCCATTCTTGCCTAGGCTTTCAGCGCGGCAGATAAGTTTCAGGCTTGCTTCATCCAAACGTTGGACACAAGAATGGGAATATGCGATAAGTTAGGCAGTTAGTTTGTCTAATGCTGTGGCAGCTTATTTTTGTTGGAAAGTTACATGTTTGCATAAAATAATTTTTTGGTCGAAAGTCCACACCTACATGGCTACAGCGACTTTATTTGTAGACTGTTGCTTGATTACGAATTGTATCATGTTTTATGGGTCCCTCAAAATAGAATGCCAGTTATCATCAGCATATTATGATTCAACTGAAGCAATAGCTTAATCAGTGGCATCCTACTTTTCAGAGTAGTTTGGTCACCTTTGTTCAGTAGCAACAGTGAATATAGAAACAGAAGTTGCTTGATCTCTCTTACATTTTAGTTTTTTGACTTCAATGAAAAGCATACCGATACTTTGGCTTGTTTGTGCTTAGATAAATATGCTTATTCCAGCCCATGATAAATAATTATCTAGGTCAAATGGAACTAATGTTGACTATCGGCAAACTATTTGTTGTGATGTTGACAAGGCACAAATTCTGTGCGGTGTTAAAAATCAAACAAGTTTTATGATCTCATTGACCTCAGATGAAAGAATTTGTAATGTGATCCTCCTTCCAAGAAGTTTATACAAGTTTATCTGCAAGGGGGGTTTAAAACTACTCTTGGTGGCAGGCAGAGTCAGCTCTAATCTTGTTGAACCAATGAACCTTAACAAAAGATTCATGTTCACTGTACTGAAATAATTTAACAGCACCAATGTGGACTTCACTCCCCCAAACACTAAAACAAACCTCTCTTGAGTCAGAATACACTGAACTAGTACTGAATGTCAGCTATATTATCTTGTACTGCTCATTTTTTTTTCATTTCTAATAATTCTGATACTCTGTTAGATGCTGGGATATCATCTAATTGTTGCACTATTTATGCCTCTCACTGACAGGTGGGACACCAACACGTCGGTCGTGTTACCGGACTCCGGGTCGACGGACCAGGTGATGTACGCGGTGGGCATCCTCCGGTCGGCGAACCCCGACGACGGGTGCTCCCACCACTGCCTCCAGGAGCTCCTCCTCCGCCACCGCCGCCTCGCCGGCGCCGCCGCGTCGGGCCTCGGCGCGAAGCAGTACCTCGCCCACCACCCGACCCCGGCCGGCTGGCGCCGCCACTTCGGCCGGCGGTGGGAGCGGTTCGCCGATCGCAAGGCCCGGTTCGACCCGAGGTGCATCCTCGGGCCAGGCCAGGGTATATTCCCCAGGGACAGCAGCAGCAGCAATGGCGCATTTGCAAGCTATAGCTGA。

Example 2

The embodiment provides a rice tillering regulation mutant, which has a gene with a nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.

Example 3

The embodiment provides a preparation method of a rice tillering control mutant, which comprises the following steps: for rice geneOsCKX3The nucleotide sequence of (2) is subjected to targeted editing to obtain a mutant with a gene with the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.

Rice geneOsCKX3Accession number at Genbank is Os10g0483500; the rice geneOsCKX3The nucleotide sequence of the rice gene is shown as SEQ ID NO.7OsCKX3The amino acid sequence of the encoded protein is shown as SEQ ID NO. 8.

SEQ ID NO.7：

ATGGAGGTTGCCATGGTCTGCACAAGAGTTAACCTGCTCATCCTCATCCTTTCTCTCTGCTCCCCATACAAGTTTATACAGAGCCCCATGGACTTTGGCCCTTTGAACCTCCTTCCCACCACCACCACTGCATCCAGTGACTTTGGTAGAATCCTCTTCCACTCCCCATCTGCAGTTCTAAAGCCCCAAGCTCCAAGGGATATATCCCTGCTTCTCAGCTTCCTCTCTGCCTCACCTCTTGGCAAGGTGACGGTGGCAGCCAGGGGAGCAGGTCACTCCATCCATGGACAGGCACAGGCCCTTGATGGCATTGTAGTGGAGATGAGCTCCTTGCCTTCTGAGATTGAATTCTACAGAAGAGGAGAAGGAGATGTTTCTTATGCTGATGTGGGTGGTGGAATCATGTGGATTGAGCTTCTGGAGCAGAGCTTGAAGCTTGGGCTGGCTCCAAGGTCTTGGACTGATTACCTCTATCTCACCATTGGTGGAACCTTGTCCAATGCTGGCATCAGTGGGCAGACATTCAAGCATGGACCTCAGATTAGCAATGTTCTACAGCTGGAAGTAGTCACAGGTGAGAGATGCATAATGAAACATTAAAAAATATTGATAAGGTCTTATATTTGCCCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTTCTCTCAACGTCTTTCTTGCCCAACGTCTTTTAACTGATGGGCAGTAGATTTTCTGTTCCTCTTTGCTTGACAGGCAGGGGTGAGATTGTGACCTGTTCACCCACCAAGGATGCAGAGCTATTCAATGCTGTTCTTGGAGGCCTTGGGCAGTTCGGCATCATTACTAGGGCAAGGATCCTGCTGCAGGAAGCTCCACAGAAAGTATGCAGCTTTAGAACTGTCCTTTGCATATTAGTATATTATTATATGATCTTAACCATTATTTCTGCATGTCATGTTTCTGACACGCATATATATAATTAAACAAATCTGGAATTTCATTTTAGGTGAAGTGGGTTAGAGCCTTCTATGATGATTTCGCCACCTTCACCAAAGACCAGGAGTTGCTGGTGTCAATGCCGGTTTTGGTGGACTATGTGGAAGGTTTCATCGTGTTGAATGAGCAATCCCTTCACAGCTCCTCCATTGCCTTCCCTACAAATGTTGACTTCAACCCAGATTTTGGCACCAAGAACAACCCTAAGATCTACTACTGCATAGAGTTTGCTGTCCACGATTACCAGAACAAGAACATCAATGTGGAACAGGTAAGCTCCAAAATTTACTCTGCTTTTTGTGAACTTATCAAGTCATGGTAATGATCAAACAAGTCTTCATCTATTTCAGGTCGTGGAAGTCATCTCAAGGCAGATGAGCCACATAGCATCCCACTTGTATAGCGTGGAGGTGTCCTACTTTGATTTTCTCAACAGGGTTAGGATGGAGGAGATGAGCCTTAGGAACAGTGGGCTCTGGGAGGTGCACCATCCATGGTTGAACATGTTCGTGCCAAGCGCTGGGATTAGTGACTTCAGGGACCTGCTCATGGACAGCATCTCACCGGATAACTTCGAGGGCCTCATCCTCATCTACCCACTCCTCAGACACAAGTAAGCAGTACTGCCCACTAATGCTAAATAGGCTTCACATTATGATATGCCAACATGAGGGCACAGCAATGCGTGGAAGCATTATTTGTGAGAAAAAGGTTATCTATTGGTTATTTAATTGATCTCTCTTTCATGATCAGCAACATTAGCTGCCAAGCAGTCCTAGTAGGACAAGTACTTGTCTGCATTATCCAACGTGGTGCCCCTTTGTGTTATAGTATTAGTCCCTTTTGGCATTAATATGTTGGTCCATTTTTTTTCAATTTTACAACTGCCCAGTGTAGAAGATATTTACATGCCTGATTAGGTTAATATTTCAGTGTGGGGCGTCTTTTTGCTCTTCTTTGGAAACATATGTTGCTCTTTTCATAGCATTTATGATTTGAGTGTGTTCTCATCGATTCTGTAAATGTTAATGTCATCTATTTATGATTTGGTAGCATCCAGCTATTTATGATATCAGGAAGAGAGCCTTTCTTTTTAATAAATGATGTTTGATTTATCCTTCTGCCTAGTCCCACTTAGGTTCTAAAGCCCTAGTATCATTACTTCATCAGTATTCTCTTGTTGGTCCCGCTTAGGTCTGTTCTTGTGGTAAGAAGTAAAGCTTCTGCTTTTCACATATTAAGAGGGCTGGGGCCTGAGGGGGATCCATTCTTGCCTAGGCTTTCAGCGCGGCAGATAAGTTTCAGGCTTGCTTCATCCAAACGTTGGACACAAGAATGGGAATATGCGATAAGTTAGGCAGTTAGTTTGTCTAATGCTGTGGCAGCTTATTTTTGTTGGAAAGTTACATGTTTGCATAAAATAATTTTTTGGTCGAAAGTCCACACCTACATGGCTACAGCGACTTTATTTGTAGACTGTTGCTTGATTACGAATTGTATCATGTTTTATGGGTCCCTCAAAATAGAATGCCAGTTATCATCAGCATATTATGATTCAACTGAAGCAATAGCTTAATCAGTGGCATCCTACTTTTCAGAGTAGTTTGGTCACCTTTGTTCAGTAGCAACAGTGAATATAGAAACAGAAGTTGCTTGATCTCTCTTACATTTTAGTTTTTTGACTTCAATGAAAAGCATACCGATACTTTGGCTTGTTTGTGCTTAGATAAATATGCTTATTCCAGCCCATGATAAATAATTATCTAGGTCAAATGGAACTAATGTTGACTATCGGCAAACTATTTGTTGTGATGTTGACAAGGCACAAATTCTGTGCGGTGTTAAAAATCAAACAAGTTTTATGATCTCATTGACCTCAGATGAAAGAATTTGTAATGTGATCCTCCTTCCAAGAAGTTTATACAAGTTTATCTGCAAGGGGGGTTTAAAACTACTCTTGGTGGCAGGCAGAGTCAGCTCTAATCTTGTTGAACCAATGAACCTTAACAAAAGATTCATGTTCACTGTACTGAAATAATTTAACAGCACCAATGTGGACTTCACTCCCCCAAACACTAAAACAAACCTCTCTTGAGTCAGAATACACTGAACTAGTACTGAATGTCAGCTATATTATCTTGTACTGCTCATTTTTTTTTCATTTCTAATAATTCTGATACTCTGTTAGATGCTGGGATATCATCTAATTGTTGCACTATTTATGCCTCTCACTGACAGGTGGGACACCAACACGTCGGTCGTGTTACCGGACTCCGGGTCGACGGACCAGGTGATGTACGCGGTGGGCATCCTCCGGTCGGCGAACCCCGACGACGGGTGCTCCCACCACTGCCTCCAGGAGCTCCTCCTCCGCCACCGCCGCCTCGCCGGCGCCGCCGCGTCGGGCCTCGGCGCGAAGCAGTACCTCGCCCACCACCCGACCCCGGCCGGCTGGCGCCGCCACTTCGGCCGGCGGTGGGAGCGGTTCGCCGATCGCAAGGCCCGGTTCGACCCGAGGTGCATCCTCGGGCCAGGCCAGGGTATATTCCCCAGGGACAGCAGCAGCAGCAATGGCGCATTTGCAAGCTATAGCTGA。

SEQ ID NO.8：

MEVAMVCTRVNLLILILSLCSPYKFIQSPMDFGPLNLLPTTTTASSDFGRILFHSPSAVLKPQAPRDISLLLSFLSASPLGKVTVAARGAGHSIHGQAQALDGIVVEMSSLPSEIEFYRRGEGDVSYADVGGGIMWIELLEQSLKLGLAPRSWTDYLYLTIGGTLSNAGISGQTFKHGPQISNVLQLEVVTGRGEIVTCSPTKDAELFNAVLGGLGQFGIITRARILLQEAPQKVKWVRAFYDDFATFTKDQELLVSMPVLVDYVEGFIVLNEQSLHSSSIAFPTNVDFNPDFGTKNNPKIYYCIEFAVHDYQNKNINVEQVVEVISRQMSHIASHLYSVEVSYFDFLNRVRMEEMSLRNSGLWEVHHPWLNMFVPSAGISDFRDLLMDSISPDNFEGLILIYPLLRHKWDTNTSVVLPDSGSTDQVMYAVGILRSANPDDGCSHHCLQELLLRHRRLAGAAASGLGAKQYLAHHPTPAGWRRHFGRRWERFADRKARFDPRCILGPGQGIFPRDSSSSNGAFASYS。

Example 4

The embodiment provides a preparation method of the rice tillering control mutant of the embodiment 3, which comprises the following specific steps:

step 1, selecting a target spot.

Will beOsCKX3The genome nucleotide sequence of (2) is imported into a website (http:// skl. Scau. Edu. Cn /), target design is carried out in the website, full genome retrieval is carried out according to the target position provided by the website, the selected target is prevented from having higher homology with other genes, and then the target position is selected on an exon.

Step 2, atOsCKX3Primers are designed on the gene.

At the position ofOsCKX3Two targets are selected on the first exon on the gene, primers are designed according to different selected promoters, and the promoters are selected as U6a and U6b.

The primer is OsCKX3-T1-F with a nucleotide sequence shown as SEQ ID NO.3, osCKX3-T1-R with a nucleotide sequence shown as SEQ ID NO.4, osCKX3-T2-F with a nucleotide sequence shown as SEQ ID NO.5 and OsCKX3-T2-R with a nucleotide sequence shown as SEQ ID NO. 6.

SEQ ID NO.3：gccgCTCTGCCTCACCTCTTGGCA；

SEQ ID NO.4：aaacTGCCAAGAGGTGAGGCAGAG；

SEQ ID NO.5：gccgAGGTAATCAGTCCAAGACCT；

SEQ ID NO.6：aaacAGGTCTTGGACTGATTACCT。

And 3, connecting the primer with the sgRNA expression cassette.

Taking 1 mug of pYLgRNA-OsU a/LacZ and 1 mug of pYLgRNA-OsU b respectively, performing enzyme digestion with 10UBsaI for 20min in 25 mu l reaction, inactivating enzyme at 70 ℃ for 5min, and freezing for preservation; the digested plasmid is connected with the corresponding connector of each primer for reaction: 1 μl of 10x T4 DNA ligase buffer, 0.5 μl of adaptor is added with double distilled water to 10 μl, and finally 20-35 UT4 DNA ligase is added and connected at room temperature for 10-15 min.

And 4, amplifying the sgRNA expression cassette connected with the primer to obtain a target gRNA expression cassette.

The PCR procedure at the time of amplification was: the PCR product was amplified by pre-denaturing at 98℃for 5min, denaturing at 98℃for 30s, annealing at 55℃for 15s, extending at 58℃for 30s, cycling for 30 times, and extending at 68℃for 5 min. And separating the amplified PCR product by agarose gel electrophoresis, and then performing gel cutting recovery by using a gel recovery kit to obtain a recovery fragment, namely the target gRNA expression cassette.

Step 5, connecting the target gRNA expression cassette with pYLCRISPR/Cas9-MH (B) to obtainOsCKX3A gene knockout vector.

BsaI endonuclease and T4 ligase are selected for edge trimming and the amplification product of step 4 is ligated to the kanamycin-resistant expression vector pYLCRISPR/Cas9-MH (B).

FIG. 1 shows a map of the expression vector pYLCRISPR/Cas9-MH (B). In the figure, bsaI cleavage site, vector resistance and the like are marked.

Step 6, willOsCKX3The gene knockout vector is transformed into competent cells of escherichia coli, and colony PCR and sequencing are carried out.

Thawing competent cells of escherichia coli on ice, adding 10 μl of a connection product into each centrifuge tube, blowing, standing on the ice for 30min, then placing the centrifuge tubes into a water bath kettle preheated to 42 ℃, carrying out heat shock for 90s, transferring into the ice, and cooling for 1-2 min; adding LB liquid culture medium without antibiotics into each centrifuge tube to supplement 1ml, transferring the centrifuge tubes to 37 ℃, carrying out shaking table incubation for 45min at 150rpm, coating bacterial liquid onto LB solid culture medium with antibiotics for culturing for 12-16 h, and then carrying out colony PCR and sequencing, wherein colony PCR and sequencing primers are as follows:

SP-L：GCGCGGTGTCATCTATGTTACT（SEQ ID NO.9）；

SP-R：CCCGACATAGATGCAATAACTTC（SEQ ID NO.10）。

step 7, correct sequencingOsCKX3The gene knockout vector is used for transforming agrobacterium and infecting mature rice callus, and then resistance screening is carried out to obtain the resistant callus.

7.1, preparation of mature callus of rice.

After mature seeds of wild Nippon Paddy (NIP) are dehulled, selecting seeds with full, smooth and sterile spots, placing the seeds into a beaker, and sterilizing the seeds with 70% alcohol for 2min; pouring out alcohol, and adding 30% (v/v) sodium hypochlorite aqueous solution for disinfection for 30min; pouring out sodium hypochlorite aqueous solution, cleaning with sterile water for 5 times, and soaking in sterile water for 30min for the last time; pouring out the sterile water, and placing the seeds on sterile filter paper to suck the seeds to dryness to obtain the pretreated rice seeds. Placing the pretreated rice seeds in a japonica rice mature embryo induction culture medium, and culturing in dark at 28 ℃ for about 10 days; opening a culture dish on an ultra-clean workbench, removing buds and endosperm by using sterile forceps, leaving embryogenic callus (light yellow, compact and irregular), transferring into a japonica rice subculture medium, and culturing at 28 ℃ in dark for 5-10 days to obtain the mature embryo callus of rice.

7.2, culturing agrobacterium.

Will beOsCKX3The gene knockout vector of the gene was transformed into Agrobacterium (Agrobacterium) EHA105 competent cells using freeze thawing. The specific method comprises the following steps: taking the constructed materialsOsCKX32. Mu.L of gene knockout vector was added to 100. Mu.L of Agrobacterium competent cells; mixing, placing on ice for 15min, placing in liquid nitrogen 90s, placing at 37deg.C for 5min, adding 800 μl of YEP liquid culture medium without antibiotics, and shaking at 28deg.C for 2 hr; mu.L of the bacterial liquid was plated on a YEP solid medium having 50mg/L spectinomycin (Spec) and 50mg/L streptomycin (Str), kanamycin (Kan); after growing for 2 days, EHA105 monoclonal bacterial plaque is picked up and cultured in 5ml of YEP liquid culture medium at 28 ℃ and shaking table at 250rpm for overnight until the bacterial liquid OD600 is about 2.0; sucking 500 mu L of the bacterial liquid into 30ml of YEP liquid culture medium, and carrying out shaking culture on a shaking table at 28 ℃ and 250rpm for 14h until the bacterial liquid OD600 = 1.0 to obtain agrobacterium bacterial liquid; the YEP liquid medium consists of: yeast Extract 10g/L, peptone 10g/L, naCl g/L, and water as solvent, pH7.0. The YEP solid culture medium is prepared by adding 15g/L agar into a YEP liquid culture medium.

7.3, screening of co-cultured and resistant calli.

Placing 15ml of the agrobacterium tumefaciens bacteria liquid cultured in the step 7.2 into a 50ml centrifuge tube, centrifuging at 4 ℃ and 4000rpm for 10min, and collecting thalli; an Agrobacterium suspension was prepared with 30ml of AAM-sensitive bacteria solution containing 200. Mu. Mol/L acetosyringone (As) to give a final concentration of OD600 of 0.6.

Picking out the mature embryo callus of the rice in the step 7.1, cutting into particles, and putting into agrobacterium suspension for shake culture at 28 ℃ for 30min; carefully taking out the callus, and draining on sterile filter paper for 30min; then placing the callus on a japonica rice co-culture medium containing 200 mu mol/L acetosyringone (As) and provided with a layer of sterilizing filter paper; after dark culture at 25 ℃ for 2.5 days, taking out the callus, washing with sterile water for 5-6 times, and continuously oscillating during the period; cleaning with sterile water for 1-2 times; finally, placing the mixture on sterile filter paper for draining for 2 hours; transferring the dried calli into a selection medium containing 250mg/L of carbenicillin sodium and 50mg/L of hygromycin for first round of resistance screening, carrying out dark culture at 28 ℃ for 14 days, transferring the obtained initial calli into a selection medium containing 250mg/L of carbenicillin sodium and 50mg/L of hygromycin for second round of selection, carrying out dark culture at 28 ℃ for 14 days, and taking the grown calli as the resistant calli with hygromycin resistance genes.

Step 8, differentiating the resistant callus to obtain seedlingsOsCKX3Mutant transgenes (rice tillering control mutant) rice seedlings.

Adding differentiation medium into 500ml differentiation tank (1/3 volume), picking 2-3 resistant calli of the same calli from step 7.3, placing on differentiation medium, covering, light culturing at 25deg.C (16 h/8h photoperiod, light intensity 2000 LUX) for about 30 days until calli differentiate into young seedlings, transferring into rooting medium test tube when young seedlings grow to about 3-5cm, sealing with sealing film, and light culturing at 25deg.C (16 h/8h photoperiod, light intensity 2000 LUX).

And 9, training and transplanting the transgenic seedlings.

In step 8OsCKX3After rooting the mutant transgenic rice seedlings for 14 days, picking out test tubes with root parts and stem leaves of the seedlings differentiated to be relatively intact to obtain seedlings, opening a sealing film, adding 30ml of sterile water at 30 ℃ and hardening the seedlings in a culture room for 2-3 days; washing off agar, culturing in rice nutrient solution, and collecting seeds.

And step 10, screening and identifying T0 generation transgenic plants.

The T0 generation transgenic plant DNA is extracted, CAS9 is identified, and the identification primers are as follows:

Cas9-F：CTGACGCTAACCTCGACAAG（SEQ ID NO.11）；

Cas9-R：CCGATCTAGTAACATAGATGACACC（SEQ ID NO.12）。

breeding T0 generation transgenic plant containing CAS9 in the ground, harvesting T1 generation for genotyping, designing primer at 500bp upstream and downstream of target point, amplifying target sequence, and PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s, extension at 72 ℃ for 50s, circulation for 38 times, extension at 72 ℃ for 5min, and preservation at 4 ℃; and (3) after amplification, carrying out 1% agarose gel electrophoresis, verifying correct colony amplification culture of the strip, extracting plasmids, and sequencing to obtain the genotype of the mutant.

FIG. 2 is a gene structure diagram (wild type NIP) and mutation sites of OsCKX3 gene of rice in which the rectangles represent exons and in which the mutants are shown in the embodiment of the present inventionosckx3-1Knocking out 8 bases (the nucleotide sequence is shown as SEQ ID NO. 1),osckx3-2deletion of 19 bases (nucleotide sequence shown in SEQ ID NO. 2).

FIG. 3 shows rice gene according to an embodiment of the present inventionOsCKX3Two mutantsosckx3-1Andosckx3-2as can be seen from the histogram, compared with the expression level of the wild type (NIP),osckx3-1andosckx3-2compared with wild typeOsCKX3Almost no expression of the gene, indicatingosckx3-1Andosckx3-2a kind of electronic deviceOsCKX3The gene function is completely inactivated, and the mutant is a loss-of-function mutant.

Example 5

The two homozygous mutants with different mutation types obtained in the example 4 and the Japanese wild seeds are soaked, germinated and spread in a seedling bed, planted in a Nanjing white horse teaching scientific research base, and 8×18 total 146 rice seedlings in a 1.5m×6m square area are planted and managed by a general rice planting method in the presence of a safety protection facility. After 130 days of growth, the effective tillering numbers of the two mutants and the wild type are counted, 20 rice seedlings are randomly selected (the edge parts of the squares are removed) for seed collection by each square, and the seeds are dried at 37 ℃ for 1 week and are subjected to agronomic trait statistics. Two independent mutant strains and about 10 seedlings are taken from the wild type mutant strains, 4 snapping seeds are taken from each seedling, and the number of primary branches of 80 snapping seeds is counted. And removing the shrunken seeds, threshing by taking each plant as a unit, weighing, and counting wild plants by the same method.

FIG. 4 shows the wild type and the wild type according to the embodiment of the present inventionosckx3-1Andosckx3-2collecting whole plant samples of 10 rice seedlings randomly selected (excluding the edge portions of squares) at each square for each material at maturity, as shown in FIG. 4, which is a plot of field phenotype and yield analysis provided by an embodiment of the present invention, wherein rice is wild-type andosckx3-1andosckx3-2maturity field phenotype comparison graph and individual yield comparison graph. It is phenotypically known that the number of the cells,osckx3-1andosckx3-2compared with the nip wild type, the plant height is shortened, tillers are increased, the single plant yield pie chart analysis shows that,osckx3-1andosckx3-2compared with the NIP wild type, the single plant yield is obviously increased.

FIG. 5 shows the wild type and the wild type according to the embodiment of the present inventionosckx3-1Andosckx3-2collecting whole plant samples of 10 rice seedlings randomly selected (excluding edge parts of squares) from each square by each material in maturity, and counting wild NIP,osckx3-1Andosckx3-2as can be seen from the plant height histogram of (c),osckx3-1andosckx3-2compared with the NIP wild type, the plant height is obviously shortenedp<0.01）。

FIG. 6 shows the wild type and the wild type according to the embodiment of the present inventionosckx3-1Andosckx3-2collecting whole plant samples of 10 rice seedlings randomly selected (excluding edge parts of squares) from each square by each material in maturity, and counting wild NIP,osckx3-1Andosckx3-2from the histogram of the tiller number of the individual plants,osckx3-1andosckx3-2compared with the NIP wild type, the single plant tillering number is obviously higher than that of the wild type, and the statistical analysis difference is extremely obviousp<0.001）。

FIG. 7 shows the wild type and the wild type according to the embodiment of the present inventionosckx3-1Andosckx3-2collecting whole plant samples of 10 rice seedlings randomly selected (excluding edge parts of squares) from each square by each material in maturity, and counting wild NIP,osckx3-1Andosckx3-2from the individual yield histogram of (c), it can be seen from the graph,osckx3-1andosckx3-2compared with the NIP wild type, the yield of the single plant is obviously higher than that of the wild type, and the statistical analysis difference is extremely obviousp<0.001）。

FIG. 8 shows the wild type and the wild type according to the embodiment of the present inventionosckx3-1Andosckx3-2collecting whole samples of 10 rice seedlings randomly selected (except for edge parts of squares) from each material in the mature period, and shooting wild NIP,osckx3-1Andosckx3-2as can be seen from the individual ear pattern map of (2),osckx3-1andosckx3-2compared with the NIP wild type, the single rice spike is obviously larger than the wild type, and the seeds are plump.

FIG. 9 shows the wild type and the wild type according to the embodiment of the present inventionosckx3-1Andosckx3-2collecting whole plant samples of 10 rice seedlings randomly selected (excluding edge parts of squares) from each square by each material in maturity, and counting wild NIP,osckx3-1Andosckx3-2from the histogram of the number of the first-stage branches of the single spike,osckx3-1andosckx3-2compared with the NIP wild type, the first branch is obviously more than the wild type, and the statistical analysis difference is obviousp<0.01）。

FIG. 10 shows the wild type and the wild type according to the embodiment of the present inventionosckx3-1Andosckx3-2collecting whole plant samples of 10 rice seedlings randomly selected (excluding edge parts of squares) from each square by each material in maturity, and counting wild NIP,osckx3-1Andosckx3-2the histogram of the number of secondary branches on the single spike of (a) can be known from the graph,osckx3-1andosckx3-2compared with the NIP wild type, the secondary branch is obviously more than the wild type, and the statistical analysis difference is obviousp<0.01）。

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the technical principles of the present invention, and such modifications and variations should also be regarded as being within the scope of the invention.

Claims (10)

1. A rice tillering regulating gene is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO.1 or SEQ ID NO. 2.

2. A rice tillering regulation mutant is characterized by comprising a gene with a nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.

3. A method for preparing a rice tillering control mutant, comprising: for rice geneOsCKX3The nucleotide sequence of (2) is subjected to targeted editing to obtain a mutant with a gene with the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.

4. The method for preparing a rice tillering control mutant according to claim 3, comprising the steps of:

at the position ofOsCKX3Selecting target spots on genes and designing primers;

connecting the primer with the sgRNA expression cassette, and then amplifying to obtain a target gRNA expression cassette;

connecting the target gRNA expression cassette with pYLCRISPR/Cas9-MH (B) to obtainOsCKX3A gene knockout vector;

will beOsCKX3Transforming agrobacterium with the gene knockout vector, infecting mature rice callus, and then carrying out resistance screening to obtain resistant callus;

differentiation of the resistant callus into seedlings is carried out to obtain rice seedlings of the rice tillering regulation mutant.

5. The method for preparing rice tillering control mutant according to claim 4, wherein, in the following stepsOsCKX3Selecting a target point on the gene, and designing a primer according to the promoter;

the promoters are U6a and U6b;

the primer is OsCKX3-T1-F with a nucleotide sequence shown as SEQ ID NO.3, osCKX3-T1-R with a nucleotide sequence shown as SEQ ID NO.4, osCKX3-T2-F with a nucleotide sequence shown as SEQ ID NO.5 and OsCKX3-T2-R with a nucleotide sequence shown as SEQ ID NO. 6.

6. The method for preparing a rice tillering control mutant according to claim 4, wherein the method for ligating the primer to the sgRNA expression cassette comprises:

and taking plasmid pYLgRNA-OsU a/LacZ and pYLgRNA-OsU b for enzyme digestion, and connecting the digested plasmid with a corresponding joint of the primer.

7. The method for preparing a rice tillering control mutant according to claim 4, wherein the method for amplifying the sgRNA expression cassette after the primer ligation comprises:

pre-denaturing at 98 ℃ for 5min, denaturing at 98 ℃ for 30s, annealing at 55 ℃ for 15s, extending at 58 ℃ for 30s, circulating for 30 times, extending at 68 ℃ for 5min to obtain amplified PCR products;

and separating the amplified PCR product by agarose gel electrophoresis, and then performing gel cutting recovery by using a gel recovery kit to obtain a recovery fragment, namely the target gRNA expression cassette.

8. The method of preparing a rice tillering control mutant according to claim 4, wherein the method of ligating the target gRNA expression cassette to the pYLCRISPR/Cas9-MH (B) comprises:

BsaI endonuclease and T4 ligase are selected for edge ligation, and the target gRNA expression cassette is ligated to the kanamycin-resistant expression vector pYLCRISPR/Cas9-MH (B).

9. The method for preparing a rice tillering control mutant according to claim 4, further comprising screening and identifying T0 generation transgenic plants for rice seedlings of the rice tillering control mutant:

extracting DNA of the T0 generation transgenic plant, and identifying CAS9 to obtain the T0 generation transgenic plant seed containing CAS 9;

breeding T0 generation transgenic plant seeds containing CAS9 in the ground, harvesting T1 generation for genotyping, designing primers at 500bp upstream and downstream of the target point, and amplifying the target sequence;

and (3) after amplification, agarose gel electrophoresis is carried out, correct colony amplification culture of the strip is verified, plasmids are extracted, and the mutant genotype is obtained after sequencing.

10. Use of the rice tillering control gene according to claim 1, or the rice tillering control mutant according to claim 2, or the mutant prepared by the method according to any one of claims 3 to 9, for controlling rice tillering.