CN112899303A

CN112899303A - Method for obtaining non-transgenic directional gene mutant plant by utilizing endosperm specific suicide and application thereof

Info

Publication number: CN112899303A
Application number: CN202110171762.8A
Authority: CN
Inventors: 和玉兵; 占华东
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2021-02-08
Filing date: 2021-02-08
Publication date: 2021-06-04

Abstract

The invention belongs to the field of plant genetic engineering, and relates to a method for obtaining a non-transgenic directed gene mutant plant by utilizing endosperm specificity suicide and application thereof, wherein a method for quickly and efficiently obtaining the non-transgenic directed gene mutant plant (TEKE system for short, gene editing of transgenic plant endosperm suicide) is developed by utilizing a promoter capable of driving gene endosperm specific expression to drive toxic protein, and comprises the following steps: a) constructing a construct for genetic transformation of a plant comprising a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule is a gene editing element; the second nucleic acid molecule is an endosperm lethal or developmental arresting element. b) The plants to be treated are transgenic by introducing foreign nucleic acid molecules. The system can autonomously eliminate seeds containing transgenic components in transgenic offspring, and can be used for simply, conveniently and quickly obtaining site-directed mutant plants without transgenic fragments.

Description

Method for obtaining non-transgenic directional gene mutant plant by utilizing endosperm specific suicide and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to a method for obtaining a non-transgenic directional gene mutant plant by utilizing endosperm specific suicide and application thereof.

Background

In recent years, with the development of biotechnology, genome site-directed mutagenesis techniques have been gradually established, and mainly rely on the functional analysis and application of Sequence-specific nucleases (SSNs). The method mainly comprises three SSNs: zinc Finger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), and Clustered regularly interspaced short palindromic repeats (CRISPR/Cas systems) and systems related thereto. A common feature of these SSNs is the ability to cleave specific DNA sequences, inducing DNA Double Strand Breaks (DSBs). Then the self-repair mechanism in the organism will self-initiate, and the repair of the broken DNA can be divided into non-homologous recombination connection (NHEJ) and homologous recombination repair (HDR) according to the different repair modes (Symington LS, gateway J. double-strand end repair and repair path chlorine. Annu Rev Gene, 2011,45: 247-. The NHEJ repair is mainly performed by directly linking chromosomes at the break site, but this linking repair does not guarantee very precise repair, so that deletion or insertion of nucleotides at the break site occurs to cause mutation of the gene. HDR repair occurs mainly in the presence of homologous sequences, and when repairing DSBs, organisms can use the homologous sequences as templates to complete the repair of the fracture position, and this way, because of the existence of the templates, can generate accurate repair, and if some artificial mutations are designed in the templates, the mutations can be accurately introduced into the genome of the organisms.

Among the SSN-based gene site-directed mutagenesis technologies, the CRISPR/CAS9 technology is simple in operation and low in cost, and can recognize different target sites by only changing a small segment of RNA sequence, so that the CRISPR/CAS9 technology is widely applied to efficient targeted editing of DNA in almost any transformable organism, and provides an unprecedented tool for agricultural improvement. CRISPR/Cas 9-identified editing events (Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. multiple gene engineering CRISPR/Cas systems science,2013,339(6121):819 and 823) were reported for the first time in eukaryotes, and CRISPR gene editing techniques have also been widely applied in plants. Timely clearance of transgenic fragments from edited plants is a key step in assessing genetic and phenotypic stability of CRISPR edited plants, and is critical for crop improvement. First, in the breeding field, if transgenic fragments are present in a crop variety, it is very difficult to obtain commercial planting approval from government regulatory agencies, and clearance of the transgene is a prerequisite for CRISPR edited crops to obtain regulatory approval for commercial use. Second, in the research field, the presence of gene editing elements on transgenic fragments greatly increases the risk of off-target effects, making phenotypic stability a problem. Furthermore, for genetic studies, the presence of gene editing elements on transgenic fragments makes it difficult to determine whether the detected mutation was inherited from a previous generation or newly generated by a current generation.

Sexual reproduction of a plant requires the passage of alternate generations of sporozoites and gametophytes, the sporocytes (2n) of the sporozoites (2n) undergoing meiosis to produce haploid gametophytes (n); the male and female gametophytes are combined through fertilization to generate a zygote (2n), and the zygote continues to grow and grow into sporophytes (2 n). The sporocytes of the transgenic plant undergo meiosis to produce gametocytes with the transgene construct and gametocytes without the transgene construct, and the male and female embryos are free to combine to produce zygotes following fertilization. According to Mendelian's law of free combination, when the copy number of the transgene construct is 1, 75% (theoretical) of the plants in the resulting zygote are with the transgene construct, when the copy number of the transgene construct is greater than 1, the number of the resulting zygotes with the transgene construct is greater than 75%, and the ratio continues to increase as the copy number increases. This increases the difficulty of obtaining a construct that does not contain the transgene. At present, the methods for obtaining the transgenic-free fragment from the gene editing system mainly comprise the following methods:

1) the non-transgenes are identified by using multi-generation selfing or backcrossing and traditional methods such as genetic segregation and the like, and the method needs multi-generation planting or hybridization on plants and is very labor-consuming and time-consuming. 2) The mCherry fluorescent reporter gene or color reporter gene specifically expressed in the seed is used as a marker for the presence of the transgenic fragment. This approach is currently used mainly in Arabidopsis, although fluorescent marker assisted selection of non-transgenic plants reduces the screening to identify non-transgenic mutant plants by around 75%, this Strategy is still relatively time and labor intensive (Gao X, Chen J, Dai X, Zhang D, Zhao Y. an Effective Strategy for regeneration of isolated mutant and Cas9-Free Arabidopsis produced by CRISPR/Cas9-Mediated Genome edition. Plant physiology, 2016,171(3): 1794. minus 1800; He Y, Zhu M, Wu J, Ouyang L, Wang R, Sun H, Yan L, Wang L, Xu M, Zhan H, Zhao Y. reproduction of transgenic synthesis for Plant 19. edition (front 2. edition) and 19)). Furthermore, this method did not enrich or increase the proportion of transgenic-free plants in the T2 generation. 3) The chemical screening marker gene is used as the mark of the existence of the transgenic segment, and the non-transgenic gene editing plant is obtained through the screening of special chemical substances. Such as coupling the CRISPR construct to an RNA interference element targeting a herbicide resistant P450 enzyme. Plants which do not contain transgenic fragments can be screened for by specific herbicides (Lu HP, Liu SM, Xu SL, Chen WY, Zhou X, Tan YY, Huang JZ, Shu QY. CRISPR-S: an active interference element for a Rapid and dependent selection of gene-estimated, transgenic-free plants. plant Biotechnol J,2017,15(11): 1371-1373). However, this strategy still does not enrich the non-transgenic plants, and needs to plant and screen the progeny, which increases the labor input. 4) Cas9 protein and gRNA were introduced directly into plant cells using transient transformation methods, avoiding DNA integration. Such as using gene guns, PEG, Agrobacterium, nanomaterial-mediated transient transformation (He Y, ZHao Y. technical break through hs in generating transformation-free and genetic stable CRISPR-edited plants. aBIOTECH,2020,1(1): 88-96). Since transient transformation does not allow efficient screening, many non-mutated plants will be adulterated to differentiated seedlings. Liang's study showed that only about 4% of plants underwent single gene editing (Liang Z, Chen K, Li T, Zhang Y, Wang Y, ZHao Q, Liu J, Zhang H, Liu C, Ran Y, Gao C. effective DNA-free genome editing of branched while using CRISPR/Cas9 ribosomal proteins complexes. Nat Commun,2017,8: 14261). Although progeny can be identified by PCR to screen for mutant plants, it is also a labor-intensive task, and especially for systematic study of multiple genes, the time and labor cost for obtaining available materials will become rate-limiting factors in research and development. 5) TKC transgenic suicide technology was used. The method uses the promoter of rice early embryonic development gene REG2 (Sun JL, Nakagawa H, Karita S, Ohmiya K, Hattori T.Rice embryo globulins: amino-terminal amino acid sequences, cDNA cloning and expression. plant Cell physiology, 1996,37(5): 612. sup. 620) to drive Bacillus subtilis ribonuclease BARNASE (HartleRW. BARNASE and expression of a cloned plasmid. J. Mol Biol,1988,202(4): 913. sup. 915), while the cytoplasmic sterility gene CMS2 of rice is driven by the 35S promoter. BARNASE can kill cells by degrading RNA in the cells (Lannenpaa M, Hassines M, Ranki A, Holta-Vuori M, Lemmetyinen J, Keinonen K, Sopanen T.Presence of flower definition in biological and other plants using a BpFULL1:: BARNASE restriction. plant Cell, 2005,24(2):69-78), while CMS2 can lead to pollen abortion by hindering the function of mitochondria during pollen development (Wang Z, Zou Y, Li X, Zhang Q, Chen L, Wu H, Su D, Chen Y, Guo J, Luo D, Long Y, Zhong Y, Liu YG. The two expression cassettes are integrated with a normal CRISPR/Cas9 gene editing element into a linked vector. The technology can kill pollen and embryo carrying transgenic segment specifically, so that transgenic T0 plant can grow only non-transgenic seed including descendant with edited DNA. However, this method has certain limitations and is not suitable for some plants transformed with young embryos.

Disclosure of Invention

The technical scheme combines a gene Editing element and an element for specifically killing Endosperm into a linked system, and the linked system is named as a TEKE (transgenic Endosperm Killer edition) system. The gene editing element in the TEKE system can perform the function of gene editing, and the element for specifically killing endosperm can destroy the endosperm carrying the transgenic construct, thereby achieving the effect that the transgenic seed can not grow normally and die. Thus, the transgenic plant can eliminate the self transgenic offspring and screen out the site-directed mutant offspring without the transgenic construct. The system can independently destroy endosperm carrying the transgenic construct in the seed growth stage after gene editing occurs, so that the effect that the transgenic seed can not grow normally and die is achieved, and a simple, effective, time-saving and labor-saving method is provided for cultivating non-transgenic plants through gene editing. The TEKE system artificially links two key elements to one T-DNA. 1) A CRISPR/Cas9 gene editing element comprising a Cas9 protein expression cassette and a gRNA transcription cassette. 2) The endosperm-specific lethal element uses an endosperm-specific promoter OsEP kinesin BARNAE, the REG2 promoter plays a role in the development process of the endosperm of the seeds very specifically, when the BARNAE gene is placed under the control of the rice OsEP promoter, the BARNAE toxoprotein is not generated in the callus or the vegetative growth period and is only generated in the development process of the endosperm of the seeds, so that the BARNAE driven by the OsEP promoter can ensure that the endosperm of the seeds containing the transgenic element is damaged, and the development of the rice seeds is defective. OsEP-BARNASE was introduced into a plasmid pCUCas9 carrying Cas9 gene, thereby completing the vector construction of the TEKE system.

Specifically, the invention is realized by the following technical scheme:

the applicant provides a method for obtaining non-transgenic targeted gene mutant plants by utilizing endosperm-specific suicide, which is used for creating targeted gene mutant non-transgenic plants (TEKE system for short) and comprises the following steps:

a) constructing a construct for genetic transformation of a plant, said construct comprising a first nucleic acid molecule and a second nucleic acid molecule, said first nucleic acid molecule being a gene editing element; the second nucleic acid molecule is an endosperm lethal or developmental arresting element;

b) the plant to be treated is transgenic by introducing the construct with an exogenous nucleic acid molecule.

Further, the plants applicable to the above steps a) and b) are seed plants; plants which can obtain transgenic materials by agrobacterium-mediated infection, such as gramineae, leguminosae, cruciferae, compositae and solanaceae; preferably rice, maize, sorghum, barley, oats, wheat, millet, brachypodium distachyon, teosintes, sugarcane, soybean, oilseed rape, Arabidopsis, safflower, tomato, tobacco, alfalfa, potato, sweet potato, sunflower and cotton.

Further, the first nucleic acid molecule is a genetic element capable of editing nucleic acids.

Further, the gene element of the editing nucleic acid is selected from the group consisting of gene elements of any gene editing system.

Further, the gene editing system preferably: a ZFN gene editing system, a TALEN gene editing system, a CRISPR/CAS9 gene editing system, or a CRISPR/CPF1 gene editing system.

Further, the gene elements of the CRISPR/CAS9 gene editing system comprise a CAS9 gene (the nucleotide sequence of the CAS9 gene is shown as the nucleotide sequence shown in SEQ ID NO: 1) and a gRNA gene (the nucleotide sequence of the core skeleton of the gRNA gene is shown as SEQ ID NO: 2).

Further, the second nucleic acid molecule is a genetic element where the endosperm is lethal or developmentally arrested.

Further, the nucleotide sequence of the second nucleic acid molecule is shown as SEQ ID NO: 3.

The specific implementation steps can be seen in the examples in detail.

The invention also provides application of the method in plant breeding.

Further, the plant is a plant belonging to the phylum Seedunculata.

Sequence listing SEQ ID NO: 1 is the nucleotide sequence of CAS9 gene, and the sequence length is 4131 bp.

Sequence listing SEQ ID NO: 2 is the core skeleton nucleotide sequence of gRNA gene, and the sequence length is 76 bp.

Sequence listing SEQ ID NO: 3 is the nucleotide sequence of the second nucleic acid molecule, and the sequence length is 4522 bp.

Sequence listing SEQ ID NO: 4 is the nucleotide sequence of the pea rbcS-E9 gene terminator, and the sequence length is 635 bp.

Sequence listing SEQ ID NO: 5 is the nucleotide sequence of BARNASE gene, the sequence length is 854 bp.

Sequence listing SEQ ID NO: 6 is the nucleotide sequence of OsEP promoter, and the sequence length is 3018 bp.

Sequence listing SEQ ID NO: 7 is OsU6 nucleotide sequence of DNA 6P, and the sequence length is 457 bp.

The invention has the beneficial effects that:

the method of the invention can quickly obtain the site-directed mutant rice without transgenic fragments, and can also be used for quickly and efficiently constructing non-transgenic plants with multiple gene mutations. The method is beneficial to accelerating the functional research of the rice gene, is convenient for carrying out complex multi-gene interaction research, and is also beneficial to accelerating the breeding progress of good genes in the rice transformation and polymerization.

The invention can ensure that the development of transgenic seeds born by the transgenic plants has defects on the basis of ensuring the high-efficiency gene editing of the current generation of the transgenes, and the transgenic plants can autonomously eliminate the filial generation containing the transgenic constructs. The present invention greatly reduces the time and labor required for isolating a gene-editing plant free of a transgenic fragment, greatly accelerates the progress of obtaining a transgene-free mutant, and can prevent the drift of a transgene caused by the drift of seeds, providing a very useful tool for crop improvement.

Drawings

FIG. 1 is a diagram of TEKE plasmid constructed in the present invention.

Fig. 2 is a schematic diagram of suicide transgene-mediated self-elimination of the CRISPR/Cas9 construct after editing of the target gene.

Reference numerals illustrate a) schematic representation of two key components of the TEKE (transgenic killer CRISPR) plasmid. NOS refers to the terminator of nopaline synthase gene derived from Agrobacterium tumefaciens. OsEP is an endosperm-specific promoter used to drive the BARNASE gene, which encodes a toxic enzyme, from plant cells. The rbcS-E9 terminator was originally cloned from the pea rbcS-E9 gene. Codon optimized Cas9 was placed under the control of the ubiquitin promoter UBQ in maize. b) TEKE-mediated isolation of rice plants without transgene and CRISPR/Cas9 editing.

FIG. 3 is a test of transgenic fragments of T1 plants generated by TEKE-Gn1 a.

FIG. 4 is a schematic diagram of the segregation pattern of mutant and non-transgenic plants in T1 plants produced by TEKE-Gn1 a.

Description of reference numerals: a) the PAM site "CCT" required for Cas9 cleavage was marked in light color. WT refers to the middle flower 11 (ZH 11 for short) of a wild-type plant rice variety. "-" means that one base pair is deleted. "a" and "T" in the light and superscripts indicate the insertions "A" and "T", respectively. The upper light color "A" indicates that base substitution occurred.

Detailed Description

Now, taking the gene element of the CRISPR/CAS9 gene editing system as an example of the first nucleic acid molecule, the feasibility verification gene of the technical scheme of the invention is exemplified by the rice Gn1a gene (GeneBank number XM _ 015773930). Transgenic methods take the example of Agrobacterium-mediated stable transformation of rice (genetic transformation of rice is now a common practice in the field of rice transgenesis, where the detailed transformation procedures and the various medium formulations used are described in Hiei Y, Ohta S, Komari T, Kumashiro T. efficient transformation of rice (Oryza sativa L.) formulated by Agrobacterium and sequence analysis of the bases of the T-DNA plant J,1994,6(2): 271-282).

Example 1 construction of vector pCUCas9 with Cas protein expression cassette

In this example, a Cas protein expression cassette, i.e., Cas9 gene encoding Cas protein, was ligated into pCXUN vector to obtain vector pCUCas 9. The method comprises the following specific construction steps:

(1) cas9 gene DNA was obtained by PCR amplification. Cas9 gene derived from rice codon optimization, the sequence of which is shown as SEQ ID NO.1, is taken as template DNA. And (3) performing primer pair:

UCas9-F：

5’-CCCGGGGGATCCCCAATACTATGGCCCCAAAGAAGAAGCGCAAGG-3’

UCas9-R：

5'-GAAATTCGGATCCCCAATACTTCAATCGCCGCCGAGTTGTGAGAGG-3' PCR amplification was performed.

And (3) PCR reaction system:

2×PCR Buffer	10μl
		2.5mM dNTP	2μl
10μM UCas9-F	0.6μl
		10μM UCas9-R	0.6μl
template DNA	0.5μl
		KOD-FX polymerase	0.2μl
Supplementing double distilled water and adding water to supplement	20μl

PCR amplification procedure:

the PCR product was: cas9 gene DNA, the size is 4172bp, which includes Cas9 gene sequence of SEQ ID NO.1, and as the primer used in PCR is added with an additional linker sequence for Gibson connection, the PCR product is slightly longer than the sequence length of Cas9 gene shown in SEQ ID NO. 1.

(2) Constructing a pCUCas9 vector: the pCXUN vector plasmid was digested with Xcm I, and the PCR product of Cas9 gene DNA obtained in step (1) of this example was ligated to the pCXUN vector (GenBank accession No. FJ905215) at Xcm I by Gibson ligation, to obtain pCUCas9 vector.

Example 2 construction of TEKE vectors

pCUCas 9A TEKE system of the invention was validated by adding a second nucleic acid molecule to the pCUCas9 vector. Wherein the second nucleic acid molecule is exemplified by an OsEP-BARNASE gene expression cassette (the nucleotide sequence of which is shown in SEQ ID NO: 3).

The specific construction steps are as follows:

1) the terminator of the amplified pea rbcS-E9 gene has a sequence shown in SEQ ID NO.4, is taken as a template, and is added with a primer pair: full-rE9T-F

(5'-CTGCAGGAATTCGATATCATTTAAATAGAGCTTTCGTTCGTATCATCGGTT-3') and full-rE9T-R

(5'-GTAAAACGACGGCCAGTGCCAAGCTTGTTGTCAATCAATTGGCAAGTCAT-3') the terminator DNA of rbcS-E9 gene was amplified by primer, and then it was recovered by cutting gel and ligated into HindIII-cut pCUCas9 vector to obtain pCXR 9T.

2) Amplifying BARNASE gene DNA with sequence shown in SEQ ID NO.5 as template, and primer pair: BAR-F

(5'-CTGCAGGAATTCGATATCATTTAAATATGGCACAGGTTATCAACACG-3') and BAR-R

(5'-ACCGATGATACGAACGAAAGCTCTTTTAATTTTAAGAAAGTATGATGGTGATGTCGCAG-3') is a primer, and BARNASE DNA is amplified; the DNA is cut into gel and recovered, and then is connected with a pCXR9T carrier cut by Swa I enzyme to obtain a BARNASE-pCXR9T carrier,

3) amplifying OsEP promoter DNA with the sequence shown in SEQ ID NO.6 as a template, and performing amplification by using a primer pair: OsEPF

(5'-CACTGCAGGAATTCGATATCATTTGTGGAAGTGTTACTACATGGCAGGTACAC-3') and OsEPR

(5'-GTTGATAACCTGTGCCATATTTAAATGCTCTCTCAAGTCTCAATGACCTGTACT-3') OsEP promoter DNA is amplified for primer, and after gel cutting and recovery, the OsEP promoter DNA is connected into the Swa I cut BARNASE-pCXR9T vector to obtain the OsEP-BARNASE-pCXR9T vector which is TEKE plasmid (figure 1).

Example 3 gRNA transcription cassettes capable of targeting target DNA were constructed onto TEKE vectors.

To examine the effectiveness of the present invention, applicants designed a specific gRNA with the Gn1a gene as the target gene, with the target sequence 5'-GGGCTCGGTCCACCTGAACCAGG-3'. The final vector TEKE obtained in example 2 (see FIG. 1) was digested with Pme I into linear DNA, and DNA capable of transcribing gRNA was introduced by overlap PCR (conventional method) amplification. In this example, OsU6 promoter was used as the promoter of the gRNA transcription unit, and the specific steps were as follows:

the carrier gRNA with OsU6P DNA (shown in SEQ ID NO. 7) and gRNA skeleton DNA (shown in SEQ ID NO. 2) constructed in the laboratory is taken as template DNA, and OsU6PF is respectively taken as

(5'-GTCGTTTCCCGCCTTCAGTTTATGTACAGCATTACGTAGG-3') and Gn1a-U6R

(5'-GCAGGTACTCCGGGCGCGACAACCTGAGCCTCAGCGCAGC-3') primer set, and OsU6TR

(5'-CTGTCAAACACTGATAGTTTAAACGATGGTGCTTACTGTTTAG-3') and Gn1a-U6F

(5'-GTCGCGCCCGGAGTACCTGCGTTTTAGAGCTAGAAATAGCAAGTTA-3') amplifying two DNAs by taking the primer pair as a template, mixing the two DNAs after cutting and recovering the gel as the template, amplifying complete gRNA transcription unit DNA by taking OsU6PF and OsU6TR as primers, then recovering the DNA cutting gel, and connecting the recovered DNA cutting gel into a TEKE vector which is cut by Pme I enzyme to obtain TEKE-Gn1 a.

Example 4 transformation of Rice callus with TEKE-Gn1a vector

The positive plasmid TEKE-Gn1a after sequencing was electrically transformed into Agrobacterium (EHA105) and infected into rice calli. The transformed variety is "Zhonghua 11" of rice (also known as ZH11, from the institute of crop science, college of agricultural sciences of China), (genetic transformation of rice has been a common practice in the field of rice transgenesis, wherein the detailed transformation procedures and the various medium formulations used are described in Hiei Y, Ohta S, Komari T, Kumashiro T.efficient transformation of rice (Oryza sativa L.) and the detailed culture of microorganism and sequence analysis of the boundary of the rice plant of the T-DNA plant J,1994,6(2) 271 and 282).

As shown in fig. 2, TEKE plasmid was transformed into rice calli by agrobacterium-mediated transformation. During callus growth and vegetative growth, the BARNASE gene is not expressed and the target gene may be edited by Cas 9. However, when generating progeny, seeds containing Cas9 fail to germinate because the endosperm is killed by BARNASE. Thus, only non-transgenic seeds from the T0 plant develop normally.

Example 5 Positive detection of TEKE-Gn1 a-transformed Rice T0 Material

DNA was extracted separately from different calli using the conventional CTAB method (He Y, Yan L, Ge C, Yao XF, Han X, Wang R, Xiong L, Jiang L, Liu CM, Zhao Y. PINOID Is Required for the Formation of the plasmid and plasmid in Rice plant Physiol,2019,180(2): 926-.

By using primer pairs: quality detection of rice genome DNA F: 5'-CTCAACCCCAAGGCTAACAG-3' (SEQ ID NO.28) + quality test of rice genomic DNA R: 5'-ACCTCAGGGCATCGGAAC-3' (SEQ ID NO.29), the size of the PCR product is 526bp, and the PCR product is used for detecting the DNA quality of the sample.

Using a C9-F: 5'-CCCTGCCTTCATACGCTATTT-3' (SEQ ID NO.30) + C9-R: 5'-GACTTGAAGTTCGGGGTGAG-3' (SEQ ID NO.31) is used as a primer pair to carry out PCR amplification to detect whether the gene C9 is transformed into rice or not, and the size of a PCR product is 887 bp.

And (3) PCR reaction system:

10×PCR Buffer	2μl
		2.5mM dNTP	2μl
10 mu M F primer	0.3μl
		10 mu M R primer	0.3μl
10 mu M C-F primer	0.3μl
		10 mu M C-R primer	0.3μl
Rice genome DNA	2μl
		rTaq polymerase	0.15μl
Supplementing double distilled water and adding water to supplement	20μl

PCR amplification procedure:

the lower band in the electrophoresis detection gel chart of two transformation events shows the result of PCR amplification by using a primer capable of amplifying a section of specific DNA fragment on the rice genome through electrophoresis detection, and the amplification result reflects that the extracted sample DNA plasmids are good. The top band shows the result of PCR amplification using specific primers that amplify gene C9.

The transgenic positive statistics are shown in table 1.

TABLE 1 Positive test results for T0 transgene

Example 6 Positive detection of TEKE-Gn1 a-transformed Rice T1 Material

Seeds were harvested from each individual T0 plant and progeny from 4 independent positive T0 plants (T1 generation) were analyzed.

The method comprises the following specific steps:

And (3) PCR reaction system:

PCR amplification procedure:

In addition, positive detection was repeated using primers that amplify another position on the T-DNA.

BAR-377F：5’-AATTCAGACCGGATTCTTTACTCA-3’，BAR-377R：5’-GTCGCTGATACTTCTGATTTGTTC-3’；

The amplification product size is 377 bp.

And (3) PCR reaction system:

10×PCR Buffer	2μl
		2.5mM dNTP	2μl
10μM CC-F	0.3μl
		10μM CC-R	0.3μl
rice genome DNA	2μl
		rTaq polymerase	0.1μl
Supplementing double distilled water and adding water to supplement	20μl

PCR amplification procedure:

the positive transgenic plants were identified by PCR amplification of genomic DNA of T1 generation plants transformed with the ordinary CRISPR vector pCUCas9 using primers Hyg-280F (5'-ACGGTGTCGTCCATCACAGTTTGCC-3') and Hyg-280R (5'-TTCCGGAAGTGCTTGACATTGGGGA-3').

Randomly selecting 4 positive plants (the numbers are 402-8, 402-10, 402-12 and 402-13 respectively) of T0 generations, harvesting seeds, germinating to obtain seedlings of T1 generations, and detecting transgenic fragments. The results showed that 51 seedlings of the 4T 1 lines were transgenic negative plants except 4 of the 14 plants of 402-8 contained the transgenic fragment. The test results of T1 generation plants of TEKE are shown in FIG. 3. The transgenic positive statistics are shown in table 2.

TABLE 2 positive test results for T1 transgene

From table 2, when using the conventional CRISPR/Cas9 construct, at least 75% of the T1 generation transgenic plants had the CRISPR/Cas9 construct; when using the TEKE plasmid approach, all T1 transgenic plants (55 total) from the 3 independent T0 transgenic plants did not contain the CRISPR construct, and one numbered T1 plant contained 28.6% of the transgenic plants and was also much less than 75% of the conventional method. The technical scheme of the TEKE plasmid is very effective in eliminating transgenes.

Example 7 Targeted mutation detection of TEKE-Gn1a transformed Rice T1 Material

Gn1a-GT1 (5'-AGCTGCCTTCCATCGTCAGCACACA-3') and Gn1a-GT2 were used

(5'-GCGTGCGTAAGCGTAAGCCCCAGG-3') As genotype detection primers, PCR amplification was performed on all plants in 4 independent T1 generation families. The PCR products were then sequenced and the sequencing results were analyzed by the Dsdecode website (http:// Dsdecode. gene. com /) to identify mutant forms of individual T1 generation plants.

After sequencing 55T 1 plants of 4 independent transgenic progeny of T0 generations: all transgenic plants contained mutations at the target site with a mutation efficiency of 100%, and the specific mutation forms and mutation efficiencies are shown in FIG. 4. The TEKE system can efficiently and independently eliminate transgenic T1 generation plant transgenic constructs and ensure that the offspring can generate efficient site-directed mutagenesis. The invention can greatly reduce the time and labor required for separating the rice without transgenic site-specific DNA editing, is a meaningful upgrade of the first version TKE technology developed earlier, and provides a potentially available gene editing tool for efficiently removing transgenic elements for some crops depending on immature embryo transformation.

Sequence listing

<110> Nanjing university of agriculture

<120> a method for obtaining non-transgenic directed gene mutant plants by utilizing endosperm specific suicide and application thereof

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atggccccaa agaagaagcg caaggtcgac aagaagtact ccatcggcct cgacatcggc 60

accaattctg ttggctgggc cgtgatcacc gacgagtaca aggtgccgtc caagaagttc 120

aaggtcctcg gcaacaccga ccgccactcc atcaagaaga atctcatcgg cgccctgctg 180

ttcgactctg gcgagacagc cgaggctaca aggctcaaga ggaccgctag acgcaggtac 240

accaggcgca agaaccgcat ctgctacctc caagagatct tctccaacga gatggccaag 300

gtggacgaca gcttcttcca caggctcgag gagagcttcc tcgtcgagga ggacaagaag 360

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aagtccaact tcgacctcgc cgaggacgcc aagctccagc tttccaagga cacctacgac 840

gacgacctcg acaatctcct cgcccagatt ggcgatcagt acgccgatct gttcctcgcc 900

gccaagaatc tctccgacgc catcctcctc agcgacatcc tcagggtgaa caccgagatc 960

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acactcctca aggccctcgt gagacagcag ctcccagaga agtacaagga gatcttcttc 1080

gaccagtcca agaacggcta cgccggctac atcgatggcg gcgcttctca agaggagttc 1140

tacaagttca tcaagccgat cctcgagaag atggacggca ccgaggagct gctcgtgaag 1200

ctcaatagag aggacctcct ccgcaagcag cgcaccttcg ataatggctc catcccgcac 1260

cagatccacc tcggcgagct tcatgctatc ctccgcaggc aagaggactt ctacccgttc 1320

ctcaaggaca accgcgagaa gattgagaag atcctcacct tccgcatccc gtactacgtg 1380

ggcccgctcg ccaggggcaa ctccaggttc gcctggatga ccagaaagtc cgaggagaca 1440

atcaccccct ggaacttcga ggaggtggtg gataagggcg cctctgccca gtctttcatc 1500

gagcgcatga ccaacttcga caagaacctc ccgaacgaga aggtgctccc gaagcactca 1560

ctcctctacg agtacttcac cgtgtacaac gagctgacca aggtgaagta cgtgaccgag 1620

gggatgagga agccagcttt ccttagcggc gagcaaaaga aggccatcgt cgacctgctg 1680

ttcaagacca accgcaaggt gaccgtgaag cagctcaagg aggactactt caagaaaatc 1740

gagtgcttcg actccgtcga gatctccggc gtcgaggata ggttcaatgc ctccctcggg 1800

acctaccacg acctcctcaa gattatcaag gacaaggact tcctcgacaa cgaggagaac 1860

gaggacatcc tcgaggacat cgtgctcacc ctcaccctct tcgaggaccg cgagatgatc 1920

gaggagcgcc tcaagacata cgcccacctc ttcgacgaca aggtgatgaa gcagctgaag 1980

cgcaggcgct ataccggctg gggcaggctc tctaggaagc tcatcaacgg catccgcgac 2040

aagcagtccg gcaagacgat cctcgacttc ctcaagtccg acggcttcgc caaccgcaac 2100

ttcatgcagc tcatccacga cgactccctc accttcaagg aggacatcca aaaggcccag 2160

gtgtccggcc aaggcgattc cctccatgag catatcgcca atctcgccgg ctccccggct 2220

atcaagaagg gcattctcca gaccgtgaag gtggtggacg agctggtgaa ggtgatgggc 2280

aggcacaagc cagagaacat cgtgatcgag atggcccgcg agaaccagac cacacagaag 2340

ggccaaaaga actcccgcga gcgcatgaag aggatcgagg agggcattaa ggagctgggc 2400

tcccagatcc tcaaggagca cccagtcgag aacacccagc tccagaacga gaagctctac 2460

ctctactacc tccagaacgg ccgcgacatg tacgtggacc aagagctgga catcaaccgc 2520

ctctccgact acgacgtgga ccatattgtg ccgcagtcct tcctgaagga cgactccatc 2580

gacaacaagg tgctcacccg ctccgacaag aacaggggca agtccgataa cgtgccgtcc 2640

gaagaggtcg tcaagaagat gaagaactac tggcgccagc tcctcaacgc caagctcatc 2700

acccagagga agttcgacaa cctcaccaag gccgagagag gcggcctttc cgagcttgat 2760

aaggccggct tcatcaagcg ccagctcgtc gagacacgcc agatcacaaa gcacgtggcc 2820

cagatcctcg actcccgcat gaacaccaag tacgacgaga acgacaagct catccgcgag 2880

gtgaaggtca tcaccctcaa gtccaagctc gtgtccgact tccgcaagga cttccagttc 2940

tacaaggtgc gcgagatcaa caactaccac cacgcccacg acgcctacct caatgccgtg 3000

gtgggcacag ccctcatcaa gaagtaccca aagctcgagt ccgagttcgt gtacggcgac 3060

tacaaggtgt acgacgtgcg caagatgatc gccaagtccg agcaagagat cggcaaggcg 3120

accgccaagt acttcttcta ctccaacatc atgaatttct tcaagaccga gatcacgctc 3180

gccaacggcg agattaggaa gaggccgctc atcgagacaa acggcgagac aggcgagatc 3240

gtgtgggaca agggcaggga tttcgccaca gtgcgcaagg tgctctccat gccgcaagtg 3300

aacatcgtga agaagaccga ggttcagacc ggcggcttct ccaaggagtc catcctccca 3360

aagcgcaact ccgacaagct gatcgcccgc aagaaggact gggacccgaa gaagtatggc 3420

ggcttcgatt ctccgaccgt ggcctactct gtgctcgtgg ttgccaaggt cgagaagggc 3480

aagagcaaga agctcaagtc cgtcaaggag ctgctgggca tcacgatcat ggagcgcagc 3540

agcttcgaga agaacccaat cgacttcctc gaggccaagg gctacaagga ggtgaagaag 3600

gacctcatca tcaagctccc gaagtacagc ctcttcgagc ttgagaacgg ccgcaagaga 3660

atgctcgcct ctgctggcga gcttcagaag ggcaacgagc ttgctctccc gtccaagtac 3720

gtgaacttcc tctacctcgc ctcccactac gagaagctca agggctcccc agaggacaac 3780

gagcaaaagc agctgttcgt cgagcagcac aagcactacc tcgacgagat catcgagcag 3840

atctccgagt tctccaagcg cgtgatcctc gccgatgcca acctcgataa ggtgctcagc 3900

gcctacaaca agcaccgcga taagccaatt cgcgagcagg ccgagaacat catccacctc 3960

ttcaccctca ccaacctcgg cgctccagcc gccttcaagt acttcgacac caccatcgac 4020

cgcaagcgct acacctctac caaggaggtt ctcgacgcca ccctcatcca ccagtctatc 4080

acaggcctct acgagacacg catcgacctc tcacaactcg gcggcgattg a 4131

<210> 2

<211> 76

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60

ggcaccgagt cggtgc 76

<210> 3

<211> 4522

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gtggaagtgt tactacatgg caggtacacg ttaccacatg tttatacagt aacactattc 60

ataaattgca gtaagacgca ttgttactac aaaacttatg gtttttacta cacacttaca 120

gtaacgtcgc gatgggattg tagtaacgca taacagatag tactaacaag tagtaatgct 180

atagtattgc tacttgtgga gagttaagat caaaaaaccc aagcttcgaa taacaatatc 240

tctcatgtga tgtatgattt tttaaaaccg tttgcaccat gatgttagaa aaaaatgctt 300

atcaaaaata gatccgtcat gactatattt caactaaatt tgaatgttgt tactgcacgt 360

aagacacagt gttactgcac gatggctgtc gctttactgc actgtttcgc aagacgagag 420

ctgagaagag gtgggtggtg ggggagttgg taggcagttt gaccgggttt tgaccgacgt 480

cgggggtgct ttgactgacc tttgactgag gtgggaggtg ggtttggccc ttgggaggga 540

gggggggata tagaatagct attctatacc cgggtgtaga atagtttgtg tatgtgtgtg 600

tgtgtgtata tatatatata tataaaatca aagtgtggga tattatattt catgtacaat 660

ctcataaaga gtctactaaa tatacttctc tctgaatata gtttgacaaa ttagttttgt 720

gtgttgctaa taactaatat aattttttac ttttagctcg attattactt aggaatttcc 780

gcagcaaagt gcggggaatc acctagttaa ttaaagaatt gagtcctcat aaaatacctt 840

ttcaatatgg cctgaatcat tgtttggttt tttggccttc tgttctattt ggtccaaaag 900

gccatctttc taagttggac tgggcccatg gtttcgttat gggccaaaaa caacaatagc 960

attttgtttt attctcccct tttgaaaaca gtatgcaggg gtgatgcttg ttcattataa 1020

gaggcaaagg gtagcatatg atatataagt ttataattta ttatagatga caacctcaaa 1080

ggtaactatc gttgacatat gtaaattatg ctacattaca aaagaaataa tgtgaacatt 1140

gacattggta catacgtgga cattatctat aattatagac tgacatctac attccctatg 1200

gttatcaatc ggaatgtcgg ctcttgtagc cttgtgaaca tacgtactct ctcgcactat 1260

atgtgcattg atgtgaatcc tccaatttgc tataggaatg tcgtaggttt gagtagccta 1320

tgtattcatt gtcagcaaat gcgcaccgtg aactctttac gtcagaggat ctacgtcagt 1380

ccgctagtac gctcatgaca tattccccct gtttttgaag atgcggttag ttactccttc 1440

tgtacactcc ataaaatgtg ctatcctaca atacattccc cgcttcaatg tatgagatat 1500

ttttccccat tttgttagaa aatacttaag caatatgttc caaatataat gttatattgg 1560

gttgatgttg tgttaaaatg gagtggttaa gcatgagcct tcatttccct cataactgtt 1620

tttttagaac tcctcgcaac tgttgcactc aacaatttca ttttccttcc tgttattcaa 1680

ttaaggttct ccttaaaaat aagtctttca tgaataattt agtattttga aggtactatt 1740

gaaagaatta tttggagatg tctaatgctt gaatgataag taatttggaa ggcatctact 1800

cctacaaagt tgaatcttct aaatactcct tcgcttttaa atatacggca ttgttaactt 1860

ttgaatatga caatatttaa caatttatct tgtacaaaga tttagtgcaa atatgcacaa 1920

tgcgtaaagt acctttaatg taaaacaagc cacaacaaaa caaatgatac ttagagcatt 1980

ttttaataag ccgattggtc gcatcatgtt taaaagtcaa ccatgtcata tataaaaaaa 2040

gggagatagt ttcatacata gctctaatat attttgaagg tactaaattt taaaaattgt 2100

tgttgcagat tgtaagtctt gcatataatt tattgtttct tttacttagc ttattagctc 2160

taattaatca caccctatct ctcctatatt tatataataa aaacatcccg acacaccaac 2220

gtacattttc tcatctattt ctaaggtatg gatggtattg tcatgtcaac ttatcttctt 2280

tctagttatg caccctcctt aattattgta ccaacttaaa ataagccata tattttgaaa 2340

ttatcatacc aactcacaat atgccatata ttttaaaatt attatgtcaa ctcacaatat 2400

gctatatatt ttgatattat cgtgccaact cacaatatac catatatttt gaaatgaagg 2460

tcatatttaa gatgtagagt aaatgtatga aatcctccca tccatatcct cattattaat 2520

tgcttccacc taccccaagc atttatagtt agagtagaac agttgtgctc ttgcaccgaa 2580

gggatgacat gtaggagcca tgcctacgtc ccctggccac ccaagcccgt tgaattaaca 2640

tggtccctca acagaaacat ccatttttga tggtgtgctt ccttcttggc gaccttactg 2700

gtaggtgaca tgctaacaaa aactgcacag tcatgcaagg caggggccgg tcaaaattga 2760

agtccctcct accttgattg taacgctaat actatggctc accgcctctg cggcaatctg 2820

gcagccactt ctcctcctct tggtgccaca gctgcacctt tgacgtggct ggctgcttgc 2880

atttgtccaa gccggcgcag ccctgatgta agtcctttgg tataaatact gcccaagcat 2940

agagcgagag agagccatca caatccaata caagttcaca acataagcat agtacaggtc 3000

attgagactt gagagagcat ttaaatatgg cacaggttat caacacgttt gacggggttg 3060

cggattatct tcagacatat cataagctac ctgataatta cattacaaaa tcagaagcac 3120

aagccctcgg ctgggtggca tcaaaaggga accttgcaga cgtcgctccg gggaaaagca 3180

tcggcggaga catcttctca aacagggaag gcaaactccc gggcaaaagc ggacgaacat 3240

ggcgtgaagc ggatattaac tatacatcag gcttcagaaa ttcagaccgg attctttact 3300

caagcgactg gctgatttac aaaacaacgg accattatca gacctttaca aaaatcagat 3360

aacgaaaaaa acggcttccc tgcgggaggc cgtttttttc agctttacat aaagtgtgta 3420

ataaattttt cttcaaactc tgatcggtca atttcacttt ccggctctag agctctagag 3480

tccggtccaa tctgcagccg tccgagacag gaggacatcg tccagctgaa accggggcag 3540

aatccggcca tttctgaaga gaaaaatggt aaactgatag aataaaatca taagaaagga 3600

gccgcacatg aaaaaagcag tcattaacgg ggaacaaatc agaagtatca gcgacctcca 3660

ccagacattg aaaaaggagc ttgcccttcc ggaatactac ggtgaaaacc tggacgcttt 3720

atgggattgt ctgaccggat gggtggagta cccgctcgtt ttggaatgga ggcagtttga 3780

acaaagcaag cagctgactg aaaatggcgc cgagagtgtg cttcaggttt tccgtgaagc 3840

gaaagcggaa ggctgcgaca tcaccatcat actttcttaa aattaaaaga gctttcgttc 3900

gtatcatcgg tttcgacaac gttcgtcaag ttcaatgcat cagtttcatt gcgcacacac 3960

cagaatccta ctgagtttga gtattatggc attgggaaaa ctgtttttct tgtaccattt 4020

gttgtgcttg taatttactg tgttttttat tcggttttcg ctatcgaact gtgaaatgga 4080

aatggatgga gaagagttaa tgaatgatat ggtccttttg ttcattctca aattaatatt 4140

atttgttttt tctcttattt gttgtgtgtt gaatttgaaa ttataagaga tatgcaaaca 4200

ttttgttttg agtaaaaatg tgtcaaatcg tggcctctaa tgaccgaagt taatatgagg 4260

agtaaaacac ttgtagttgt accattatgc ttattcacta ggcaacaaat atattttcag 4320

acctagaaaa gctgcaaatg ttactgaata caagtatgtc ctcttgtgtt ttagacattt 4380

atgaactttc ctttatgtaa ttttccagaa tccttgtcag attctaatca ttgctttata 4440

attatagtta tactcatgga tttgtagttg agtatgaaaa tattttttaa tgcattttat 4500

gacttgccaa ttgattgaca ac 4522

<210> 4

<211> 635

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

agagctttcg ttcgtatcat cggtttcgac aacgttcgtc aagttcaatg catcagtttc 60

attgcgcaca caccagaatc ctactgagtt tgagtattat ggcattggga aaactgtttt 120

tcttgtacca tttgttgtgc ttgtaattta ctgtgttttt tattcggttt tcgctatcga 180

actgtgaaat ggaaatggat ggagaagagt taatgaatga tatggtcctt ttgttcattc 240

tcaaattaat attatttgtt ttttctctta tttgttgtgt gttgaatttg aaattataag 300

agatatgcaa acattttgtt ttgagtaaaa atgtgtcaaa tcgtggcctc taatgaccga 360

agttaatatg aggagtaaaa cacttgtagt tgtaccatta tgcttattca ctaggcaaca 420

aatatatttt cagacctaga aaagctgcaa atgttactga atacaagtat gtcctcttgt 480

gttttagaca tttatgaact ttcctttatg taattttcca gaatccttgt cagattctaa 540

tcattgcttt ataattatag ttatactcat ggatttgtag ttgagtatga aaatattttt 600

taatgcattt tatgacttgc caattgattg acaac 635

<210> 5

<211> 854

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggcacagg ttatcaacac gtttgacggg gttgcggatt atcttcagac atatcataag 60

ctacctgata attacattac aaaatcagaa gcacaagccc tcggctgggt ggcatcaaaa 120

gggaaccttg cagacgtcgc tccggggaaa agcatcggcg gagacatctt ctcaaacagg 180

gaaggcaaac tcccgggcaa aagcggacga acatggcgtg aagcggatat taactataca 240

tcaggcttca gaaattcaga ccggattctt tactcaagcg actggctgat ttacaaaaca 300

acggaccatt atcagacctt tacaaaaatc agataacgaa aaaaacggct tccctgcggg 360

aggccgtttt tttcagcttt acataaagtg tgtaataaat ttttcttcaa actctgatcg 420

gtcaatttca ctttccggct ctagagctct agagtccggt ccaatctgca gccgtccgag 480

acaggaggac atcgtccagc tgaaaccggg gcagaatccg gccatttctg aagagaaaaa 540

tggtaaactg atagaataaa atcataagaa aggagccgca catgaaaaaa gcagtcatta 600

acggggaaca aatcagaagt atcagcgacc tccaccagac attgaaaaag gagcttgccc 660

ttccggaata ctacggtgaa aacctggacg ctttatggga ttgtctgacc ggatgggtgg 720

agtacccgct cgttttggaa tggaggcagt ttgaacaaag caagcagctg actgaaaatg 780

gcgccgagag tgtgcttcag gttttccgtg aagcgaaagc ggaaggctgc gacatcacca 840

tcatactttc ttaa 854

<210> 6

<211> 3018

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtggaagtgt tactacatgg caggtacacg ttaccacatg tttatacagt aacactattc 60

ataaattgca gtaagacgca ttgttactac aaaacttatg gtttttacta cacacttaca 120

gtaacgtcgc gatgggattg tagtaacgca taacagatag tactaacaag tagtaatgct 180

atagtattgc tacttgtgga gagttaagat caaaaaaccc aagcttcgaa taacaatatc 240

tctcatgtga tgtatgattt tttaaaaccg tttgcaccat gatgttagaa aaaaatgctt 300

atcaaaaata gatccgtcat gactatattt caactaaatt tgaatgttgt tactgcacgt 360

aagacacagt gttactgcac gatggctgtc gctttactgc actgtttcgc aagacgagag 420

ctgagaagag gtgggtggtg ggggagttgg taggcagttt gaccgggttt tgaccgacgt 480

cgggggtgct ttgactgacc tttgactgag gtgggaggtg ggtttggccc ttgggaggga 540

gggggggata tagaatagct attctatacc cgggtgtaga atagtttgtg tatgtgtgtg 600

tgtgtgtata tatatatata tataaaatca aagtgtggga tattatattt catgtacaat 660

ctcataaaga gtctactaaa tatacttctc tctgaatata gtttgacaaa ttagttttgt 720

gtgttgctaa taactaatat aattttttac ttttagctcg attattactt aggaatttcc 780

gcagcaaagt gcggggaatc acctagttaa ttaaagaatt gagtcctcat aaaatacctt 840

ttcaatatgg cctgaatcat tgtttggttt tttggccttc tgttctattt ggtccaaaag 900

gccatctttc taagttggac tgggcccatg gtttcgttat gggccaaaaa caacaatagc 960

attttgtttt attctcccct tttgaaaaca gtatgcaggg gtgatgcttg ttcattataa 1020

gaggcaaagg gtagcatatg atatataagt ttataattta ttatagatga caacctcaaa 1080

ggtaactatc gttgacatat gtaaattatg ctacattaca aaagaaataa tgtgaacatt 1140

gacattggta catacgtgga cattatctat aattatagac tgacatctac attccctatg 1200

gttatcaatc ggaatgtcgg ctcttgtagc cttgtgaaca tacgtactct ctcgcactat 1260

atgtgcattg atgtgaatcc tccaatttgc tataggaatg tcgtaggttt gagtagccta 1320

tgtattcatt gtcagcaaat gcgcaccgtg aactctttac gtcagaggat ctacgtcagt 1380

ccgctagtac gctcatgaca tattccccct gtttttgaag atgcggttag ttactccttc 1440

tgtacactcc ataaaatgtg ctatcctaca atacattccc cgcttcaatg tatgagatat 1500

ttttccccat tttgttagaa aatacttaag caatatgttc caaatataat gttatattgg 1560

gttgatgttg tgttaaaatg gagtggttaa gcatgagcct tcatttccct cataactgtt 1620

tttttagaac tcctcgcaac tgttgcactc aacaatttca ttttccttcc tgttattcaa 1680

ttaaggttct ccttaaaaat aagtctttca tgaataattt agtattttga aggtactatt 1740

gaaagaatta tttggagatg tctaatgctt gaatgataag taatttggaa ggcatctact 1800

cctacaaagt tgaatcttct aaatactcct tcgcttttaa atatacggca ttgttaactt 1860

ttgaatatga caatatttaa caatttatct tgtacaaaga tttagtgcaa atatgcacaa 1920

tgcgtaaagt acctttaatg taaaacaagc cacaacaaaa caaatgatac ttagagcatt 1980

ttttaataag ccgattggtc gcatcatgtt taaaagtcaa ccatgtcata tataaaaaaa 2040

gggagatagt ttcatacata gctctaatat attttgaagg tactaaattt taaaaattgt 2100

tgttgcagat tgtaagtctt gcatataatt tattgtttct tttacttagc ttattagctc 2160

taattaatca caccctatct ctcctatatt tatataataa aaacatcccg acacaccaac 2220

gtacattttc tcatctattt ctaaggtatg gatggtattg tcatgtcaac ttatcttctt 2280

tctagttatg caccctcctt aattattgta ccaacttaaa ataagccata tattttgaaa 2340

ttatcatacc aactcacaat atgccatata ttttaaaatt attatgtcaa ctcacaatat 2400

gctatatatt ttgatattat cgtgccaact cacaatatac catatatttt gaaatgaagg 2460

tcatatttaa gatgtagagt aaatgtatga aatcctccca tccatatcct cattattaat 2520

tgcttccacc taccccaagc atttatagtt agagtagaac agttgtgctc ttgcaccgaa 2580

gggatgacat gtaggagcca tgcctacgtc ccctggccac ccaagcccgt tgaattaaca 2640

tggtccctca acagaaacat ccatttttga tggtgtgctt ccttcttggc gaccttactg 2700

gtaggtgaca tgctaacaaa aactgcacag tcatgcaagg caggggccgg tcaaaattga 2760

agtccctcct accttgattg taacgctaat actatggctc accgcctctg cggcaatctg 2820

gcagccactt ctcctcctct tggtgccaca gctgcacctt tgacgtggct ggctgcttgc 2880

atttgtccaa gccggcgcag ccctgatgta agtcctttgg tataaatact gcccaagcat 2940

agagcgagag agagccatca caatccaata caagttcaca acataagcat agtacaggtc 3000

attgagactt gagagagc 3018

<210> 7

<211> 457

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tatgtacagc attacgtagg tacgttttct ttttcttccc ggagagatga tacgataatc 60

atgtaaaccc agaatttaaa aaatattctt tactataaaa attttaatta gggaacgtat 120

tattttttac atgacacctt ttgagaaaga gggacttgta atatgggaca aatgaacaat 180

ttctaagaaa tgggcatatg actctcagta caatggacca aattccctcc agtcggccca 240

gcaatacaaa gggaaagaaa tgagggggcc cacaggccac ggcccacttt tctccgtggt 300

ggggagatcc agctagaggt ccggcccaca agtggccctt gccccgtggg acggtgggat 360

tgcagagcgc gtgggcggaa acaacagttt agtaccacct cgctcacgca acgacgcgac 420

cacttgctta taagctgctg cgctgaggct caggttg 457

Claims

1. A method for obtaining a non-transgenic directed gene mutant plant by utilizing endosperm specific suicide is characterized by comprising the following steps:

constructing a construct for genetic transformation of a plant, said construct comprising a first nucleic acid molecule and a second nucleic acid molecule, said first nucleic acid molecule being a gene editing element; the second nucleic acid molecule is an endosperm lethal or developmental arresting element; the plant to be treated is transgenic by introducing the construct with an exogenous nucleic acid molecule.

2. The method of claim 1, wherein the first nucleic acid molecule is a genetic element capable of editing nucleic acids.

3. The method of claim 2, wherein the genetic element of the editing nucleic acid is selected from the group consisting of genetic elements of any one of the gene editing systems.

4. The method of claim 3, wherein the gene editing system is selected from the group consisting of a ZFN gene editing system, a TALEN gene editing system, a CRISPR/CAS9 gene editing system, and a CRISPR/CPF1 gene editing system.

5. The method of claim 4, wherein the genetic elements of the CRISPR/CAS9 gene editing system comprise a CAS9 gene and a gRNA gene; the nucleotide sequence of the CAS9 gene is shown as SEQ ID NO: 1 is shown.

6. The method of claim 5, wherein the nucleotide sequence of the core backbone of the gRNA gene is set forth in SEQ ID NO: 2, respectively.

7. The method of claim 1, wherein the second nucleic acid molecule is a genetic element that is lethal or developmentally arrested by the endosperm.

8. The method of claim 1, wherein the nucleotide sequence of the second nucleic acid molecule is as set forth in SEQ ID NO: 3, respectively.

9. Use of the method of any one of claims 1 to 8 in plant breeding.

10. Use according to claim 9, characterized in that the plant is a seed plant.