CN111100852B - Directional mutation method of OsALS1 and crop endogenous gene directed evolution method - Google Patents

Abstract

The invention provides a method for directional mutation of OsALS1 and directional evolution of crop endogenous genes. The amino acid sequence of the OsALS1 is obtained by subjecting the sequence 78 to at least one of a first mutation, a second mutation and a third mutation, wherein the first mutation is one of P171F, P171S and P171L, the second mutation is L158F, and the third mutation is R190H. The method comprises the following steps: selecting endogenous genes of crops as target genes; designing a plurality of target nucleotide sequences based on the target gene to obtain a plurality of directed evolution cloning sequences, and cloning the directed evolution cloning sequences to plasmids containing gRNAs respectively to obtain a directed evolution gRNA plasmid library; integrating a related sequence in the directed evolution gRNA library and a Cas9 expression cassette into the same vector, and transforming the crop to obtain a mutation library of the crop; screening the mutant library of the crop plant for a desired trait associated with the target gene.

Description

Directional mutation method of OsALS1 and crop endogenous gene directed evolution method

Technical Field

The invention relates to directed mutation of OsALS1 and a method for directed evolution of crop endogenous genes, in particular to directed mutation of herbicide resistance of OsALS1 and a method for directed evolution of rice endogenous genes.

Background

In nature, a large amount of genetic diversity exists among different species and different varieties of the same crop, and the genetic diversity determines the diversity and commodity attribute of key agronomic traits of various crops, so that the method has an important promoting effect on crop breeding improvement and commercial application in the long-term domestication process of the crops. Therefore, the excavation and utilization of new genes for controlling important agronomic traits are always the first task for genetic improvement and new variety breeding of various crops. Moreover, the excavation of a key new gene can cause great change in agriculture, such as the rice dwarf gene sd-1 and the wheat dwarf gene Rht causing the green revolution of agriculture for the second time. However, in practice, the variation of genes in nature is limited, huge manpower and material resources are needed for mining new genes from the natural germplasm resource library, and the time consumption is long, so that the huge demands of the human society with the growing population on grains and crops cannot be met.

Rice, as an important food crop, lives more than half of the world's population. The safe production of rice and the stable supply of food are important to human society. Modern rice production is already intensive and large-scale planting, and the harm of weeds in rice fields is more and more serious. The traditional manual weeding and mechanical weeding are time-consuming, labor-consuming, high in cost and low in efficiency, and the weeding method by using chemical agents is remarkable in effect and saves more manpower and material resources, so that the demand on commercial varieties of rice with herbicide resistance is stronger. In recent years, the herbicide is widely popularized and applied in agricultural production, and the stable production of rice in China is effectively guaranteed.

Acetolactate synthase (ALS), also known as acetohydroxyacid synthase (AHAS), is the first key enzyme in the biosynthesis of branched-chain amino acids (valine, leucine and isoleucine) required for plant growth. ALS is a common target site for five herbicides of Sulfonylureas (SU), imidazolinones (IMI), Triazolopyrimidines (TP), Pyrimidinylsalicylates (PTB) and Sulfonamides (SCT) that are commonly used in production. These types of herbicides are all potent inhibitors of ALS, ultimately leading to plant death by preventing the synthesis of branched chain amino acids. The excellent characteristics of ALS inhibitor herbicides that control a wide variety of weeds, are low-toxicity to mammals, and selective for major crops, enable such herbicides to be used in a wide variety of different crops worldwide. However, since the use of herbicides is liable to cause phytotoxicity to crops, it is prudent to put them into practical use in production, and therefore, in order to solve this problem, it is necessary to develop herbicide-resistant rice. The traditional method for developing herbicide-resistant rice is mainly a natural screening and mutation breeding method, such as EMS mutation, gamma ray and other physicochemical methods, mutation treatment is carried out on rice to mutate ALS genes in the rice, and then the rice with ALS herbicide resistance is screened from the offspring generated by mutation, but a large amount of manpower and material resources are required to be invested to separate germplasm in the nature through large-scale screening.

In recent years, increasingly developed genome editing technologies have brought new directions to the precise breeding of modern crops. The technology realizes the genetic improvement of the crops by carrying out site-directed editing (such as deletion, insertion or substitution of DNA fragments and the like) on target genes in the crop genome, and subverts the traditional breeding mode. Especially, newly developed single base editing technologies (cytosine editing technology CBE and adenine base editing technology ABE) successfully realize directional conversion (base pair G/C and A/T interchange) of four bases in crop genomes, which provides technical support for accurate crop breeding and gene function correction and accelerates the breeding process of crops. However, these base editor-mediated molecular breeding are limited to genetic engineering under conditions where certain gene functional sites are well defined. Based on the prior art, most of the excavated functional sites of key agronomic trait control genes are unknown, so that the genetic modification of the genes is not from the beginning.

Disclosure of Invention

The invention provides a directed mutant protein of OsALS1, wherein the amino acid sequence of the directed mutant protein is at least one of a first mutation, a second mutation and a third mutation of an amino acid sequence shown as SEQ ID No.78, the first mutation is one of P171F, P171S and P171L, the second mutation is L158F, and the third mutation is R190H. Wherein P171F and L158F may be mutated simultaneously; P171S and L158F may be mutated simultaneously; P171L and L158F may be mutated simultaneously.

The second invention provides a directional mutant nucleic acid of OsALS1, wherein the directional mutant nucleic acid is at least one of a first directional mutant nucleic acid, a second directional mutant nucleic acid and a third directional mutant nucleic acid;

the first directional mutation nucleic acid is one of amino acid capable of coding the amino acid sequence shown as SEQ ID No.78 to generate P171F mutation, amino acid capable of coding the amino acid sequence shown as SEQ ID No.78 to generate P171S mutation and amino acid capable of coding the amino acid sequence shown as SEQ ID No.78 to generate P171L mutation;

the second directional mutant nucleic acid is a directional mutant nucleic acid which can code an amino acid of which the amino acid sequence shown as SEQ ID No.78 has L158F mutation;

the third directional mutant nucleic acid is a directional mutant nucleic acid which can code an amino acid of which the amino acid sequence shown as SEQ ID No.78 has R190H mutation.

When P171F and L158F are mutated simultaneously, the targeted mutation nucleic acid can encode the amino acid of the amino acid sequence shown as SEQ ID No.78 with mutations of P171F and L158F; when P171S and L158F are mutated simultaneously, the targeted mutation nucleic acid can encode the amino acid of the amino acid sequence shown as SEQ ID No.78 with mutations of P171S and L158F; when P171L and L158F are mutated simultaneously, the targeted mutation nucleic acid can encode the amino acid of the amino acid sequence shown as SEQ ID No.78 with mutations of P171L and L158F.

In one embodiment, when the amino acid is the amino acid sequence shown as SEQ ID No.78 mutated with P171F: the first directional mutant nucleic acid is directional mutant nucleic acid which mutates cytosine at the 511 th position of a nucleotide sequence shown as SEQ ID No.77 into thymine and mutates cytosine at the 512 th position into thymine; or the first directional mutant nucleic acid is a directional mutant nucleic acid in which the 511 th cytosine of the nucleotide sequence shown as SEQ ID No.77 is mutated into thymine, the 512 th cytosine is mutated into thymine, and the 513 th cytosine is mutated into thymine.

In one embodiment, when the amino acid is the amino acid sequence shown as SEQ ID No.78 mutated with P171S: the first directional mutant nucleic acid is a directional mutant nucleic acid which mutates cytosine at the 511 th position of a nucleotide sequence shown as SEQ ID No.77 into thymine.

In one embodiment, when the amino acid is the amino acid sequence shown as SEQ ID No.78 mutated with P171L: the first directional mutant nucleic acid is directional mutant nucleic acid which enables cytosine at the 511 th position of a nucleotide sequence shown as SEQ ID No.77 to be mutated into thymine, cytosine at the 512 th position to be mutated into thymine and cytosine at the 513 th position to be mutated into guanine.

In one embodiment, when the amino acid is the amino acid sequence shown as SEQ ID No.78 mutated at L158F: the second directional mutant nucleic acid is a directional mutant nucleic acid which mutates cytosine at the 472 th site of the nucleotide sequence shown as SEQ ID No.77 into thymine.

In one embodiment, the amino acid is a sequence obtained by mutating the amino acid sequence shown as SEQ ID No.78 with R190H: the third directional mutant nucleic acid is a directional mutant nucleic acid which is obtained by mutating guanine at the 569 th site of the nucleotide sequence shown as SEQ ID No.77 into adenine.

The invention also provides a method for directional evolution of endogenous genes of crops, which comprises the following steps:

1) selecting endogenous genes of the crops as target genes for directed evolution;

2) designing a plurality of target nucleotide sequences for CRISPR/Cas9 (such as cytosine base editor CBE and adenine base editor ABE) based on the target gene to obtain a plurality of directed evolution clone sequences;

3) cloning the directed evolution cloning sequences to plasmids containing gRNAs respectively, wherein the directed evolution cloning sequences are connected with the gRNAs and are arranged at the 5' ends of the gRNAs to obtain directed evolution gRNA plasmid libraries;

4) integrating a promoter, a target nucleotide sequence, a gRNA scaffold and a terminator in the directed evolution gRNA library and a Cas9 expression cassette to the same vector to obtain a directed evolution plasmid library;

5) transforming the directed evolution plasmid library into the crops to obtain a mutation library of the crops;

6) screening the mutant library of the crop plant for a desired trait associated with the target gene.

In one embodiment, a homology arm sequence capable of fusion (infusion) with a vector is ligated to each of the target nucleotide sequences at the 5 'end and the 3' end, respectively.

In one embodiment, a linker sequence SEQ ID No.69 is attached to the 5 'end of each target nucleotide sequence and a linker sequence SEQ ID No.70 is attached to the 3' end of each target nucleotide sequence.

In one embodiment, 5 'to 3' SEQ ID No.71 and 69 are ligated to the 5 'end of each target nucleotide sequence, and 5' to 3 'SEQ ID No.72 and 70 are ligated to the 3' end of each target nucleotide sequence.

In one embodiment, the homology arm at the 5 'end of each target nucleotide sequence is shown in SEQ ID No.14 and the homology arm at the 3' end of each target nucleotide sequence is shown in SEQ ID No. 68.

In a specific embodiment, the nucleotide sequence of the target gene is shown in SEQ ID No. 77.

In a specific embodiment, the plurality of directed evolution clone sequences are SEQ ID Nos.3-13 and SEQ ID Nos. 16-17.

In a specific embodiment, the crop plant is a graminaceous plant.

Preferably, in one embodiment, the crop plant is rice.

Has the advantages that:

the invention obtains the directional mutation of OsALS1 resistant to herbicide (such as bispyribac-sodium). In addition, the invention also develops a brand-new molecular breeding technology, namely a base editor-mediated crop endogenous gene directed evolution technology (BEMGE). The technology utilizes a series of single base editors to simulate gene evolution in crop cells, concentrates the gene evolution process for hundreds of millions of years into one year through manual interference, creates tens of thousands or even hundreds of thousands of new genes, and can quickly separate crop plants with better characters through agricultural character investigation. Different from the traditional transgenic crops, the base editing germplasm obtained by the method does not contain exogenous T-DNA, has no biosafety problem, and can be directly applied to conventional breeding of rice; the sgRNA dug correspondingly can be used for genetic improvement and upgrading of other commercial rice varieties. In a word, the base editor-mediated crop endogenous gene directed evolution technology can carry out saturation mutation on any cloned important agronomic trait control gene of rice, excavate novel materials and become a key technology of crop breeding in the foreseeable future. It can be said that BEMGE fills the gap between basic research and applied research, and theoretically BEMGE could potentially genetically modify any functional gene identified from model plants or commercial crops. The human does not need to borrow genes from nature, can directly carry out manual large-scale creation, and is more efficient.

The invention specifically takes OsALS1 as a target gene, designs a gRNA library, performs saturation mutation on OsALS1, obtains a large amount of new alleles of OsALS1 from rice at one time, and identifies new herbicide resistance genes and new sites from the new alleles.

In conclusion, the base editor-mediated crop endogenous gene directed evolution technology has a great promoting effect on modern crop breeding.

Drawings

FIG. 1 shows the results of high-throughput sequencing analysis of each of the 11 target nucleotides in pool-2. Wherein the shades represent different target nucleotides.

FIG. 2 shows the results of high throughput sequencing of the types of mutations that occur in the target gene region in Bank-2. Wherein, the color intensity represents the mutation of different target nucleotide sites, which indicates that the mutation types are various and have efficiency.

FIG. 3 shows a phenotypic resistance chart of P171F mutant plants after 10 days of culture after bispyribac-sodium addition.

FIG. 4 shows a phenotypic resistance chart of P171L mutant plants after 10 days of culture after bispyribac-sodium addition.

FIG. 5 shows a phenotypic resistance chart of P171S mutant plants after 10 days of culture after bispyribac-sodium addition.

FIG. 6 shows a phenotypic resistance chart of L158F mutant plants after 10 days of culture after bispyribac-sodium addition.

FIG. 7 shows a phenotypic resistance chart of R190H mutant plants after 10 days of culture after bispyribac-sodium addition.

FIG. 8 shows a phenotypic resistance chart of P171F mutant plants after 21 days of cultivation after bispyribac-sodium spraying.

Detailed Description

The above-described aspects of the invention are explained in more detail below by means of preferred embodiments, but they are not intended to limit the invention.

The reagents in the examples of the present invention were all commercially available unless otherwise specified.

Example 1: construction of pENTR4-gRNA10 recombinant plasmid

The technical route for constructing the vector is as follows:

primers ccdB-F1(SEQ ID No.1, restriction enzyme BsaI cleavage site introduced at positions 24-29) and ccdB-R1(SEQ ID No.2, restriction enzyme BsaI cleavage site introduced at positions 25-30) were synthesized using Hi-Fi enzyme I-5^TM2 × High-Fidelity Master Mix (from Clauing, Beijing Biotechnology Ltd.) was subjected to PCR amplification using pUbi: rBE14 plasmid (CN201810069129.6) as a template to obtain a target fragment of 800bp in size (ccdB gene, genbank accession number KR233518.1, nucleotide sequence 3289 to 3594). Meanwhile, the sequence of the U6 promoter, the nucleotide sequence containing two BsaI cleavage sites, gRNA scaffold and (T)8 termination sequence (CN201810069129.6) which are connected in sequence is artificially synthesized according to the direction from the 5 'end to the 3' end, the sequence is cloned into a pENTR4 vector (purchased from Invitrogen corporation of America) and named as pENTR4-gRNA3, then pENTR4-gRNA3 is subjected to BsaI cleavage, a linearized vector skeleton (the main elements comprise the U6 promoter, gRNA scaffold and (T)8 termination sequence) with the size of about 2.75kb is recovered, and the linearized vector skeleton is further utilized

II One Step Cloning reaction of One Step Cloning Kit (purchased from Nyvowed Biotech Co., Ltd., Nanjing) and the obtained 800bp ccdB PCR fragment to obtain the vector pENTR4-gRNA 10. Wherein the main part of the constitution of the plasmid pENTR4-gRNA10 is as follows: u6 promoter, ccdB gene, gRNA scaffold, (T)8 termination sequence.

Example 2: design and cloning of recognition sequence aiming at OsALS1 gene

The transcript sequence and the genome sequence of the OsALS1 gene are obtained from an MSU/TIGR rice genome database (http://rice.plantbiology.msu.edu/)。

For the OsALS1 gene, the synthetic oligonucleotide sequences were 63 in total, divided into 6 pools, each pool containing 10-11 gRNA sequences.

For pool-1, 11 oligonucleotide sequences were included:

the synthetic sequences were as follows: OsALS1-Pool01-1(SEQ ID No.3, the target nucleotide sequence of OsALS1-Pool01-1 at positions 23 to 41), OsALS1-Pool01-2(SEQ ID No.4, the target nucleotide sequence of OsALS1-Pool01-2 at positions 23 to 40), OsALS1-Pool01-3(SEQ ID No.5, the target nucleotide sequence of OsALS1-Pool01-3 at positions 23 to 41), OsALS1-Pool01-4(SEQ ID No.6, the target nucleotide sequence of OsALS1-Pool01-4 at positions 23 to 41), OsALS1-Pool01-5(SEQ ID No.7, the target nucleotide sequence of OsALS1-Pool01-5 at positions 23 to 40), OsALS1-Pool 366-Pool 01 (SEQ ID No.72, SEQ ID No. 01-01, the target nucleotide sequence of OsALS 01-01), target nucleotide sequence of OsALS1-Pool01-7 at positions 23 to 41), OsALS1-Pool01-8(SEQ ID No.10, target nucleotide sequence of OsALS1-Pool01-8 at positions 23 to 41), OsALS1-Pool01-9(SEQ ID No.11, target nucleotide sequence of OsALS1-Pool01-9 at positions 23 to 41), OsALS1-Pool01-10(SEQ ID No.12, target nucleotide sequence of OsALS1-Pool01-10 at positions 23 to 41), OsALS1-Pool01-11(SEQ ID No.13, target nucleotide sequence of OsALS1-Pool01-11 at positions 23 to 41).

The 11 oligonucleotide sequences are mixed in equal volume and equal concentration to be used as a template, and the Array-F1(SEQ ID No.14) and the Array-R1(SEQ ID No.15) are used as primers, and I-5 is utilized^TMThe 11 oligonucleotide fragments were subjected to mixed amplification at 2 Xhigh-Fidelity Master Mix, and about 140bp of PCR product was recovered to obtain 11 mixed DNA fragments containing the target nucleotide. At the same time, the vector pENTR4-gRNA10 was digested with BsaI, and the vector backbone of about 5.3kb (releasing ccdB gene fragment) was recovered and used

II, randomly replacing the ccdB gene by 1 DNA fragment in 11 fragments containing the target nucleotide aiming at the OsALS1 gene (the homologous arm sequence at the 5 'end of the target nucleotide sequence for fusion (infusion) is shown as SEQ ID No.14, the homologous arm sequence at the 3' end of the target nucleotide sequence for fusion (infusion) is shown as SEQ ID No.68 (namely the reverse complementary sequence of SEQ ID No. 15)), transforming the ligation product into Escherichia coli to obtain an Escherichia coli library-1, randomly selecting 20 colonies for sequencing, wherein the accuracy is as high as 100%, and the 11 target nucleotides are contained. The plasmid library was named pENTR4-gRNA10-POOL 1.

For pool-2, there were 11 oligonucleotide sequences:

the synthetic sequences were as follows: OsALS1-Pool02-12(SEQ ID No.16, target nucleotide sequence of OsALS1-Pool02-12 at positions 23 to 41), OsALS1-Pool02-13(SEQ ID No.17, target nucleotide sequence of OsALS1-Pool02-13 at positions 23 to 41), OsALS1-Pool02-14(SEQ ID No.18, target nucleotide sequence of OsALS1-Pool02-14 at positions 23 to 41), OsALS1-Pool02-15(SEQ ID No.19, target nucleotide sequence of OsALS1-Pool02-15 at positions 23 to 41), OsALS1-Pool02-16(SEQ ID No.20, target nucleotide sequence of OsALS1-Pool02-16 at positions 23 to 40), OsALS1-Pool 3617-Pool 02 (SEQ ID No. 02, target nucleotide sequence of OsALS 02-02), OsALS 02-02 (SEQ ID No. 02, target nucleotide sequence of OsALS 02-02, target nucleotide sequence of OsALS1-Pool02-18 at positions 23 to 41), OsALS1-Pool02-19(SEQ ID No.23, target nucleotide sequence of OsALS1-Pool02-19 at positions 23 to 41), OsALS1-Pool02-20(SEQ ID No.24, target nucleotide sequence of OsALS1-Pool02-20 at positions 23 to 41), OsALS1-Pool02-21(SEQ ID No.25, target nucleotide sequence of OsALS1-Pool02-21 at positions 23 to 41), OsALS1-Pool02-22(SEQ ID No.26, target nucleotide sequence of OsALS1-Pool02-22 at positions 23 to 41).

The 11 oligonucleotide sequences are mixed in equal volume and equal concentration to be used as a template, and the Array-F1(SEQ ID No.14) and the Array-R1(SEQ ID No.15) are used as primers, and I-5 is utilized^TM2 × High-Fidelity Master Mix on the above 11 oligoThe nucleotide fragments were mixed and amplified, and about 140bp of the PCR product was recovered to obtain 11 mixed DNA fragments containing the target nucleotide. At the same time, the vector pENTR4-gRNA10 was digested with BsaI, and the vector backbone of about 5.3kb (releasing ccdB gene fragment) was recovered and used

II One Step Cloning Kit replaces the ccdB gene randomly with any 1 DNA fragment in 11 fragments containing the target nucleotides aiming at the OsALS1 gene, converts the ligation product into Escherichia coli to obtain an Escherichia coli bank-2, randomly selects 20 colonies for sequencing, and has the accuracy rate of 100 percent and 11 target nucleotides. The plasmid library was named pENTR4-gRNA10-POOL 2.

For pool-3, 11 oligonucleotide sequences were included:

the synthetic sequences were as follows: OsALS1-Pool03-23(SEQ ID No.27, target nucleotide sequence of OsALS1-Pool03-23 at positions 23 to 41), OsALS1-Pool03-24(SEQ ID No.28, target nucleotide sequence of OsALS1-Pool03-24 at positions 23 to 41), OsALS1-Pool03-25(SEQ ID No.29, target nucleotide sequence of OsALS1-Pool03-25 at positions 23 to 41), OsALS1-Pool03-26(SEQ ID No.30, target nucleotide sequence of OsALS1-Pool03-26 at positions 23 to 41), OsALS1-Pool03-27(SEQ ID No.31, target nucleotide sequence of OsALS1-Pool03-27 at positions 23 to 41), OsALS1-Pool 03-03 (SEQ ID No. 03, target nucleotide sequence of OsALS 03-03, target nucleotide sequence of OsALS1-Pool03-29 at positions 23 to 40), OsALS1-Pool03-30(SEQ ID No.34, target nucleotide sequence of OsALS1-Pool03-30 at positions 23 to 40), OsALS1-Pool03-31(SEQ ID No.35, target nucleotide sequence of OsALS1-Pool03-31 at positions 23 to 41), OsALS1-Pool03-32(SEQ ID No.36, target nucleotide sequence of OsALS1-Pool03-32 at positions 23 to 40), OsALS1-Pool03-33(SEQ ID No.37, target nucleotide sequence of OsALS1-Pool03-33 at positions 23 to 40).

The 11 oligonucleotide sequences are mixed in equal volume and equal concentration to be used as a template, and the Array-F1(SEQ ID No.14) and the Array-R1(SEQ ID No.15) are used as primers, and I-5 is utilized^TM 2×HighAfter mixed amplification of the 11 oligonucleotide fragments by the Fidelity Master Mix, approximately 140bp of PCR products were recovered, and 11 mixed DNA fragments containing the target nucleotide were obtained. At the same time, the vector pENTR4-gRNA10 was digested with BsaI, and the vector backbone of about 5.3kb (releasing ccdB gene fragment) was recovered and used

II One Step Cloning Kit replaces the ccdB gene randomly with any 1 DNA fragment in 11 fragments containing the target nucleotides aiming at the OsALS1 gene, converts the ligation product into escherichia coli to obtain an escherichia coli library-3, randomly selects 20 colonies for sequencing, and has the accuracy rate of 100 percent and 11 target nucleotides. The plasmid library was named pENTR4-gRNA10-POOL 3.

For pool-4, 10 oligonucleotide sequences were included:

the synthetic sequences were as follows: OsALS1-Pool04-34(SEQ ID No.38, target nucleotide sequence of OsALS1-Pool04-34 at positions 23 to 41), OsALS1-Pool04-35(SEQ ID No.39, target nucleotide sequence of OsALS1-Pool04-35 at positions 23 to 41), OsALS1-Pool04-36(SEQ ID No.40, target nucleotide sequence of OsALS1-Pool04-36 at positions 23 to 41), OsALS1-Pool04-37(SEQ ID No.41, target nucleotide sequence of OsALS1-Pool04-37 at positions 23 to 41), OsALS1-Pool04-38(SEQ ID No.42, target nucleotide sequence of OsALS1-Pool04-38 at positions 23 to 41), OsALS1-Pool04 (SEQ ID No. 04, target nucleotide sequence of OsALS 04-04), OsALS 04-Pool 04 (SEQ ID No. 04, target nucleotide sequence of OsALS 04-04, target nucleotide sequence of OsALS1-Pool04-40 at positions 23 to 41), OsALS1-Pool04-41(SEQ ID No.45, target nucleotide sequence of OsALS1-Pool04-41 at positions 23 to 41), OsALS1-Pool04-42(SEQ ID No.46, target nucleotide sequence of OsALS1-Pool04-42 at positions 23 to 41), OsALS1-Pool04-43(SEQ ID No.47, target nucleotide sequence of OsALS1-Pool04-43 at positions 23 to 40).

The 10 oligonucleotide sequences are mixed in equal volume and equal concentration to be used as a template, and the primers are Array-F1(SEQ ID No.14) and Array-R1(SEQ ID No.15) and I-5^TM2 × High-Fidelity Master Mix the above 10 oligonucleotide fragmentsThe PCR product was recovered by approximately 140bp after the amplification, to obtain 10 mixed DNA fragments containing the target nucleotides. At the same time, the vector pENTR4-gRNA10 was digested with BsaI, and the vector backbone of about 5.3kb (releasing ccdB gene fragment) was recovered and used

II One Step Cloning Kit replaces the ccdB gene randomly with 1 DNA fragment of 10 fragments containing target nucleotides aiming at the OsALS1 gene, converts the ligation product into Escherichia coli to obtain an Escherichia coli bank-4, randomly selects 20 colonies for sequencing, and has the accuracy rate of 100 percent and 10 target nucleotides. The plasmid library was named pENTR4-gRNA10-POOL 4.

For pool-5, 10 oligonucleotide sequences were included:

the synthetic sequences were as follows: OsALS1-Pool05-44(SEQ ID No.48, target nucleotide sequence of OsALS1-Pool05-44 at positions 23 to 41), OsALS1-Pool05-45(SEQ ID No.49, target nucleotide sequence of OsALS1-Pool05-45 at positions 23 to 40), OsALS1-Pool05-46(SEQ ID No.50, target nucleotide sequence of OsALS1-Pool05-46 at positions 23 to 40), OsALS1-Pool05-47(SEQ ID No.51, target nucleotide sequence of OsALS1-Pool05-47 at positions 23 to 41), OsALS1-Pool05-48(SEQ ID No.52, target nucleotide sequence of OsALS1-Pool05-48 at positions 23 to 41), OsALS1-Pool05 (SEQ ID No. 05, target nucleotide sequence of OsALS 05-05), OsALS 05-05 (SEQ ID No. 05, target nucleotide sequence of OsALS 05-05, target nucleotide sequence of OsALS1-Pool05-50 at positions 23 to 41), OsALS1-Pool05-51(SEQ ID No.55, target nucleotide sequence of OsALS1-Pool05-51 at positions 23 to 41), OsALS1-Pool05-52(SEQ ID No.56, target nucleotide sequence of OsALS1-Pool05-52 at positions 25 to 41), OsALS1-Pool05-53(SEQ ID No.57, target nucleotide sequence of OsALS1-Pool05-53 at positions 23 to 41).

The 10 oligonucleotide sequences are mixed in equal volume and equal concentration to be used as a template, and the primers are Array-F1(SEQ ID No.14) and Array-R1(SEQ ID No.15) and I-5^TM2 x High-Fidelity Master Mix was mixed to amplify the above 10 oligonucleotide fragments, and PCR products were recovered at approximately 140bp to obtain 10 containingThere is a mixed DNA fragment of the target nucleotide. At the same time, the vector pENTR4-gRNA10 was digested with BsaI, and the vector backbone of about 5.3kb (releasing ccdB gene fragment) was recovered and used

II One Step Cloning Kit replaces the ccdB gene randomly with 1 DNA fragment of 10 fragments containing target nucleotides aiming at the OsALS1 gene, converts the ligation product into Escherichia coli to obtain an Escherichia coli bank-5, randomly selects 20 colonies for sequencing, and has the accuracy rate of 100 percent and 10 target nucleotides. The plasmid library was named pENTR4-gRNA10-POOL 5.

For pool-6, 10 oligonucleotide sequences were included:

the synthetic sequences were as follows: OsALS1-Pool06-54(SEQ ID No.58, the target nucleotide sequence of OsALS1-Pool06-54 at positions 23 to 41), OsALS1-Pool06-55(SEQ ID No.59, the target nucleotide sequence of OsALS1-Pool06-55 at positions 23 to 41), OsALS1-Pool06-56(SEQ ID No.60, the target nucleotide sequence of OsALS1-Pool06-56 at positions 23 to 41), OsALS1-Pool06-57(SEQ ID No.61, the target nucleotide sequence of OsALS1-Pool06-57 at positions 23 to 41), OsALS1-Pool06-58(SEQ ID No.62, the target nucleotide sequence of OsALS1-Pool06-58 at positions 23 to 41), OsALS1-Pool 3663-Pool 3659 (SEQ ID No. 72-06, the target nucleotide sequence of OsALS 06-06 at positions 23 to 41), target nucleotide sequence of OsALS1-Pool06-60 at positions 23 to 41), OsALS1-Pool06-61(SEQ ID No.65, target nucleotide sequence of OsALS1-Pool06-61 at positions 23 to 41), OsALS1-Pool06-62(SEQ ID No.66, target nucleotide sequence of OsALS1-Pool06-62 at positions 23 to 41), OsALS1-Pool06-63(SEQ ID No.67, target nucleotide sequence of OsALS1-Pool06-63 at positions 23 to 40).

The 10 oligonucleotide sequences are mixed in equal volume and equal concentration to be used as a template, and the primers are Array-F1(SEQ ID No.14) and Array-R1(SEQ ID No.15) and I-5^TMThe 10 oligonucleotide fragments were mixed and amplified with 2 Xhigh-Fidelity Master Mix, and about 140bp of PCR products were recovered to obtain 10 mixed DNA fragments containing the target nucleotides. At the same time, to the vector pENTR4-gRNA10 was digested with BsaI, and the vector backbone (with the release of ccdB gene fragment) of about 5.3kb was recovered and used

II One Step Cloning Kit replaces the ccdB gene randomly with 1 DNA fragment of 10 fragments containing target nucleotides aiming at the OsALS1 gene, converts the ligation product into Escherichia coli to obtain an Escherichia coli bank-6, randomly selects 20 colonies for sequencing, and has the accuracy rate of 100 percent and 10 target nucleotides. The plasmid library was named pENTR4-gRNA10-POOL 6.

Wherein the upstream (i.e., 5 'end) linker sequence of the oligonucleotide sequence which is linked to the target nucleotide sequence is shown in SEQ ID No.69, and the downstream (i.e., 3' end) linker sequence of the oligonucleotide sequence which is linked to the target nucleotide sequence is shown in SEQ ID No. 70. After amplification with Array-F1(SEQ ID No.14) and Array-R1(SEQ ID No.15) as primers, the upstream (i.e., 5 '-end) sequences thereof, such as SEQ ID Nos. 71 and 69 from 5' to 3 ', which are ligated to the target nucleotide sequence, and the downstream (i.e., 3' -end) linker sequences thereof, such as SEQ ID Nos. 72 and 70 from 5 'to 3', which are ligated to the target nucleotide sequence.

Example 3: construction of plasmid libraries of pUbi-ccdB-rBE9-gRNA10-POOL1/2/3/4/5/6 and pUbi-ccdB-rBE14-gRNA10-POOL1/2/3/4/5/6 and rice transformation

1. Construction of pUbi-ccdB-rBE9-gRNA10-POOL1/2/3/4/5/6 plasmid library and pUbi-ccdB-rBE14-gRNA10-POOL1/2/3/4/5/6 plasmid library

pENTR4-gRNA10-POOL1 cloned in example 2 was digested with NheI for linearization, and then the common fragments of the U6 promoter, the target nucleotide sequence, gRNA scaffold and (T)8 terminator were cloned into pUbi-ccdB-rBE9 and pUbi-ccdB-rBE14, respectively, by means of the LR reaction of Gateway, to obtain pUbi-ccdB-rBE9-gRNA10-POOL1 and pUbi-ccdB-rBE14-gRNA10-POOL1 plasmid libraries.

Other 5 gRNA plasmid libraries cloned in example 2 were treated in the same manner to obtain pUbi-ccdB-rBE9-gRNA10-POOL2, pUbi-ccdB-rBE9-gRNA10-POOL3, pUbi-ccdB-rBE9-gRNA10-POOL4, pUbi-ccdB-rBE9-gRNA rBE 9-POOL rBE9, pUbi-ccdB-rBE9-gRNA rBE9, pUbi-gRNA rBE 9-POOL rBE9, pUbi-ccdB-rBE9-gRNA rBE 9-POOL rBE9, and pUbi-ccdB-pCCdB-rBE 9-gRNA rBE 9-POOL rBE 9-rBE 9.

2. Kitaake japonica rice Kitaake is transformed by pUbi-ccdB-rBE9-gRNA10-POOL1/2/3/4/5/6 and pUbi-ccdB-rBE14-gRNA10-POOL1/2/3/4/5/6

1) Rice callus induction:

treating the dehulled mature rice seeds with 50% commercial sterilizing solution for 25 minutes; cleaning with sterile water for 3-5 times, transferring the seeds to a sterile culture dish, and sucking out excessive water; placing the seeds on MSD plate (4.43g/L MS powder; 30g/L sucrose; 2ml/L2, 4-D; 8g/L plant gel; pH5.7), culturing in light culture room for 10 days to induce callus formation; embryos and shoots of the seeds were removed and the calli were transferred to a new MSD petri dish and cultured for 5 days until they could be used for agrobacterium transformation.

2) And (3) agrobacterium transformation:

pUbi-ccdB-rBE9-gRNA10-POOL 1/2/3/4/5/66 plasmid libraries and pUbi-ccdB-rBE14-gRNA10-POOL 1/2/3/4/5/66 plasmid libraries are respectively transferred into an agrobacterium strain EHA105 by an electric shock method to construct an EHA105 strain containing all 63 gRNA plasmid libraries. Washing Agrobacterium with sterile water to remove Agrobacterium to obtain Agrobacterium OD₆₀₀Between 1-2, Agrobacterium was harvested by centrifugation and resuspended in MSD solution to OD₆₀₀Stand up to 0.2 for use.

3) Agrobacterium infection of rice callus:

placing the callus in the agrobacterium suspension for 30 minutes; removing the agrobacterium suspension, transferring the callus onto sterile absorbent paper to remove redundant agrobacterium liquid, transferring the callus onto a new MSD culture medium containing 100 mu M acetosyringone, and culturing at room temperature in a dark place for 2-3 days.

4) Rice resistance callus screening:

transferring the dark cultured callus onto MSD culture medium (100mg/L timentin; 50mg/L hygromycin B) for 2 weeks to 2 months until the surface of the callus shows resistant callus; the medium was changed every 2 weeks.

5) Resistant callus differentiation and rooting

Transferring the resistant callus to a regeneration culture medium (4.43g/L MS powder, 30g/L sucrose, 25g/L sorbitol, 0.5mg/L NAA, 3mg/L BA, 100mg/L timentin, 50mg/L hygromycin B, 12g/L agar powder, pH5.7) once every 7-10 days until the resistant callus grows into a plant seedling; the seedlings were then transferred to 1/2MS medium (2.21g/L MS powder; 15g/L sucrose; 8g/L plant gel; pH5.7) for rooting.

Example 4: using library-2 as an example, high throughput sequencing for detecting gene evolution efficiency

To examine the efficiency of gene evolution, pool-2 was selected for high throughput sequencing.

1) Extraction of genomic DNA

Selecting 300 calli from the library-2, mixing the 300 calli together, quickly freezing by liquid nitrogen, and grinding; adding 600 μ l2 × CTAB DNA extract (containing 1/1000 β -mercaptoethanol), shaking, mixing, and splitting at 65 deg.C for 45 min. Add 500. mu.l chloroform and shake vigorously to form an emulsion, centrifuge at 14000rpm for 10 min. Transferring the supernatant into a 1.5ml centrifuge tube after centrifugation, adding isopropanol with the same volume, reversing and mixing uniformly, centrifuging at 14000rpm for 10min, discarding the supernatant, adding 700 mu l of 70% ethanol to wash the white precipitate, centrifuging at 14000rpm for 5min, discarding the supernatant, and placing the centrifuge tube in a fume hood to air-dry for 10 min. Add 100. mu.l ddH₂O dissolves the DNA. The DNA solution was stored at-20 ℃ for further use.

2) Coverage detection of target nucleotides

The primers U6P-ALS-DS-F1(SEQ ID No.73) and NOST-ALS-DS-R1(SEQ ID No.74) were designed for library-2, and after PCR amplification of DNA from 300 calli extracted in mixture, the obtained PCR product containing the target nucleotide sequence was committed to Sowthistle-Tokyo Tech Biotech Co., Ltd for high throughput sequencing and data analysis. As a result of the sequencing analysis, as shown in FIG. 1, a certain number of reads could be detected for each target nucleotide sequence, that is, the coverage of 11 target nucleotide sequences against pool-2 was 100%.

3) Detection of mutation efficiency

After PCR amplification of the POOL-2 region of OsALS1 using primers OsALS1-POOL2-F1(SEQ ID No.75) and OsALS1-POOL2-R1(SEQ ID No.76) and mixed 300 calli DNA as templates, the PCR product was submitted to high throughput sequencing and data analysis by King Zhi Biotech, Suzhou, and as a result, as shown in FIG. 2, a large number of base mutations were detected in the POOL-2 region of OsALS1, and in particular, 11 target nucleotide sequence regions contained high numbers of reads. This shows that the method can introduce a large number of mutation events in the target gene region and the mutation types are diversified, and then the directional evolution of the crop endogenous gene is further realized by combining various phenotype screening identification methods.

Example 5: screening and identification of OsALS1 rice herbicide-resistant mutant

Screening of OsALS1 rice herbicide-resistant mutant

The resistant calli of example 3 step 4) were transferred to regeneration medium (4.43g/L MS powder; 30g/L sucrose; 25g/L sorbitol; 0.5mg/L NAA; 3mg/L BA; 100mg/L timentin; 50mg/L hygromycin B; 12g/L agar powder; pH5.7) for 7-10 days, the cells were transferred to a regeneration medium containing bispyribac-sodium at a concentration of 0.25 μ M (purchased from bio-technologies ltd, bingda, beijing), and rice calli resistant to bispyribac-sodium were selected and transferred every 7-10 days until plantlets were grown.

Identification of OsALS1 rice herbicide-resistant mutant

1) PCR amplification and sequencing detection of mutation sites

Aiming at OsALS1 gene (the nucleotide sequence is shown as SEQ ID No.77, and the amino acid sequence is shown as SEQ ID No. 78.), the full-length amplification primer of the gene is designed as follows: OsALS1-F1(SEQ ID No.79) and OsALS1-P56-R1(SEQ ID No.80), T0-generation plants screened on a herbicide medium were subjected to PCR amplification using Phanta Max Super-Fidelity DNA Polymerase (purchased from Nanjing Novisan Biotech Co., Ltd.) to obtain a PCR product having a fragment size of 2.25kb, and the PCR product was directly subjected to Sanger sequencing with the wild type as a control. Sanger sequencing results showed: the plants screened from the herbicide have base mutation when being compared with wild plants. Wherein 1 mutation was found in pool-1; 12 mutations were found in pool-2; 3 mutations were found in pool-3 and 5 mutations were found in pool-5.

2) Herbicide identification experiments are carried out on the P171F, P171S, P171L, L158F and R190H mutation sites obtained by screening

Five mutation sites were randomly selected from pool-2, respectively: L158F (the cytosine mutation at the 472 th site is thymine), P171F (the cytosine mutation at the 511 th site is thymine, and the cytosine mutation at the 512 th site is thymine; or the cytosine mutation at the 511 th site is thymine, the cytosine mutation at the 512 th site is thymine, and the cytosine mutation at the 513 th site is thymine), P171S (the cytosine mutation at the 511 th site is thymine), P171L (the cytosine mutation at the 511 th site is thymine, the cytosine mutation at the 512 th site is thymine, and the cytosine mutation at the 513 th site is guanine), R190H (the guanine mutation at the 569 th site is adenine), and carrying out herbicide resistance verification experiments.

The T2 generation mature seeds of the P171F (the cytosine at the 511 th position is mutated into thymine, and the cytosine at the 512 th position is mutated into thymine), P171S, P171L, L158F and R190H mutant plants obtained by screening are subjected to a drug-loading plate experiment. Treating the hulled mature rice seeds with 50% commercial disinfectant (purchased from Beijing Rui Co., Ltd.) for 25 min, washing with sterile water for 3-5 times, transferring the seeds to a sterile petri dish, and sucking out excess water; the seeds were grown in 1/2MS (2.21g/L MS powder; 15g/L sucrose; 8g/L plant gel; pH5.7) plus 0.6. mu.M bispyribac-sodium petri dishes and after 10 days the phenotype was observed and the genotype was identified. The wild type with the same dose of bispyribac-sodium as a negative control, the wild type without bispyribac-sodium as a wild type blank control, and the mutant without bispyribac-sodium as a mutant blank control. The results are shown in FIGS. 3 to 7, where it can be seen that at the concentration of 0.6. mu.M, the seeds of the negative control became black and dead after germination, while the mutant plants of P171F, P171S, P171L, L158F and R190H all continued to grow after bispyribac-sodium treatment, indicating that the mutations of P171F, P171S, P171L, L158F and R190H were all resistant to bispyribac-sodium (0.6. mu.M).

In order to further verify the reliability of the experiment, the agricultural bispyribac-sodium is used for carrying out the herbicide spraying experiment on the rice seedlings in the 2-4 leaf stage with the P171F mutation (the cytosine at the 511 th position is mutated into thymine, and the cytosine at the 512 th position is mutated into thymine). The method comprises the steps of performing pregermination on mature rice seeds and wild rice seeds of T2 generation, potting the seeds in soil, culturing in an incubator, after the seeds grow to 2-4 leaves for about 14 days, selecting 20 plants of P171F mutant plants and wild plants with consistent growth vigor, spraying agricultural double-grass ether herbicide (purchased from Huaian bioscience, Huainan, 5% of effective components and 30-40 g/mu of recommended product dosage, wherein the dosage of the experiment is 1680 g/mu, namely 42 times of the recommended highest dosage) on 10P 171F mutant plants and 10 wild plants to obtain a P171F mutant double-grass ether treatment group and a wild type double-grass ether negative control group, taking 10 wild type plants without application of double-grass ether as wild type blank controls, and taking 10P 171F plants without application of double-grass ether as mutant blank controls. The phenotype was investigated after 21 days and the results are shown in FIG. 8. As is evident from FIG. 8, the 10 negative controls all died after growth had ceased, whereas the 10P 171F bispyribac-sodium treated mutant both grew well compared to the wild-type blank as well as the mutant blank, thus again indicating that the P171F mutation has very good bispyribac-sodium resistance.

Fig. 3 and 8 show the resistance phenotype results obtained from two different experimental designs, and it is evident that the P171F mutant bispyribac-sodium treatment group in fig. 3 has some inhibitory effect although it can grow, while in fig. 8, no significant inhibitory effect can be observed, which may be due to three reasons: 1) the two methods of treatment by using bispyribac-sodium are different, namely the root action is probably more influenced on the growth of plants than the foliage spraying; 2) the concentrations of bispyribac-sodium used in the two were different, although the concentration of bispyribac-sodium used in fig. 8 was already 42 times higher (i.e., sufficiently high) than the highest concentration of the recommended dose, whereas the concentration of bispyribac-sodium used in fig. 3 is relatively likely to be higher; 3) the state of the seeds and the state of germination are not consistent in fig. 3, while fig. 8 is the result of the treatment performed after the vigor is consistent. However, none of these factors affected the conclusion that the mutation had very good bispyribac-sodium resistance.

Sequence listing

<110> institute of plant protection of Chinese academy of agricultural sciences

<120> OsALS1 directed mutation and crop endogenous gene directed evolution method

<130> LHA1960789

<160> 80

<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<210> 3

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cccgcgcgct gtcgcttgtg tggctacgac cgccgcggcc ggttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 4

<211> 73

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cccgcgcgct gtcgcttgtg tgccttgtcc gccgccgcga gttttagagc tagaaatagc 60

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<210> 5

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cccgcgcgct gtcgcttgtg tgcgctggtg gttcttacgg cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 6

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cccgcgcgct gtcgcttgtg tgaccacgtc cttcccgctc ggttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 7

<211> 73

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cccgcgcgct gtcgcttgtg tgcccccacc cggcctcgag gttttagagc tagaaatagc 60

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<210> 8

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cccgcgcgct gtcgcttgtg tggcggcggt caggtgctcg ggttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 9

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cccgcgcgct gtcgcttgtg tgccgccgtc cccggcgccg cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 10

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cccgcgcgct gtcgcttgtg tgccgctccg gccgtggggg cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 11

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cccgcgcgct gtcgcttgtg tgcgcccttg cggggctcgg cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 12

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cccgcgcgct gtcgcttgtg tgccgctcca gcgcctccac ggttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 13

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cccgcgcgct gtcgcttgtg tgtacccggg cggcgcgtcc agttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 14

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cacattgccc agctaactcg aacgcgacca acttataaac ccgcgcgctg tcgcttgtgt 60

<210> 15

<211> 70

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt gctatttcta 60

gctctaaaac 70

<210> 16

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cccgcgcgct gtcgcttgtg tgcaggcgct gacgcgctcc cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 17

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cccgcgcgct gtcgcttgtg tgcacctctt ccgccacgag cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 18

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cccgcgcgct gtcgcttgtg tgcgaggcgt tcgcggcgtc cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 19

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cccgcgcgct gtcgcttgtg tgcgcgcgcg tccggccgcg tgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 20

<211> 73

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cccgcgcgct gtcgcttgtg tgcgtcgcca cctccggccc gttttagagc tagaaatagc 60

aagttaaaat aag 73

<210> 21

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

cccgcgcgct gtcgcttgtg tggcgagcgc ggacacgagg tgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 22

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

cccgcgcgct gtcgcttgtg tgctgctcga ctccgtcccg agttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 23

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

cccgcgcgct gtcgcttgtg tgaggtcccc cgccgcatga tgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 24

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

cccgcgcgct gtcgcttgtg tgatcggcac cgacgccttc cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 25

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

cccgcgcgct gtcgcttgtg tggagcgggt gacctcgact agttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 26

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cccgcgcgct gtcgcttgtg tgtaccttgt ccttgatgtg ggttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 27

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

cccgcgcgct gtcgcttgtg tggacatccc ccgcgtcata cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 28

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

cccgcgcgct gtcgcttgtg tgccttcttc ctcgcgtcct cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 29

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

cccgcgcgct gtcgcttgtg tggtgctggt cgacatcccc agttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 30

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

cccgcgcgct gtcgcttgtg tgcagcagca gatggctgtg cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 31

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

cccgcgcgct gtcgcttgtg tgacacctcg atgaatctac cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 32

<211> 73

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

cccgcgcgct gtcgcttgtg tgcgacagaa ttgcttgagc gttttagagc tagaaatagc 60

aagttaaaat aag 73

<210> 33

<211> 73

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

cccgcgcgct gtcgcttgtg tgcgtctggt tggcgagtca gttttagagc tagaaatagc 60

aagttaaaat aag 73

<210> 34

<211> 73

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

cccgcgcgct gtcgcttgtg tgcccgattc tctatgtcgg gttttagagc tagaaatagc 60

aagttaaaat aag 73

<210> 35

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

cccgcgcgct gtcgcttgtg tgcaaccact ctgatgggcc tgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 36

<211> 73

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

cccgcgcgct gtcgcttgtg tgttgtccct gcgcatgctt gttttagagc tagaaatagc 60

aagttaaaat aag 73

<210> 37

<211> 73

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

cccgcgcgct gtcgcttgtg tgcaaattat gcggtggata gttttagagc tagaaatagc 60

aagttaaaat aag 73

<210> 38

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

cccgcgcgct gtcgcttgtg tgctgacctg ttgcttgcat tgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 39

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

cccgcgcgct gtcgcttgtg tgcacgccaa atgcaagcaa cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 40

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

cccgcgcgct gtcgcttgtg tgatgtcaat gtgcacaatc tgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 41

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

cccgcgcgct gtcgcttgtg tgctgcgcaa attgacacat ggttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 42

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

cccgcgcgct gtcgcttgtg tgagtgcatg gcacaatgag tgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 43

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

cccgcgcgct gtcgcttgtg tgctctgggg tacaagactt tgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 44

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

cccgcgcgct gtcgcttgtg tgccaccgca atatgctatt cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 45

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

cccgcgcgct gtcgcttgtg tgagctcatc cagcacctga agttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 46

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

cccgcgcgct gtcgcttgtg tgaatcatcg ctactggtgt tgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 47

<211> 73

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

cccgcgcgct gtcgcttgtg tggacagcac cagatgtggg gttttagagc tagaaatagc 60

aagttaaaat aag 73

<210> 48

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

cccgcgcgct gtcgcttgtg tgccgaagac agccactgcc ggttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 49

<211> 73

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

cccgcgcgct gtcgcttgtg tggctgtctt cggctggtct gttttagagc tagaaatagc 60

aagttaaaat aag 73

<210> 50

<211> 73

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

cccgcgcgct gtcgcttgtg tgctgcagct ggtgcttctg gttttagagc tagaaatagc 60

aagttaaaat aag 73

<210> 51

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

cccgcgcgct gtcgcttgtg tgtcacagtt gttgatattg agttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 52

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

cccgcgcgct gtcgcttgtg tgagcttcct catgaacatt cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 53

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

cccgcgcgct gtcgcttgtg tgcaccggga ggttctcaat ggttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 54

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

cccgcgcgct gtcgcttgtg tgaacctccc ggtgaaggtg agttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 55

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

cccgcgcgct gtcgcttgtg tgcacaacca tacccaaatg tgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 56

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

cccgcgcgct gtcgcttgtg tgtgggagga taggttttac agttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 57

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

cccgcgcgct gtcgcttgtg tgaatagggc gcatacatac tgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 58

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

cccgcgcgct gtcgcttgtg tgtatctcgc tctcacattc cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 59

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 59

cccgcgcgct gtcgcttgtg tgtagcaata gtcacaaaat cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 60

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 60

cccgcgcgct gtcgcttgtg tgttcactct tctttgttac agttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 61

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 61

cccgcgcgct gtcgcttgtg tgcatcttct tgatggcggc agttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 62

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 62

cccgcgcgct gtcgcttgtg tgtatccaac aagtatggcc cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 63

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 63

cccgcgcgct gtcgcttgtg tgaccccagg gccatacttg tgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 64

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 64

cccgcgcgct gtcgcttgtg tgcagcacat gctcctggtg cgttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 65

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 65

cccgcgcgct gtcgcttgtg tgtgctgcct atgatcccaa ggttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 66

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 66

cccgcgcgct gtcgcttgtg tgtcaaggac atgatcctgg agttttagag ctagaaatag 60

caagttaaaa taag 74

<210> 67

<211> 73

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 67

cccgcgcgct gtcgcttgtg tgatcctgga tggtgatggc gttttagagc tagaaatagc 60

aagttaaaat aag 73

<210> 68

<211> 70

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 68

gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60

ggcaccgagt 70

<210> 69

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 69

cccgcgcgct gtcgcttgtg tg 22

<210> 70

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 70

gttttagagc tagaaatagc aagttaaaat aag 33

<210> 71

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 71

cacattgccc agctaactcg aacgcgacca acttataaa 39

<210> 72

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 72

gctagtccgt tatcaacttg aaaaagtggc accgagt 37

<210> 73

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 73

gaggcgggag gaacagttta g 21

<210> 74

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 74

cgccagtgtg atggatatct g 21

<210> 75

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 75

cgacgtgttc gcctacc 17

<210> 76

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 76

gccatctgct gctggat 17

<210> 77

<211> 1935

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 77

atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac ggccaagacc 60

ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt gggggcggcg 120

gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180

ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240

ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300

caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360

gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420

acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480

gtcccgatgg tcgccatcac gggccaggtc ccccgccgca tgatcggcac cgacgccttc 540

caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600

gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660

cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc tgtgccagtc 720

tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc acccgcgaca 780

gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840

ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct gaccggcatc 900

ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc gttgtccctg 960

cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa ggctgacctg 1020

ttgcttgcat ttggcgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080

agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa gaacaagcaa 1140

ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa tgctctgcta 1200

gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260

cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc accgcaatat 1320

gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac tggtgttgga 1380

cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440

tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500

aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560

gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt gaacaaccaa 1620

catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag ggcgcataca 1680

tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac tattgctaaa 1740

gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800

aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca ccaggagcat 1860

gtgctgccta tgatcccaag tgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920

aggactatgt attaa 1935

<210> 78

<211> 644

<212> PRT

<213> Rice (Oryza sativa L.)

<400> 78

Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala

1 5 10 15

Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro

20 25 30

Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser

35 40 45

Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro

50 55 60

Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala

65 70 75 80

Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala

85 90 95

Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn

100 105 110

His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr

115 120 125

Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro

130 135 140

Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser

145 150 155 160

Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly

165 170 175

Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile

180 185 190

Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val

195 200 205

Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val

210 215 220

Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val

225 230 235 240

Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys

245 250 255

Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu

260 265 270

Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly

275 280 285

Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr

290 295 300

Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu

305 310 315 320

Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp

325 330 335

Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val

340 345 350

Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile

355 360 365

Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser

370 375 380

Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu

385 390 395 400

Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn

405 410 415

Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe

420 425 430

Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu

435 440 445

Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met

450 455 460

Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser

465 470 475 480

Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly

485 490 495

Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp

500 505 510

Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu

515 520 525

Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met

530 535 540

Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr

545 550 555 560

Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val

565 570 575

Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys

580 585 590

Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro

595 600 605

Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met

610 615 620

Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly

625 630 635 640

Arg Thr Met Tyr

<210> 79

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 79

ccacacgatc ccatccgag 19

<210> 80

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 80

taccatgcca agcacatcaa ac 22

Claims (4)

1. The directional mutant protein of OsALS1 has an amino acid sequence of one of a first mutation and a second mutation of an amino acid sequence shown as SEQ ID number 78, wherein the first mutation is P171F, and the second mutation is L158F.
2. A targeted mutant nucleic acid of OsALS1, which targeted mutant nucleic acid is one of a first targeted mutant nucleic acid or a second targeted mutant nucleic acid;

   the first directional mutant nucleic acid is an amino acid which can code the mutation P171F of an amino acid sequence shown as SEQ ID number 78;

   the second directional mutant nucleic acid is a directional mutant nucleic acid which can code an amino acid of which the amino acid sequence shown as SEQ ID number 78 has L158F mutation.
3. 3. The targeted mutant nucleic acid of OsALS1, according to claim 2, wherein when the amino acid sequence is the amino acid sequence shown as SEQ ID number 78 with the P171F mutation: the first directional mutant nucleic acid is directional mutant nucleic acid which mutates cytosine at the 511 th position of a nucleotide sequence shown as SEQ ID number 77 into thymine and mutates cytosine at the 512 th position into thymine; or the first directional mutant nucleic acid is a directional mutant nucleic acid in which the 511 th cytosine of the nucleotide sequence shown as SEQ ID number 77 is mutated into thymine, the 512 th cytosine is mutated into thymine, and the 513 th cytosine is mutated into thymine.
4. 4. The targeted mutant nucleic acid of OsALS1, according to claim 2, wherein when the amino acid sequence is the amino acid sequence shown as SEQ ID number 78 with the L158F mutation: the second directional mutant nucleic acid is a directional mutant nucleic acid which can mutate the 472 th cytosine of the nucleotide sequence shown as SEQ ID number 77 into thymine.

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