CN105504031B - From the grain weight GAP-associated protein GAP and its relevant biological material of soybean and application - Google Patents
From the grain weight GAP-associated protein GAP and its relevant biological material of soybean and application Download PDFInfo
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Abstract
The invention discloses the grain weight GAP-associated protein GAP for deriving from soybean and its relevant biological material and applications.Protein name provided by the present invention is GmHSFA2, and be following protein a) or b): a) amino acid sequence is the protein of SEQ ID No.2；B) by amino acid sequence shown in SEQ ID No.2 in sequence table by replacing and/or being deleted and/or added one or several amino acid residues and the protein as derived from a) relevant to phanerogamous grain weight.The experiment proves that GmHSFA2 and biomaterial relevant to GmHSFA2 can be used for regulating and controlling phanerogamous grain weight, can be especially useful for improving phanerogamous grain weight.
Description
Technical field
The present invention relates to the grains that soybean is derived from field of biotechnology to weigh GAP-associated protein GAP and its relevant biological material and answer
With.
Background technique
Soybean is important traditional crop, contains nutrition abundant, is to provide the Important Economic of edible oil and vegetable protein
Crop.Soybean oil also has many purposes in the industrial production, such as bio-fuel, surfactant, softening agent etc..Simultaneously
Soybean is the very friendly crop of a kind of pair of environment, passes through rhizobium in atmosphere with 100 kilograms of per hectare of efficiency every year
Nitrogen is transformed among soil, and plant can be absorbed, and is suitably applied in the Agricultural Sustainable Developments mould such as crop rotation, continuous cropping and interplanting
In formula.Although soybean only accounts for the one third of demand originating from China, domestic soybean yields, China is caused to become the whole world most
Big Soybean import state, total import value account for the half of global soybean general export volume.In the past 50 years, Soybean production only increases
62%, and other cereal crops have increased 5 times or more, the production of soybean lags far behind domestic other cereal crops production
Paces, have been unable to meet national demand, therefore improve soybean per unit area yield and have become current urgent problem to be solved.
The weight (grain weight) of seed is an important indicator in crop production, is the important agronomy for influencing crop yield
One of shape, plant can achieve the purpose that volume increase again by increasing seed grain.Mass of 1000 kernel is 1,000 seeds in grams
Weight, it be embody seed size and turgor a Xiang Zhibiao, be the content examined the quality of seeds with crop species test,
Important evidence when being field forecast production.It is to count three 1,000 seeds at random when general measurement small-sized seed mass of 1000 kernel,
It weighs, is averaged respectively.100-grain weight is the weight of 100 seeds in grams, such as beautiful commonly used in large seed
Rice, soybean, peanut, cotton etc..
Soybean yields is made of factors such as plant type, Pod Bearing Percentage, pod grain number, hundred grain weights, wherein grain be again influence power most
High factor.Grain is not limited to leguminous plant to the influence of yield again, also has important influence to other single dicotyledons, because
This becomes important selection traits in need of consideration in crop varieties Breeding Process.Existing research shows that seed grain is cultivated again
The influence of environment and genetics.Under normal cultivation condition, heredity namely related gene play an important role, it is considered that, seed
Grain is quantitative character again, is determined by multiple genes, therefore the research of molecular mechanism relevant to grain weight becomes hot spot.
Summary of the invention
The technical problem to be solved by the present invention is to how improve phanerogamous grain weight.
In order to solve the above technical problems, present invention firstly provides seed plant grain weight GAP-associated protein GAPs.
Seed plant grain weight GAP-associated protein GAP provided by the present invention, entitled GmHSFA2 derive from soybean (Glycine
Max), it is following protein a) or b):
A) amino acid sequence protein as shown in SEQ ID No.2；
B) by amino acid sequence shown in SEQ ID No.2 in sequence table by replacing and/or being deleted and/or added one
Or several amino acid residues and to the relevant protein as derived from a) of seed grain weight.
In above-mentioned seed plant grain weight GAP-associated protein GAP, the substitution of one or several amino acid residues and/or missing and/
Or it is added to substitution and/or deletion and/or addition no more than 10 amino acid residues.
Wherein, SEQ ID No.2 is made of 368 amino acid residues in sequence table.
It is above-mentioned b) in protein can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain.
It is above-mentioned b) in protein encoding gene can by will in DNA sequence dna shown in SEQ ID No.1 lack one
Or the codon of several amino acid residues, and/or carry out the missense mutation of one or several base-pairs and obtain.
Biomaterial relevant to GmHSFA2 also belongs to protection scope of the present invention.
Biomaterial relevant to GmHSFA2 provided by the present invention is following B1) any one of to B5):
B1 the nucleic acid molecules of the GmHSFA2) are encoded；
B2) contain B1) expression cassettes of the nucleic acid molecules；
B3) contain B1) recombinant vectors of the nucleic acid molecules or contain B2) recombinant vector of the expression cassette；
B4) contain B1) recombinant microorganisms of the nucleic acid molecules or contain B2) recombinant microorganism of the expression cassette or
Contain B3) recombinant microorganism of the recombinant vector；
B5) contain B1) the transgenic plant cells systems of the nucleic acid molecules or contain B2) transgenosis of the expression cassette
Plant cell contains B3) the transgenic plant cells system of the recombinant vector or contain B4) recombinant microorganism
Transgenic plant cells system.
Above, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules
It can be RNA, such as mRNA or hnRNA.B1) the nucleic acid molecules concretely following B11) or B12) or B13) shown in base
Cause:
B11) its coded sequence is the DNA molecular or cDNA molecule of SEQ ID No.1；
B12) hybridize under strict conditions with the B11) DNA molecular limited or cDNA molecule and encode the GmHSFA2's
DNA molecular or cDNA molecule；
B13 described in DNA molecular or cDNA the molecule identity and coding with 90% or more) and B11) limited
The DNA molecular or cDNA molecule of GmHSFA2.
Wherein, SEQ ID No.1 is made of 1107 nucleotide, and coded sequence is the 1-1107 of SEQ ID No.1
Position nucleotide, encodes protein shown in SEQ ID No.2.
Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " can be with the naked eye
Or computer software is evaluated.Using computer software, the identity between two or more sequences can use percentage
(%) is indicated, can be used to evaluate the identity between correlated series.
In said gene, the stringent condition can be as follows: 50 DEG C, 7% lauryl sodium sulfate (SDS),
0.5MNaPO4Hybridize in the mixed solution of 1mM EDTA, is rinsed in 50 DEG C, 2 × SSC, 0.1%SDS；May be used also are as follows: 50 DEG C,
In 7%SDS, 0.5M NaPO4Hybridize in the mixed solution of 1mM EDTA, is rinsed in 50 DEG C, 1 × SSC, 0.1%SDS；Also
It can are as follows: 50 DEG C, in 7%SDS, 0.5M NaPO4Hybridize in the mixed solution of 1mM EDTA, at 50 DEG C, 0.5 × SSC, 0.1%
It is rinsed in SDS；May be used also are as follows: 50 DEG C, in 7%SDS, 0.5M NaPO4Hybridize in the mixed solution of 1mM EDTA, at 50 DEG C,
It is rinsed in 0.1 × SSC, 0.1%SDS；May be used also are as follows: 50 DEG C, in 7%SDS, 0.5M NaPO4In the mixed solution of 1mM EDTA
Hybridization, rinses in 65 DEG C, 0.1 × SSC, 0.1%SDS；It can also are as follows: in 6 × SSC, the solution of 0.5%SDS, at 65 DEG C
Hybridization, then with 2 × SSC, 0.1%SDS and 1 × SSC, it is primary that 0.1%SDS respectively washes film.
In above-mentioned biomaterial, B2) described in the nucleic acid molecules containing coding GmHSFA2 expression cassette (GmHSFA2 gene
Expression cassette), it is the DNA for referring to express GmHSFA2 in host cell, which not only may include that starting GmHSFA2 gene turns
The promoter of record may also include the terminator for terminating GmHSFA2 transcription.Further, the expression cassette may also include enhancer sequence
Column.Promoter for use in the present invention includes but is not limited to: constitutive promoter, organizes, the promoter that organ and development are special,
And inducible promoter.The example of promoter includes but is not limited to: the constitutive promoter 35S of cauliflower mosaic virus: coming from
The wound-inducible promoter of tomato, leucine aminopeptidase (" LAP ", Chao et al. (1999) Plant Physiol
120:979-992)；Chemical inducible promoter from tobacco, pathogenesis correlation 1 (PR1) is (by salicylic acid and BTH (benzo
Thiadiazoles -7- carbothioic acid S-methyl ester) induction)；Tomato protease inhibitors II promoter (PIN2) or LAP promoter are (
It can be induced with methyl jasmonate)；Heat-shock promoters (United States Patent (USP) 5,187,267)；Tetracycline inducible promoter (the U.S.
Patent 5,057,422)；Seed specific promoters, such as Millet Seed specificity promoter pF128 (CN101063139B (China
Patent 200710099169.7)), the special promoter of seed storage protein matter is (for example, phaseolin, napin, oleosin
With the promoter (Beachy et al. (1985) EMBO is J.4:3047-3053) of soybean beta conglycin).They can be independent
It is used in combination using or with other plant promoters.All references cited herein is cited in full text.Suitable transcription
Terminator includes but is not limited to: Agrobacterium nopaline syntase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S
Terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator (see, e.g.:
Odell et al. (I985)Nature313:810；Rosenberg et al. (1987) Gene, 56:125；Guerineau et al.
(1991)Mol.Gen.Genet,262:141；Proudfoot(1991)Cell,64:671；Sanfacon et al. Genes
Dev.,5:141；Mogen et al. (1990) Plant Cell, 2:1261；Munroe et al. (1990) Gene, 91:151；
Ballad et al. (1989) Nucleic Acids Res.17:7891；Joshi et al. (1987) Nucleic Acid Res.,
15:9627)。
The recombinant expression carrier of the GmHSFA2 expression casette, institute can be contained with existing plant expression vector construction
Stating plant expression vector includes Gateway systemic vectors and double base agrobacterium vector etc., as pGWB405, pBin438,
PCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or
PCAMBIA1391-Xb (CAMBIA company) etc..The plant expression vector also may include 3 ' end non-translational regions of foreign gene
Domain, i.e., comprising polyadenylation signals and any other DNA fragmentation for participating in mRNA processing or gene expression.The polyadenylic acid letter
Number bootable polyadenylic acid is added to 3 ' ends of mRNA precursor, as (such as kermes closes Agrobacterium crown gall nodule induction (Ti) plasmid gene
At enzyme Nos gene), plant gene (such as soybean storage protein genes) 3 ' end transcription non-translational region all have similar functions.Make
When with gene constructed plant expression vector of the invention, enhancer, including translational enhancer or transcriptional enhancer also can be used, this
A little enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but must be with the reading of coded sequence
Frame is identical, to guarantee the correct translation of entire sequence.The source of the translation control signal and initiation codon be it is extensive, can
Be it is natural, be also possible to synthesis.Translation initiation region can come from transcription initiation region or structural gene.For the ease of
Transgenic plant cells or plant are identified and screened, plant expression vector used can be processed, it can be as being added
The coding expressed in plant can produce the enzyme of color change or the gene (gus gene, luciferase genes etc.) of luminophor,
The marker gene of antibiotic (if assigned the nptII gene to kanamycins and associated antibiotic resistance, is assigned to herbicide phosphine silk
The bar gene of rhzomorph resistance assigns the hph gene to antibiotic hygromycin resistance, assigns to methatrexate resistance
Dhfr gene and assign to the EPSPS gene of glyphosate), anti-chemical reagent marker gene etc. (such as anti-herbicide gene) or
The mannose-6-phosphate isomerase gene of metabolism mannose ability is provided.
The recombinant microorganism concretely bacterium, yeast, algae and fungi.Wherein, bacterium may be from Escherichia
(Escherichia), Erwinia (Erwinia), Agrobacterium tumefaciems category (Agrobacterium), Flavobacterium
(Flavobacterium), Alcaligenes (Alcaligenes), pseudomonas (Pseudomonas), Bacillus
(Bacillus) etc..The transgenic plant cells system is non-plant propagation material.
In order to solve the above technical problems, the present invention also provides GmHSFA2 or above-mentioned biomaterials relevant to GmHSFA2
Application in regulation seed plant seed grain weight, or the application in preparation regulation seed plant seed grain weight product.
In above-mentioned application, the regulation seed plant grain weight can be raising seed plant grain weight.The receptor seed plant
Concretely angiosperm；Further, the angiosperm can be dicotyledon or monocotyledon；The angiosperm can
For crucifer.
In order to solve the above technical problems, the present invention also provides a kind of encoding genes using GmHSFA2 to cultivate high grain weight
Transgenic seed plant method.
The method of the transgenic seed plant provided by the present invention for cultivating high grain weight, including led into receptor seed plant
The encoding gene for entering GmHSFA2 obtains the transgenic seed plant that grain is higher than the receptor seed plant grain weight again.
In the above method, the receptor seed plant concretely angiosperm；Further, the angiosperm can be double
Cotyledon plant or monocotyledon；The angiosperm can be crucifer.
In the above method, the encoding gene of the GmHSFA2 can be above-mentioned B11) or B12) or B13) shown in gene；On
It states in method, wherein the GmHSFA2 gene can be modified first as follows, then imports in receptor seed plant, to reach more preferable
Expression effect:
1) it modifies and optimizes according to actual needs, so that gene efficient expression；For example, can be according to receptor seed plant
The codon being had a preference for changes its codon while keeping the amino acid sequence of GmHSFA2 gene of the present invention to accord with
Close plant-preference；In optimization process, it is desirable that certain G/C content is kept in the coded sequence after optimization, with best real
The high level expression of quiding gene in existing plant, wherein G/C content can be 35%, be more than 45%, more than 50% or more than about
60%；
2) gene order of neighbouring initial methionine is modified, so that translation effectively starting；For example, using in plant
The effective sequence known is modified；
3) it is connect with the promoter of various plants expression, in favor of its expression in plant；The promoter may include
Composing type, induction type, timing adjusting, growth adjustment, Chemical Regulation, tissue are preferably and tissue-specific promoter；Promoter
Selection will need with expression time and space and be changed, and also depend on target kind；Such as the specificity of tissue or organ
Promoter is expressed, receptor as needed is depending on what period of development；
4) it is connect with suitable transcription terminator, can also be improved the expression efficiency of gene of the present invention；Such as from
The tml of CaMV, from the E9 of rbcS；Any known available terminator to work in plant can be with the present invention
Gene is attached；
5) enhancer sequence is introduced, such as intron sequences (such as from Adhl and bronzel) and viral leader sequence
(such as from TMV, MCMV and AMV).
In the above method, the GmHSFA2 gene passes through the recombinant expression carrier containing GmHSFA2 expression casette
(GmHSFA2 expression vector) imports in the receptor seed plant, in the GmHSFA2 expression casette, starting
The promoter of GmHSFA2 genetic transcription is cauliflower mosaic virus 35 S promoter.
The GmHSFA2 expression vector can be by using Ti-plasmids, plant viral vector, directly delivered DNA, micro- note
It penetrates, the standard biologics technical method such as electroporation, mediated by agriculture bacillus imports plant cell or tissue.
Above, the transgenic seed plant is interpreted as not only comprising obtaining the genetic transformation receptor seed plant
First generation genetically modified plants, also include its filial generation.For genetically modified plants, the gene can be bred in the species, it can also
The gene transfer is entered to other kinds of same species with traditional breeding techniques, particularly including in commercial variety.It is described to turn base
Because plant includes seed, callus, intact plant and cell.
The experiment proves that GmHSFA2 gene is transferred to transgenic arabidopsis kind obtained in wildtype Arabidopsis thaliana
Son grain be significantly higher than again wildtype Arabidopsis thaliana (turn GmHSFA2 arabidopsis strain be overexpressed strain OE-1 seed mass of 1000 kernel be
The seed mass of 1000 kernel of 22.1 ± 0.9g, OE-45 are 18.2 ± 1.2g, and the seed mass of 1000 kernel of OE-55 is 19.2 ± 0.8g, wild type
The seed mass of 1000 kernel of arabidopsis (Col-0) is 15.6 ± 0.4g), illustrate that GmHSFA2 and its encoding gene can regulate and control plant species
The grain weight of son improves plant species seed weight after overexpression.The gene can be used for improving crop yield, breed high-yield variety.
The present invention is described in further details combined with specific embodiments below.
Detailed description of the invention
Fig. 1 is cloning vectorSchematic diagram.
Fig. 2 is plant expression vector pGWB405-GmHSFA2 schematic diagram.
Fig. 3 is the phenotypic evaluation for being overexpressed GmHSFA2 plant: A is the life for compareing and being overexpressed under normal operation strain
Long situation；B is that the lotus throne diameter before compareing and being overexpressed under normal operation strain bolting compares；C is to compare and be overexpressed strain
It is thousand grain weigth statistics.Control indicates wildtype Arabidopsis thaliana (Col-0).
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
In following embodiments the black agriculture 44 (HN44) of soybean (be completely group etc., the breeding and difference of the black agriculture 44 of new soybean varieties
Influence of the planting patterns to its yield and kind, Exploitation of Agriculture in Heilongjiang science 5 phases in 2004,1-5), the public can be from the Chinese Academy of Sciences
Heredity is obtained with Developmental Biology research, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as it
Its purposes uses；It is obtained from Exploitation of Agriculture in Heilongjiang academy of sciences soybean research institute within the soybean 2006；By Exploitation of Agriculture in Heilongjiang academy of sciences soybean
Research institute's soybean varieties through Heilongjiang Province's crop varietal approval committee in 2002, first, which is bred as artificial Du Weiguang, grinds
The person of studying carefully, kind power number are as follows: CNA20020216.2, authorization number are as follows: black careful beans 2002003.
Expression carrier used thereof pGWB405 (Department of Molecular and in following embodiments
Functional Genomics,Shimane University,Aatsue,Shimane 690-8504,Japan,E.mail:
tnakagaw@life.shimane-u.ac.jp Isuyoshi Nakagawa,et al.,Improved Gatway binary
Vectors:high-performance vectors for creation for fusion constructs in
Transgenic, Biosci.Biotechnol.Biochem., 2007,71 (8), 2095-2100).By Tsuyoshi
Doctor Nakagawa provides, and the public obtains can be from the heredity of institute, the Chinese Academy of Sciences and hair after Tsuyoshi doctor Nakagawa agrees to
Biological study is educated to be obtained.The biomaterial is only attached most importance to used in the related experiment of duplicate invention, and not can be used as other purposes makes
With.
Agrobacterium tumefaciems GV3101 (Lee CW etc., Agrobacterium tumefaciens used in following embodiments
promotes tumor induction by modulating pathogen defense in Arabidopsis
Thaliana, Plant Cell, 2009,21 (9), 2948-62), the public can grind from Chinese Academy of Sciences's heredity with Developmental Biology
Study carefully and obtained, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as other purposes and uses.
For below using arabidopsis as receptor seed plant, technical solution of the present invention is illustrated.
Embodiment 1, the cDNA gene that Protein G mHSFA2 is closed with Soybean Species seed heavy phase prepare and the structure of plant expression vector
It builds
1, the building of transcription factor GmHSFA2 gene cloning and plant expression vector
RNA reverse transcriptase reverse transcription is synthesized cDNA by the total serum IgE for extracting black 44 seedling of agriculture of soybean.Using the cDNA as mould
Plate, with GmHSFA2-up (5 '-ATGACGACTCACGTAGTTAAAGA-3 ') and GmHSFA2-dp (5 '-
TCAACTGGTTTGTTCCACCAATC-3 ') it is primer, PCR amplification is carried out, the PCR product of about 1.1Kb is obtained.By being sequenced,
The PCR product is 1107bp, and nucleotides sequence is classified as SEQ ID No.1, and DNA molecular shown in the nucleotide sequence is
The protein of the cDNA gene (GmHSFA2) of GmHSFA2, gene coding is named as GmHSFA2, the amino acid sequence of GmHSFA2
It is classified as SEQ ID No.2.
The Gateway system that gene cloning is provided using invitrogen company, 3 '-T jag of carrier, for directly connecting
Connect the PCR product of Taq enzyme amplification.
By the PCR product (the cDNA gene of GmHSFA2) of 1107bp obtained above with TA clone principle clone with
CarrierUpper (TA Cloning Kit,Catalog number:K2500-
20, Invitogen Corporation, Carlsbad, CA, USA carrier schematic diagram such as Fig. 1) connection, by the cDNA of GmHSFA2
Gene insertionObtain intermediate vector -GmHSFA2。
It willIt is anti-that-GmHSFA2 and carrier pGWB405 carries out LR recombination under the action of recombinase
It answers, finally target gene GmHSFA2 is successfully building up on carrier pGWB405, obtaining recombinant vector, (operation is shown in that company mentions in detail
The specification of confession).Sequencing result shows that the recombinant vector is that DNA molecular shown in SEQ ID No.1 in sequence table is homologous heavy
GmHSFA2 expression vector obtained in group to carrier pGWB405 is named as pGWB405-GmHSFA2 (part-structure signal
Figure is as shown in Figure 2).
Embodiment 2, the transgenic seed plant that high grain weight is cultivated using GmHSFA2
1, the acquisition of recombinational agrobacterium
Embodiment 1 is obtained into the electric shocking method of the recombinant vector pGWB405-GmHSFA2 containing GmHSFA2 and imports crown gall agriculture bar
Bacterium GV3101 obtains the recombinational agrobacterium GV3101/GmHSFA2 containing pGWB405-GmHSFA2.
2, turn the acquisition and identification of GmHSFA2 arabidopsis
Recombinational agrobacterium GV3101/GmHSFA2 is cultivated to logarithmic phase, is then converted Colombia with vacuumizing method
In Arabidopsis thaliana ecotype (Col-0) (seed is purchased from Arabidopsis Biological Resource Center, ABRC), obtain
To T0In generation, turns GmHSFA2 arabidopsis.Seed (T is harvested after cultivating1Generation), seed is sowed at the MS containing kanamycins (50mg/L)
On screening and culturing medium, obtained T to be screened1It moves on on vermiculite and grows when for plant length to 4-6 leaf, harvest T1For single plant, each single plant
Seed is sowed respectively, continues screening with identical MS screening and culturing medium to observe T2In the separation situation in generation, such repeat number generation, are straight
To the transgenic homozygous strain for obtaining inheritance stability, obtains 11 and turn GmHSFA2 arabidopsis pure lines (T5In generation, turns the quasi- south GmHSFA2
Mustard strain).Extract above-mentioned 11 T5In generation, turns GmHSFA2 arabidopsis strain seedling RNA, and reverse transcription obtains cDNA as template, primer
Are as follows: 5 '-GTCCATAAGATTAGGCGAAAAAGA and 5 '-GGTGCTCCATCCTACCAGTGTT carry out Real Time-PCR mirror
It is fixed, it is control with wildtype Arabidopsis thaliana (Col-0).Arabidopsis AtActin2 gene be internal standard, the primer Primer-TF:
5 '-ATGCCCAGAAGTCTTGTTCC and Primer-TR:5 '-TGCTCATACGGTCAGCGATA-3 '.Choose T5In generation, turns
Number in GmHSFA2 arabidopsis strain is that the strain of OE-1, OE-45 and OE-55 make further research.With internal standard AtActin2
The expression quantity of gene is 1, measures the relative expression quantity of GmHSFA2.
Real Time-PCR qualification result shows that the relative expression quantity of GmHSFA2 in OE-1 is in 0.6, OE-45
The relative expression quantity that the relative expression quantity of GmHSFA2 is GmHSFA2 in 0.5, OE-18 is 0.5, in wildtype Arabidopsis thaliana (Col-
0) fail to detect the relative expression quantity of GmHSFA2 in.
The above results further prove that GmHSFA2 is transferred in arabidopsis, and are expressed.
Empty carrier pGWB405 is transferred in wildtype Arabidopsis thaliana using same method, obtains T0In generation, turns the quasi- south of empty carrier
Mustard, sowing, sowing, until obtaining turning empty carrier pure lines arabidopsis (T5In generation, turns empty carrier arabidopsis strain).
3, turn the phenotypic analysis of GmHSFA2 gene arabidopsis
Detect wildtype Arabidopsis thaliana (Col-0), the number of step 2 is the T of OE-1, OE-45 and OE-555In generation, turns GmHSFA2
Arabidopsis strain (being referred to as overexpressed strain) and T5In generation, turns the phenotype of empty carrier arabidopsis strain under normal operation.Phenotypic examination
The result shows that under normal operation, wildtype Arabidopsis thaliana (Col-0), the T that number is OE-1, OE-45 and OE-555In generation, turns
GmHSFA2 arabidopsis strain and T5The phenotype that generation turns empty carrier arabidopsis strain is showed no notable difference (A in Fig. 3).It counts
Wildtype Arabidopsis thaliana (Col-0), the T that number is OE-1, OE-45 and OE-555In generation, turns GmHSFA2 arabidopsis strain and T5In generation, turns sky
Lotus throne diameter before each 24 plants of boltings of carrier arabidopsis strain, the results showed that wildtype Arabidopsis thaliana (Col-0), number be OE-1,
The T of OE-45 and OE-555In generation, turns GmHSFA2 arabidopsis strain and T5In generation, turns the lotus throne diameter before empty carrier arabidopsis strain bolting
40 millimeters are each about, no significant difference (B in Fig. 3).
Measurement wildtype Arabidopsis thaliana (Col-0), the T that number is OE-1, OE-45 and OE-555In generation, turns GmHSFA2 arabidopsis
Strain and T5In generation, turns the seed mass of 1000 kernel of empty carrier arabidopsis strain.Thoroughly dry seed mass of 1000 kernel to be measured.Each strain takes 24
The seed of strain is tested in triplicate, ± standard deviation that results are averaged.The calculating of variance is carried out with following equation:
Seed mass of 1000 kernel measurement result shows that the seed mass of 1000 kernel of wildtype Arabidopsis thaliana (Col-0) is 15.6 ± 0.4g, T5
The seed mass of 1000 kernel that generation turns GmHSFA2 arabidopsis strain OE-1 is 22.1 ± 0.9g, T5In generation, turns GmHSFA2 arabidopsis strain OE-
45 seed mass of 1000 kernel is 18.2 ± 1.2g, T5Generation turn GmHSFA2 arabidopsis strain OE-55 seed mass of 1000 kernel be 19.2 ±
0.8g.In three transgenic lines, the seed mass of 1000 kernel of OE-1 and OE-55 with compare between difference in extremely significant, and OE-45 with it is right
According to also there were significant differences.The above results show that 3 transgenic line seed mass of 1000 kernel are apparently higher than wildtype Arabidopsis thaliana.It is wild
The seed mass of 1000 kernel of type arabidopsis (Col-0) and T5In generation, turns empty carrier arabidopsis strain and is not significantly different.
It is in positive regulating and controlling effect to seed weight that above-mentioned experiment, which shows that the grain heavy phase from soybean closes Protein G mHSFA2,
Normal growth of the weight of transgenic plant seed without influencing plant can be improved in the overexpression of encoding gene GmHSFA2.
Claims (10)
1. protein is amino acid sequence protein as shown in SEQ ID No.2.
2. it is following B1 biomaterial relevant to protein described in claim 1) at least one of to B4):
B1 the nucleic acid molecules of protein described in claim 1) are encoded；
B2) contain B1) expression cassettes of the nucleic acid molecules；
B3) contain B1) recombinant vectors of the nucleic acid molecules or contain B2) recombinant vector of the expression cassette；
B4) contain B1) recombinant microorganisms of the nucleic acid molecules or contain B2) recombinant microorganism of the expression cassette or contain
B3) the recombinant microorganism of the recombinant vector.
3. relevant biological material according to claim 2, it is characterised in that: B1) nucleic acid molecules for coded sequence are
The DNA molecular or cDNA molecule of SEQ ID No.1.
4. relevant biological material described in protein or Claims 2 or 3 described in claim 1 is in regulation seed plant grain weight
Application, or preparation regulation seed plant seed grain weight product in application.
5. application according to claim 4, it is characterised in that: the seed plant is angiosperm.
6. application according to claim 5, it is characterised in that: the angiosperm is crucifer.
7. a kind of method for the transgenic seed plant for cultivating high grain weight, including claim 1 is imported into receptor seed plant
The encoding gene of the protein obtains the step of grain is higher than the transgenic seed plant of the receptor seed plant grain weight again.
8. according to the method described in claim 7, it is characterized by: the receptor seed plant is angiosperm.
9. according to the method described in claim 8, it is characterized by: the angiosperm is crucifer.
10. according to the method any in claim 7-9, it is characterised in that: the coding base of protein described in claim 1
Because coded sequence is the DNA molecular or cDNA molecule of SEQ ID No.1.
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| CN111197047B (en) * | 2018-11-19 | 2022-03-01 | 中国科学院遗传与发育生物学研究所 | Soybean protein GmUBCa related to seed weight regulation and application of soybean protein GmUBCa and related biological material thereof |
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| WO2005059147A2 (en) * | 2003-12-17 | 2005-06-30 | Cropdesign N.V. | Plants having modified growth characteristics and method for making the same |
| CN1934259A (en) * | 2004-03-22 | 2007-03-21 | 克罗普迪塞恩股份有限公司 | Plants having improved growth characteristics and method for making the same |
| WO2008015263A3 (en) * | 2006-08-02 | 2008-05-15 | Cropdesign Nv | Plants having improved characteristics and a method for making the same |
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| WO2005059147A2 (en) * | 2003-12-17 | 2005-06-30 | Cropdesign N.V. | Plants having modified growth characteristics and method for making the same |
| CN1934259A (en) * | 2004-03-22 | 2007-03-21 | 克罗普迪塞恩股份有限公司 | Plants having improved growth characteristics and method for making the same |
| WO2008015263A3 (en) * | 2006-08-02 | 2008-05-15 | Cropdesign Nv | Plants having improved characteristics and a method for making the same |
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