CN107937409A - The clone of rice tillering angle gene TAC3 and application - Google Patents
The clone of rice tillering angle gene TAC3 and application Download PDFInfo
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- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
Abstract
The invention belongs to field of plant genetic.Clone and application more particularly to rice tillering angle gene TAC3.The sequence of TAC3 genes such as SEQ ID NO：Shown in 1, cDNA sequence such as SEQ ID NO：Shown in 2, the protein sequence such as SEQ ID NO of coding：Shown in 3 and 4.The relevant QTL site of rice tillering angle is positioned using GWAS, is analyzed with reference to LD and reduces qTA3 scopes, candidate gene TAC3 is determined with reference to reverse genetics means.TAC3 gene functions are verified by gain-of-function type mutant tac3D 1, gene expression quantity in tac3D 1 significantly improves, compared with wild type ZH11, tillering stage tillering angle improves significantly to 19.4 ° by 10.4 °, and florescence tillering angle improves significantly to 17.6 ° by 8.4 °.There are significant difference for tillering angle in different haplotype long-grained nonglutinous rice materials for TAC3 genes.
Description
Technical field
The present invention relates to field of plant genetic.Clone more particularly to rice tillering angle gene TAC3 and should
With the gene is located on rice trisome, which controls the tillering angle of rice plant.
Background technology
The tillering angle of rice determines that the grown per area of plant is close as one of major traits for moulding ideotype
Degree and crop yield.Preferable tillering angle had not only been avoided that angle too small caused some diseases induced by high humility, but also can keep away
Exempt from photosynthetic efficiency reduction and the decline of yield per unit area caused by sprawl growth, therefore, in the long-term domestication of rice
With the selection that the mankind are received during genetic improvement.However, on rice tillering angle genetic molecule mechanism research until
Just comparatively fast developed within nearly 30 years.
The end of last century, researcher have excavated some control rice tillering angles using classical double parents mapping
QTL (Quantitative Trait Loci) site (Li et al., 2006, New phytologist 170:185-194；Li
Deng 1999, Euphytica 109:79-84；Qian etc., 2000, Yi chuan xue bao=Acta genetica
Sinica 28:29-32；Thomson etc., 2003, Theoretical and applied genetics107:479-493；Xu
Deng, 1995, Science in China Series B, Chemistry, life sciences＆earth sciences 38:
422-428；Yan etc., 1999, Crop science 39:538-544).In the past 10 years, map based cloning and reverse genetics are passed through
Technology, cloned the tillering angle such as LAZY1, TAC1, PROG1, LPA1, SOLs related gene (Li et al., 2007, Cell
research 17:402-410；Yoshihara etc., 2007, Plant and cell physiology 48:678-688；Yu
Deng 2007, The Plant Journal 52:891-898；Jin etc., 2008, Nature genetics 40:1365-1369；
Tan etc., 2008, Nature genetics 40:1360-1364；Wu etc., 2013, Plant physiology161:317-
329；Sang etc., 2014, Proceedings of the National Academy of Sciences 111:11199-
11204), this has preferable understanding to the molecular mechanism for controlling rice tillering angle.
Tillering angle between Different Rice Varieties is there are larger natural variation, however, being currently known control tillering angle
Gene it is very limited, especially control tillering angle natural variation related gene.Therefore, it is badly in need of excavating more points at present
Tiller angle relevant gene preferably improves its hereditary basis, so that assistant breeding.In recent years, whole-genome association
(GWAS) quoted extensively in crop as a kind of method of effective detectable complex character natural variation (Huang and
Han, 2014, Annual review of plant biology 65:531-551), this method be based on substantial amounts of DNA marker and
Association between phenotypic character, sufficiently make use of the phenotypic variation enriched between crop varieties and hereditary variation (Rafalski,
2010, Current opinion in plant biology 13:174-180).
The application report in adjusting and controlling rice tillering angle in relation to TAC3 genes is had no so far.
The content of the invention
The defects of it is an object of the invention to overcome the prior art, separation clone one is located at trisome from rice
The gene of upper control tillering angle, which is TAC3 (Tiller Angle Control 3) gene by applicant, profit
With the tillering angle of this improvement of genes rice, so as to achieve the purpose that to control plant type of rice and yield.
Technical scheme is as described below：
TAC3 genes according to the present invention, are to be based on State Key Laboratory of Crop Genetic Improvent
The 529 parts of cultivated rices (Oryza sativa) having SNPs mark (document referring to：Zhao etc., 2015, Nucleic acids
research 43:D1018-D1022) and corresponding florescence tillering angle phenotypic data carries out GWAS analyses, further combined with
The mutant 05Z11AZ62 of State Key Laboratory of Crop Genetic Improvent report (publish by the mutant information
In State Key Laboratory of Crop Genetic Improvent Rice mutant pool, network address is downloaded：http://
rmd.ncpgr.cn/.Document referring to：Wu etc., 2003, The Plant Journal 35:418-427；Zhang etc., 2007,
The Plant Journal 49:947-959), the base for the control rice tillering angle on trisome being cloned into
Because of TAC3, the conservative hypothesis albumen of the gene code one.The separation clone of TAC3 genes helps to understand rice tillering angle
Genetic mechanism, for cultivate ideotype new genetic resources is provided.
Application of one separated TAC3 gene in rice tillering angle is controlled, the nucleotide sequence such as SEQ of the gene
ID NO：Shown in 1.
Application of one separated TAC3 gene in rice tillering angle is controlled, the protein sequence of the gene code is such as
SEQ ID NO：Shown in 3.
Application of one separated TAC3 gene in rice tillering angle is controlled, the protein sequence of the gene code is such as
SEQ ID NO：Shown in 4.
The nucleotide sequence and protein sequence of the TAC3 genes of the present invention can also be in plant type of rice improvement and rice
Further applied in breeding.
More detailed technical solution is as described below：
The present invention positions the relevant QTL site of rice tillering angle using the method for whole-genome association (GWAS),
Analyzed further combined with linkage disequilibrium (LD) and reduce QTL scopes, finally determine candidate gene with reference to reverse genetics means.
In addition to the application in rice tillering angle character is controlled, other are multiple for the colony of the invention for being used for GWAS analyses
Application in character (such as characters such as metabolism, heading stage, fringe types) has been reported (Chen etc., 2014, Nature genetics
46:714-721；Yang etc., 2014, Nature communications 44：92-96).
QTA3 is navigated to trisome 29,504,013~29 by the present invention using 295 parts of long-grained nonglutinous rice materials therein,
On 791,496 physical locations (MSU.V6), it is candidate gene that further LD analyses, which are narrowed down to 2 genes, finally by sieve
The mutant of anthology invention, LOC_ is determined by separating identification, gene expression spectrum analysis and haplotype analysis
Os03g51660 is the candidate gene that TAC3 is the site.Mutant 05Z11AZ62 is that the gain-of-function type of TAC3 genes is dashed forward
This is had the function of that new mutant 05Z11AZ62 is named as tac3D-1 by variation, the present invention, and TAC3 genes are in tac3D-1
Middle expression quantity significantly improves, and corresponding tillering stage tillering angle is significantly improved compared with 11 (ZH11) are spent in wild type by 10.4 °
To 19.4 °, florescence tillering angle improves significantly to 17.6 ° by 8.4 °.Between these 295 parts of long-grained nonglutinous rice material TAC3 difference haplotypes
For tillering angle there are significant difference, this provides molecular basis for later genetic breeding.
The present invention has the advantages that following prominent and effect compared with prior art：
(1) present invention reports the gene TAC3 of an adjusting and controlling rice tillering angle first, to cultivate Ideal Rice Plant Type
Provide new genetic resources and hereditary basis.
(2) present invention combines the method that GWAS, LD, gene expression spectrum analysis and mutant isolate identification, rapidly and efficiently
Clone gene, technological borrowing is provided for the research and gene cloning of correlated traits genetic mechanism in other crops.
Brief description of the drawings
SEQ ID NO：1 is the DNA sequence dna of TAC3 genes, sequence 1717bp.
SEQ ID NO：2 be the cDNA sequence of TAC3 genes, and the present invention relates to two transcripts, their transcription initiation position
Point is different, therefore in this sequence, 1-408 bit base sequences are transcripts 1, i.e., code area 1 (CDS1)；56-169 alkali
Basic sequence is transcript 2, i.e. code area 2 (CDS2).
SEQ ID NO：3 be the protein sequence of TAC3 gene Cs DS1 codings, encodes the protein sequence of 135 amino acid
Row.
SEQ ID NO：4 be the protein sequence of TAC3 gene Cs DS2 codings, encodes the protein sequence of 37 amino acid.
Fig. 1：The GWAS analysis results of 295 parts of long-grained nonglutinous rice flowering period tillering angles.Description of reference numerals：
A figures in Fig. 1 are to pass through linear regression model (LRM) according to 295 parts of China Hainan long-grained nonglutinous rice florescence tillering angle data
(LR) (left figure) manhattan and (right figure) quantile-quantile figures that GWAS is analyzed are carried out；B figures in Fig. 1 are
(left figure) analyzed according to 295 parts of long-grained nonglutinous rice florescence tillering angle data of Wuhan, China by LR progress GWAS
Manhattan and (right figure) quantile-quantile schemes.
Fig. 2：The LD analyses of TAC3 candidate genes.Description of reference numerals:Calculate all polymorphic positions in two candidate genes
Point (SNP) two-by-two between r2Value (method for weighing LD values), numerical values recited is indicated by scale.Heavy black line indicates lead SNP
Put.
Fig. 3：Tac3D-1 mutation type surfaces picture and tillering angle statistic analysis result.Description of reference numerals：
A figures in Fig. 3 are tillering stage tac3D-1 mutation type surface picture；B figures in Fig. 3 are florescence tac3D-1 mutation
Body surface type picture；C figures in Fig. 3 are 2015 and Wuhan, China summer tac3D-1 mutant tillering angle statisticals in 2016
Analyse result.In figure 3：WT, represents wild type ZH11；Tac3D-1H, represents heterozygous mutation body；Tac3D-1M, represents homozygous
Type mutant；* * expressions mutability compares tillering angle with wild type, and there are significant difference, p<0.001；* is represented
Mutability compares tillering angle with wild type, and there are significant difference, p<0.01.Fig. 4：Tac3D-1 mutant isolates identification
As a result.Description of reference numerals：
A figures in Fig. 4 are to isolate identification on DNA level.L, R represent that the identification on rice genome should respectively
Left and right primer on mutant, N represent the primer on carrier.B figures in Fig. 4 be expression quantity it is horizontal isolate identification.W and
WT, represents wild type ZH11；H and tac3D-1H, represents heterozygous mutation body；M and tac3D-1M, represents homozygous prominent
Variation；* * expressions mutability compares TAC3 gene expression amounts with wild type, and there are significant difference, p<0.001.
Fig. 5：TAC3 genes are in rice at whole growth periods spatial and temporal expression spectrum analysis.Description of reference numerals：
Material therefor is to spend 11 (ZH11) in wild rice kind；Mark 1 represents tillering stage tissue sample, identifies 2 tables
Show florescence tissue sample.
Embodiment
Embodiment 1：The GWAS analyses of rice anthesis tillering angle
1. the investigation of rice anthesis tillering angle
Respondent includes local varieties and the Asian Cultivated Rice germ plasm resource of improved seeds for 529 parts, is divided into 9 small Asias
Group：IndI, indII, indica osculant, Tej, Trj, japonica osculant, Aus, VI and osculant.Wherein indI,
3 small subgroups of indII and indica osculants belong to long-grained nonglutinous rice subgroup, by 295 parts of kinds (Zhao etc., 2015, Nucleic
acids research 43:D1018-D1022).The colony has abundant phenotypic variation, using it to metabolic trait, agronomy
The character such as character such as florescence, plant height, fringe type carries out phenotypic analysis and GWAS genetic analyses, and the phenotype of rice natural population is become
Different and hereditary basis has more fully parsing (Chen etc., 2014, Nature genetics 46:714-721；Yang etc.,
2014, Nature communications 44：92-96；Han etc., 2016, Front.Plant Sci.7:1270；Bai etc.,
2016, Plant Genome).Its tillering angle is investigated after present invention selection Rice Flowering within about 5 days or so, and phase is investigated with protractor
Away from the angle between two farthest tillers, the half of numerical value is the tillering angle of the single plant.The colony was respectively at 2013
Winter plants in Wuhan, China in China Hainan and summer in 2014 and investigates.
2. the GWAS analyses of tillering angle
529 parts of Asian Cultivated Rices Illumina Genome Analyzer II sequencing analysis, whole gene group DNA
Sequence coverage is about 2.5 times and is used for genotyping (Chen etc., 2014, Nature genetics 46:714-721).
In FaST-LMM programs, 3916415,2767159 and 1857845 SNPs are shared in total group and indica rice subgroup respectively at the same time
Meet MAF (minor allele frequency) >=0.05 and minor allele type kinds number no less than 6 conditions and pass through
LMM (linear mixture method) and LR (linear regression) two methods are used for GWAS analyses (Lippert
Deng 2011, Nature Methods 8:833-835；Chen etc., 2014, Nature genetics 46:714-721).When logical
Cross and help Consideration supplemented by group structure and affiliation (kinship) when LMM methods carry out GWAS analyses.In total group and Xian
Respectively obtained in japonica rice subgroup 757578,571843 and 245348 effectively independent SNPs (Li et al., 2009,
Bioinformatics 25:2078-2079).P value is respectively 1.3 × 10 in total group and indica rice subgroup-6、1.8×10-6With 4.1 × 10-6As the effective of the conspicuousness correlation signal being detected simultaneously by with LMM methods in China Hainan and Wuhan, China
Threshold value；Since LMM can cause false negative to reduce number of sites, LR can cause false positive site to occur, and therefore, p value is 1.0 × 10-8
Effective threshold value of correlation signal is then detected as LR methods, and selects to meet that first 5 of the condition are used for follow-up study.This hair
QTA3/TAC3 in bright is to carry out GWAS by LR methods in long-grained nonglutinous rice subgroup to analyze the conspicuousness detected association site,
The p value of the lead SNP (being sf0329582676) of two annual data of China Hainan and Wuhan, China is respectively 3.9 × 10-16With
1.1×10-23(Fig. 1).
Embodiment 2：QTA3 association site candidate genes determine
1. linkage disequilibrium (LD) is analyzed
Between unbalance factor (D ') and the SNP site of pairing that linkage disequilibrium is standardized by TASSEL5.0 softwares
Square (the r of allelotype coefficient2) weigh research.Average LD decline of the research colony of the present invention in the range of full-length genome
Value has been reported, in total group and Xian round-grained rice subgroups be respectively 167kb, 93kb and 171kb (Xie etc., 2015, Proceedings
of the National Academy of Sciences of the United States of America 112:5411-
5419).We calculate the LD decay section of covering lead SNPs, and method is as follows：First, calculate lead SNP and its above and below
Swim the r between all SNPs in 2Mb regions2Value；Then lead SNP and in upstream and downstream 1.5Mb to 2Mb sections preceding 10% r are used2Value
Average value as background value；The counted r of the last first step2It is lead that value, which subtracts continuum of the background value more than 0.2,
The decay section of SNP.Wherein, the LD decay section of the qTA3/TAC3 in the present invention is 29504013~29791496.Further
The LOC_Os03g51670 and LOC_ for including lead SNP (sf0329582676) are drawn by Haploview4.2 software analysis
The r between all SNP between Os03g516602Value, which part SNPs show as height linkage disequilibrium, LOC_Os03g51660
5 ' ends with LOC_Os03g51670 positioned at (Fig. 2) in LD cell.Result above prompt our LOC_Os03g51660 and
LOC_Os03g51670 may be the candidate gene of qTA3/TAC3.
2.tac3D-1 mutant isolates identification
In order to quickly determine candidate gene, in RiceGE databases (http://signal.salk.edu/cgi-bin/
RiceGE the generation of LOC_Os03g51660 and LOC_Os03g51670 genes) is have found in the rice mutant information announced respectively
The mutant of T-DNA insertion mutations.Wherein, mutant 05Z11AZ62 spends 11 (ZH11) backgrounds in being, from Hua Zhong Agriculture University
Mutant library (the mutant library website of crop genetic improvement National Key Laboratory：http://rmd.ncpgr.cn/.Text
Offer and see Wu etc., 2003, The Plant Journal 35:418-427；Zhang etc., 2007, The Plant Journal 49:
947-959), its T-DNA insertion point is the promoter region that distance LOC_Os03g51660 initiation codons are 991bp.Will
The mutant is planted in Wuhan, China Hua Zhong Agriculture University reality respectively at summer in 2015 (60 single plant) and summer in 2016 (96 single plant)
Field is tested, investigates the tillering angle in its tillering stage and florescence, statistical observation finds that tiller angle occurs in repetition plantation in 2 years
Spend the separation single plant (the b figures in a figures and Fig. 3 in Fig. 3) of increase.
To prove whether tillering angle causes compared to the single plant that wild type significantly increases since T-DNA is inserted into, it is prominent to choose this
The fresh blade extracting DNA of all single plants of variation family, proves whether gene isolates with phenotype on DNA level.DNA is extracted
Method is with reference to CTAB methods (Zhang etc., 1992, Theor Appl Genet, 83,495-499).Utilize 2 couples of PCR primer (L1+R1
Totally 3 primers, primer sequence information have the offer of RiceGE databases with N1+R1；L1 and R1 primers is on rice genomes
Primer, N1 are vector primer, primer sequence：L1-CGAGTAGCTACGGATGAGGC, R1-TTCTTCAACTCTGATGGGGC,
N1-AATCCAGATCCCCCGAATTA), PCR amplification is carried out from the genome of all single plants of mutation family using r-Taq
(PCR reaction total systems are 20 μ l, are with method specifically：2 μ l, 10xPCR buffer of the first chains of DNA template, 2 μ l, 10mM dNTP
1.5 μ l, two-way each 0.2 μ l of 0.3 μ l, r-Taq enzyme of primer, add distilled water to 20 μ l.Used PCR buffer, dNTP, r-
Taq enzyme etc. is purchased from precious bioengineering Dalian Co., Ltd product.PCR reaction conditions are as follows：1. 94 DEG C 4 minutes, 2. 94 DEG C 30
Second, 3. 58 DEG C 30 seconds, 4. 72 DEG C 60 seconds, the 5. step be 6. 72 DEG C 7 minutes from 2. step -4. step circulation 35 times, 7. 4 DEG C of preservations).
PCR product is detected with agarose gel electrophoresis, and the correspondence single plant that only combination of L1+R1 primers can amplify band is no T-
The WT lines of DNA insertions, represent that the correspondence single plant that only combination of N1+R1 primers can amplify band is to have T-DNA with W
The homozygous mutation plant of insertion, is represented with M, and the correspondence single plant that two pairs of primer combinations can amplify band is to have T-DNA
The heterozygous mutant single plant of insertion, is represented (Fig. 4 a) with H.The tillering angle of the single plant of 3 kinds of genotype is counted respectively, and carries out t surveys
Test detection (Fig. 3 c), the results showed that the tillering stage of 2 years and the homozygous mutant M tillering angles average value in florescence are maximum, heterozygosis
Saltant type H tillering angle average values are placed in the middle, and wild type W is minimum, and there are the difference of conspicuousness between saltant type and wild type,
Therefore, which is named as tac3D-1, concrete outcome such as table 1 below：
The tillering angle in table 1tac3D-1 mutant family tillering stages and florescence counts
* *, represent p<0.001；*, represents p<0.01
To prove whether the mutant is gain-of-function mutant, i.e., whether because of LOC_Os03g51660 expression quantity
Improving causes the increase of mutant plant tillering angle, and the present invention chooses in summer in 2016 in Wuhan has just started tillering occur
Blade extracting RNA, each genotype, which chooses single plant number and is used for expression quantity no less than 9 plants, to be detected.Pass through conventional RT-PCR side
Method (referring to：J. Pehanorm Brooker, not Ritchie, T Mannies A Disi write EF, Huang Peitang, and Wang Jiaxi etc. is translated, and Molecular Cloning: A Laboratory refers to
Southern (third edition), Beijing, Science Press, 2002 editions) amplification obtains cDNA sequence.The specific amplification method of cDNA is as follows：
1) first extracting just starts the RNA for the blade for tillering occur, and RNA extractings use Invitrogen companies
Trizol extraction agents box (concrete operation step is shown in kit specification)；
2) reverse transcription synthesizes the first chains of cDNA in RT-PCR：With mixed liquor 1：Total serum IgE 4 μ g, DNaseI 2U, 10x
DNAseI buffer1 μ l, add DEPC (pyrocarbonic acid diethyl ester, a kind of strong inhibition agent of RNase) to handle water (0.01%DEPC)
To 10 μ l, mixed liquor 1 is placed 20 minutes to remove DNA at 37 DEG C after mixing, mixed liquor 1 is placed in 65 DEG C of water-baths after twenty minutes
Middle warm bath is subsequently placed in 5 minutes on ice to remove DNAse I activity, 1 μ l, 500 μ g/ml is 3. added into mixed liquor 1 for 10 minutes
OligdT, 4. will be immediately placed on warm bath 10 minutes in 65 DEG C of water-baths in the mixed liquor 1 of cooled on ice, be thoroughly denatured RNA,
It is subsequently placed in 5 minutes on ice, 5. with mixed liquor 2：110 μ l, 5x first strand buffer of mixed liquor, 4 μ l, 0.1M DTT
(mercaptoethanol) 2 μ l, 10mM dNTP mixture 1.5 μ l, DEPC processing 0.5 μ l of water, 2 μ l of reverse transcriptase, will be mixed after mixing
When closing liquid 2 and being placed in that warm bath 1.5 is small in 42 DEG C of water-baths, mixed liquor 2 is 6. placed in 90 DEG C of dry baths 3 minutes after reaction, 7. -20
DEG C preserve reaction final product.The reagent used in reaction is all purchased from Invitrogen companies；
3) and then according to TIGR databases (http://rice.plantbiology.msu.edu/) announce the gene it is complete
Long cDNA sequence, designs specific primer PCR amplifying specific fragment.The qRT-PCR primer sequences of LOC_Os03g51660 gene specifics
Row are as described below：
L2-CTTTGCTCCTCATCGCTGCT
R2-AGGCTCCTTGATCTGGTGATG
Using UBQ as reference gene, its special qRT-PCR primer sequence is as described below：
L-AACCAGCTGAGGCCCAAGA
R-ACGATTGATTTAACCAGTCCATGA
4) expression quantity of LOC_Os03g51660 is detected with the method for real-time fluorescence quantitative PCR.Reagent uses Roche reagent
(Roche, Mannheim, Germany), 10 μ l, cDNA2.5 μ l of overall reaction system, 0.25 μM of gene-specific primer, 5 μ l
Fast Start Universal SYBR Green Master(Rox)superMIX.PCR instrument is QuantStudio (TM)
6Flex System, PCR parameter are 95 DEG C of pre-degenerations 10 minutes, into circulation after 95 DEG C be denatured 10 seconds, 60 DEG C of annealing extensions 40
Second, 45 circulations.The result is shown in Fig. 4 b figure, compared with wild type control (ZH11W), heterozygous mutation body (tac3D-1H) and
The expression quantity of LOC_Os03g51660 significantly rises in homozygous mutant (tac3D-1M), and gene expression in homozygous single plant
The average value of amount is higher than heterozygosity.
Result above shows that the increase of tac3D-1 mutant tillering angles and T-DNA are inserted into LOC_Os03g51660 bases
Corresponded because promoter is related, and with the raising of the gene expression amount, therefore, the corresponding TAC3 genes in qTA3 sites are
LOC_Os03g51660, encodes a kind of conservative hypothesis albumen.
3.TAC3 genes are in rice at whole growth periods spatial and temporal expression spectrum analysis
According to report has been studied, control the gene of rice tillering angle specific can be expressed in tiller base portion, such as
TAC1 (Yu etc., 2007, The Plant Journal 52:891-898), therefore, can to the expression pattern analysis of TAC3 genes
It controls the function of tillering angle to secondary proof, and helps to understand its molecular mechanism, so as to reach assistant breeding purpose.This hair
Tissue site in bright for gene expression spectrum analysis includes：The rice plant stem apex (length 1-1.5cm) just sprouted, tiller
Tiller base portion, internode, blade, leaf sheath and the root of phase, and the internode in florescence, blade and leaf sheath etc..Use real time fluorescent quantitative
The expression quantity of the method detection TAC3 of PCR, specific operating method are isolated with Section 2 tac3D-1 mutant of embodiment 2
Whether the proof tac3D-1 mutant described in identifying is gain-of-function mutant.Specific testing result as shown in figure 5,
Gene expression quantity in each tissue of rice is not high, but is significantly higher than other tissue sites in the expression of tiller base portion, this
Indicate the regulating and controlling effect of the gene pairs rice tillering.
Embodiment 3：The haplotype analysis of TAC3 genes
In order to be better understood by the natural variation of TAC3 genes, so as to achieve the purpose that assistant breeding, we according to
RiceVarMap databases (http://ricevarmap.ncpgr.cn/) provide SNPs information, be extracted TAC3 genes and exist
Totally 13 SNPs information, selection wherein meet totally 10 single times of SNPs structures of MAF >=0.05 present in 295 parts of rice varieties
Type, and (meet the haplotype at least containing 10 kinds) between main haplotype and test progress multiple ratio using Duncan
Compared with as a result as shown in table 2 below：
The Multiple range test of tillering angle is controlled between main haplotypes of 2 TAC3 of table in 295 portions of long-grained nonglutinous rices
The explanation of table 2：Duncan is test, p<0.01.
Can be drawn by 2 result of table, have TAC3-Hap2 (such as rice varieties " Guangdong perfume (or spice) accounts for ") and TAC3-Hap3 (such as
Rice varieties " 9311 ", Approved variety is named as Yang No.6 rice) rice variety of genotype compares TAC3-Hap1 genotypic varieties
With less tillering angle, be conducive to dense planting and make full use of illumination.It can be selected with TAC3-Hap2 in breeding process
It is polymerize with the rice variety of TAC3-Hap3 genotype with other merits, cultivates and meet the excellent of people's greater demand
Non-defective unit kind.
Claims (3)
1. application of the separated TAC3 gene in rice tillering angle is controlled, it is characterised in that the nucleotide of the gene
Sequence such as SEQ ID NO：Shown in 1.
2. application of the separated TAC3 gene in rice tillering angle is controlled, it is characterised in that the egg of the gene code
White matter sequence such as SEQ ID NO：Shown in 3.
3. application of the separated TAC3 gene in rice tillering angle is controlled, it is characterised in that the egg of the gene code
White matter sequence such as SEQ ID NO：Shown in 4.
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Cited By (2)
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| CN114350685A (en) * | 2022-01-27 | 2022-04-15 | 中国烟草总公司郑州烟草研究院 | Application of tobacco NtTAC1 gene in leaf angle regulation |
| CN117304288A (en) * | 2023-09-05 | 2023-12-29 | 三峡大学 | Rice tillering angle related protein OsITAND and encoding gene and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114350685A (en) * | 2022-01-27 | 2022-04-15 | 中国烟草总公司郑州烟草研究院 | Application of tobacco NtTAC1 gene in leaf angle regulation |
| CN114350685B (en) * | 2022-01-27 | 2023-10-24 | 中国烟草总公司郑州烟草研究院 | Application of tobacco NtTAC1 gene in leaf angle regulation and control |
| CN117304288A (en) * | 2023-09-05 | 2023-12-29 | 三峡大学 | Rice tillering angle related protein OsITAND and encoding gene and application thereof |
| CN117304288B (en) * | 2023-09-05 | 2024-04-19 | 三峡大学 | Rice tillering angle related protein OsITAND, coding gene and application thereof |
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| CN107937409B (en) | 2020-11-24 |
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