一种编辑水稻巨胚基因ge培育巨胚粳稻品种的方法

Method for cultivating giant embryo japonica rice variety by editing rice giant embryo gene GE

文档序号：3338 发布日期：2021-09-17 浏览：65次中文

1. Editing rice giant embryo geneGEThe method for cultivating giant embryo japonica rice is characterized by comprising the following steps: the method comprises the following steps: editing rice giant embryo gene by using CRISPR-Cas9 multi-target technologyGEAttenuation or inhibition of giant embryo genes in riceGESo as to cultivate giant embryo japonica rice variety with enlarged seed embryo;

constructing a knock-out recombinant vector CRISPR-3GE by using a CRISPR-Cas9 multi-target technology;

the knock-out recombinant vector CRISPR-3GE is specific to rice giant embryo genesGEThe sgRNA target sequence of the first exon for site-directed editing is: GE 1: 5'-GCGGTGATGTCGCTCTCCGT-3', GE 2: 5'-GAGGGAGATCCTCGTCAGCC-3', GE 3: 5'-GGCGACATCTCGATCCAGGA-3' are provided.

2. The method of claim 1, wherein:

said attenuation or inhibition of giant embryo genes in riceGEThe method of expressing of (1), comprising the steps of:the agrobacterium tumefaciens containing the knock-out recombinant vector CRISPR-3GE is transferred into rice varieties, and the rice giant embryo genes are subjected toGEPerforming gene editing, identifying regenerated plant, and screening homozygous plantsGEThe base mutation of the gene resulting in amino acid variationgeGenotypic mutants, and/or homozygous in which the base deletion or insertion is not an integral multiple of 3 resulting in a frame shiftgeA genotypic mutant.

3. The method of claim 2, wherein:

the identification of the regeneration plant is the identification of the mutation plant, and the method specifically comprises the following steps: first of all resistHPTDetecting the positive transformed plant, and passingGESpecific primers of the gene are amplified and sequenced;

resist againstHPTThe primers used for detection were as follows:

HPT-F：5′-TACTTCTACACAGCCATCGG-3′，

HPT-R：5′-AATGTCCTGACGGACAATGG-3′；

GEthe specific primers of the gene have the following sequences:

GE-g-F：5′-ATGGCGCTCTCCTCCATGGC-3′，

GE-g-R：5′-GGCCCTAGCCACGGCCTTGC-3′。

4. giant embryo geneGEMutant 66-5, characterized by: the coded amino acid sequence is shown as SEQ ID NO: 4, respectively.

5. A giant embryo gene according to claim 4GEMutant 66-5, characterized by: the nucleotide sequence of the coding region is shown as SEQ ID NO: 3, respectively.

6. Giant embryo geneGEMutant 66-2, characterized by: the coded amino acid sequence is shown as SEQ ID NO: and 6.

7. A giant embryo gene according to claim 6GEMutant 66-2, characterized by: coding thereofThe nucleotide sequence of the region is shown as SEQ ID NO: 5, respectively.

8. Giant embryo geneGEMutant 63-1, characterized in that: the coded amino acid sequence is shown as SEQ ID NO: shown in fig. 8.

9. A giant embryo gene according to claim 8GEMutant 63-1, characterized in that: the nucleotide sequence of the coding region is shown as SEQ ID NO: shown at 7.

10. A giant embryo gene as claimed in any one of claims 4 to 5GEMutant 66-5 and giant embryo gene according to any one of claims 6 to 7GEA mutant 66-2 or a giant embryo gene according to any one of claims 8 to 9GEApplication of mutant 63-1 in culturing giant embryo japonica rice variety.

Background

Rice is an important food crop in China and is staple food for more than 50% of people in China, and in recent years, people have a higher pursuit on the nutritional quality of the rice along with the improvement of the living standard of people. The functional rice is special and health-care rice which is rich in certain physiological active substances besides the nutrient components of common rice and is used for regulating the physiological metabolism of a human body and meeting the requirements of special consumer groups such as hypertension, hyperglycemia and the like. Various functional physiological active substances such as gamma-aminobutyric acid, vitamin E, dietary fibers, essential amino acids, phenols, inositol, oryzanol, N-dehydroceramide and the like are enriched in the embryo.

Giant embryo rice is a rice variety with enlarged embryo, and belongs to functional rice. The embryo weight of the giant embryo rice is 2-3 times of that of the normal rice, and the embryo weight of some giant embryo rice is even larger, so that the giant embryo brown rice is rich in gamma-aminobutyric acid, vitamin E, essential amino acid and the like and is obviously higher than that of the common embryo rice brown rice. The nutritive value of giant embryo rice is greatly improved due to the enlargement of the embryo of the giant embryo rice, and the giant embryo rice has unique and potential economic utilization value. Gamma-aminobutyric acid (GABA) rich in giant embryo rice is also called Gamma-aminobutyric acid (GABA) and is a naturally-occurring non-protein constitutive functional amino acid, and has important medical value. GABA exists in plant tissues, also exists in central nervous systems of mammals and human beings, is an important inhibitory neurotransmitter in the central nervous systems, participates in a plurality of metabolism and physiological activities in human bodies, has the effects of reducing blood pressure, resisting aging, activating liver and kidney, promoting ethanol metabolism, improving brain cell metabolism and the like, can improve symptoms of insomnia, anxiety, depression and the like of climacteric women to a certain extent, and is known as a natural sedative of the brain. More recent studies have shown that GABA can improve the symptoms of diabetes. In addition, vitamin E rich in giant embryo rice is an important antioxidant, can effectively exert antioxidant capacity in human body, can remove free radicals in human body, can delay aging, and has obvious curative effect on prevention and treatment of hyperlipidemia.

The GABA content in common rice grains is low, and the GABA content in grains can be obviously increased by obtaining giant embryo rice varieties. The method comprises the steps of firstly obtaining a giant embryo mutant by treating zygotic cells of a Nanfeng variety of rice by a Japanese scholars with a chemical mutagen MNU (Methyl-niseurea); subsequently, the korean scholars also applied MNU chemical mutagens to treat fertilized eggs of rice and selected giant embryo rice from the variant population; china researchers mainly utilize Japanese giant embryo germplasm and then adopt mutagenesis technologies such as Co 60-gamma ray irradiation and the like to breed giant embryo rice strains through selfing and hybridization. It has been found that the giant embryo character in rice is controlled by recessive monogene, and the cloned giant embryo gene for controlling rice kernel embryo sizeGE（Giant embryo) I.e., Os07g0603700, encodes a protein in the cytochrome P450 family, which is designated CYP78a 13. At present, giant embryo rice is mainly obtained by mutagenesis and hybridization breeding techniques of the gene, however, the breeding methods have high cost, high labor intensity and long breeding period, and the obtained giant embryo rice has embryo size distributionAnd (4) limiting. CRISPR (clustered regulated short palindromic repeats) -Cas9 genome editing technology is mature and applied to various organisms for genome editing, and is also widely used for creating directional rice gene mutation materials for rapid and effective rice genetic improvement.

Disclosure of Invention

In order to overcome the defects of the traditional breeding and mutation breeding technology, the invention mainly aims to provide a method for editing rice giant embryo genesGEA method for cultivating giant embryo japonica rice variety. The method specifically comprises the step of editing rice giant embryo genes by using CRISPR-Cas9 multi-target technologyGEObtain a plurality ofgeThe gene type mutant can produce giant embryo material with several kinds of embryo larger than wild type, and is used in breeding rice variety without transgenic segment inserted.

Control of rice embryo size by CRISPR-Cas9 multi-target systemGEThe giant embryo gene is edited at a fixed point, japonica rice genetic transformation is carried out to obtain a giant embryo transgenic plant, and the transgenic insert of the rice is detected for the offspring, so that the breeding period can be shortened, japonica rice varieties of various embryos without transgenic insert can be rapidly obtained, and rice varieties with different embryo weights can meet the requirements of different consumer groups for extracting GABA with different contents.

Another object of the present invention is to provide a rice giant embryo genegeAnd (3) mutants.

Another object of the present invention is to provide the rice giant embryo genegeThe use of the mutants.

The purpose of the invention is realized by the following technical scheme:

editing rice giant embryo geneGEThe method for cultivating giant embryo japonica rice variety comprises the following steps: editing rice giant embryo gene by using CRISPR-Cas9 multi-target technologyGEObtain a plurality ofgeThe genotype mutant can produce giant embryo material with several kinds of embryo larger than wild type, and can be used for breeding rice variety without transgenic fragment inserted into giant embryo. The rice giant embryo geneGEThe nucleotide sequence is shown as SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 2, respectively.

The method specifically comprises the following steps:

1) the japonica rice middle flower 11 is a wild small embryo variety, and refers to the genome sequence of the japonica rice middle flower 11 rice, and the japonica rice middle flower 11 giant embryo geneGESelecting 3 sgRNA target sequences in the first exon of (a); the sgRNA target sequence is as follows: GE 1: 5'-GCGGTGATGTCGCTCTCCGT-3', GE 2: 5'-GAGGGAGATCCTCGTCAGCC-3', GE 3: 5'-GGCGACATCTCGATCCAGGA-3', respectively;

2) synthesizing a primer sequence constructed by the vector according to the target sequence and by referring to a method for constructing a homologous recombination vector;

3) construction of CRISPR-Cas9 Multi-target editing techniques with reference to Xie et al (Xie Kabin, Minkenberg Basitan, and Yang Yin Yang, Boosting CRISPR/Cas9 multiplex editing capabilities with the endogenesis tRNA-processing system, PNAS, 2015)GEKnocking out a recombinant vector CRISPR-3GE of 3 target points of the gene; wherein, the starting vector for knocking out the recombinant vector CRISPR-3GE is pRGEB 32;

4) transferring the CRISPR-3GE knockout recombinant vector into a conventional medium japonica rice flower 11 of a small embryo by adopting an agrobacterium-mediated genetic transformation method to obtain a transgenic rice T0 generation plant;

5) carrying out positive mutation detection on the transgenic plant, and extracting DNA from each T0-generation plant leaf to carry out hygromycin resistance geneHPT(Hygromycin B phosphotransferase) detecting positive transformed plant, designing primer for positive transformed plant target sequence, and pairingHPTCarrying out PCR amplification on the positive transformed plant, sequencing, analyzing the mutation condition, and screeningGEThe base mutation of the gene resulting in amino acid variationgeGenotypic mutants, and in which a deletion or insertion of a base other than an integral multiple of 3 results in a frame shiftgeAnd (3) genotyping mutant strains, obtaining T0 generation homozygous mutant single strains, and analyzing CRISPR-Cas9 multi-target technology mutation efficiency of target sequences. Detection ofHPTThe primer base sequences of the genes are as follows: HPT-F: 5'-TACTTCTACACAGCCATCGG-3', HPT-R: 5'-AATGTCCTGACGGACAATGG-3', respectively; reference toGEPhysical location of the target sequence of a gene, designed to contain the target sequenceGEA pair of specificities of the geneAnd (3) a sex primer for detecting the positive mutant material, wherein the base sequence of the primer is as follows: GE-g-F: 5'-ATGGCGCTCTCCTCCATGGC-3', GE-g-R: 5'-GGCCCTAGCCACGGCCTTGC-3' are provided.

6) Screening giant embryo mutant material with stable and homozygous genotype without inserted transgenic segment. Planting the homozygous mutant strain of the T0 generation in the T1 generation, and using the hygromycin-resistant geneHPTThe primers of the gRNA-tRNA sequence and the Cas9 protein sequence on the pGTR vector detect the transgenic fragment of the T1 homozygous single plant genome DNA so as to detectActinThe gene is used as an internal reference, the correctness and the integrity of the DNA of the homozygous giant embryo mutant material are detected, the PCR is amplified again on the target sequence of the transgenic material without the insertion of the transgenic segment, and the mutation condition of the target site is sequenced, so that the giant embryo mutant material without the insertion of the transgenic segment with stable genotype and homozygous is obtained.

Detecting gRNA-tRNA sequences, Cas9 fragments, andActinthe primer sequences of the genes are as follows:

gt-F：5′-GTTTTAGAGCTAGAAATAGC-3′，

gt-R：5′-TGCACCAGCCGGGAATCGAA-3′；

Cas9-F：5′-GAGAATGCTGGCCTCTGCC-3′，

Cas9-R：5′-GCTCTCTGATGGGCTTATCCCG-3′，

Actin-F：5′-TGCTATGTACGTCGCCATCCAG-3′，

Actin-R：5′-AATGAGTAACCACGCTCCGTCA-3′。

7) planting genotype stable homozygous giant embryo mutant material without transgenic fragment insertion for T2 generations, performing phenotype investigation on the size of the seed embryo, and counting the size of the seed embryo to obtain 3 homozygous mutant strains without transgenic fragment insertion with different genotype and stable phenotype of the seed embryo.

8) The GABA content of the homozygous mutant strain without the transgene fragment insertion with different genotypes and stable phenotype of 3 plant embryos is measured.

The invention edits rice by using CRISPR-Cas9 multi-target technologyGEThe rice variety with various embryos of different sizes is obtained by gene and application thereof, and medium flower 11 is obtained by knocking out a carrier CRISPR-3GE of 3 targets and genetically transforming the medium flower 11GEThe deletion mutation of the gene in different degrees generates mutant materials of embryos in different sizes, and the mutant strains of the embryos in different sizes contain different GABA contents so as to meet the application of different requirements.

Giant embryo geneGEMutant 66-5, the nucleotide sequence of its coding region is set forth in SEQ ID NO: 3, and the amino acid sequence is shown as SEQ ID NO: 4, respectively. The giant embryo gene can be utilizedGEThe mutant 66-5 has the function of obviously increasing the seed embryo and can be used for cultivating giant embryo japonica rice varieties.

Giant embryo geneGEMutant 66-2, the nucleotide sequence of its coding region is set forth in SEQ ID NO: 5, and the amino acid sequence is shown as SEQ ID NO: and 6. The giant embryo gene can be utilizedGEThe mutant 66-2 has the function of obviously increasing the seed embryo, and can be used for cultivating giant embryo japonica rice varieties.

Giant embryo geneGEMutant 63-1, the nucleotide sequence of its coding region is shown in SEQ ID NO: 7, the amino acid sequence is shown as SEQ ID NO: shown in fig. 8. The giant embryo gene can be utilizedGEThe mutant 63-1 has the function of obviously increasing the seed embryo and can be used for cultivating giant embryo japonica rice varieties.

Compared with the prior art, the invention has the following advantages and effects:

1. the present invention utilizes riceGEThe gene can control the size of the rice embryo, and the CRISPR-Cas9 multi-target technology can be used for targeted editing of the genomeGEDesigning 3 target point sequences on the gene can make site-directed mutagenesisGECompared with the traditional mutation and hybridization methods for giant embryo variety breeding, the method can quickly and directionally obtain the japonica rice variety without the insertion of the transgenic segment, shortens the breeding period and provides an efficient breeding mode.

2. The invention knocks out by CRISPR-Cas9 multi-target technologyGEThe gene can generate different mutations by directionally editing 3 sites simultaneously, and the traditional rice giant embryo gene edited by the CRISPR-Cas9 multi-target technology is obtainedGEThe target site with high mutation efficiency can obtain 3 embryos with different sizesgeGenotypic germplasmThe material can be used for production and application with different GABA content requirements.

Drawings

FIG. 1 is the sequencing results of 3 mutants 66-5, 66-2 and 63-1 in example; wherein WT is a wild-type material;

FIG. 2 shows the results of the detection of T1 transgenic material insert in the examples; wherein 1-8 are different T1 generation transgenic materials, and the T1 generation with the 1 st and 2 nd pore canals 63-1 contains or does not containHPTA gRNA-tRNA sequence and a Cas9 fragment; the T1 generations having 66-5 of the No. 3, No. 4 and No. 5 pore channels, respectively, do not contain, contain and do not containHPTA gRNA-tRNA sequence and a Cas9 fragment; the T1 generation with the 6 th, 7 th and 8 th pore passages being 66-2 respectively does not contain, contains and containsHPTA gRNA-tRNA sequence and a Cas9 fragment, wherein CG is a recombinant vector CRISPR-3GE, and WT is a wild type control;

FIG. 3 is seed morphology of wild type and mutant material in the examples; wherein (1) is wild type, (2) mutant strain 63-1, (3) is mutant strain 66-5, (4) is mutant strain 66-2, and the scale bar =1000 μm;

FIG. 4 shows grain weight phenotype of wild type and mutant in examples; the statistical number of each phenotype was n = 6;

FIG. 5 is grain volume phenotype of wild type and mutant in the examples; the statistical number of each phenotype was n = 6;

FIG. 6 shows GABA content of brown rice in wild type and mutant in examples; the statistical number of each phenotype was n = 6.

Note: error bars in all figures of the above specification are standard deviations, which indicate significant differences between mutant and wild-type phenotypes,P<0.01。

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.

Example 1

The invention constructs riceGEThe CRISPR-Cas9 multi-target vector CRISPR-3GE of the gene is introduced into the middle flower 11 of japonica rice by an agrobacterium-mediated genetic transformation method to obtain transgenic rice, and a primer is designed for a transgenic materialGEPCR amplification and sequencing of the gene, screeningGEMutant strains in which mutation of the base of the gene results in amino acid variation, and mutant strains in which deletion or insertion of a base other than an integral multiple of 3 results in frame shift of the reading frame, for homozygosisgeThe genotype non-transgenic fragment is inserted into the mutant strain to examine the size of the embryo, the mutant strain is planted to obtain the offspring giant embryo japonica rice with stable phenotype, and the GABA content of the japonica rice brown rice with 3 embryo sizes is measured to obtain the giant embryo japonica rice variety which can be produced and applied for different GABA content requirements. The method specifically comprises the following steps:

1. design and synthesis of sgRNA target primers

(1) Selecting to obtain multiple mutant materials with different fragment deletion or insertion mutantsGEEditing gene with 3 target sites on the first exon of gene, and selectingGE3 20bp sequences on the first exon of the gene are used as 3 sgRNA target sites, GE 1: 5'-GCGGTGATGTCGCTCTCCGT-3', GE 2: 5'-GAGGGAGATCCTCGTCAGCC-3', GE 3: 5'-GGCGACATCTCGATCCAGGA-3', corresponding to the physical positions on the reference genome of flower 11 in japonica rice, which are +277. +296bp, +339. +358bp, +655. +674bp, respectively (A in the initiation codon ATG is +1bp, as shown in SEQ ID NO: 1).

(2) Referring to Xie et al (Xie Kabin, Minkenberg Basian, and Yang Yin Yang, Boosting CRISPR/Cas9 multiplex editing capability with the endogenic tRNA-processing system, PNAS, 2015), the CRISPR-Cas9 multi-target editing technology is selected to construct a vector in a homologous recombination mode, vector sequence joints are respectively added at two ends of a sgRNA target sequence, and the 3 sgRNA target sequences are artificially synthesized into forward and reverse primer sequences which respectively are as follows:

GE1-F：5′-TAGGTCTCCGTCGCTCTCCGTGTTTTAGAGCTAGAA-3′，

GE1-R：5′-CGGGTCTCACGACATCACCGCTGCACCAGCCGGGAA-3′；

GE2-F：5′-TAGGTCTCCTCCTCGTCAGCCGTTTTAGAGCTAGAA-3′，

GE2-R：5′-CGGGTCTCAAGGATCTCCCTCTGCACCAGCCGGGAA-3′；

GE3-F：5′-TAGGTCTCCCTCGATCCAGGAGTTTTAGAGCTAGAA-3′，

GE3-R：5′-CGGGTCTCACGAGATGTCGCCTGCACCAGCCGGGAA-3′。

(3) the primers on both sides of the vector construction are L5AD5-F and L3AD5-R respectively, and the sequences of the primers are as follows:

L5AD5-F：5′-CGGGTCTCAGGCAGGATGGGCAGTCTGGGCAACAAAGCACCAGTGG-3′，

l3AD 5-R: 5'-TAGGTCTCCAAACGGATGAGCGACAGCAAACAAAAAAAAAAGCACCGACTCG-3' are provided. The primer synthesis was completed by the primer synthesis company.

2.3 target sites are connected

(1) Knock-out fragment amplification

50 muL PCR amplification system: pGTR (BioVector-063143) 0.1ng, 5 xPhusion HF buffer 10 muL, dNTPs (10mM) 1 muL, 10 muM forward/reverse primers each 2.5 muL, polymerase Phusion (2U/muL, NEB) 0.5 muL, plus ddH₂O to the PCR reaction system is 50 muL. PCR reaction procedure: at 98 ℃ for 2 min; 98 ℃, 10sec, 50 ℃, 20sec, 72 ℃, 20sec, 35 cycles; 72 ℃ for 2.5 min; 4 ℃ for 10 sec. Knock-out fragment 1: l5AD5-GE1, and PCR forward/reverse primers are L5AD5-F and GE1-R, respectively; knock-out fragment 2: GE1-GE2, and GE1-F and GE2-R are PCR forward/reverse primers respectively; knock-out fragment 3: GE2-GE3, and GE2-F and GE3-R are PCR forward/reverse primers respectively; knock-out fragment 4: GE3-L3AD5, and the PCR forward/reverse primers were GE3-F and L3AD5-R, respectively. The sizes of all PCR amplification products are about 150-200 bp.

(2) Purification and recovery of knock-out fragment

And (3) taking 5 mu L of PCR product, detecting a positive strip of the PCR product by using 1% agarose electrophoresis, and recovering the remaining positive PCR product by using a DNA recovery kit.

(3) Joining of knock-out fragments

20 mu L ligation reaction system: 4 PCR products, 25-50 ng; 2xT7 DNA ligase Buffer (NEB), 10 μ L; bovine serum albumin (1mg/mL)2 μ L; restriction enzyme Bsa I (10U/muL, NEB), 0.5 muL; ligase T7 DNA Ligase (3000U/. mu.L, NEB), 0.5. mu.L. And (3) connection reaction: 35 cycles of 37 deg.C, 5min, 20 deg.C, 10 min; 20 ℃ and 1 h.

(4) PCR amplification of knock-out fragment ligation product

Adding 180 mu L ddH to the 4 PCR product ligation products in the step₂And after O dilution, sucking 1 mu L for PCR amplification. 50 muL PCR amplification system: diluted ligation product 1 μ L, 10 XPCR buffer (Mg)²⁺plus) 5 μ L, dNTPs (10mM) 1 μ L, 10 μ M forward primer S5AD5-F (sequence: 5'-CGGGTCTCAGGCAGGATGGGCAGTCTGGGCA-3') and the reverse primer S3AD5-R (sequence: 5'-TAGGTCTCCAAACGGATGAGCGACAGCAAAC-3'), 1 muL each, polymerase rTaq (5U/muL, TaKaRa) 0.5 muL, add ddH₂O to the PCR reaction system is 50 muL. PCR reaction procedure: at 95 ℃ for 2 min; 95 deg.C, 10sec, 55 deg.C, 30sec, 72 deg.C, 1min, 35 cycles; 72 ℃ for 10 min; 4 ℃ for 10 sec.

(5) PCR product recovery

And (3) taking 5 mu L of PCR product, detecting a positive strip of the PCR product by using 1% agarose electrophoresis, and recovering the remaining positive PCR product by using a DNA recovery kit.

3. Construction of knock-out vector CRISPR-3GE

(1) Enzyme digestion PCR product and final vector

And (3) carrying out enzyme digestion on the PCR product in the step by using FokI restriction enzyme, wherein the enzyme digestion system is as follows: the product recovered in the previous step plus ddH₂And O to 44 muL, adding enzyme digestion buffer 5 muL, FokI 1 muL, and carrying out enzyme digestion for 1h at 37 ℃. Meanwhile, BsaI restriction enzyme is used for cutting the vector pRGEB32, and the cutting system is as follows: pRGEB 321 μ g, Buffer 5 μ L, BsaI 1 μ L, ddH₂And supplementing O to 50 mu L, and carrying out enzyme digestion at 37 ℃ for 60 min. Among them, vector pRGEB32 was a conventional commercially available plasmid.

(2) Recovery of the digestion product

And (3) taking 5 mu L of enzyme digestion product, detecting a positive strip of the PCR product by using 1% agarose electrophoresis, and recovering the remaining positive enzyme digestion product by using a DNA recovery kit.

(3) The target site sequence is linked to a final vector

10 mu L connection reaction system: carrying out enzyme digestion on a PCR product of 6 muL; 2 muL of the enzyme-digested final vector, 10x T4 DNA ligase Buffer (NEB), 1 muL; ligase T4 DNA Ligase (2000U/. mu.L, NEB), 1. mu.L. And (3) connection reaction: ligation was performed overnight at 16 ℃.

(4) The ligation product is transformed into competent cells of Escherichia coli JM109 by a heat shock method, and a positive clone is obtained by culturing, picking and sequencing a single clone

Adding 10 muL of the ligation product into 100 muL of thawed escherichia coli JM109 competent cells, gently sucking and uniformly mixing by using a pipette gun, placing the mixture on ice for 30min, carrying out water bath at 42 ℃ for 45s, quickly transferring the mixture onto the ice for 2min, adding 700 muL of LB culture medium without antibiotics, shaking the mixture on a shaker at 37 ℃ at 200rpm for 1h, centrifuging the mixture at 5000rpm for 1min, leaving 100 muL of supernatant, discarding the rest, suspending escherichia coli, coating the suspension on an LB culture medium plate containing kanamycin antibiotics, and inversely placing the suspension in an incubator at 37 ℃ for 18 h. Selecting monoclonal culture, carrying out colony PCR verification, selecting positive bacteria, sending to a company for sequencing, wherein sequencing primers are C-GE: 5'-AGTACCACCTCGGCTATCCACA-3', select positive clones that sequenced correctly for the next experiment. Among them, Escherichia coli JM109 competent cells are conventional commercially available products.

(5) Small quantity extraction of colibacillus plasmid DNA by alkali cracking method

Coli containing positive clones were cultured at 37 ℃ for 16h for propagation and centrifuged at 12000rpm in a 1.5mL centrifuge tube for 1 min. Discarding the supernatant, collecting the bacterial solution repeatedly for 3 times, removing the supernatant by suction, adding 300 μ L of solution I (50 mL PH8.01M Tris-HCl, 20mL 0.5M EDTA, 920mL ddH)₂O), shaking violently, adding 300 mu L of newly configured solution II (equivolumetrically configured by 0.4M NaOH and 2% SDS), slowly turning the centrifuge tube up and down for several times, mixing uniformly, standing at room temperature for 2min, adding precooled solution III (129.69 g potassium acetate, adding 600mL ddH)₂O, 100mL glacial acetic acid, adjusted pH to 4.8 with acetic acid, ddH₂O constant volume to 1L) 300 muL, slowly turning the centrifuge tube up and down for several times, uniformly mixing, carrying out ice bath for 10min, centrifuging for 15min at 12000rpm, sucking supernatant fluid 900 muL, adding 600 muL isopropanol, placing at room temperature for 5min, centrifuging for 15min at 12000rpm, discarding supernatant fluid, washing twice with 500 muL 70% ethanol, centrifuging for 2min at low speed, pouringAnd (3) removing the supernatant, centrifuging at 12000rpm for 1min, sucking the supernatant, air-drying for 10min, adding 40 mu L of TE buffer solution (1 mL PH8.01M Tris-HCl, 0.2 mL PH8.00.5M EDTA) to dissolve the plasmid to obtain the plasmid CRISPR-3GE, and storing at 4 ℃ for later use.

4. Genetic transformation and obtaining of giant embryo japonica rice

(1) Adding 5 mu L of positive clone plasmid CRISPR-3GE into agrobacterium EHA105 competent cells, placing on ice for 30min, quickly freezing in liquid nitrogen for 1min, immediately placing in a 37 ℃ water bath or a metal bath for 5min, adding 1mL of liquid LB culture medium, culturing at 28 ℃, 200rpm for 2h, centrifuging at 5000rpm for 1min, uniformly coating the bacterial liquid on an LB culture medium plate containing kanamycin antibiotic, and culturing at 28 ℃ for 2 d. And (3) selecting a monoclonal colony for PCR amplification and sequencing, and culturing the positive clone with correct sequencing by shaking and plating for 2 d. Wherein, the agrobacterium EHA105 competent cell is a conventional commercial product.

(2) And (3) genetic transformation of introducing CRISPR-3GE into japonica rice middle flower 11. Selecting the callus with good activity of japonica rice mid-flower 11 for genetic transformation experiment of agrobacterium. (preculture) from the secondary callus, the pale yellow, granular, dry, vigorous callus was picked up and transferred to a preculture medium (300. mu.L acetosyringone, 5mL 40% glucose per 250mL medium) and placed on NB medium for dark culture at 27 ℃ for 3 days. (infection and co-culture) marking the agrobacterium strain containing the positive clone plasmid CRISPR-3GE on a plate containing kanamycin antibiotic, and activating; scraping the streak-cultured Agrobacterium into 250mL 1/2N 6 suspension medium, adding 100 μ L acetosyringone, 2mL 50% glucose, shaking at 28 deg.C and 200rpm for 30min until it is slightly turbid (OD 600 ≈ 0.4); pouring the agrobacterium liquid into a 250mL sterile triangular flask containing callus, soaking for 30min, pouring out the liquid, drying the callus, uniformly spreading the fully dried callus on a co-culture medium (250 mL of N6 culture medium added with 300 mu L of acetosyringone and 5mL of 50% glucose) by using a spoon, and performing dark culture at 19 ℃ for 3 d. (washing and screening S1) transferring the co-cultured callus into a water washing cup, pouring sterile distilled water until the callus is completely immersed, covering the water washing cup, shaking for 20-30S, and pouring the sterile distilled water. Repeating the above steps for 2-3 times. Adding sterilized distilled water until the callus is completely immersed, covering a cover, violently oscillating for 20-30 s, and standing for 10 min. Then, observation is carried out, if the water in the water washing cup is clear, the agrobacterium is washed clean, otherwise, the water washing is required to be continued. Finally, the sterilized distilled water was poured off, and sterilized distilled water containing 500mg/L of carbenicillin was added thereto, followed by shaking at 200rpm for 30 min. Water was poured off, the callus was dried, and the well-dried callus was uniformly applied onto selection medium S1 (250 mL of N6 medium supplemented with 400. mu.L of carbenicillin, 250. mu.L of hygromycin, 5mL of 50% glucose) with a spoon, and cultured in the dark at 19 ℃ for 20 days. (screening S2) Dry, Agrobacterium-free callus transfer dishes were selected from S1 medium, sparsely plated on S2 medium (300. mu.L carbenicillin, 250. mu.L hygromycin, 5mL 50% glucose in 250mL N6 medium), dark cultured for 20 days, and observed to see if a fresh yellow-tender resistant callus was grown, if no resistant callus was transferred onto the dishes as S3. The selection medium was supplemented with carbenicillin reduced to 200. mu.L. The resistant callus can be grown by twice screening of the japonica rice, namely S2. The method comprises the following steps of (differentiation, rooting and seedling hardening) selecting faint yellow, compact and dry, placing resistant callus small blocks attached to a culture medium into a differentiation culture medium, carrying out light culture for 40 days, differentiating seedlings (about 5-10 cm in height), then inoculating the differentiated seedlings into a rooting tube for rooting culture, carrying out light culture for 15-20 days in a light culture chamber, after roots grow fully, uncovering a root canal sealing film, adding a certain amount of tap water, carrying out light culture for 4-7 days, and then carrying out seedling hardening to obtain T0 generation transgenic seedlings.

(3) Transgenic seedling planting and mutant identification

Transplanting transgenic T0 seedlings to field or bread box, transplanting to survival to obtain 34 transgenic seedlings, extracting DNA from leaves of each transgenic seedling, and detecting hygromycin gene to obtain positive transformed plant, hygromycinHPTThe gene detection PCR primer has the following base sequence, HPT-F: 5'-TACTTCTACACAGCCATCGG-3', HPT-R: 5'-AATGTCCTGACGGACAATGG-3' are provided. Identified byHPTAnd (3) sequencing the transgenic positive plant by using a target site design primer to identify the mutation condition, and sequencing and identifying the PCR primer GE-g-F by using the target site: 5'-ATGGCGCTCTCCTCCATGGC-3', GE-g-R: 5'-GGCCCTAGCCACGGCCTTGC-3' are provided.

(4) And analyzing the positive mutation situation of the T0 generation transgenic material target site. Using hygromycinHPTAnalyzing the target point mutation condition of the T0 generation 34 transgenic material according to the gene detection and sequencing result of the rice giant embryoGEIs changed intogeThe homozygous mutant and the wild type control seed embryo phenotype become giant embryo, 10 single-site homozygous mutant strains are found, 18 single-site heterozygous mutant strains (mutation sites are all at the 2 nd target site), and 6 negative non-mutation single strains. Analyzing 10 homozygous mutant strains to find that a deoxynucleotide T is inserted at the 2 nd target point +355 to generate frame shift mutation and cause the premature termination of protein translation at the 328 nd amino acid, and 4 mutant strains 66-5, 67-3, 67-4 and 68-6 are provided; deletion of deoxynucleotide A at 2 nd target site +355, frame shift mutation, early termination of protein translation at 126 th amino acid, and 5 mutants of 66-2, 67-1, 70-2, 70-5 and 71-1; the deoxynucleotide is mutated from A to C at the 3 rd target point +661, the corresponding codon is converted from ATC (amino acid Ile) to CTC (amino acid Leu), and a mutant strain 63-1 is provided. The results of genotype sequencing of 3 of these giant embryo homozygous mutants 66-5, 66-2 and 63-1 are shown in FIG. 1. Sequencing results show that (1) the CRISPR-Cas9 multi-target editing technology can realize site-directed mutation on the 2 nd target site and the 3 rd target site of a giant embryo gene, and the 1 st target site does not produce mutant materials; (2) a large amount of single-site homozygous mutant materials can be obtained at the T0 generation, but mutation caused by deletion or insertion of large fragments is not realized, and the single-site mutation is realized, wherein the mutation efficiency of the 2 nd target site is very high, the mutation sites of 27 mutant materials in 34 transgenic materials are all generated at the 2 nd target site, and the mutation efficiency reaches 79.41 percent; whereas the mutation efficiency of the 3 rd target is 2.94% lower, and the mutation efficiency of the 1 st target is 0. The mutation efficiency of the 3 target sites in the CRISPR-Cas9 multi-target editing system can be realized by other CRISPR-Cas9 editing systems for editing rice megablast genesGEReference is provided.

(5) Detection of no transgene insert. Planting the transgenic positive homozygous mutant strain T1 generation, and detecting the transgenic insert on the segment of the transgenic recombinant vector. Extracting T1 generation transgenic plant DNA fragmentHygromycin geneHPT(HPT-F/R), gRNA-tRNA sequence on pGTR vector (gt-F/R) and Cas9 protein sequence (Cas 9-F/R) toActinThe gene (Actin-F/R) is used as an internal reference to detect the correctness and the integrity of the homozygous giant embryo mutation material DNA, and 3 target sites of the positive transgenic material without the transgenic fragment inserted in the T1 generation are subjected to re-amplification PCR and the mutation condition of the target sites is sequenced. The primers are used for carrying out PCR amplification on T1 generation transgenic plant DNA and carrying out detection by using 1% agarose gel electrophoresis, wherein mutants without transgenic fragment insertion are obtained in T1 generation separated plants of 3 giant embryo homozygous mutant strains 66-5, 66-2 and 63-1 (figure 2), and target site sequencing is carried out on the mutants without transgenic fragment insertion to analyze mutant strains with stable genotypes, so that giant embryo mutant materials with stable genotypes and homozygous and without transgenic fragment insertion are obtained.

gt-F：5′-GTTTTAGAGCTAGAAATAGC-3′，

gt-R：5′-TGCACCAGCCGGGAATCGAA-3′；

Cas9-F：5′-GAGAATGCTGGCCTCTGCC-3′，

Cas9-R：5′-GCTCTCTGATGGGCTTATCCCG-3′，

Actin-F：5′-TGCTATGTACGTCGCCATCCAG-3′，

Actin-R：5′-AATGAGTAACCACGCTCCGTCA-3′。

Wherein, the gRNA-tRNA sequence on the pGTR vector is shown as SEQ ID NO: shown at 9.

(6) Giant embryogeAnd (3) inspecting the weight and the volume of the genotype mutant brown rice and seed embryos thereof. Planting T2 generation of genotype stable homozygous giant embryo mutant materials 66-5, 66-2 and 63-1 without transgenic fragment insertion, harvesting and airing, preserving for 3 months under room temperature and dry conditions, selecting 6 families from each giant embryo mutant material for phenotype investigation, and simultaneously finding that 3 giant embryo mutant materials show different sizes of embryo (figure 3). Removing rice glumes by a small brown rice machine, selecting the brown rice which normally develops from each material, and measuring the thousand grain weight of the brown rice by an electronic balance. Selecting 100 homozygous giant embryo mutant materials with normal development for each family, measuring weight with electronic balance, recording brown rice weight, and using tweezersCarefully peeling off the embryo, scraping the joint of the brown rice and the embryo by using a blade, measuring the weight by using an electronic balance, recording the weight of the endosperm, calculating the thousand seed weight of the brown rice, the thousand seed weight of the endosperm and the thousand seed weight of the embryo by using the mean value, and dividing the thousand seed weight of the embryo by the thousand seed weight of the brown rice to obtain the embryo/brown rice weight ratio. The analysis statistics data show that the embryo thousand seed weight, the brown rice thousand seed weight, the endosperm thousand seed weight and the embryo/brown rice weight ratio of 66-5, 66-2 and 63-1 respectively have very significant difference compared with the phenotype of wild type WT, wherein the embryo thousand seed weight and the embryo/brown rice weight ratio of the family 66-2 are the highest (figure 4), and the embryo thousand seed weight of 63-1, 66-5 and 66-2 respectively increase by 95.38%, 130.77% and 169.23% compared with the WT; the thousand grain weight of the brown rice of 63-1, 66-5 and 66-2 is respectively reduced by 11.18 percent, 35.18 percent and 34.81 percent compared with that of the WT; the thousand kernel weight of endosperm of 63-1, 66-5 and 66-2 is respectively reduced by 14.89%, 40.95% and 41.91% compared with that of WT; the embryo/brown rice weight ratios of 63-1, 66-5 and 66-2 were increased by 119.98%, 256.00% and 313.00%, respectively, compared with WT. The brown rice and the degerming brown rice are screened by the method, a suction nozzle of a 5mL pipette is sealed, a certain amount of industrial alcohol is added to read the initial volume of the brown rice, 100 pieces of brown rice are added to read the final volume, the difference value of the two volume readings can be used for calculating the average volume of each brown rice, the 100 pieces of degerming brown rice are also added to read the final volume, the difference value of the two volume readings can be used for calculating the average volume of each degerming brown rice, the volume of the embryo is the difference value of the brown rice and the degerming brown rice, the volume of the hundred pieces of endosperm and the volume of the hundred pieces of embryo are calculated by the average value, and the volume of the embryo is divided by the volume of the brown rice to obtain the embryo/brown rice volume ratio. Analyzing the volume related data, the phenotype hundred-grain embryo volume and the embryo/brown rice volume ratio of 63-1, 66-5 and 66-2 show extremely significant difference and have a trend of increasing change when compared between the mutant strain and the WT; hundred endosperm volume 3 mutant lines all have very significant difference with WT, but have no increasing or decreasing trend; the volume of the hundred-grain brown rice, the mutant strain families and the WT have no difference (figure 5), wherein the volumes of the hundred-grain embryos of 63-1, 66-5 and 66-2 are respectively increased by 160.87%, 284.78% and 332.39% compared with the WT; volume of 63-1, 66-5 and 66-2 hundred grains of brown rice and WT phaseThe ratio is respectively reduced by 1.38 percent, 1.84 percent and 2.30 percent; the hundred kernel endosperm volumes of 63-1, 66-5 and 66-2 are respectively reduced by 6.57%, 11.01% and 13.01% compared with WT; the embryo/brown rice volume ratios of 63-1, 66-5 and 66-2 were increased by 164.51%, 292.00% and 342.58%, respectively, compared with WT.

(7) GABA content was determined for homozygous mutants with stable genotype and phenotype of different embryo sizes without transgene fragment insertion. And (3) carrying out T2 generation on genotype-stable homozygous giant embryo mutation materials 63-1, 66-5 and 66-2 without transgene fragments inserted planted in the steps, harvesting and airing, preserving for 3 months under a room temperature drying condition, removing rice glumes by using a small brown rice machine, grinding brown rice into powder capable of passing through a 80-mesh sieve by using a sample freeze grinding instrument, quickly putting the powder into liquid nitrogen, quickly weighing 0.050 +/-0.001 g of powder on an analytical balance into a 2mL centrifuge tube, adding 0.75mL of 50% methanol solution, carrying out low-temperature water bath ultrasonic treatment for 15min, centrifuging for 5min at 12000g at 4 ℃, taking supernatant, carrying out vacuum concentration and drying, carrying out silanization derivatization treatment by using MSTFA, taking 1 mu L of powder for GC-MS mass spectrum detection of GABA content, and sending the sample to an agricultural biological gene research center of Guangdong province agricultural science research institute for absolute quantitative detection. Statistical data show that the GABA content is gradually increased along with the increase of embryo volume, the GABA content of the brown rice of 63-1, 66-5 and 66-2 is respectively 0.0155mg/g, 0.0416 mg/g and 0.0434 mg/g, and compared with the GABA content of WT (0.0056 mg/g), the GABA content is respectively increased by 176.79%, 642.86% and 675.00% (FIG. 6).

Therefore, by the method provided by the invention, after the japonica rice middle flower 11 is knocked out through the CRISPR-Cas9 multiple targets, multiple rice varieties with different embryo sizes can be generated, and each rice variety with different embryo sizesgeThe GABA content of brown rice of the genotype mutant strain is also different, and the GABA content is increased along with the increase of embryos. The homozygous mutant strain without the inserted transgenic fragment and with stable genotype and phenotype, which is obtained by the method provided by the invention, giant embryo japonica rice varieties with different embryo sizes can be produced and applied as different GABA content requirements.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> Rice research institute of Guangdong province academy of agricultural sciences

<120> method for cultivating giant embryo japonica rice variety by editing rice giant embryo gene GE

<160> 33

<170> SIPOSequenceListing 1.0

<211> 1578

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CDS sequence of gene GE of 11 giant embryos of japonica rice medium flower

<400> 1

atggcgctct cctccatggc cgcggcgcaa gagagctccc tcctcctctt cctcctcccg 60

acgtcggccg cctccgtgtt cccgccgctc atctccgtgg tcgtcctcgc cgcgctcctc 120

ctgtggctct cgccgggtgg ccccgcgtgg gcgctgtccc gttgccgtgg cacgccgccg 180

ccgccgggcg tggcgggggg cgcggccagc gcgctgtccg gccctgccgc gcaccgcgtg 240

ctcgccggga tttcgcgcgc cgtcgagggc ggcgcggcgg tgatgtcgct ctccgtcggc 300

ctcacccgcc tcgtcgtggc gagccggccg gagacggcga gggagatcct cgtcagcccg 360

gcgttcggcg accgccccgt gaaggacgcg gcgaggcagc tgctgttcca ccgcgccatg 420

gggttcgccc cgtcgggcga cgcgcactgg cgcgggctcc gccgcgcctc cgcggcgcac 480

ctcttcggcc cgcgccgcgt ggccgggtcc gcgcccgagc gcgaggccat cggcgcccgc 540

atagtcggcg acgtcgcctc cctcatgtcc cgccgcggcg aggtccccct ccgccgcgtc 600

cttcacgccg cgtcgctcgg ccacgtcatg gcgaccgtct tcggcaagcg gcacggcgac 660

atctcgatcc aggacggcga gctcctggag gagatggtca ccgaagggta cgacctcctc 720

ggcaagttca actgggccga ccacctgcca ttgctcaggt ggctcgacct ccagggcatc 780

cgccgccggt gcaacaggct agtccagaag gtggaggtgt tcgtcggaaa gatcatacag 840

gagcacaagg cgaagcgagc tgccggaggc gtcgccgtcg ccgacggcgt cttgggcgac 900

ttcgtcgacg tcctcctcga cctccaggga gaggagaaga tgtcagactc cgacatgatc 960

gctgttcttt gggagatgat ctttagaggg acggacacgg tggcgatctt gatggagtgg 1020

gtgatggcga ggatggtgat gcacccggag atccaggcga aggcgcaggc ggaggtggac 1080

gccgccgtgg ggggacgccg cggccgcgtc gccgacggcg acgtggcgag cctcccctac 1140

atccagtcca tcgtgaagga gacgctgcgc atgcacccgc cgggcccgct cctgtcgtgg 1200

gcgcgcctcg ccgtgcacga cgcgcgcgtc ggtggccacg ccgtccccgc cgggacgacg 1260

gcgatggtga acatgtgggc gatcgcccac gacgccgccg tctggccgga gccggatgcg 1320

ttccgcccgg agcgcttctc ggagggggag gacgtcggcg tgctcggcgg cgacctccgc 1380

ctcgcgccgt tcggcgccgg ccgccgcgtc tgccctggca ggatgctggc gctcgccacc 1440

gcccacctct ggctcgccca gctgctgcac gccttcgact ggtcccccac cgccgccggc 1500

gtcgacctgt ccgagcgcct cggcatgtcg ctggagatgg cggcgccgct cgtgtgcaag 1560

gccgtggcta gggcctga 1578

<211> 525

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence coded by gene GE of medium flower 11 giant embryo of japonica rice

<400> 2

Met Ala Leu Ser Ser Met Ala Ala Ala Gln Glu Ser Ser Leu Leu Leu

1 5 10 15

Phe Leu Leu Pro Thr Ser Ala Ala Ser Val Phe Pro Pro Leu Ile Ser

20 25 30

Val Val Val Leu Ala Ala Leu Leu Leu Trp Leu Ser Pro Gly Gly Pro

35 40 45

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

50 55 60

Ala Gly Gly Ala Ala Ser Ala Leu Ser Gly Pro Ala Ala His Arg Val

65 70 75 80

Leu Ala Gly Ile Ser Arg Ala Val Glu Gly Gly Ala Ala Val Met Ser

85 90 95

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

100 105 110

Ala Arg Glu Ile Leu Val Ser Pro Ala Phe Gly Asp Arg Pro Val Lys

115 120 125

Asp Ala Ala Arg Gln Leu Leu Phe His Arg Ala Met Gly Phe Ala Pro

130 135 140

Ser Gly Asp Ala His Trp Arg Gly Leu Arg Arg Ala Ser Ala Ala His

145 150 155 160

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

165 170 175

Ile Gly Ala Arg Ile Val Gly Asp Val Ala Ser Leu Met Ser Arg Arg

180 185 190

Gly Glu Val Pro Leu Arg Arg Val Leu His Ala Ala Ser Leu Gly His

195 200 205

Val Met Ala Thr Val Phe Gly Lys Arg His Gly Asp Ile Ser Ile Gln

210 215 220

Asp Gly Glu Leu Leu Glu Glu Met Val Thr Glu Gly Tyr Asp Leu Leu

225 230 235 240

Gly Lys Phe Asn Trp Ala Asp His Leu Pro Leu Leu Arg Trp Leu Asp

245 250 255

Leu Gln Gly Ile Arg Arg Arg Cys Asn Arg Leu Val Gln Lys Val Glu

260 265 270

Val Phe Val Gly Lys Ile Ile Gln Glu His Lys Ala Lys Arg Ala Ala

275 280 285

Gly Gly Val Ala Val Ala Asp Gly Val Leu Gly Asp Phe Val Asp Val

290 295 300

Leu Leu Asp Leu Gln Gly Glu Glu Lys Met Ser Asp Ser Asp Met Ile

305 310 315 320

Ala Val Leu Trp Glu Met Ile Phe Arg Gly Thr Asp Thr Val Ala Ile

325 330 335

Leu Met Glu Trp Val Met Ala Arg Met Val Met His Pro Glu Ile Gln

340 345 350

Ala Lys Ala Gln Ala Glu Val Asp Ala Ala Val Gly Gly Arg Arg Gly

355 360 365

Arg Val Ala Asp Gly Asp Val Ala Ser Leu Pro Tyr Ile Gln Ser Ile

370 375 380

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

385 390 395 400

Ala Arg Leu Ala Val His Asp Ala Arg Val Gly Gly His Ala Val Pro

405 410 415

Ala Gly Thr Thr Ala Met Val Asn Met Trp Ala Ile Ala His Asp Ala

420 425 430

Ala Val Trp Pro Glu Pro Asp Ala Phe Arg Pro Glu Arg Phe Ser Glu

435 440 445

Gly Glu Asp Val Gly Val Leu Gly Gly Asp Leu Arg Leu Ala Pro Phe

450 455 460

Gly Ala Gly Arg Arg Val Cys Pro Gly Arg Met Leu Ala Leu Ala Thr

465 470 475 480

Ala His Leu Trp Leu Ala Gln Leu Leu His Ala Phe Asp Trp Ser Pro

485 490 495

Thr Ala Ala Gly Val Asp Leu Ser Glu Arg Leu Gly Met Ser Leu Glu

500 505 510

Met Ala Ala Pro Leu Val Cys Lys Ala Val Ala Arg Ala

515 520 525

<211> 987

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CDS sequence of giant embryo gene GE mutant 66-5

<400> 3

atggcgctct cctccatggc cgcggcgcaa gagagctccc tcctcctctt cctcctcccg 60

acgtcggccg cctccgtgtt cccgccgctc atctccgtgg tcgtcctcgc cgcgctcctc 120

ctgtggctct cgccgggtgg ccccgcgtgg gcgctgtccc gttgccgtgg cacgccgccg 180

ccgccgggcg tggcgggggg cgcggccagc gcgctgtccg gccctgccgc gcaccgcgtg 240

ctcgccggga tttcgcgcgc cgtcgagggc ggcgcggcgg tgatgtcgct ctccgtcggc 300

ctcacccgcc tcgtcgtggc gagccggccg gagacggcga gggagatcct cgtcatgccc 360

ggcgttcggc gaccgccccg tgaaggacgc ggcgaggcag ctgctgttcc accgcgccat 420

ggggttcgcc ccgtcgggcg acgcgcactg gcgcgggctc cgccgcgcct ccgcggcgca 480

cctcttcggc ccgcgccgcg tggccgggtc cgcgcccgag cgcgaggcca tcggcgcccg 540

catagtcggc gacgtcgcct ccctcatgtc ccgccgcggc gaggtccccc tccgccgcgt 600

ccttcacgcc gcgtcgctcg gccacgtcat ggcgaccgtc ttcggcaagc ggcacggcga 660

catctcgatc caggacggcg agctcctgga ggagatggtc accgaagggt acgacctcct 720

cggcaagttc aactgggccg accacctgcc attgctcagg tggctcgacc tccagggcat 780

ccgccgccgg tgcaacaggc tagtccagaa ggtggaggtg ttcgtcggaa agatcataca 840

ggagcacaag gcgaagcgag ctgccggagg cgtcgccgtc gccgacggcg tcttgggcga 900

cttcgtcgac gtcctcctcg acctccaggg agaggagaag atgtcagact ccgacatgat 960

cgctgttctt tgggagatga tctttag 987

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> giant embryo gene GE mutant 66-5 coded amino acid sequence

<400> 4

Met Ala Leu Ser Ser Met Ala Ala Ala Gln Glu Ser Ser Leu Leu Leu

1 5 10 15

Phe Leu Leu Pro Thr Ser Ala Ala Ser Val Phe Pro Pro Leu Ile Ser

20 25 30

Val Val Val Leu Ala Ala Leu Leu Leu Trp Leu Ser Pro Gly Gly Pro

35 40 45

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

50 55 60

Ala Gly Gly Ala Ala Ser Ala Leu Ser Gly Pro Ala Ala His Arg Val

65 70 75 80

Leu Ala Gly Ile Ser Arg Ala Val Glu Gly Gly Ala Ala Val Met Ser

85 90 95

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

100 105 110

Ala Arg Glu Ile Leu Val Met Pro Gly Val Arg Arg Pro Pro Arg Glu

115 120 125

Gly Arg Gly Glu Ala Ala Ala Val Pro Pro Arg His Gly Val Arg Pro

130 135 140

Val Gly Arg Arg Ala Leu Ala Arg Ala Pro Pro Arg Leu Arg Gly Ala

145 150 155 160

Pro Leu Arg Pro Ala Pro Arg Gly Arg Val Arg Ala Arg Ala Arg Gly

165 170 175

His Arg Arg Pro His Ser Arg Arg Arg Arg Leu Pro His Val Pro Pro

180 185 190

Arg Arg Gly Pro Pro Pro Pro Arg Pro Ser Arg Arg Val Ala Arg Pro

195 200 205

Arg His Gly Asp Arg Leu Arg Gln Ala Ala Arg Arg His Leu Asp Pro

210 215 220

Gly Arg Arg Ala Pro Gly Gly Asp Gly His Arg Arg Val Arg Pro Pro

225 230 235 240

Arg Gln Val Gln Leu Gly Arg Pro Pro Ala Ile Ala Gln Val Ala Arg

245 250 255

Pro Pro Gly His Pro Pro Pro Val Gln Gln Ala Ser Pro Glu Gly Gly

260 265 270

Gly Val Arg Arg Lys Asp His Thr Gly Ala Gln Gly Glu Ala Ser Cys

275 280 285

Arg Arg Arg Arg Arg Arg Arg Arg Arg Leu Gly Arg Leu Arg Arg Arg

290 295 300

Pro Pro Arg Pro Pro Gly Arg Gly Glu Asp Val Arg Leu Arg His Asp

305 310 315 320

Arg Cys Ser Leu Gly Asp Asp Leu

325

<211> 381

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CDS sequence of giant embryo gene GE mutant 66-2

<400> 5

atggcgctct cctccatggc cgcggcgcaa gagagctccc tcctcctctt cctcctcccg 60

acgtcggccg cctccgtgtt cccgccgctc atctccgtgg tcgtcctcgc cgcgctcctc 120

ctgtggctct cgccgggtgg ccccgcgtgg gcgctgtccc gttgccgtgg cacgccgccg 180

ccgccgggcg tggcgggggg cgcggccagc gcgctgtccg gccctgccgc gcaccgcgtg 240

ctcgccggga tttcgcgcgc cgtcgagggc ggcgcggcgg tgatgtcgct ctccgtcggc 300

ctcacccgcc tcgtcgtggc gagccggccg gagacggcga gggagatcct cgtcgcccgg 360

cgttcggcga ccgccccgtg a 381

<211> 126

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence encoded by giant embryo gene GE mutant 66-2

<400> 6

Met Ala Leu Ser Ser Met Ala Ala Ala Gln Glu Ser Ser Leu Leu Leu

1 5 10 15

Phe Leu Leu Pro Thr Ser Ala Ala Ser Val Phe Pro Pro Leu Ile Ser

20 25 30

Val Val Val Leu Ala Ala Leu Leu Leu Trp Leu Ser Pro Gly Gly Pro

35 40 45

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

50 55 60

Ala Gly Gly Ala Ala Ser Ala Leu Ser Gly Pro Ala Ala His Arg Val

65 70 75 80

Leu Ala Gly Ile Ser Arg Ala Val Glu Gly Gly Ala Ala Val Met Ser

85 90 95

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

100 105 110

Ala Arg Glu Ile Leu Val Ala Arg Arg Ser Ala Thr Ala Pro

115 120 125

<211> 1578

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CDS sequence of giant embryo gene GE mutant 63-1

<400> 7