CN109161554A - The controlling gene and application thereof of the crisp bar mutant DBC1 of rice dwarf - Google Patents
The controlling gene and application thereof of the crisp bar mutant DBC1 of rice dwarf Download PDFInfo
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Abstract
The invention belongs to gene engineering technology fields, and in particular to the controlling gene and application thereof of the crisp bar mutant DBC1 of rice dwarf.The present invention is by cloning the crisp bar mutant DBC1 of rice dwarf, being sequenced, excavate the controlling gene of the crisp bar of rice dwarf, and the experimental verification gene has the function of that adjusting and controlling rice downgrades crisp bar, strong tool is provided for Transgenic Rice research, can promote the breeding research of high yield and high quality rice.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to the controlling gene of the crisp bar mutant DBC1 of rice dwarf and
Its purposes.
Background technique
Rice is the most important cereal crops in China, and the population more than about half is using it as staple food；Meanwhile rice Yin Jiyin
Group homology smaller, with higher is the cereal grain crop being sequenced earliest, and it is raw to have become monocotyledon molecule
The model plant of object research.The application of " green revolution " and hybrid rice greatly improves rice list amount, to solution China's grain
Food safety produces significance.Straw-returning is the well stimulation of a culture fertility of most attention in the world today,
There are also getting fat production-increasing functions while having prevented atmosphere pollution caused by crop straw burning.In addition, straw-returning can increase soil
Organic matter improves soil texture, makes loosing soil, and porosity increases, and capacity mitigates, and promotes microbial activity and crop root
Development.Straw-returning getting fat production-increasing function is significant, can generally increase production 5%-10%.Since stalk has toughness, chopping and returning is not
Part easy thing, stalk processing are always thorny problem.Crisp bar mutant stalk moderately becomes fragile, and is conducive to straw-returning, is to solve this
One of one contradictory important technique measure.Brittle Culm Mutant of Rice normally behaves as the content of cellulose and lignin in cell wall
Variation, they can usually change stalk mechanical strength, and then influence lodging resistance in rice and environmental suitability.Therefore, stem is identified
Stalk moderately becomes fragile, economical character is unaffected or impacted lesser mutant, carries out Molecular level study to it, not only helps
In the biosynthesis mechanism for illustrating rice cell wall, and have to the environment-friendly type new rice variety of breeding " crisp without falling " important
Meaning.
Summary of the invention
The present invention is using the crisp bar mutant of rice dwarf as research object, by carrying out to the crisp bar mutant DBC1 of rice dwarf
Clone, sequencing, excavate the controlling gene of the crisp bar of rice dwarf, and that there is adjusting and controlling rice to downgrade is crisp for the experimental verification gene
The effect of bar.
Specifically, the present invention includes several aspects:
One aspect of the present invention is related to a kind of crisp bar mutant DMC1 gene of rice dwarf, has such as SEQ ID No.1
Shown in nucleotide sequence, the protein of coding has the amino acid sequence as shown in SEQ ID No.2.
Another aspect of the present invention is related to the crisp bar mutant DBC1 gene of rice dwarf applied to environment-friendly type water rice varieties
Purposes in breeding.
Another aspect of the invention is related to a kind of environment-friendly type water rice varieties selection, and the method includes by rice dwarf
Crisp bar mutant DBC1 gene or genophore are transformed into rice.
Another aspect of the invention is related to a kind of reagent of adjusting and controlling rice plant height, and main active is that rice dwarf is crisp
The protein of bar mutant DBC1 gene coding, the carrier of the crisp bar mutant DBC1 gene coding of expression rice dwarf or host are thin
Born of the same parents.
The invention has the benefit that excavating water outlet by being cloned, being sequenced to the crisp bar mutant DBC1 of rice dwarf
Rice downgrades the controlling gene of crisp bar, provides strong tool for Transgenic Rice research, can promote educating for high yield and high quality rice
Kind research.By experimental verification, which has the fissional effect of adjusting and controlling rice, to improve the plant type of rice side of providing
To.
Detailed description of the invention
Fig. 1 is the Morphological Identification figure of wild type and mutant DBC1；
Figure 1A is wild type and the mutant dbc1 for sowing 7 days；
Figure 1B is seedling stage wild type and mutant；
Fig. 1 C is tillering stage wild type and mutant；
Fig. 1 D is maturity period wild type and each internode length of mutant；
Fig. 1 E is wild type and each panel length statistics of mutant；
Fig. 1 F is that wild type and mutant plant height compare；
Fig. 2 is the histologic analysis of wild type (WT) and mutant (DBC1)；
Fig. 2A is the longitudinal sectional figure of the second section of heading stage wild type stem；
Fig. 2 B is the longitudinal sectional figure of the second section of heading stage mutant stem；
Fig. 2 C is the second leaf leaf sheath endepidermis scanning electron microscope (SEM) photograph of wild type；
Fig. 2 D is the second leaf leaf sheath endepidermis scanning electron microscope (SEM) photograph of mutant；
Fig. 2 E is wild type and mutant the second section stem cell size statistical chart；
Fig. 2 F is wild type and mutant the second leaf leaf sheath endepidermis cell size statistical chart；
Fig. 2 G is the two section stem sectional views of falling of wild type；
Fig. 2 H is the two section stem sectional views of falling of mutant；
Fig. 2 I is the enlarged drawing of Fig. 2 G red frame region；
Fig. 2 J is the enlarged drawing of Fig. 2 H red frame region；
Fig. 3 is the map based cloning of gene DBC1；
Fig. 3 A is the molecule positioning on the 2nd chromosome；
Fig. 3 B is the gene structure of DBC1；
Fig. 3 C is the amplified fragments size of wild type (WT) and dbc1；
Fig. 3 D is that the sequencing of wild type and mutant compares；
Fig. 3 E is that wild type and variant amino acid sequence compare；
Fig. 4 is the expression pattern analysis of DBC1 gene；
Fig. 5 is transgenic plant identification and phenotypic analysis；
Fig. 5 A is maturity period wild type, mutant and transgenic plant；
Fig. 5 B is wild type, the Brittleness Analysis of mutant and transgenic plant；
Fig. 5 C is expression analysis of the DBC1 gene in wild type, mutant and transgenosis overexpression strain；
Fig. 6 is response and analysis of related genes of the mutant DBC1 to gibberellin GA3；
Fig. 6 A is that wild type the second leaf leaf sheath responds experiment to gibberellin；
Fig. 6 B is the response experiment of the second leaf of mutant DBC1 leaf sheath gibberellin；
Fig. 6 C is that wild type and mutant the second leaf leaf sheath test gibberellin GA3 growth response；
Fig. 6 D is the expression analysis of gibberellin biological signal transduction related gene；
Fig. 6 E is the expression analysis of gibberellin biological synthesis related gene；
Fig. 6 F is that gibberellin GA3 influences the expression of GA2ox3 in wild type and mutant DBC1；
Fig. 7 is the Meristernatic zone cellular morphology and cyclin expression analysis of wild type and mutant；
Fig. 7 A is wild type Meristernatic zone cellular morphology；
Fig. 7 B is mutant Meristernatic zone cellular morphology；
Fig. 7 C is the enlarged drawing of red frame region in Fig. 7 A；
Fig. 7 D is the enlarged drawing of red frame region in Fig. 7 B；
Fig. 7 E is the unicellular number statistical figure that the tip of a root contains two nucleus；
Fig. 7 F is cyclin expression analysis；
Detailed description of the invention: " * " indicates that, in 0.05 horizontal upper significant difference, " * * " is indicated in 0.01 horizontal upper significant difference.
Specific embodiment
The present invention is clearly and completely described below by specific embodiment, it is clear that described embodiment is only
It is a part of the embodiments of the present invention, instead of all the embodiments.Based on the embodiment of this practical invention, the common skill in this field
Art personnel every other embodiment obtained, belongs to protection scope of the present invention.
Test method without specific conditions in embodiment, usually according to normal condition, such as Molecular Cloning: A Laboratory refers to
Condition described in south (third edition, J. Pehanorm Brooker etc. write, and Huang Peitang etc. is translated, Science Press, 2002), or according to system
Make condition proposed by manufacturer.
Material used in embodiment: extensive No. 10 of wild type red silk and the crisp bar mutant DBC1 of rice dwarf are by Southwest University's water
Rice research institute provides；M-MLV reverse transcriptase, high-fidelity DNA polymerase PFU, T4DNA ligase, Trizol kit, DNA are solidifying
Plastic recovery kit, plasmid extraction kit are purchased from TaKaRa company；Ampicillin (Ampicillin, Amp) is Sigma public
Take charge of product；Primer synthesis and DNA sequencing are completed by the handsome Bioisystech Co., Ltd in Shanghai；Other chemical reagent are purchased from Beijing ancient cooking vessel
Biotechnology Co., Ltd of state；Bacillus coli DH 5 alpha is provided by Southwest University's rice research；Expression vector PTCK303 is equal
It is provided by Southwest University's rice.
In previous research work, Southwest University's rice research is provided: utilizing EMS mutagenesis Elite restorer line red silk extensive 10
It is obvious compared to wild type to show as plant height for the crisp bar mutant (being named as DBC1) of rice dwarf for number obtaining an inheritance stability
It downgrades, brittleness obviously increases, and it is horizontal up to significant difference, see Figure 1A-F, wherein in 1E, PL represents spike length, and NL represents neck length of spike,
FL represents long between Section 1, and SL represents long, Bar=5cm between Section 2, and " * " indicates that " * * " is indicated in 0.05 horizontal upper significant difference
In 0.01 horizontal upper significant difference.11 are spent to hybridize with mutant DBC1 in the normal sterile line of phenotype, F1 generation phenotype is normal, says
The bright mutant is controlled by recessive gene.F2 shows parents' character for occurring apparent separation in group respectively.It is surveyed through card side
It tests, normal Zhu ﹕ mutant strain meets 3: 1 segregation ratio, shows that DBC1 mutant is controlled by Recessive genes.Pass through paraffin section and electricity
Sub- microscope (referring to Ph.D. Dissertation: the map based cloning of Rice Vascular Bundle development related gene AVB gene and functional analysis,
2016,33-34 pages) cytological observation discovery is carried out, the size of cell without significant difference, that is, passes through influence compared with wild type
Cell number is shown in Fig. 2A-F so as to cause plant height dwarfing, wherein in 2A-2D, Bar=100 μm, and in 2E-2F, n=50.In addition,
The gene affects the structure of vascular bundle in stem, and xylem relative area increases, and sees Fig. 2 G-J, wherein 2G-J kind, Bar=
200 μm, in 2I-J, Bar=50 μm.Using the F2 group of middle colored 11/DBC1 hybridization as target group, 600 mutation are obtained altogether
Strain is used for the assignment of genes gene mapping.400 pairs of SSR markers being uniformly distributed on 12 chromosome are selected to be mutated F2 for target group single
Strain progress single plant verifying (referring to master thesis: the genetic analysis and the assignment of genes gene mapping of rice leaf margin albino mutant mal,
2014, page 22), as a result Linkage mapping is mutated in the Ind label Indel02-2 and Indel02-7 and DBC1 on 2 chromosomes
Site performance is chain.Two label between further design Indel primer, wherein Indel02-1-Indel02-7, between two parents
Polymorphism is all shown, primer sequence is auspicious to be shown in Table 1, and DBC1 is finally located in Indel label Indel02-4 and Indel02-5
Between, physical distance 86.7kb is shown in Fig. 3 A.
1 gene finely positioning the primer of table and its sequence
Clone, sequencing and the functional verification of the crisp bar mutated gene DBC1 of 1 rice dwarf of embodiment
1, the clone of the crisp bar gene DBC1 of rice dwarf and sequencing
On the basis of early period is to mutated gene DBC1 finely positioning, the present invention by online predictive genes (http: //
Mendel.cs.rhul.ac.uk), BLAST compares (http://blast.ncbi.nlm.nih.gov/) and the super table of gene online
Up to functional analysis.By map based cloning, sequence alignment discovery, which is since the 5th of gene LOC_Os02g50550 includes
5th base of son sports T by normal C.Due to being modified, being sheared and being spliced after genetic transcription, i.e., introne is prominent
Become the shearing editor that may will affect precursor mRNA.Therefore, a pair of primer P1 at a distance of 550bp or so is designed on cDNA,
P2 includes mutational site, sees Fig. 3 B, and wherein gray box represents exon, and black line represents introne, underscore mark the
Five exons, red base represent mutational site.Pcr amplified fragment shows that the band of mutant DBC1 is opposite through gel electrophoresis
Lower than wild type, there may be the missing of large fragment, see Fig. 3 C-D.Clone gene DBC1 is transferred to expression vector pMD19-T, chooses spot,
Detection send positive bacterium solution to be sequenced.Comparison result shows that mutant has the missing of 62bp, the missing of the segment compared to wild type
It is placed exactly at the 5th exon, sees Fig. 3 E.That is, the single base mutation of the 5th introne of mutant DBC1 leads to the 5th exon
Whole missing.Amino acid alignment discovery to wild type and mutant, the gene mutation cause GTP structural domain to become
Change.
2, the functional verification of the short crisp gene DBC1 of rice
Expression analysis hair is carried out by LOC_Os02g50550 of the real time fluorescent quantitative qPCR to wild type and mutant
It is existing, (referring to Ph.D. Dissertation: the map based cloning of rice Spikelet development related gene MFS1 and functional analysis, 2013,29
Page) the expression quantity versus wild type of LOC_Os02g50550 gene is obviously lowered in mutant DBC1, and LOC_Os02g50550
There is expression at each position of rice, expresses highest in plant stalk and apical meristem, see Fig. 4.Then, structure of the present invention
It has built using the cDNA of the extensive LOC_Os02g50550 gene of wild type red silk as template, the segment of amplification is connected to expression vector
On pTCK303, pTCK303-DBC1OE gene overexpression vector is constructed, Agrobacterium tumefaciens-mediated Transformation to mutant DBC1 is passed through
In.The discovery of transgenic line phenotype is observed, their phenotype is similar to wild type, returns to normal.LOC_ is primarily determined
Os02g50550 is the candidate wild type gene of rice dwarf brittleness mutated gene DBC1, sees Fig. 5 A-C.
The hormone of the short crisp mutated gene DBC1 of 2 rice of embodiment responds experimental analysis
1) it by mutant and wild type seeds vernalization, chooses the consistent seed that shows money or valuables one carries unintentionally and broadcasts respectively and containing 0,10-10、10-8、
10-6With 10-4In the nutrient solution of μM gibberellin (GA), it is placed in constant incubator and cultivates.Add the nutrition of equivalent respectively daily
The nutrient solution of liquid and the gibberellin containing respective concentration.Every the length variation of 24 hours measurement the second leaf sheaths of statistics, until two leaves are wholeheartedly
Phase.And the blade under (the about the 7th day) takes each processing in one heart stage an of leaf, RNA is extracted, and reverse transcription is saved backup at cDNA.Knot
Fruit discovery, when gibberellin concentration is lower than 10-8μM when, the growth of wild type and mutant the second leaf leaf sheath and blank control group are almost
Indifference；When concentration rises to 10-6μM, plant starts to increase rapidly, and wild type and mutant leaf sheath length difference increase；With
Concentration continues to rise, the second leaf leaf sheath length difference of wild type and mutant also by increase, see Fig. 6 A-C.
2) quantitative PCR analysis
The growth of plant is inseparable with hormone sensitive lipase gene metabolism.Take the overground part of a leaf one heart stage wild type and mutant
Point, RNA is extracted, reverse transcription is at the first chain of cDNA, using participating in gibberellin biosynthesis genes in quantitative PCR analysis rice
GA2ox-1, GA2ox-3, GA20ox-1, GA20ox-2/SD1, GA3ox-2/D18, carbamyl phosphate synthetase (CPS1), and letter
Number differential expression of conduction path gene M YB61, NAC29, NAC31, SLR1 and GID1 in wild type and mutant.Meanwhile
Detect the expression of gibberellin inactivation gene GA2ox-3 in wild type and the mutant under various concentration processing.Quantitative primer
Sequence is shown in Table 2.As the result is shown: the expression quantity of MYB61, GID1 and NAC29 in mutant reduces, SLR1 and NAC31 expression quantity
It increases, sees Fig. 6 D.In addition, the GA2ox-3 expression quantity in mutant DBC1 is significantly higher than wild type, in addition to this, GA20ox-1
It is substantially less than wild type with expression quantity of the GA20ox-2/SD1 in mutant, sees Fig. 6 E, wherein ACTIN gene is as internal reference
Gene.GA2ox-3 is gibberellin inactivation gene, applies gibberellin outside or endogenous gibberellins content increases, the expression meeting of GA2ox-3
It increases.Therefore, show money or valuables one carries unintentionally consistent wild type and mutant seeds are handled with the gibberellin of water and various concentration, and in light
It is cultivated under conditions of being suitble to according to, temperature and humidity.Analyze GA2ox-3 differential expression therebetween and expression under various concentration
Trend.It is by Fig. 6 F it is found that consistent with gibberellin response experimental result trend, it is 10 in concentration-6μM when, expression quantity start occur it is poor
It is different.Rise with concentration, expression of the GA2ox-3 in wild type and mutant all increases, and in the expression in mutant
It adjusts faster.This may explain Exogenous gibberellic acid increase, the slow phenomenon of change of height.
2 quantification PCR primer sequence of table
The cell splitting rate of 3 mutant DBC1 of embodiment is tested
Paraffin section analyzes tip of a root growing point cell, under same cell number, the single celled par of double-core in DBC1
It is 7.4, is 4.1 in wild type, is in the level of signifiance, sees Fig. 7 A-E.Which imply that mutant cells split speed slows down.
RT-PCR analyzes wild type, mutant and the expression variation for overexpressing cyclin related gene in strain OE-1, RT-
PCR quantifies primer sequence and is shown in Table 3, the results showed that, the expression quantity of the associated period protein gene in mutant is dropped compared to wild type
It is low；The expression of CycB, CycD, CycP and CycT are all restored in transgenic line, see Fig. 7 F.This is just further illustrated
The cell splitting rate of mutant DBC1 slows down.That is, the mutation of the gene causes cell splitting rate to slow down.
3 cyclin RT-PCR of table quantifies primer sequence
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, all any modification, equivalent substitution, improvement and etc. be should all be included in the protection scope of the present invention.
Sequence table
<110>Southwest University
<120>controlling gene and application thereof of the crisp bar mutant DBC1 of rice dwarf
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<210> 3
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<213>artificial sequence (Artificial Sequence)
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<211> 3
<212> DNA
<213>artificial sequence (Artificial Sequence)
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Claims (4)
1. the controlling gene of the crisp bar mutant DBC1 of rice dwarf has the nucleotide sequence as shown in SEQ ID No.1, coding
Protein have the amino acid sequence as shown in SEQ ID No.2.
2. the controlling gene of the crisp bar mutant DBC1 of rice dwarf is applied to the purposes in environment-friendly type rice variety selective.
3. a kind of environment-friendly type water rice varieties selection, the method includes the crisp bar of rice dwarf described in claim 1 is dashed forward
The controlling gene or genophore of modification D BC1 is transformed into rice.
4. the reagent of adjusting and controlling rice plant height, the controlling gene that main active is the crisp bar mutant DBC1 of rice dwarf is encoded
Protein, expression the crisp bar mutant DBC1 of rice dwarf controlling gene coding carrier or host cell.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111471697A (en) * | 2020-03-20 | 2020-07-31 | 西南大学 | Rice crisp stalk regulatory gene DBC2 and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1488643A (en) * | 2002-10-10 | 2004-04-14 | 中国科学院遗传与发育生物学研究所 | Paddy rice fragile straw controlling gene BC1 and use thereof |
| KR100658183B1 (en) * | 2005-03-02 | 2006-12-14 | 인제대학교 산학협력단 | Method for detection of genetic variants of breast cancer resistance protein gene |
| US7605310B2 (en) * | 2007-05-30 | 2009-10-20 | Limagrain Verneuil Holding Sa | Inbred corn line BC5 |
| CN103242436A (en) * | 2012-02-08 | 2013-08-14 | 中国科学院遗传与发育生物学研究所 | mechanical strength controlling gene BC15 of rice stem and application thereof |
| CN108913668A (en) * | 2018-07-27 | 2018-11-30 | 西南大学 | Rice albefaction turns albumen and the application of greenery gene VAL1 and its coding |
-
2018
- 2018-09-30 CN CN201811154661.4A patent/CN109161554B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1488643A (en) * | 2002-10-10 | 2004-04-14 | 中国科学院遗传与发育生物学研究所 | Paddy rice fragile straw controlling gene BC1 and use thereof |
| KR100658183B1 (en) * | 2005-03-02 | 2006-12-14 | 인제대학교 산학협력단 | Method for detection of genetic variants of breast cancer resistance protein gene |
| US7605310B2 (en) * | 2007-05-30 | 2009-10-20 | Limagrain Verneuil Holding Sa | Inbred corn line BC5 |
| CN103242436A (en) * | 2012-02-08 | 2013-08-14 | 中国科学院遗传与发育生物学研究所 | mechanical strength controlling gene BC15 of rice stem and application thereof |
| CN108913668A (en) * | 2018-07-27 | 2018-11-30 | 西南大学 | Rice albefaction turns albumen and the application of greenery gene VAL1 and its coding |
Non-Patent Citations (8)
| Title |
|---|
| BIN WU等: ""Brittle culm15 encodes a membrane-associated chitinase-like protein required for cellulose biosynthesis in rice"", 《PLANT PHYSIOL》 * |
| KIKUCHI,S等: ""Oryza sativa Japonica Group cDNA clone:J013074K16,full insert sequence"", 《GENBANK DATABASE》 * |
| KO HIRANO等: ""Rice BRITTLE CULM3(BC3) encodes a classical dynamin OsDRP2B essential for proper secondary cell wall synthesis"", 《PLANTA》 * |
| XIANCHUN SANG等: ""DBC1，an allelic OsDRP2B gene,regulating plant height by controlling cell division in Oryza sativa L."", 《MOL BREEDING》 * |
| 桑贤春等: ""水稻矮杆脆性突变体dbc1的鉴定与基因定位"", 《作物学报》 * |
| 舒亚洲: ""水稻脆秆突变体bc16的遗传分析和基因精细定位"", 《中国优秀硕士学位论文全文数据库（电子期刊）》 * |
| 许作鹏等: ""水稻脆秆突变体bc1-wu3的鉴定与基因克隆"", 《中国水稻科学》 * |
| 许作鹏等: ""水稻脆秆突变体bc-wy7的鉴定与基因定位"", 《植物生理学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111471697A (en) * | 2020-03-20 | 2020-07-31 | 西南大学 | Rice crisp stalk regulatory gene DBC2 and application thereof |
| CN111471697B (en) * | 2020-03-20 | 2022-02-01 | 西南大学 | Rice crisp stalk regulatory gene DBC2 and application thereof |
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