CN109439670B

CN109439670B - A kind of preparation method of the gene for improved seed size and quality and application

Info

Publication number: CN109439670B
Application number: CN201811611337.0A
Authority: CN
Inventors: 苏英华; 李英菊; 张宪省; 于洋; 姚汪劲松; 彭静
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2018-12-27
Filing date: 2018-12-27
Publication date: 2019-09-27
Anticipated expiration: 2038-12-27
Also published as: CN109913473A; CN109439670A; CN109913473B

Abstract

The invention discloses a kind of preparation method of gene for improved seed size and quality and applications.Present invention finds the gene M EE45 and BnaC04g36220D of being capable of improved seed size and quality.The DNA sequence dna of full length fragment of the present invention comprising said gene constructs over-express vector, then by MEE45 gene overexpression vector introduction agrobacterium strains, and infects arabidopsis floral with agrobacterium-mediated transformation, establishes transgenic arabidopsis strain.Transgenic line is compared by inventor after further cultivating with WT lines, and discovery is using the resulting transgenic line of the gene with foliage organ becomes larger, seed becomes larger, mass of 1000 kernel increases and fat content increases isophenous.The gene overexpression is applied to the Main Agronomic Characters such as production, the size of improved seed, mass of 1000 kernel and fat content, for improving the yield and quality of crop, there is important economic value and social benefit.

Description

A kind of preparation method of the gene for improved seed size and quality and application

Technical field

The present invention relates to plant genetic engineerings and technical field of cell biology, and in particular to one kind is big for improved seed The preparation method of small and quality gene and application.

Background technique

The seed size of plant be mark one of most important feature of yield (Alonso-Blanco C et al, PNAS, 1999,96:4710-4717；Moles AT et al, PNAS, 2005,102:10540-10544).In angiosperm, seed is logical It often include embryo, endosperm and kind skin, they are respectively from the development of fertilized eggs, the central cell being fertilized and integument.The nearly more than ten years Come, although people obtain certain progress, the regulation seed size of discovery in terms of the molecular mechanism for understanding control seed size Key gene and few.Following significant challenge is the further key gene for excavating regulation seed size, and is established perfect Molecular regulation network, to use modern biotechnology (genomics correlative study, gene editing, protein science, metabolism group With mathematics model foundation etc.) Crop Improvement provides important genetic resources.

Arabidopsis is a kind of crucifer, is widely used in Plant genetics, Developmental Biology and molecular biology Research, it has also become a kind of typical model plant.MEE45 (MATERNAL EFFECT EMBRYO ARREST 45), is quasi- Containing there are four the transcription factors of B3-domain in southern mustard.There is the research to other members of the gene family in the prior art, still Its research for primarily focusing on generation of the genoid in plant somatocyte embryo.However, MEE45 and improved seed size and product There is not been reported for the related functional analysis of matter.

Summary of the invention

For the above-mentioned prior art, the present invention is studied for a long period of time and is explored, it was found that being capable of improved seed size and quality Gene M EE45 and BnaC04g36220D.The DNA sequence dna of full length fragment of the present invention comprising said gene, which constructs, to be overexpressed Carrier then by MEE45 gene overexpression vector introduction agrobacterium strains, and infects arabidopsis floral with agrobacterium-mediated transformation, Establish transgenic arabidopsis strain.Transgenic line is compared by inventor after further cultivating with WT lines, hair Now using the resulting transgenic line of the gene with foliage organ becomes larger, seed becomes larger, mass of 1000 kernel increases and fat content increases Isophenous.The gene overexpression is applied to the important agronomy such as production, the size of improved seed, mass of 1000 kernel and fat content Shape has important economic value and social benefit for improving the yield and quality of crop.

Based on said gene, the first aspect of the present invention provides the gene for improved seed size and quality, is as follows 1) any gene in -4):

1) its nucleotide sequence is as shown in SEQ ID NO.1；

2) its nucleotide sequence is as shown in SEQ ID NO.3；

3) encoding gene for the albumen that the amino acid sequence shown in SEQ ID NO.2 forms；

4) encoding gene for the albumen that the amino acid sequence shown in SEQ ID NO.4 forms.

The second aspect of the present invention provides recombinant expression carrier, transgenic cell line or the gene work for carrying said gene Journey bacterium.

Wherein, the recombinant expression carrier for carrying MEE45 gene is as shown in Figure 1；Carry the recombination of BnaC04g36220D gene Expression vector is as shown in Figure 2.

Recombinant expression carrier, transgenic cell line or the genetic engineering bacterium of said gene are carried in improved seed size and product Application in matter is also protection scope of the present invention.

In above-mentioned application, the improved seed size and quality include: that foliage organ becomes larger, seed becomes larger, mass of 1000 kernel becomes larger Increase with fat content.

The third aspect of the present invention provides following a)-f) any one of described in DNA fragmentation in improved seed size and product Application in matter；

A) DNA fragmentation shown in SEQ ID NO.1；

B) DNA fragmentation of amino acid sequence shown in SEQ ID NO.2 is encoded；

C) DNA fragmentation has 75% or 75% or more identity, and the albumen encoded with DNA fragmentation a) or b) limited It is functionally of equal value with albumen shown in SEQ ID NO.2；

D) DNA fragmentation shown in SEQ ID NO.3；

E) DNA fragmentation of amino acid sequence shown in SEQ ID NO.4 is encoded；

F) DNA fragmentation has 75% or 75% or more identity, and the albumen encoded with DNA fragmentation d) or e) limited It is functionally of equal value with albumen shown in SEQ ID NO.4.

The fourth aspect of the present invention provides following 1) -6) any one of described in albumen in improved seed size and quality In application；

1) amino acid sequence is albumen shown in SEQ ID NO.2；

2) by amino acid sequence shown in SEQ ID NO.2 by one, several or dozens of amino acid replacements, deletion Or insertion obtain with albumen albumen with the same function shown in SEQ ID NO.2；

3) fusion protein that the N-terminal of the albumen shown in SEQ ID NO.2 and/or C-terminal connection label obtain；

4) amino acid sequence is albumen shown in SEQ ID NO.4；

5) by amino acid sequence shown in SEQ ID NO.4 by one, several or dozens of amino acid replacements, deletion Or insertion obtain with albumen albumen with the same function shown in SEQ ID NO.4；

6) fusion protein that the N-terminal of the albumen shown in SEQ ID NO.4 and/or C-terminal connection label obtain.

The fifth aspect of the present invention provides a kind of promotion seed and increases and/or the method for oil synthesis, including with as follows A)-f) described in any item polynucleotides conversion plant and the step of express that the polynucleotides in the plant；

A) DNA fragmentation shown in SEQ ID NO.1；

B) DNA fragmentation of amino acid sequence shown in SEQ ID NO.2 is encoded；

D) DNA fragmentation shown in SEQ ID NO.3；

E) DNA fragmentation of amino acid sequence shown in SEQ ID NO.4 is encoded；

In agricultural production, it is sometimes desirable to fat content is reduced in vegetable seeds to obtain more favorable character, such as In the soybean for production of albumen powder.Based on this, the sixth aspect of the present invention provides fat content in a kind of reduction seed Method, include generate make in the seed and plant containing the described in any item polynucleotides of following a)-f) it is described more The step of nucleotide expression is reduced or is not expressed；

A) DNA fragmentation shown in SEQ ID NO.1；

B) DNA fragmentation of amino acid sequence shown in SEQ ID NO.2 is encoded；

D) DNA fragmentation shown in SEQ ID NO.3；

E) DNA fragmentation of amino acid sequence shown in SEQ ID NO.4 is encoded；

F) DNA fragmentation has 75% or 75% or more identity, and the albumen encoded with DNA fragmentation d) or e) limited It is functionally of equal value with albumen shown in SEQ ID NO.4；

Or include to generate the seed and containing as following 1) -4) any one of described in albumen plant in make The step of protein active reduces or loses；

1) amino acid sequence is albumen shown in SEQ ID NO.2；

3) amino acid sequence is albumen shown in SEQ ID NO.4；

4) by amino acid sequence shown in SEQ ID NO.4 by one, several or dozens of amino acid replacements, deletion Or insertion obtain with albumen albumen with the same function shown in SEQ ID NO.4.

Preferably, the method for making the polynucleotides expression reduce or not express includes: mutation or the knockout multicore glycosides All or part of sequence of acid；Or the expression of the polynucleotides is interfered using RNA interfering；Or use gene silencing system Make the expression silencing of the polynucleotides.

The seventh aspect of the present invention provides a kind of method of genetically modified plants for cultivating seed size and quality-improving, is The described in any item polynucleotides of following a)-f) are imported into purpose plant and obtain the transgenosis plant of seed size and quality-improving Object；

A) DNA fragmentation shown in SEQ ID NO.1；

B) DNA fragmentation of amino acid sequence shown in SEQ ID NO.2 is encoded；

D) DNA fragmentation shown in SEQ ID NO.3；

E) DNA fragmentation of amino acid sequence shown in SEQ ID NO.4 is encoded；

At least one of in the above method, the seed size and quality-improving are following 1) -4):

1) seed of the genetically modified plants is greater than the purpose plant；

2) foliage organ of the genetically modified plants is greater than the purpose plant；

3) mass of 1000 kernel of the seed of the genetically modified plants is greater than the purpose plant；

4) fat content is greater than the purpose plant in the seed of the genetically modified plants.

Preferably, the purpose plant is dicotyledon or monocotyledon；The monocotyledon be selected from rice, Wheat or corn；The dicotyledon is selected from arabidopsis, soybean, rape, peanut or sunflower.

Beneficial effects of the present invention:

The present invention provides a kind of gene for improved seed size and quality: arabidopsis transcription factor gene for the first time The MEE45 and its homologous gene BnaC04g36220D in cabbage type rape, has using the resulting genetically modified plants of the gene Foliage organ, which becomes larger, seed becomes larger, mass of 1000 kernel becomes larger increases isophenous with fat content.The gene is applied to production, improvement kind The Main Agronomic Characters such as size, mass of 1000 kernel and the fat content of son have important warp for improving the yield and quality of crop Ji value and social benefit.

Detailed description of the invention

Fig. 1: plant expression vector pMEE45::MEE45-GFP carrier structure figure.

Fig. 2: plant expression vector 35S::BnaC04g36220D carrier structure figure.

Fig. 3: wild type (WT), mee45 mutant (mee45), mee45 recovery-type (pMEE45::MEE45mee45) and MEE45 is overexpressed in the comparison and wild type and mee45 mutant seeds of plant (pMEE45::MEE45) seed size and grain weight The measurement of fat content.

Fig. 4: wild type, mee45 mutant, mee45 recovery-type and MEE45 are overexpressed the embryonic development of plant different times Compare；

The reciprocal cross that Fig. 5: mee45 mutant and WT lines carry out Parent is tested；

The subcellular localization in ovule and embryo's expression pattern and albumen of Fig. 6: MEE45 gene；

Fig. 7: wild type, mee45 mutant and overexpression 35S::MEE45 seed Oil Content Analysis；

Fig. 8: it is overexpressed ratio of the cabbage type rape homologous gene BnaC04g36220D in mee45 mutant and wild type Compared with.

Specific embodiment

It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field The identical meanings of understanding.

As background technology part is introduced, the key gene of the regulation seed size of existing report is simultaneously few.Future Significant challenge be the further key gene for excavating regulation seed size.

The present inventor passes through long-term research and exploration, it was found that a kind of gene for improved seed size and quality, The gene is arabidopsis transcription factor gene MEE45, which guards containing there are four from arabidopsis gene group, the gene B3 structural domain belongs to LEC2 family, and as shown in SED ID NO.1, which is nucleotide sequence 1587bp；Amino acid sequence encodes 528 amino acid as shown in SED ID NO.2.With the overall length piece comprising the MEE45 gene The DNA sequence dna of section constructs over-express vector, then by MEE45 gene overexpression vector introduction agrobacterium strains, and uses Agrobacterium Mediated method infects arabidopsis floral, establishes transgenic arabidopsis strain.Inventor further cultivate after by transgenic line with WT lines are compared, discovery using the resulting transgenic line of the gene have foliage organ becomes larger, seed becomes larger, thousand Increase again and fat content increases isophenous.By the gene overexpression be applied to production, the size of improved seed, mass of 1000 kernel and The Main Agronomic Characters such as fat content have important economic value and social benefit for improving the yield and quality of crop.

By phylogenetic analysis and tetraploid rice, the present invention also have found in cabbage type rape with MEE45 affiliation Closer homology is up to 56% gene BnaC04g36220D, and the nucleotide sequence of the gene is as shown in SED ID NO.3, the base Because overall length is 1530bp, amino acid sequence encodes 509 amino acid as shown in SED ID NO.4.Research finds to be overexpressed BnaC04g36220D gene turns arabidopsis mee45 mutant, can restore the phenotype that mee45 seed becomes smaller；It is overexpressed BnaC04g36220D gene turns arabidopsis wild type, and transgenic plant seed becomes larger.Therefore, BnaC04g36220D gene can also To be used for improved seed size and quality.

MEE45 gene and BnaC04g36220D gene based on above-mentioned discovery, protection scope of the present invention further include with it is upper The DNA fragmentation of two DNA homologs is stated, as long as egg shown in albumen and SEQ ID NO.2 or SEQ ID NO.4 that they are encoded White function equivalence.It is as referred to herein to mean target " with the equivalence of protein function shown in SEQ ID NO.2 or SEQ ID NO.4 " The encoded albumen of DNA fragmentation biological function and in terms of with SEQ ID NO.2 or SEQ in the present invention Albumen shown in ID NO.4 is same or similar.The typical biology function of albumen shown in SEQ ID NO.2 or SEQ ID NO.4 Can be promote vegetable seeds increase, foliage organ increase, mass of 1000 kernel increase and seed in fat content improve.By raising SEQ ID The expression quantity and/or activity of albumen shown in NO.2 or SEQ ID NO.4, can be in improved seed size and quality.

These include nucleotide sequence of the present invention with the DNA fragmentation of MEE45 gene and BnaC04g36220D DNA homolog (SEQ ID NO.1, SEQ ID NO.3) corresponding allele, homologous gene, mutated gene and derivative gene；They are encoded Albumen be similar to albumen shown in SEQ ID NO.2 or SEQ ID NO.4 of the present invention, or there are one, several or dozens ofs Replacement, deletion or the insertion phenomenon of amino acid, belong to the content of present invention.

Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation Method is mutated the nucleotide sequence of MEE45 gene and BnaC04g36220D gene of the invention.Those are by manually repairing Decorations, have and the nucleotide sequence 75% or higher identity of MEE45 gene of the present invention and BnaC04g36220D gene Nucleotide is derived from as long as protein function shown in the albumen and SEQ ID NO.2 or SEQ ID NO.4 of coding is of equal value Nucleotide sequence of the invention and it is equal to sequence of the invention.

Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair Nucleotide sequence shown in bright SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5 have 75% or higher or 85% or Higher or 90% or higher or 95% or higher identity nucleotide sequence.The equivalent rate of amino acid or nucleotide sequence can (Altschul et al.1990.Journal of Molecular Biology 215:403- is measured using BLAST algorithm 410；Karlin and Altschul.1993.Proceedings of the National Academy of Sciences 90:5873-5877)。

Above-mentioned 75% or 75% or more identity can be 80%, 85%, 90% or 95% or more identity.

It in one embodiment of the invention, is from after fertilization three days silique genomes of the arabidopsis Col ecotype Amplification obtains the gene, the specific steps of which are as follows:

(1) CTAB method extracts arabidopsis fruit genome；

(2) clone of MEE45 gene

Using genomic DNA as template, PCR amplification is carried out using following primer pair:

Upstream primer: 5'-TAGGATCCATGGCGCATCAACATTTCTTCT-3', as shown in SEQ ID NO.5；

Downstream primer: 5'-TAGGTACCTCATTTGATTTCTAGCTTCACCTT-3', as shown in SEQ ID NO.6；

Wherein drawing horizontal line part is I restriction enzyme site of BamH I and Kpn；

PCR amplification system is 4 μ L upstream primers (50pmol/ μ L), 4 μ L downstream primers (50pmol/ μ L), 10 μ L 10 × PCR buffer, 4 μ L dNTP mixed liquors (10mmol/L), 4 μ L EVO archaeal dna polymerases (5U), 2 μ L cDNA templates add ddH₂Total volume is supplemented to 50 μ L by O；

Amplification condition are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 56 DEG C of annealing 15s, 72 DEG C of extension 1min, circulation 32 It is secondary；72 DEG C of extension 7min.

4 μ L PCR products are taken to connect with pEasy-Blunt Simple Vector carrier, operating procedure is according to TransGen Biotech Products pEasy-Blunt Simple Vector specification carries out.Then connection product converts Escherichia coli DH5 α competent cell, the overnight incubation on the LB solid medium containing ampicillin (100mg/L).Picking white bacterium It falls, the overnight incubation in the LB liquid medium containing ampicillin (100mg/L).Alkalinity extraction Plasmid DNA, digestion identification After carry out sequencing.For amplified production through sequencing analysis, sequence shows that amplified production is MEE45 as shown in SEQ ID NO.1 Gene.

Also there is the research of other members of the gene family in the prior art, but it primarily focuses on the genoid in plant The research of the generation of somatic embryo does not find that it has the function similar with the present invention, and inventor thinks this and the present invention Gene and existing gene function have biggish difference related.

After obtaining above-mentioned target gene, inventor constructs plant expression vector pMEE45::MEE45-GFP using it, The carrier is the MEE45 gene DNA fragment expanded using restriction enzyme BamH I and I digestion of Kpn, and is connected It is obtained in PROK2 empty expression vector；Inventor further converts Agrobacterium using the carrier obtained.

Last inventor utilizes agrobacterium mediation converted arabidopsis floral, and finally screens and obtain resistant plant Strain, and finally verifying finds the plant with foliage organ becomes larger, seed becomes larger (shown in Fig. 3) and fat content increases (Fig. 3 institute Show) isophenous.The gene is applied to production, the Main Agronomic Characters such as size, mass of 1000 kernel and fat content of improved seed are used In the yield and quality for improving crop, there is important economic value and social benefit.

In another embodiment of the present invention, the Cytological Basis that mee45 mutant and MEE45 are overexpressed is had studied, Specific step is as follows:

(1) using clarifier by plant tissue it is transparent after observation photograph to mee45 mutant, MEE45 be overexpressed and it is wild Ovule before type pollination, the embryogenesis after pollination carry out detailed comparisons' research (shown in Fig. 4), and research finds that mee45 is prominent Variant ovule and embryo become smaller, and seed becomes smaller；Opposite overexpression plant pMEE45::MEE45 ovule and embryo become larger, and seed becomes Greatly.

(2) reciprocal cross that mee45 mutant and WT lines carry out Parent is tested, observes the embryo of filial generation Pearl, embryo and endosperm phenotype, analysis MEE45 whether pass through maternal tissue or zygote tissue modulation seed size development (Fig. 5 institute Show), research shows that MEE45 is to control seed size by matrilinear inheritance.

In another embodiment of the invention, the expression pattern of MEE45 gene and the subcellular localization of albumen are had studied, It is specific as follows:

PMEE45::MEE45-GFP fusion expression vector and transformed plant are constructed, the expression pattern of MEE45 albumen is carried out It analyzes (shown in Fig. 6), research shows that MEE45 is expressed in ovule and embryo, station-keeping mode is nuclear location.

In another embodiment of the invention, MEE45 gene regulation fat content is studied, specific as follows:

Inventor has surveyed wild type Col, mutant mee45 and has been overexpressed the content of total grease in 35S::MEE45 seed, It was found that the content of grease is reduced compared to wild type in mutant, and the content of grease is risen (Fig. 7 institute in being overexpressed Show).

In another embodiment of the invention, the cabbage type rape homologous gene BnaC04g36220D of MEE45 is carried out Research, specific as follows:

By phylogenetic analysis and tetraploid rice, have found closer same with MEE45 affiliation in cabbage type rape The gene cloning is come out up to 56% gene BnaC04g36220D and turns arabidopsis mee45 mutant and wild type by source property, is observed Whether the development (Fig. 8 shown in) of seed size is influenced, and research finds that being overexpressed BnaC04g36220D gene turns arabidopsis mee45 Mutant can restore the phenotype that mee45 seed becomes smaller；It is overexpressed BnaC04g36220D gene and turns arabidopsis wild type, turn base Because plant seed becomes larger.We are also converting the cabbage type rape of the gene overexpression, observe after obtaining transgenic plant The variation of the size and quality of seed.For its nucleotide sequence as shown in SED ID NO.3, which is 1530bp, amino Acid sequence encodes 510 amino acid as shown in SED ID NO.4.

The gene is expanded from cabbage type rape 10 days seedling spire cDNA and is obtained, the specific steps of which are as follows:

1. the extraction and purifying of rape RNA

The synthesis of the first chain of 2.cDNA

The clone of 3.BnaC04g36220D gene

Using cDNA as template, PCR amplification is carried out using following primer pair:

Upstream primer: 5'-TAGGATCCATGGTGAACAAACGTTTCTTCAAGC-3', as shown in SEQ ID NO.7；

Downstream primer: 5'-TAGAGCTCTCATTCGGTCTCATGCTTTACCTT-3', as shown in SEQ ID NO.8；

Wherein drawing horizontal line part is I restriction enzyme site of BamH I and Sac；

4 μ L PCR products are taken to connect with pEasy-Blunt Simple Vector carrier, operating procedure is according to TransGen Biotech Products pEasy-Blunt Simple Vector specification carries out.Then connection product converts Escherichia coli DH5 α competent cell, the overnight incubation on the LB solid medium containing ampicillin (100mg/L).Picking white bacterium It falls, the overnight incubation in the LB liquid medium containing ampicillin (100mg/L).Alkalinity extraction Plasmid DNA, digestion identification After carry out sequencing.For amplified production through sequencing analysis, sequence shows that amplified production is as shown in SEQ ID NO.3 BnaC04g36220D gene.

In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool The technical solution of the application is described in detail in the embodiment of body.

Test material used in the embodiment of the present invention and comparative example is the test material of this field routine, can be passed through Commercial channel is commercially available.The experimental method that detailed conditions are not specified is according to conventional methods or according to proposed by supplier Operational manual carry out.

Embodiment 1, MEE45 and BnaC04g36220D gene cloning and plant expression vector construction

The extraction and purifying of 1.1 arabidopsis RNA

The fresh tender silique of arabidopsis children is ground in liquid nitrogen, and ground sample is moved into 15mL CTAB extracting solution rapidly In (2% (W/V) CTAB, 2% (W/V) PVP, 100mM Tris-HCl (pH8.0), 25mM EDTA, 2.0M NaCl, 2% (W/ V) beta -mercaptoethanol, 0.5g/L spermidine), be vortexed concussion 30s immediately, in 65 DEG C of water-bath a moments, adds isometric with CTAB extracting solution Chloroform: the mixture of isoamyl alcohol, wherein chloroform: the volume ratio of isoamyl alcohol is 24:1, and oscillation mixes；At room temperature 10000rpm from The heart 15 minutes, supernatant was shifted into another clean pipe, is added and the isometric chloroform of supernatant: iso pentane alcohol mixture (volume Than 24:1) standing is mixed, it repeats to extract once, 10000rpm is centrifuged 15 minutes at room temperature, and transfer water phase is into another centrifuge tube. 8M LiCl is added according to liquor capacity, makes the final concentration of 2M of LiCl, 4 DEG C of overnight precipitations (most 16 hours).4 DEG C are centrifuged 20 points Clock removes supernatant, is rinsed and is precipitated with 500 μ L, 70% ethyl alcohol, with 500 μ L SSTE liquid (1.0M NaCl, 0.5% (W/V) SDS, 10mM Tris-HCl (pH8.0), 1mM EDTA (pH8.0)) dissolution precipitating, it transfers the solution into 1.5mL centrifuge tube, Isometric chloroform: iso pentane alcohol mixture (volume ratio 24:1) is added, then extracts primary.The second of 2 times of volumes is added in supernatant Alcohol, -70 DEG C precipitate 2 hours for precipitating 30 minutes or -20 DEG C, and 4 DEG C of 12000rpm are centrifuged 20 minutes, DNA are precipitated, first with 400 μ L 70% ethyl alcohol rinsing precipitating adds 400 μ L, 100% ethyl alcohol rinsing precipitating, after drying precipitated, with 50 μ L DEPC ddH₂O is molten Solution precipitating.

It is required to ensure that RNA mass reaches sequencing, respectively using spectrophotometer and agarose gel electrophoresis detection purifying RNA sample purity and concentration, moderate purity and concentration standard afterwards are as follows: RNA purity is that OD260/280 and OD260/230 exists Within the scope of 1.8-2.0, RNA concentration is within the scope of 1.0-2.0 μ g/ μ L.

The synthesis of the first chain of 1.2cDNA

It is total that 1 μ L 50 μM of Oligo dT Primer and 15.5 μ L are sequentially added in the centrifuge tube of 0.2mL DEPC processing RNA, 70 DEG C are denaturalized 6 minutes, rapid ice bath 10 minutes.2 μ L dNTP Mixture are sequentially added in above-mentioned centrifuge tube (10mM), 5 μ L 5 × Primer Script Buffer, 0.5 μ L RNase Inhibitor (40U), 1 μ L Primer Script RTase (200U), adds DEPC-H₂O to 25 μ L.It is 25 DEG C, 10 minutes that program is run in PCR instrument；42 DEG C, 90 points Clock；95 DEG C, 5 minutes.After EP (end of program), sample freezes for use in -80 DEG C.

1.3MEE45 the clone with BnaC04g36220D gene

Using the cDNA of reverse transcription as template, PCR amplification is carried out using following primer pair:

MEE45 carries out PCR amplification using genome as template, using following primer pair:

Amplification condition are as follows: 94 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 56 DEG C of annealing 15s, 72 DEG C of extension 1min, circulation 32 It is secondary；72 DEG C of extension 7min.

Cabbage type rape gene carries out PCR amplification using cDNA as template, using following primer pair:

PCR amplification system be 4 μ L upstream primers (50pmol/ μ L), 4 μ L downstream primers (50pmol/ μ L), 10 μ L10 × PCR buffer, 4 μ L dNTP mixed liquors (10mmol/L), 4 μ L EVO archaeal dna polymerases (5U), 2 μ L cDNA templates add ddH₂Total volume is supplemented to 50 μ L by O；

Amplification condition are as follows: 95 DEG C of denaturation 3min；95 DEG C of denaturation 15s, 56 DEG C of annealing 15s, 72 DEG C of extension 1min, circulation 32 It is secondary；72 DEG C of extension 7min.

The acquisition of 1.4 plant expression vector pMEE45::MEE45-GFP and 35S::BnaC04g36220D genes

MEE45DNA segment is cut with restriction enzyme BamH I and Kpn I.With restriction enzyme BamH I and Kpn I II empty expression vector of PROK is cut, the two passes through electrophoresis detection and recycles target fragment.After connecting digestion using T4 ligase Segment, operating procedure are carried out according to Fermentas Products T4DNA ligase specification.Then connection product converts large intestine Bacillus DH5 α competent cell, the overnight incubation on the LB solid medium of (50mg/L) containing kanamycin.Picking white bacterium It falls, the overnight incubation in the LB liquid medium containing spectinomycin (50mg/L).Alkalinity extraction Plasmid DNA simultaneously carries out digestion mirror It is fixed.Digestion is identified correct expression vector pMEE45::MEE45-GFP conversion Agrobacterium GV3101 and obtained to be made for conversion Agrobacterium strains.The present invention converts Agrobacterium using electric shocking method.

BnaC04g36220D DNA fragmentation is cut with restriction enzyme BamH I and Sac I.With restriction enzyme BamH I and Sac I cuts II empty expression vector of PROK, and the two passes through electrophoresis detection and recycles target fragment.It is connected using T4 ligase Segment after digestion, operating procedure are carried out according to Fermentas Products T4DNA ligase specification.Then connection product Convert bacillus coli DH 5 alpha competent cell, the overnight incubation on the LB solid medium of (50mg/L) containing kanamycin.It chooses Extracting waste bacterium colony, the overnight incubation in the LB liquid medium containing spectinomycin (50mg/L).Alkalinity extraction Plasmid DNA is gone forward side by side Row digestion identification.Correct expression vector 35S::BnaC04g36220D conversion Agrobacterium GV3101 is identified into digestion and acquisition can For converting the agrobacterium strains used.

Embodiment 2, the conversion of the arabidopsis floral of mediated by agriculture bacillus and the acquisition of resistant plant

Dip dyeing the previous day picking Agrobacterium (the Agrobacterium single colonie for carrying recombinant plasmid) be inoculated in containing 50mg/L card that In the YEP culture medium of mycin, 28 DEG C, 220rpm shakes bacterium and stays overnight, and precipitates within second day and is resuspended early in the morning in pre- dip dyeing culture medium.So Disseminate the arabidopsis floral tissue bloomed afterwards, inflorescence group, which is woven in dip dyeing liquid for shell, disseminates 15s, then dark culture for 24 hours, then 16h illumination 8h dark culturing grows into arabidopsis floral and grows into arabidopsis silique maturation, all seeds has finally been received, for sieving Select positive plant.Because the plant resistance that II empty carrier of PROK has is to block that resistance, with the GM training containing that resistance of card It supports base and screens seed, what can be survived is possible to design one end from gene for the sake of rigorous for positive plant and draw Object designs other end primer from carrier framework, with this pair of of nested primers screening-gene group, can show that correct band It is positive plant.

The nested primers sequence of pMEE45::MEE45-GFP and 35S::BnaC04g36220D positive plant is as follows:

MEE45 upstream primer: 5'-TAGGATCCATGGCGCATCAACATTTCTTCT-3', as shown in SEQ ID NO.9；

BnaC04g36220D upstream primer: 5'-TAGGATCCATGGTGAACAAACGTTTCTTCAAGC-3', such as SEQ Shown in ID NO.10；

Downstream primer sequence: 5'-CAAGAGTCCACTATTAAAGAACGTGG-3', as shown in SEQ ID NO.11；(two A gene can share).

The phenotypic analysis of embodiment 3, mutant and positive transgenic plant

The seed size of mutant and positive transgenic plant and WT lines maturation is measured, mass of 1000 kernel and length and width are counted (Fig. 3)；And it observes ovule and Seed development and measures the content of grease in seed.As seen from Figure 3, mee45 is mutated Body seed becomes smaller compared to wild type seeds, and mass of 1000 kernel reduces；The opposite plant pMEE45::MEE45-GFP seed that is overexpressed becomes larger, Mass of 1000 kernel increases.As seen from Figure 4, mee45 mutant ovule and embryo become smaller, and fat content reduces；It is opposite to be overexpressed The ovule and embryo of pMEE45::MEE45-GFP plant become larger.

The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.

SEQUENCE LISTING

<110>Shandong Agricultural University

<120>a kind of preparation method of gene for improved seed size and quality and application

<130> 2018

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 1587

<212> DNA

<213>MEE45 gene

<400> 1

atggcgcatc aacatttctt caagcctctt cttcctggct tccacgcctc cttgacaatt 60

cctgtagcct tcttcttgaa gtatatagaa ggaagatatg agcagaagac ggcgaagctg 120

agatcagacg cgtcaaagag aacttgggaa gtgaagatag atggccagag actcaccgac 180

ggttggaaag agtttgctgt ctcacatgat cttcgaatcg gtgacattgt tgttttcaga 240

caagagagtg acttggcttt ccatgtaaca ctgttgggac ctagttgttg tgggattcaa 300

tatggttcgt gttcagtcga aaagaacaac ctcggtgacg agaaaaagaa agtgaaggag 360

aatccaaatg gagaagcaga gtcttcttca cgagatccct cttgttttgt ggctaatgtt 420

gcgccttcga gtctacgtta tgacttgatg agatttccaa ggggttttgt gagggataat 480

ggtgtagtcg gatctggaga gattgttctg atgaatgaaa agggcagatc atggaatttt 540

aacttgagac aaaagccatc aaacggaaca gtttatgtta gaggagggtg ggtgagtttt 600

tgtgatgcca atgggcttaa agctggagat aactacactt tcaaactgat caaaagagca 660

ggaactcttg ttctacgttt gttacccaat gagccaaaag aggaagctaa tgaagtgtct 720

cttcccgaag aaccggaaag cgatgcagag cgcaaccttg aaaagattca aaggaaggag 780

aaagtgaaga agaatgtaac aagagaggca gagtcttctt cacaagatcc ctcttgtttt 840

gtggctaatg tctccccttc gagtctacgc tatgacacac tgtatcttcc aaagcgtttt 900

atgagggaaa atggtgtaga caaaagatgt ggagagatga ttctgattaa tgaaaaggga 960

aaatcatgga ctttagattt gaaagtaaag aaatcatccg gaacttctct catcaaacga 1020

ggatggagaa gtttctgtag tgccaatgga ctaagagctg gaagtatcat aactctcaaa 1080

ctgataaaga aaagagcaac tcttgttcta cgtttgatcc ccaacgagcc agaagaagct 1140

aatgaagtag tctctctttc gacagagcaa gaaagcgatg aagagagtat ccacgacgag 1200

aaaatctcaa gaagaaagtc tttactatcc gaaaaccgat ttgtgacatt aactctaaca 1260

ccttatacaa tccaaagttc tctactgaat gagaatcttt tgtgtgaatc tatgtttcag 1320

cgtcttccgg ttcctttcac gaggatgaat ggtatcaatg aagaaactaa aatgactctg 1380

ttggataaac atggtgtgaa gtggttaacg actctgcggt tcgaggacga caaaagaaaa 1440

agactacgaa tggtaggagg atggcaagga ttcatccaag ctaacgatgt gaaggcaaac 1500

gaatccatca tgttggaact gatttgggaa gaagaaacaa gttgcgtcct taagttctgc 1560

tccaaggtga agctagaaat caaatga 1587

<210> 2

<211> 528

<212> PRT

<213>MEE45 gene coded protein

<400> 2

Met Ala His Gln His Phe Phe Lys Pro Leu Leu Pro Gly Phe His Ala

1 5 10 15

Ser Leu Thr Ile Pro Val Ala Phe Phe Leu Lys Tyr Ile Glu Gly Arg

20 25 30

Tyr Glu Gln Lys Thr Ala Lys Leu Arg Ser Asp Ala Ser Lys Arg Thr

35 40 45

Trp Glu Val Lys Ile Asp Gly Gln Arg Leu Thr Asp Gly Trp Lys Glu

50 55 60

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

65 70 75 80

Gln Glu Ser Asp Leu Ala Phe His Val Thr Leu Leu Gly Pro Ser Cys

85 90 95

Cys Gly Ile Gln Tyr Gly Ser Cys Ser Val Glu Lys Asn Asn Leu Gly

100 105 110

Asp Glu Lys Lys Lys Val Lys Glu Asn Pro Asn Gly Glu Ala Glu Ser

115 120 125

Ser Ser Arg Asp Pro Ser Cys Phe Val Ala Asn Val Ala Pro Ser Ser

130 135 140

Leu Arg Tyr Asp Leu Met Arg Phe Pro Arg Gly Phe Val Arg Asp Asn

145 150 155 160

Gly Val Val Gly Ser Gly Glu Ile Val Leu Met Asn Glu Lys Gly Arg

165 170 175

Ser Trp Asn Phe Asn Leu Arg Gln Lys Pro Ser Asn Gly Thr Val Tyr

180 185 190

Val Arg Gly Gly Trp Val Ser Phe Cys Asp Ala Asn Gly Leu Lys Ala

195 200 205

Gly Asp Asn Tyr Thr Phe Lys Leu Ile Lys Arg Ala Gly Thr Leu Val

210 215 220

Leu Arg Leu Leu Pro Asn Glu Pro Lys Glu Glu Ala Asn Glu Val Ser

225 230 235 240

Leu Pro Glu Glu Pro Glu Ser Asp Ala Glu Arg Asn Leu Glu Lys Ile

245 250 255

Gln Arg Lys Glu Lys Val Lys Lys Asn Val Thr Arg Glu Ala Glu Ser

260 265 270

Ser Ser Gln Asp Pro Ser Cys Phe Val Ala Asn Val Ser Pro Ser Ser

275 280 285

Leu Arg Tyr Asp Thr Leu Tyr Leu Pro Lys Arg Phe Met Arg Glu Asn

290 295 300

Gly Val Asp Lys Arg Cys Gly Glu Met Ile Leu Ile Asn Glu Lys Gly

305 310 315 320

Lys Ser Trp Thr Leu Asp Leu Lys Val Lys Lys Ser Ser Gly Thr Ser

325 330 335

Leu Ile Lys Arg Gly Trp Arg Ser Phe Cys Ser Ala Asn Gly Leu Arg

340 345 350

Ala Gly Ser Ile Ile Thr Leu Lys Leu Ile Lys Lys Arg Ala Thr Leu

355 360 365

Val Leu Arg Leu Ile Pro Asn Glu Pro Glu Glu Ala Asn Glu Val Val

370 375 380

Ser Leu Ser Thr Glu Gln Glu Ser Asp Glu Glu Ser Ile His Asp Glu

385 390 395 400

Lys Ile Ser Arg Arg Lys Ser Leu Leu Ser Glu Asn Arg Phe Val Thr

405 410 415

Leu Thr Leu Thr Pro Tyr Thr Ile Gln Ser Ser Leu Leu Asn Glu Asn

420 425 430

Leu Leu Cys Glu Ser Met Phe Gln Arg Leu Pro Val Pro Phe Thr Arg

435 440 445

Met Asn Gly Ile Asn Glu Glu Thr Lys Met Thr Leu Leu Asp Lys His

450 455 460

Gly Val Lys Trp Leu Thr Thr Leu Arg Phe Glu Asp Asp Lys Arg Lys

465 470 475 480

Arg Leu Arg Met Val Gly Gly Trp Gln Gly Phe Ile Gln Ala Asn Asp

485 490 495

Val Lys Ala Asn Glu Ser Ile Met Leu Glu Leu Ile Trp Glu Glu Glu

500 505 510

Thr Ser Cys Val Leu Lys Phe Cys Ser Lys Val Lys Leu Glu Ile Lys

515 520 525

<210> 3

<211> 1530

<212> DNA

<213>BnaC04g36220D gene

<400> 3

atggtgaaca aacgtttctt caagcctctt cttcctggct tccacagcca cttgacaatt 60

cctgtagcct tcttcgtcaa gtatatagaa ggaaaaaacg agcaccatac gacgaagcta 120

agatcagacg cgtcaaagat aacctgggaa gtgaaaatag aagatggcca gaaactcact 180

gacggttgga aagagttcgc tcttgcacac gatcttcgta tcggcgacat tctcattttc 240

aagcaagaga aagacatggc tttccacgta acactcttgg gacccagtgg ctgtgagatt 300

caatatgagt cgtgttcaga agaagagaac aacttcggga atattccaaa gaagaagaat 360

tcaaaaagag aagcagagtc ttcttcacta gatccttctt gtttcttggc taatatctgg 420

ccttcgtcct tacgctatga ctcattgaac cttccaagga gttttgtgag ggcaaatggt 480

ctagagacaa gatgtggaag agagatcgtt ctgatcaatg aaaagggtaa atcatggact 540

ttggctttaa aacaaaagct atctggacct acttacatca gacgagggtg gagaagtttc 600

tgtattgcca atggtcttaa aactggaggc gtctacactt tcaaactaat caagagaggg 660

agagctccgg ttcttcgttt gtcctccaca gagtcagagt tagaagagag aaacatcgag 720

aagattcaga ggaacaaagc agagtcttcc tcactaaatc cctcttgttt tgtggctaat 780

atctcgcgtg caaccctacg ttatgacaca ctgggtcttc caatgaaatt ttcaagggaa 840

aatggtctag aggcaagatg tggagagatt gttctaatga atgaaaaggg tagatcgtgg 900

aagctaaatc tgaaacgaaa gagatcatgc ggaactatgt atatcacaca agggtggagg 960

agtttctgta gtgcaaatgg acttagagct ggaagttctt ccactttcaa actgatcaaa 1020

agaggaggaa ctctggctct acgtttgtca tctaaagaga ctgaagaaga agaagaagat 1080

tgctcattaa aagctaatga agtggagtct ctttccacag aaccagaaag cgatgaagag 1140

gggagccaag atgagaaaca aatcaagaag catagatcga catggaaagc ttcatcttca 1200

caatcccaaa accgatttgt gacacttact tttagacctt tcaatcttga aaagtattta 1260

ctgtttcttc ctttacgctt caccaggtgg cacggcatca atgaagaaac taaaatgaga 1320

ctgttggaca aaaacggtgt caagtggtct acggatctgc ggtctgggaa aactaatatt 1380

gataaaataa gattggtagg aggttggcaa gaattcttca aagctaactg tgtgaagcca 1440

ggtgaatcta tcattgtgaa gctgatatgg gatggagaca aaagttgtat cctcaagttc 1500

tgctctaagg taaagcatga gaccgaatga 1530

<210> 4

<211> 509

<212> PRT

<213>BnaC04g36220D gene coded protein

<400> 4

Met Val Asn Lys Arg Phe Phe Lys Pro Leu Leu Pro Gly Phe His Ser

1 5 10 15

His Leu Thr Ile Pro Val Ala Phe Phe Val Lys Tyr Ile Glu Gly Lys

20 25 30

Asn Glu His His Thr Thr Lys Leu Arg Ser Asp Ala Ser Lys Ile Thr

35 40 45

Trp Glu Val Lys Ile Glu Asp Gly Gln Lys Leu Thr Asp Gly Trp Lys

50 55 60

Glu Phe Ala Leu Ala His Asp Leu Arg Ile Gly Asp Ile Leu Ile Phe

65 70 75 80

Lys Gln Glu Lys Asp Met Ala Phe His Val Thr Leu Leu Gly Pro Ser

85 90 95

Gly Cys Glu Ile Gln Tyr Glu Ser Cys Ser Glu Glu Glu Asn Asn Phe

100 105 110

Gly Asn Ile Pro Lys Lys Lys Asn Ser Lys Arg Glu Ala Glu Ser Ser

115 120 125

Ser Leu Asp Pro Ser Cys Phe Leu Ala Asn Ile Trp Pro Ser Ser Leu

130 135 140

Arg Tyr Asp Ser Leu Asn Leu Pro Arg Ser Phe Val Arg Ala Asn Gly

145 150 155 160

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

165 170 175

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

180 185 190

Ile Arg Arg Gly Trp Arg Ser Phe Cys Ile Ala Asn Gly Leu Lys Thr

195 200 205

Gly Gly Val Tyr Thr Phe Lys Leu Ile Lys Arg Gly Arg Ala Pro Val

210 215 220

Leu Arg Leu Ser Ser Thr Glu Ser Glu Leu Glu Glu Arg Asn Ile Glu

225 230 235 240

Lys Ile Gln Arg Asn Lys Ala Glu Ser Ser Ser Leu Asn Pro Ser Cys

245 250 255

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

260 265 270

Leu Pro Met Lys Phe Ser Arg Glu Asn Gly Leu Glu Ala Arg Cys Gly

275 280 285

Glu Ile Val Leu Met Asn Glu Lys Gly Arg Ser Trp Lys Leu Asn Leu

290 295 300

Lys Arg Lys Arg Ser Cys Gly Thr Met Tyr Ile Thr Gln Gly Trp Arg

305 310 315 320

Ser Phe Cys Ser Ala Asn Gly Leu Arg Ala Gly Ser Ser Ser Thr Phe

325 330 335

Lys Leu Ile Lys Arg Gly Gly Thr Leu Ala Leu Arg Leu Ser Ser Lys

340 345 350

Glu Thr Glu Glu Glu Glu Glu Asp Cys Ser Leu Lys Ala Asn Glu Val

355 360 365

Glu Ser Leu Ser Thr Glu Pro Glu Ser Asp Glu Glu Gly Ser Gln Asp

370 375 380

Glu Lys Gln Ile Lys Lys His Arg Ser Thr Trp Lys Ala Ser Ser Ser

385 390 395 400

Gln Ser Gln Asn Arg Phe Val Thr Leu Thr Phe Arg Pro Phe Asn Leu

405 410 415

Glu Lys Tyr Leu Leu Phe Leu Pro Leu Arg Phe Thr Arg Trp His Gly

420 425 430

Ile Asn Glu Glu Thr Lys Met Arg Leu Leu Asp Lys Asn Gly Val Lys

435 440 445

Trp Ser Thr Asp Leu Arg Ser Gly Lys Thr Asn Ile Asp Lys Ile Arg

450 455 460

Leu Val Gly Gly Trp Gln Glu Phe Phe Lys Ala Asn Cys Val Lys Pro

465 470 475 480

Gly Glu Ser Ile Ile Val Lys Leu Ile Trp Asp Gly Asp Lys Ser Cys

485 490 495

Ile Leu Lys Phe Cys Ser Lys Val Lys His Glu Thr Glu

500 505

<210> 5

<211> 30

<212> DNA

<213>artificial sequence

<400> 5

taggatccat ggcgcatcaa catttcttct 30

<210> 6

<211> 32

<212> DNA

<213>artificial sequence

<400> 6

taggtacctc atttgatttc tagcttcacc tt 32

<210> 7

<211> 33

<212> DNA

<213>artificial sequence

<400> 7

taggatccat ggtgaacaaa cgtttcttca agc 33

<210> 8

<211> 32

<212> DNA

<213>artificial sequence

<400> 8

tagagctctc attcggtctc atgctttacc tt 32

<210> 9

<211> 30

<212> DNA

<213>artificial sequence

<400> 9

taggatccat ggcgcatcaa catttcttct 30

<210> 10

<211> 33

<212> DNA

<213>artificial sequence

<400> 10

taggatccat ggtgaacaaa cgtttcttca agc 33

<210> 11

<211> 26

<212> DNA

<213>artificial sequence

<400> 11

caagagtcca ctattaaaga acgtgg 26

Claims

1. following a)-b) any one of described in application of the DNA fragmentation in improved seed size and quality；

A) DNA fragmentation shown in SEQ ID NO.1；

B) DNA fragmentation of amino acid sequence shown in SEQ ID NO.2 is encoded；

The improved seed quality specifically: improve the fat content in seed.

2. following 1) -2) application of the albumen described in any one of in improved seed size and quality；

1) amino acid sequence is albumen shown in SEQ ID NO.2；

2) fusion protein that the N-terminal of the albumen shown in SEQ ID NO.2 and/or C-terminal connection label obtain；

The improved seed quality specifically: improve the fat content in seed.

3. a kind of promote seed to increase and/or the method for oil synthesis, which is characterized in that including with any one of following a)-b) institute The step of polynucleotides stated convert plant and express that the polynucleotides in the plant；

A) DNA fragmentation shown in SEQ ID NO.1；

B) DNA fragmentation of amino acid sequence shown in SEQ ID NO.2 is encoded.

4. a kind of method for reducing fat content in seed, which is characterized in that be included in and generate the seed and containing as follows A)-b) described in any item polynucleotides plant in the step of making the polynucleotides expression reduce or not express；

A) DNA fragmentation shown in SEQ ID NO.1；

B) DNA fragmentation of amino acid sequence shown in SEQ ID NO.2 is encoded；

It or include making the protein in the plant for generating the seed and containing the albumen as described in SEQ ID NO.2 The step of activity reduces or loses.

5. according to the method described in claim 4, it is characterized in that, the method for making the polynucleotides expression reduce or not express It include: mutation or all or part of sequence for knocking out the polynucleotides；Or the polynucleotides are interfered using RNA interfering Expression；Or make the expression silencing of the polynucleotides using gene silencing system.

6. a kind of method for the genetically modified plants for cultivating seed size and quality-improving, which is characterized in that be to appoint following a)-b) Polynucleotides described in one import purpose plant and obtain the genetically modified plants of seed size and quality-improving；

A) DNA fragmentation shown in SEQ ID NO.1；

B) DNA fragmentation of amino acid sequence shown in SEQ ID NO.2 is encoded.

7. according to the method described in claim 6, it is characterized in that, the seed size and quality-improving are following 1) -3) in At least one:

1) seed of the genetically modified plants is greater than the purpose plant；

2) mass of 1000 kernel of the seed of the genetically modified plants is greater than the purpose plant；

3) fat content is greater than the purpose plant in the seed of the genetically modified plants.