CN109776664A - A kind of gene and its application controlling rice class granule and semi-dwarf mutant - Google Patents
A kind of gene and its application controlling rice class granule and semi-dwarf mutant Download PDFInfo
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- CN109776664A CN109776664A CN201811414159.2A CN201811414159A CN109776664A CN 109776664 A CN109776664 A CN 109776664A CN 201811414159 A CN201811414159 A CN 201811414159A CN 109776664 A CN109776664 A CN 109776664A
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Abstract
The present invention relates to a kind of rice class granule and semi-short-stalked related gene and its applications.Specifically, the present inventor is using the biggish rice varieties of seed extensive 12-29 and granule kind FH212 building genetic group, discloses a kind of control rice granule and Semi dwarfism geneTGW5, gene coding tripolymer G-protein α subunit.For granule parent FH212 compared with the big extensive 12-29 of grain parental breed, the variation (A → T) of the 4th introne end single base leads to the change of introne cut mode, generates 3 kinds of transcripts for being different from extensive 12-29, and the TGW5 protein three-dimensional structure of prediction is changed.The invention further relates to missing or deficient protein function TGW5 mutain and its coded sequence, and in terms of application.
Description
Technical field
The present invention relates to plant biotechnology fields.Specifically related to it is a kind of control rice granule and semi-dwarf mutant gene and
It is applied.
Background technique
The grain size of rice is a typical quantitative character and important economic characters, which forms is imitated by close source
It answers, the various Genetic signals such as Plant hormone signal approach, G-protein and the control and influence that adjust approach.Rice paddy seed mainly by
Embryo, endosperm, pericarp, kind skin and glume are constituted.Wherein, the part eaten for the mankind is provided in internal endosperm；In outer
On the one hand the glume in portion provides protection for seed, the container of limitation seed growth is on the other hand also considered as, to the tune of particle shape
Control plays an important role.The size of glume depends on glume cell, mainly increases two ways by cell division and cell
Carry out coordinated control.In glume development early stage, glume cell is accelerated by extensive cell division；Then, cell division
Speed slows down and begins through cell expansion to increase cell；Finally by the cell quantity and cell on seed glume different dimensions
Size determines grain length, the phenotype that grain is wide and grain is thick.
Rice grain weight and particle shape are the key factors that rice yield is constituted, generally with grain length, grain is wide, grain is thick and length-width ratio etc.
Grain shape traits are measured.Rice grain weight/particle shape is typical quantitative character, by controlled by multiple genes, usually with QTL
(quantitative trait locus) research method carries out genetic analysis.Currently, identified rice grain weight/particle shape
QTL has more than 400, is all distributed (www.gramene.com) on any bar chromosome of rice genome.Particle shape
Shape has very high heritability, stablizes performance to kind genetic improvement with important in different genetic background and environment
Meaning.Over the past decade, it controls gene/QTL clone of rice grain shape character and functional study achieves impressive progress, at present
The QTL of existing 16 control rice grain shapes and grain weight is parsed by molecular cloning and function, but other than a small amount of QTL/ gene, absolutely
The QTL of most of adjusting and controlling rice particle shapes and grain weight, is still in the Primary Study stage.Therefore, it needs to reinforce to controlling the character
QTL is cloned and is parsed to the function of its candidate gene.The present invention clones a control rice using the method separation of map based cloning
The gene TGW5 of granule semi-dwarf mutant, important genetic resources are provided for rice modification breeding.
Summary of the invention
The object of the present invention is to provide a kind of gene and its applications for controlling crop particle shape and plant height.
The first aspect of the present invention, provides a kind of polypeptide of the control rice granule semi-dwarf mutant of separation, which is selected from
The following group:
(1) with the polypeptide of SEQ ID NO:2 amino acid sequence；Or
(2) SEQ ID NO:2 amino acid sequence to be formed by replacing, missing or adding for 1-10 amino acid residue
The polypeptide as derived from (1).
The second aspect of the present invention, provides a kind of polypeptide of the control rice granule semi-dwarf mutant of separation, which is selected from
The following group:
(1) with the polypeptide of SEQ ID NO:4 amino acid sequence；Or
(2) SEQ ID NO:4 amino acid sequence to be formed by replacing, missing or adding for 1-10 amino acid residue
The polypeptide as derived from (1).
The third aspect of the present invention, provides a kind of polypeptide of the control rice granule semi-dwarf mutant of separation, which is selected from
The following group:
(1) with the polypeptide of SEQ ID NO:6 amino acid sequence；Or
(2) SEQ ID NO:6 amino acid sequence to be formed by replacing, missing or adding for 1-10 amino acid residue
The polypeptide as derived from (1).
The fourth aspect of the present invention provides a kind of isolated polynucleotides, and the polynucleotides are selected from the group:
(a) polynucleotides of polypeptide described in first aspect present invention are encoded；
(b) sequence polynucleotides as shown in SEQ ID NO:1.
The fifth aspect of the present invention provides a kind of isolated polynucleotides, and the polynucleotides are selected from the group:
(a) polynucleotides of polypeptide described in second aspect of the present invention are encoded；
(b) sequence polynucleotides as shown in SEQ ID NO:3.
The sixth aspect of the present invention provides a kind of isolated polynucleotides, and the polynucleotides are selected from the group:
(a) polynucleotides of polypeptide described in third aspect present invention are encoded；
(b) sequence polynucleotides as shown in SEQ ID NO:5.
The seventh aspect of the present invention provides a kind of isolated polynucleotides, and the polynucleotides are selected from the group:
(a) polynucleotides of the transcription present invention fourth, fifth and the six aspect polypeptides；
(b) sequence polynucleotides as shown in SEQ ID NO:7.
The eighth aspect of the present invention provides a kind of Crop Improvement (it is furthermore preferred that for breeding granule semidwarf rice product
System) method, this method comprises:
(I) grain characters of rice are improved；
(II) Plant Height of Rice is adjusted.
Detailed description of the invention
Following drawings is for illustrating specific embodiments of the present invention, rather than limits and be defined by the claims
The scope of the invention.
Fig. 1 shows rice TGW5 complementation transgenic plant compared with the plant height of receptor kind " FH212 " and particle shape.
Fig. 2 shows the plant height and grain that TGW5 mutant plants and receptor kind " C815S " are obtained using crispr technology
Shape compares.
Fig. 3 shows the plant height and particle shape and receptor using the improvement plant of molecular marker assisted selection importing TGW5 gene
The comparison of kind " Wu Xiang S ".
Specific embodiment
The present inventor has found the new gene of a kind of control crop particle shape and grain weight through deeply extensive research for the first time, should
The function of gene reduces or missing can reduce particle shape and reduces plant height.Research confirms the single base mutation and clpp gene of TGW5 gene
It is reduced except particle shape can be made significantly to become smaller with plant height.Before showing that TGW5 gene is widely used in breeding granule crop lines
Scape.
Term
" crop " of the present invention includes but is not limited to: rice, wheat, corn, sorghum, soybean etc..
" separation " of the present invention refers to that substance is separated from its primal environment.As natural in active somatic cell
Polynucleotide and polypeptide under state do not isolate and purify, but same polynucleotide or polypeptide are such as from native state
In with being separated in other existing substances, then to isolate and purify.
" isolated TGW5 albumen or polypeptide " of the present invention refer to the TGW5 albumen substantially free of naturally with
Its relevant other albumen.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide.Polypeptide of the invention includes natural
The product of purifying or chemically synthesized product, or generated from protokaryon or eucaryon host by recombinant technique.
" long grain " of the present invention, " big grain " may be used interchangeably.
" TGW5 albumen " of the present invention refers to the polypeptide of the SEQ ID NO:2 sequence with TGW5 protein active.
The present invention also provides the polynucleotide sequences of coding TGW5 albumen.Polynucleotides of the invention can be DNA shape
Formula or rna form.DNA form includes: DNA, genomic DNA or artificial synthesized DNA.DNA can be coding strand or non-coding
Chain.It the code area of encoding mature polypeptide can be with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7
Shown in coding region sequence is identical or the variant of degeneracy.
The invention further relates to application of the Marker-assisted selection technology of TGW5 gene in crops granule sterile line breeding.
The present invention is with extensive 12-29 (big grain kind) and FH212 (granule kind) hybridization building genetic group, using QTL-
Seq location technology located the gene TGW5 of the control rice grain length that one is located at the 5th chromosome and grain weight, and pass through figure position
Clone technology has cloned the gene.
Main advantages of the present invention are:
(1) the new small grain gene of rice of isolated one kind for the first time, changes the transcription and translation of the gene or knocks out the gene
The seed of crop (such as rice) can be made to become smaller, strain stalk becomes short.
(2) the small grain gene TGW5 of rice of the invention can be used as a gene of control crop kernel size and plant height,
Improvement applied to variety of crops.
The acquisition of 1 rice granule Semi dwarfism gene TGW5 of embodiment
Extensive 12-29 is larger grain rice varieties, and FH212 is granule rice varieties.The present inventor with extensive 12-29 with
FH212 hybridization building genetic group, located the new gene of control a rice grain shape and plant height using QTL-seq analysis method
(or QTL) TGW5, the gene are located on the 5th chromosome.Further the gene, gene order have been cloned using map-based cloning
As shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7, the albumen of coding such as SEQ ID NO:
2, shown in SEQ ID NO:4 and SEQ ID NO:6.
The experiment of 2 TGW5 Transgenic Rice of embodiment
Genetic transformation verifies TGW5 control rice grain shape and plant height.The present embodiment, which uses, derives from plant expression vector
PCAMBIA1301 is as Transgenic Rice carrier.The vector encoded one bacterial origin of replication (ori), kalamycin resistance base
Because of (Kanr), hygromycin gene (Hygr), beta-glucosiduronatase gene (GUS), double CaMV35S promoters, NOS gene
Termination signal sequence and restriction enzyme multiple cloning sites (MCS).It can be at restriction enzyme multiple cloning sites
The DNA sequence dna of positive or reversed insertion TGW5 constructs transgene carrier.
1.TGW5 complementation transgenosis plasmid construction
In the present embodiment, using the genomic DNA from the big extensive 12-29 of grain kind as template, high fidelity enzyme is used
KOD FX Neo (TOYOBO) expands TGW5 gene order in extensive 12-29, and amplified fragments are returned with glue after 1% agargel electrophoresis
It receives.Glue recycling segment is cloned on intermediate vector pEASY-Blunt3 carrier by way of blunt end cloning, and to multiple heavy
Group is sequenced, and the correctness of sequence is verified.PCR amplification primer sequence:
5 ' end Oligonucleolide primers sequences are as follows: 5 '-CCGGAATTCCAAACCCCGTTAAAGCC-3 '
3 ' end Oligonucleolide primers sequences are as follows: 5 '-ATAGGGTACAGACCTGAACAGC-3 '
With EcoR I and Hind III digestion with restriction enzyme TGW5-pEASY-Blunt3 and pCAMBIA1301 carrier,
The postdigestive target fragment of TGW5-pEASY-Blunt3 is connected to the EcoR I and Hind of final carrier pCAMBIA1301
In III digestion site.Connection product converts coli strain T1, and screening turns on the LB culture medium there are also Kan (50 μ g/ml)
Beggar, picking individual colonies extract plasmid, whether just to carry out sequencing detection target fragment sequence to the clone with M13 universal primer
Really.So successfully construct TGW5 complementation transgenosis plasmid.
2.TGW5 rice transformation
TGW5 complementation transgenosis plasmid imports agrobacterium strains EHA105 by freeze-thaw method.Every 200 μ lEHA105 competence
Cell and 0.5-1 μ g (about 10 μ l) Plasmid DNA mix, and successively respectively place 5 points of kinds on ice, in liquid nitrogen and in 37 DEG C of water-baths: using
Fresh YEB fluid nutrient medium is diluted to 1ml, cultivates 2-4 hours in 28 DEG C of shakings；Take 200 μ l coating and Kan containing antibiotic
On the YEB plate of (50 μ g/ml), 28 DEG C are cultivated 2-3 days.The bacterium colony grown draws single bacterium on the YEB plate containing antibiotic, even
It is continuous to draw 3 times.Be inoculated into the antibiotic AB fluid nutrient medium of 3ml from picking Agrobacterium single colonie on YEB plate, 200rpm after
Continuous shaking is cultivated to OD600It is collected simultaneously for 0.6-0.8 or so by fresh Agrobacterium bacterium solution in 500rpm, 4 DEG C of centrifugations, 5 points of kinds
It is resuspended in the AAM fluid nutrient medium of 1/3 volume, can be used to the various acceptor materials of rice transformation at this time.
The present embodiment converts the rataria callus of FH212 using conventional conversion method for agrobacterium.It takes after pollinating 12-15 days
FH212 immature seed (mixes with water 1:3 in NaCLO solution after 70% ethyl alcohol impregnates 1 point of kind, adds 2-3 drop tween
200) 90 points of kinds or more are sterilized, with aseptic water washing 4-5 times, then chooses rataria with scalpel and tweezers and is inoculated in N6D2Training
Support base on evoked callus, 26 ± 1 DEG C, be protected from light under the conditions of cultivate, can be used for converting after 4 days.Rataria callus is soaked into
It shakes in fresh AAM Agrobacterium bacterium solution and frequently, rice material is removed after 20 points of kinds, excessive bacterium is sucked on aseptic paper
Liquid is transferred to N6D immediately2On C culture medium, co-cultured 3 days in 26 DEG C.When co-cultivation, acetyl is added in co-culture medium
Syringone is 100 μm of ol/L using concentration as Agrobacterium Vir gene activation object.After 3 days, taken out from co-culture medium
Callus cuts plumule and is transferred to Selective agar medium N6D2S1 (Hyg25 ㎎/l), carries out selection culture.It will resist after 7-12 days
Property callus is transferred to N6D2Continue to screen on S2 (Hyg50 ㎎/l) Selective agar medium.Eugonic resistance after 10-12 days
Callus is transferred on pre- differential medium and cultivates one week or so, then move on differential medium differentiation (illumination in 12 hours/
It).Seedling is regenerated in 1/2MS0Strong plantlets and rootage on culture medium is subsequently moved within the cultivation of phjytotron basin soil.
3 rice TGW5 complementation transgenic plant of embodiment is compared with the grain size and plant height of wild type
Method as described in Example 2 obtains rice TGW5 complementation transgenic plant, observes wild type FH212 and transgenosis
The phenotype of rice grain shape and plant height analyzes the influence of TGW5 gene pairs grain size and plant height.As a result as shown in Figure 1, rice
TGW5 complementation transgenic plant (C5) is compared with compareing FH212, and grain obviously becomes larger, and plant height dramatically increases.
Embodiment 4 obtains TGW5 mutant using crispr technology
1, TGW5 gene order is analyzed, CRIPSR-P tool (http://crispr.hzau.edu.cn/ is utilized
CRISPR2/ sgRNA target site) is designed, target sequence:
Target sequence: 5 '-aaagaggtggagaggtatatagg-3 '
2, by primer denaturation, annealing, gRNA segment is obtained, digestion connection obtains recombinant plasmid, and conversion Escherichia coli are set
37 DEG C of constant incubators, are incubated overnight, and select monoclonal, extract plasmid identification.
3,2 monoclonals, sequencing identification are sent the plasmid of extracting
4, it identifies that correct plasmid enzyme restriction is connected to expression vector, converts competent escherichia coli cell DH5 α, be coated on
LB solid medium is incubated overnight, and selects monoclonal, extracts plasmid identification.
5, it identifies correct plasmid vector, the callus of C815S is infected using Agrobacterium (EHA105) infestation method, acquisition turns base
Because of plant.
6, from T0 for tender blade is picked in transgenic plant, plant tissue DNA is extracted using CTAB method.
7, include the primer of target site using primer5 software design, obtain TGW5 segment, primer using round pcr
Sequence:
5 ' terminal nucleotide primer sequence TGW5-341 (+): 5 '-CCATTTCCCGTCATTTCTTA-3 '
3 ' terminal nucleotide primer sequence TGW5-417 (-): 5 '-TTCCTTGGTCTCCATCATTC-3 '
8,1% agarose gel electrophoresis selects target stripe gel extraction, is sent to company's sequencing.
9, sequencing result is divided using website DSDECODE (http://dsdecode.scgene.com/home/)
Analysis.
10,2 plants of TGW5 mutant C65 and C70 are obtained as the result is shown.
C65 with C70 mutant strain is compared with the target sequence of wild type (Control):
Wild type: CCTCCTACATCATACAACCTATATACCTCTCCACCTCTTT
J22:CCTCCTACATCATACAACCT---TACCTCTCCACCTCTTT
J42:CCTCCTACATCATACAACCTATA-- CCTCTCCACCTCTTT
5 rice TGW5 mutant transgenic plant of embodiment is compared with the grain size of WT lines
Method as described in Example 4 obtains rice TGW5 gene knockout plant, observes rice grain shape and plant height phenotype, point
Analyse the influence of TGW5 gene pairs grain size and plant height.As a result as shown in Fig. 2, the transgenic plant of rice TGW5 gene knockout
(J22) grain is obviously smaller than receptor rice varieties C815S, plant height also extremely significant reduction.
The molecular marker assisted selection breeding of 6 TGW5 gene of embodiment
PCR Oligonucleolide primers (SEQ ID NO:8 and SEQ ID NO:9) is designed in TGW5 gene in the present embodiment,
The DNA that the big grain kind force perfume S and granule kind FH212 of PCR amplification is carried out with Taq enzyme expands the segment of generation in restricted
I digestion of enzyme cutting Alu detects that there are DNA polymorphisms between big grain kind and granule kind by 2.5% agarose gel electrophoresis
(difference), big grain kind molecular weight 286bp, granule kind 314bp, therefore this develops into specificity to primer and identifies big grain TGW5
The molecular labeling of gene and granule TGW5 gene.As a result as shown in figure 3, in the miscellaneous of big grain kind force perfume S and granule kind FH212
It hands over seedling stage in progeny population that can rapidly be picked out the individual for carrying small grain gene with the molecular labeling, cultivates granule rice
New lines (XS1).
The primer sequence of TGW5 gene specific molecular marker:
5 ' end oligonucleotide primer sequences are as follows:
5 '-AGAGTTCTTTTGCTCCTTCATATAGTAGC-3 ' (SEQ ID NO:8)；
3 ' end Oligonucleolide primers sequences are as follows:
5 '-TCCGTACGCCGCTAGTTAGTC-3 ' (SEQ ID NO:9).
Claims (5)
1. a kind of isolated albumen, which is characterized in that the albumen is selected from the group:
(1) polypeptide as shown in SEQ ID NO:2 amino acid sequence；Or
(2) amino acid sequence shown in SEQ ID NO:2 to be formed by 1-10 replacing, missing or adding for amino acid residue
It is polymorphic as derived from (1).
2. a kind of isolated albumen, which is characterized in that the albumen is selected from the group:
1. the polypeptide as shown in SEQ ID NO:4 amino acid sequence；Or
2. amino acid sequence shown in SEQ ID NO:4 to be formed by 1-10 replacing, missing or adding for amino acid residue
The polypeptide as derived from (i).
3. a kind of isolated albumen, which is characterized in that the albumen is selected from the group:
(i) polypeptide as shown in SEQ ID NO:6 amino acid sequence；Or
(ii) amino acid sequence shown in SEQ ID NO:6 is replaced, missed or added into shape by 1-10 amino acid residue
At the polypeptide as derived from (i).
4. a kind of isolated polynucleotides, which is characterized in that the polynucleotides are selected from the group:
(a) polynucleotides of albumen described in claim 1,2 and 3 are encoded；
(b) sequence polynucleotides as shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7；
(c) nucleotide sequence and SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7 sequence it is homologous
Property >=95% (preferably >=98%) polynucleotides；
(d) with (a), (b) and (c) described in the complementary polynucleotides of polynucleotides.
5. the purposes of the polypeptide of claim 1 and 2 or its coded polynucleotide, which is characterized in that the purposes is selected from the group:
(I) polypeptide or its coded polynucleotide are for controlling crop kernel shape；
(II) polypeptide or its coded polynucleotide are for adjusting crop plant height；
(III) polypeptide or its coded polynucleotide are for adjusting grain crop weight and crop yield.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111848765A (en) * | 2020-07-22 | 2020-10-30 | 中国水稻研究所 | Rice gene OsFBK4 and mutant and application thereof |
| NL2028064B1 (en) * | 2021-04-24 | 2022-04-05 | China Nat Rice Res Inst | Gene for controlling small grain and semi-dwarf of oryza sativa and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1261101A (en) * | 1998-06-19 | 2000-07-26 | 农林水产省农业生物资源研究所所长代表的日本国 | Method for dwarfing plants |
| CN103936843B (en) * | 2013-03-25 | 2016-06-22 | 袁隆平农业高科技股份有限公司 | Rice Os 05g26890.1 albumen, the gene encoding this albumen and application thereof |
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2018
- 2018-11-26 CN CN201811414159.2A patent/CN109776664A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1261101A (en) * | 1998-06-19 | 2000-07-26 | 农林水产省农业生物资源研究所所长代表的日本国 | Method for dwarfing plants |
| CN103936843B (en) * | 2013-03-25 | 2016-06-22 | 袁隆平农业高科技股份有限公司 | Rice Os 05g26890.1 albumen, the gene encoding this albumen and application thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111848765A (en) * | 2020-07-22 | 2020-10-30 | 中国水稻研究所 | Rice gene OsFBK4 and mutant and application thereof |
| CN111848765B (en) * | 2020-07-22 | 2021-10-08 | 中国水稻研究所 | Rice gene OsFBK4 and mutant and application thereof |
| NL2028064B1 (en) * | 2021-04-24 | 2022-04-05 | China Nat Rice Res Inst | Gene for controlling small grain and semi-dwarf of oryza sativa and application thereof |
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