CN108085320A

CN108085320A - Rice Dominant negative mutants Ef-cd and its application

Info

Publication number: CN108085320A
Application number: CN201711479841.5A
Authority: CN
Inventors: 方军; 储成才; 邓晓建; 张帆涛
Original assignee: Northeast Institute of Geography and Agroecology of CAS
Current assignee: Northeast Institute of Geography and Agroecology of CAS
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2018-05-29
Anticipated expiration: 2037-12-29
Also published as: CN108085320B

Abstract

Rice Dominant negative mutants Ef cd and its application are related to a kind of paddy gene Ef cd and its application.The object of the present invention is to provide a kind of rice Dominant negative mutants Ef cd and its application, for promoting eary maturity of rice and not influencing yield, so as to further improve hybrid paddy rice yield and adapt to the short breeding time condition of high latitude area.SEQ ID NO in the nucleotide sequence of rice Dominant negative mutants Ef cd such as sequence table:Shown in 1.Applications of the rice Dominant negative mutants Ef cd in plant precocity is promoted.The Ef cd mutant of the present invention can be used for promoting eary maturity of rice and not influence yield, so as to further improve hybrid paddy rice yield and adapt to the short breeding time condition of high latitude area, while also meet the required precocious characteristic of modern simple and laborsaving cultivation.

Description

Rice Dominant negative mutants Ef-cd and its application

Technical field

The present invention relates to a kind of paddy gene Ef-cd and its applications.

Background technology

Rice (Oryza sativa L.) is important cereal crops for the survival of mankind, the whole world people of more than half Mouth is using rice as staple food.China is paddy big producer of the world and Dao Zuo history ancient country, is the largest one of Rice Production base, Rice total output ranks first in the world.In recent years, with the growth of world population, aging aggravation, Process of Urbanization Construction quickening, And some areas vegetation deterioration leads to problems such as the appearance of environmental degradation so that how in limited manpower and environmental resource item The yield and quality of rice is improved under part, ensures that grain security becomes the significant problem to involve the interests of the state and the people.

Raising unit area grain yield expands cultivated area simultaneously can efficiently solve lasting population growth to grain need It asks.First, it is exactly season more than a year to improve unit area grain yield most efficient method, shortening breeding time.According to the natural law, Crop, which wants volume increase, to be needed to accumulate carbohydrate by the photosynthesis of long period.For example, the first generation is bred as by IRRI Kind IR20, IR24, IR26 and by the country be bred as commercial hybrids rice varieties Shan excellent 1 and Shan excellent 2 be required for 160 days or Longer time is ripe or harvests.Although these kind yield are high, breeding time is very long, is planted in the torrid zone or subtropical zone also only In 1 year season of energy, seriously hinder output increased.On the other hand, there is the growing area largely having abundant water resources in northern China, but by Low in accumulated temperature, frost arrives, and early plantable kind is considerably less, and land use is very low, thus cultivates the rice varieties of kipeming high yield Shorten breeding time, expanding cultivated area becomes the emphasis of breeding research instantly.And in rice breeding, high yield and precocity are long-term Existing contradiction, i.e. high-yield variety are often late-maturing, therefore cultivate excellent early-maturing variety and meet a variety of Ecological Areas and production season The needs of section, and it is a huge challenge for Rice Production to obtain stable high yield, it may have highly important meaning.

Rice breeding man has been working hard cultivate early-maturing variety for Cross-incompatibility groups.The IR36's of IRRI issues in 1976 Maturity period is 110 days, and IR50 is 105 days, and IR58 is 100 days.The survey 64 that domestic breeder cultivates is Poa linn, is derived Kind and Shan excellent 64 are crops that can be in two seasons of one year.1991-2010, the bright brightness 77 of another Poa linn Dominant variety and The cultivated area of its derived varieties is up to 7,446,700 hectares.And control the gene of these kind kipeming high yield characteristics and exploitation can It is used in the hot spot that the mark of breeding is studied as rice science man and breeder.

The prematureness of rice, it is considered that be the quantity of the recessive character or controlled by multiple genes controlled by 1-2 to key-gene Character finds or located multiple influences breeding time (heading stage) or ripe quantitative trait locus during breeding research (QTL).System has been carried out to the positioning of rice ear sprouting period QTL for Japanese Rice genomic gene (RGP) and in-depth study, the meter It draws and Nipponbare/Kasalath Derived Populations is carried out with 14 heading stage QTLs (Hd1-Hd14) of analyzing and positioning, ground later Study carefully personnel and located Hd15-17 again, at present bases such as map based cloning Hd1, Hd3a (Hd3 sites are divided into Hd3a and Hd3b), Hd6 Cause.Research finds that Rice Flowering is mainly regulated and controled by the homologous gene RFT1 of florigen gene Hd3a and Hd3a, wherein short day condition Under controlled by Hd3a, and be then by OsSOC1/OsMADS50 the expression of Ehd1 and RFT1 to be promoted to regulate and control under long-day conditions 's.In addition 29 Heading date genes and its modifier, dominant earliness gene and modifier 18, wherein non-photo-sensing base are reported again Because such as Dominant negative mutants Ef-1, Ef-x (t), Ef-y (t), Ef-cd (t), recessive ripe gene late have ef-3 (t), ef-4 (t), lf-3(t)；Modifier has m-Ef-1, w-Ef-1 (t), Su-Ef-ed (t) etc..

According to the hybrid vigour effect and dominance action of the hereditary basis of breeding practice and hybrid vigour, i.e. Dominant gene Prevailing theory, selection and breeding precocity hybrid paddy rice requirement sterile line are that dominant precocious or restorer is recessive ripe late can be only achieved Preferable effect.The discovery of eary maturity of rice gene and using will be helpful to solve it is precocious be difficult to the contradiction taken into account with high yield, also have Beneficial to overcoming super close obstacle ripe late between indica and japonica subspecies.Therefore, excavate and identify that rice ear sprouting period gene includes, carry out heading stage The research of the assignment of genes gene mapping, clone and backcross transformation etc. is mutually tied in particular with molecular labeling auxiliary with economical character selection It closes, the breeding utilization of Heading date gene is furtherd investigate on the basis of traditional genetic breeding, there is important theory significance and answer With value.

The content of the invention

The object of the present invention is to provide a kind of rice Dominant negative mutants Ef-cd and its application, for promoting eary maturity of rice And yield is not influenced, so as to further improve hybrid paddy rice yield and adapt to the short breeding time condition of high latitude area.

Rice Dominant negative mutants Ef-cd of the present invention, from long-grained nonglutinous rice sterility system 6442S-7, the nucleotides sequence of the gene SEQ ID NO in row such as sequence table:Shown in 1.

The accession number of the corresponding Nipponbare allele of Ef-cd is Os03g0122500, in nucleotide sequence such as sequence table SEQ ID NO:Shown in 2.

The rice Dominant negative mutants Ef-cd mutant that the present invention clones is long non-coding RNA (long noncoding RNA, lncRNA), come from the antisense strand of OsSOC1 genes, eary maturity of rice is controlled by the expression for just regulating and controlling OsSOC1 genes. The mutant nucleotide sequence is included in 4 missings of Os03g0122500 promoter regions, 1 insertion, 22 SNPs and gene internal point 1 insertion that Wei Yu be in the first and second intrones and a SNP mutation.Ef-cd there are variation can remarkably promote water Rice is precocious.By the genotyping to different cultivars, it was also found that in 1479 cultivars, share 72 kinds and contain The Dominant negative mutants make a variation, wherein there is 29 to come from International Rice institute (International Rice Research Institute, IRRI)；In 39 parts of superior hybrid crosses rice into Production of Large Fields of Isosorbide-5-Nitrae of China, there are 299 parts containing heterozygosis Genotype and 16 points of homozygous genotype, all significantly promote the heading of rice.

The Ef-cd mutant of the present invention can be used for promoting eary maturity of rice and not influence yield, miscellaneous so as to further improve It hands over rice yield and adapts to the short breeding time condition of high latitude area, while it is required precocious special also to meet modern simple and laborsaving cultivation Property.

Description of the drawings

Fig. 1 is Ef-cd near isogenic lines D248 and the phenotype in recurrent parent another name for Sichuan Province extensive 881 (SH881)；

Fig. 2 is Ef-cd finely positionings section；

Fig. 3 is the Os03g0122500 gene orders that D248 is compared with another name for Sichuan Province extensive 881 (SH881)；

Fig. 4 is the phenotype of Os03g0122500 gene T-DNA insertion mutation bodies ef-cd-1 and ef-cd-2；

Fig. 5 is the RNAi mutantion line phenotypes of Os03g0122500 genes；

Fig. 6 identifies for RNAi mutantion line Os03g0122500 gene expressions；

Fig. 7 is Ef-cd-Cas9 knockout mutations body surface types；

Fig. 8 is Os03g0122500 gene structure displays；Knockout site is Os03g0122500 promoter regions；

Fig. 9 is to compare Os03g0122500 promoter regulations in D248 and its recurrent parent SH881 in protoplast Uciferase activity, * * conspicuousnesses p<0.01；

Figure 10 is relative expression quantities of the after planting 15-60 days Ef-cd in D248 and SH881；

Figure 11 is relative expression quantities of the after planting 15-60 days OsSOC1 in D248 and SH881；

Figure 12 is is after planting 45 days Ef-cd and OsSOC1 in wild type and ef-cd-2 mutant (PFG_2A- 10486.R) relative expression quantity；

Figure 13 is after planting 45 days Ef-cd and OsSOC1 in wild type and Ossoc1 mutant (PFG_3A-05349.L) Relative expression quantity；

Figure 14 is the relative expression quantity of after planting 30 days short-day sunshine treatments Hd3a and RFT1 in D248 and SH881；

Figure 15 is the relative expression quantity of after planting 40 days short-day sunshine treatments Hd3a and RFT1 in D248 and SH881；

Figure 16 is the precocious relative expression quantity with OsSOC1 genes in late variety of different genotype,^*Represent p< 0.05；^**Represent p<0.01；NS is not notable；

Figure 17 is the precocity of different genotype and the relative expression quantity of Ef-cd genes in late variety.^*Represent p<0.05；^** Represent p<0.01；NS is not notable；

Figure 18 compares for Beijing (39 ° of 54 ' N) recurrent parent heading stage；

Figure 19 compares for Sichuan Chengdu (30 ° of 42 ' N) recurrent parent heading stage；

Figure 20 compares for Hainan Lingshui (18 ° of 22 ' N) recurrent parent heading stage；

Figure 21 is eared the maturity period for Ef-cd near isogenic lines hybrid paddy rice ZS97A/D330 with compareing hybrid paddy rice ZS97A/MH63 Compare；

Figure 22 is Ef-cd near isogenic lines hybrid paddy rice E-II-32A/FH838 with compareing hybrid paddy rice II-32A/FH838 heading Maturity period compares；

Figure 23 compares for Beijing hybrid paddy rice SY63, E-SY63, II-You838, E-II-You838 yield data；

Figure 24 compares for Jiaxing hybrid paddy rice SY63, E-SY63, II-You838, E-II-You838 yield data；

Figure 25 compares for Chengdu hybrid paddy rice SY63, E-SY63, II-You838, E-II-You838 yield data；

Figure 26 compares for Foochow hybrid paddy rice SY63, E-SY63, II-You838, E-II-You838 yield data；

Figure 27 is the Pedigree information of D248；

Figure 28 is to mark analysis Ef-cd genes (neighbouring with the insertion/deletion gene loci of 36bp) with 122500indel-1 Popularization degree in rice varieties；

Figure 29 is the popularization degree with 122500indel-1 labeled analysis Ef-cd genes in rice varieties；

Figure 30 is the phenotype of Sanya Area rice varieties Ef-cd and genotype transactional analysis；

Figure 31 is the phenotype of In Hangzhou Region of Zhe Jiang Province rice varieties Ef-cd and genotype transactional analysis.

Specific embodiment

Technical solution of the present invention is not limited to act specific embodiment set forth below, further includes between each specific embodiment Any combination.

Specific embodiment one：Present embodiment rice Dominant negative mutants Ef-cd, from long-grained nonglutinous rice sterility system 6442S- 7, SEQ ID NO in the nucleotide sequence of the gene such as sequence table:Shown in 1.

Specific embodiment two：The accession number of the corresponding Nipponbare allele of present embodiment Ef-cd is SEQ ID NO in Os03g0122500, nucleotide sequence such as sequence table:Shown in 2.

Specific embodiment three：Application of the present embodiment in plant precocity is promoted.

Specific embodiment four：Present embodiment is unlike specific embodiment three：The plant is rice.It is other It is identical with specific embodiment three.

Specific embodiment five：Present embodiment is unlike specific embodiment three：The precocious tool of promotion plant Body is promotion Rice Heading.It is other identical with specific embodiment three.

Elaborate below to the embodiment of the present invention, following embodiment under based on the technical solution of the present invention into Row is implemented, and gives detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following realities Apply example.

Embodiment 1：Rice Ef-cd gene finely positionings

Sites of the Ef-cd as dominant precocity, most finds early in long-grained nonglutinous rice male sterile line 6442S-7, and is positioned at water On No. 3 chromosomes of rice.It is recurrent parent and sterile line using another name for Sichuan Province extensive 881 (SH881) for finely positioning Ef-cd genes 6442S-7 continuous backcrosses, then self cross and selection, constructs BC₅F₁₁Near isogenic lines D248, BC₅F₁₁Near isogenic lines D248 with Recurrent parent another name for Sichuan Province extensive 881 (SH881) carries out hybridization and obtains F₁Seed, F₁Selfing obtains F₂.From F₂For screening and identification in group 4, 800 plants of evenings spend individual extraction DNA, using the rice SSR marker delivered and according to Ef-cd flanks in SH881 and 6442S-7 Genomic dna sequence exploitation Indel mark (table 1) finely positioning Ef-cd genes.

Using CTAB methods extraction DNA as PCR reaction templates, the total volumes of PCR reaction systems is 10 μ l, rice genome 1 μ l (about 200ng) of DNA masterplates, 2 × Master Mix, 5 μ l, each 0.5 μ l of 10 μM of primers, add ddH₂O to 10 μ l.Reaction interval Sequence：94 DEG C of denaturation 5min；94℃30s；58℃30s；72 DEG C of 20s, 35 Xun Huans；72 DEG C of extension 10min.

1 map based cloning molecule labelled series information of table

As a result as depicted in figs. 1 and 2, near isogenic lines D248 heading stages apparent 881 (Fig. 1) extensive earlier than recurrent parent another name for Sichuan Province； Utilize F₂Mass screening identifies multiple Indel marks, Ef-cd is navigated in the section of 12.9Kb, candidate region Nei Bao Containing the sub- Os03g0122600 (OsSOC1/OsMADS50/DTH3) of activation and the Os03g0122500 genes (Fig. 2) of blooming.The pumping Ear period referred to from the time for being seeded into tassel extraction Flag Leaf Sheath.

Section is positioned to shorten, more finely positioning mutator extracts SH881 using TRIzol (Invitrogen, USA) With the total serum IgE of the blade of D248, ReverTra Ace qPCR RT Master Mix (Toyobo, Japan) reverse transcription 1 is utilized μ g total serum IgEs synthesize cDNA, and PCR expands the full-length cDNA sequence of the OsSOC1 and Os03g0122500 genes of SH881 and D248 respectively Row, recycling PCR product carry out cloning and sequencing.The total volume of PCR reaction systems is 50 μ l, and 1 μ l of oryza sativa genomic dna masterplate are (about 200ng), 25 μ l of 2XPCR buffer for KOD Fx, 10 μ l of 2mM dNTP, KOD Fx (1U/ μ l) 1 μ l, 10 μM of primers Each 1.5 μ l add ddH2O to 50 μ l.Response procedures：98 DEG C of denaturation 2min；98℃10s；60℃90s；68 DEG C of 2min, 30 are followed Ring；78 DEG C of extension 10min.PCR product is recycled, clone carries out Sanger sequencing analysis.

Sequencing result is compared discovery, the cDNA sequence of the OsSOC1 genes from SH881 and D248 is simultaneously not present The difference of SNP, and lncRNA, database (https are belonged to by the Os03g0122500 that the antisense strand of OsSOC1 is transcribed:// Shigen.nig.ac.jp/rice/oryzabase/) accession number is AK242050, in its promoter region and includes sub-district and deposits In difference.It was found that, there are the missings of 4 large fragments (wherein to wrap in Os03g0122500 promoter regions by D248 with SH881 Containing 36bp and big deletion mutation of 267bp), 1 single base insertion and 22 SNPs；And respectively intragenic One and second introne, one insertion and a SNP (Fig. 3).

In order to verify candidate gene function, from T-DNA-inserted Mutant Pool in Rice (http://signal.salk.edu/ Cgi-bin/RiceGE two mutant for) obtaining Os03g0122500 genes are named as ef-cd-1 (PFG-1D-05526.R) With ef-cd-2 (PFG-2A-10486.R).The T-DNA insertion points of ef-cd-1 and ef-cd-2 are located at Os03g0122500 respectively Gene First Intron and promoter region (sudden change region is identical with D248), the two and the phenotype that heading stage delay is all presented (Fig. 4).It these results suggest that Os03g0122500 promoter regions and included the breeding time that sub-district variation affects rice, Os03g0122500 may be the candidate gene of Ef-cd.

Embodiment 2：Promoter function is analyzed

(1) RNAi carrier is built

5 ' area 168bp of Os03g0122500 genes, amplimer sequence are expanded using the Ef-cd full-length genes of D248 as masterplate It is classified as RNAiP forward primer:5’-GTCGACTCAAAGCGATCTGTCACCTGAA-3’；RNAiP reverse primer:5 '-GGATCCTACTTGTACTCCTGCTTG-3 ' are connected using XhoI/BglII with SalI/BamHI double digestions Method is building up on pUCRNAi carriers.Finally stem ring is cloned on pCAMBIA2301 carriers.Turned using agrobacterium-mediated transformation Change rice Nipponbare.Utilize the expression quantity of the method detection Ef-cd genes of sxemiquantitative.

(2) CRISP/Cas9 vector constructions

Ef-cd-Cas9 mutant builds (Hangzhou China) by Biogle companies.Building process is as follows：It is right Os03g0122500 promoter regions separately design two different sgRNA target spots:sg1213:5’- GTCGATTCGGTGCGTGATAA-3 ' and sg1214:5’-GTCATATACAACAGGGGTAC-3’.The two sgRNAs are built Onto the BGK032-DSG carriers containing Cas9 albumen, Agrobacterium EHA105, then genetic transformation rice Nipponbare are converted.Transgenosis Plant knocks out detection：Transfer-gen plant genomic DNA is extracted, using primer Efcd-cas9 near target spot, PCR amplification is simultaneously sequenced Analysis knocks out segment.

Efcd-cas9 forward primer:5’-TCAGACTCCAGAGTTGGAAAA-3’

Efcd-cas9 reverse primer:5’-TGCGAAATGCTAGACGCATA-3’。

As a result as shown in figures 5-9, Os03g0122500 promoter region sequences are acted on using RNA perturbation techniques, caused Os03g0122500 gene expression amounts decline after, present heading stage delay the phenomenon that (Fig. 5 and Fig. 6).Utilize CRISP/Cas9 skills The Os03g0122500 gene knockout Ef-cd-Cas9 mutant of art structure, the promoter region of sequencing analysis Os03g0122500 are deposited It is knocked out in 158bp, evening flower phenotype (Fig. 7 and Fig. 8) is presented in mutant.These results also illustrate Ef-cd (Os03g0122500) base Growth period duration of rice is influenced because promoter region makes a variation, causes late-maturing phenotype.

(3) LUC assays luciferase assays

Os03g0122500 promoter regions therefore speculate that these variations may influence there are the variation of many nucleotide sites The activity of the gene promoter.

The promoter sequence of 2050 bases of the upstream region of gene is cloned respectively from D248 and its recurrent parent SH881 respectively The sequence is inserted into purpose carrier pGWB535 by row by the In-Fusion kits of Clontech companies, glimmering with firefly Light element enzyme coded sequence merges.Reporter gene luciferase is had detected with the Dual-Luciferase kit survey of Promega companies Activity.

The results are shown in Figure 9, pD248::The uciferase activity of LUC compares pSH881::The uciferase activity of LUC is higher by 7.34 times, showing the promoter of the Ef-cd in D248 has stronger activity.

Embodiment 3：QPCR comparative analysis Ef-cd and OsSOC1 expression changes

(1) Ef-cd and OsSOC1 expression changes in D248 and SH881

After planting 15-60 days rice leaf total serum IgEs are extracted using TRIzol (Invitrogen, USA).Utilize ReverTra 1 μ g total serum IgEs of Ace qPCR RT Master Mix (Toyobo, Japan) reverse transcription synthesize cDNA, design qPCR primers：

Ef-cdQ forward primer:5 '-ACTCCTCCTTTCGTCTTCCT-3 ',

Ef-cdQ reverse primer:5 '-GCTTGATTGTGGCTTGGTATTT-3 ',

OsSOC1Q forward primer:5 '-CTGCGTTGAGAAAGGAGATGAT-3 ',

OsSOC1Q reverse primer:5’-CATGGACGACAATACGGGTAAG。

QPCR is mixed according to SYBR Green Real-Time PCR Master Mix reagent (Toyobo, Japan) Reaction system, the condition of qPCR reactions：95℃2min；95℃15s；60℃30s；Goto step2 40 are cycled, 95 DEG C of 1min, 55 DEG C of 1min, 95 DEG C of 30s.Using rice Os Actin as internal reference, primer sequence is as follows：

OsActin1Q forward primer:5 '-ACCATTGGTGCTGAGCGTTT-3 ',

OsActin1Q reverse primer:5’-CGCAGCTTCCATTCCTATGAA-3’。

QPCR using compare CT methods (^ΔΔCT gene expression amount) is calculated, using rice Os Actin genes as reference gene, with not Processed sample is as control.Target gene differential expression is by treated sample compared with each time point without place The multiple of the sample of reason represents.Each sample includes 3 secondary pollutants and repeats to repeat with 3 technologies, and data take 3 secondary pollutants The average value repeated, if there is the deviation of a numerical value is bigger, takes the average value of two data.Initial data is normalized Processing.Data after standardization are examined through T carries out significance difference analysis.Relative expression quantity computational methods：2^-ΔΔCT=2^{- (Δ CT processing-Δ CT controls)}=2^{- [(CT processing intent genes-CT handles reference gene)-(CT control target gene-CT controls reference gene)]}。

As a result as shown in figures 10-13：Ef-cd starts to raise on the 30th day D248 seedling developments, reaches within the 60th day maximum Value, and in expression variation, less (Figure 10, wherein curve 1 represent SH881, and curve 2 represents during SH881 seedling developments D248)；OsSOC1 rises in the expression quantity that presents for 15-60 days of D248 and SH881 seedling developments, and the expression quantity in D248 Higher particularly rose significantly (Figure 11, wherein curve 1 represent SH881, and curve 2 represents D248) in 30-60 days expression quantity. OsSOC1 expression quantity significantly reduces (Figure 12) in ef-cd mutant (ef-cd-2)；And (the PFG_3A- in Ossoc1 mutant 05349.L) Ef-cd expression quantity reduces unobvious (Figure 13).It these results suggest that Ef-cd and OsSOC1 in D248 expression quantity more Height, the expression of particularly after planting 30-60 days Ef-cd regulation and control OsSOC1.

(2) florigen gene Hd3a and RFT1 relative expression quantity in D248 and SH881

OsSOC1 is utilized by regulating and controlling downstream florigen gene Hd3a and RFT1 Gene Expression growth period duration of rice Whether the variation of the method analysis Ef-cd of qPCR influences Hd3a and RFT1 gene relative expression quantities.

D248 and SH881 is in seedling leaves total serum IgE, reversion under after planting 30 days short-day of extraction and 40 days long-day conditions Record and qPCR analysis Hd3a and RFT1 gene relative expression quantities, analytical procedure is with embodiment 3 (1), the primer sequence of Hd3a and RFT1 Row are as follows：

Hd3aQ forward primer:5’-TCGAGGTCGGCGGCAATGAC-3’

Hd3aQ reverse primer:5’-CCTCGCGCTGGCAGTTGAAGTAGAC-3’；

RFT1Q forward primer:5 '-CCATTCGTCCGGATCACTAACCTCA-3 ',

RFT1Q reverse primer:5’-CGCGCTGGCAGTTGAAGTAGAC-3’。

Research is as shown in FIG. 14 and 15：Seedling under after planting 30 days short-day and 40 days long-day conditions, Hd3a and Expression of the RFT1 in D248 is all remarkably higher than SH881, and the variation for illustrating no matter to grow Ef-cd under the conditions of day or short day promotes The expression of Hd3a and RFT1 genes.

(3) Ef-cd and OsSOC1 expression changes in different genotype heading stage kind

Dth3 and Hd9 is the Heading date gene for being positioned at No. 3 chromosome, respectively from African rice and Japan cultivars Kasalath, different from IRRI sources and its genotype of Derivative line kind.Near isogenic lines dth3-NIL (African Dao ╳ samsaras Parent Dianjingyou1, DJY1), Hd9-NIL (Kasalath ╳ recurrent parents Nipponbare, Nip) is late-maturing table Type.

The total serum IgE of the following different precocious and late-maturing material of extraction, reverse transcription into qPCR after cDNA analyze Ef-cd and OsSOC1 gene relative expression quantities, step is the same as embodiment 3 (1).

Its Genotype of 2 different bearing of table and its control material

It is precocious	It is late-maturing
		DJY1	Dth3-NIL (African Dao ╳ recurrent parent DJY1)
Nip	Hd9-NIL (Kasalath ╳ recurrent parent Nip)
		Zaohui89 (IR50 ╳ recurrent parent IR24)	IR24
D330	Bright extensive 63 (MH63)

QPCR analysis results as shown in FIG. 16 and 17, Ef-cd and relative expression quantity of the OsSOC1 genes in early-maturing variety Apparently higher than late variety；Sequence analysis finds Ef-cd sequences, and in dth3-NIL, there are 30 SNPs and 3 large fragments to be inserted into, There are the missings of the large fragment of 11Kb by Hd9-NIL.And early extensive 89, D330 is identical with D248 genotype；IR24, bright extensive 63 with SH881 genotype is identical.It these results suggest that, the middle early characteristic of kind and the Ef-cd and OsSOC1 of different genotype are expressed Measure correlation.

Embodiment 4, Ef-cd genotype and eary maturity of rice character close linkage

According to Family data, the Ef-cd genotype for inferring D248 is to develop kind IR9761- from International Rice Research Institute 19, (Figure 27) can be detected in the breeding kind of offspring.

Using 122500indel-1 detection Ef-cd genotype is marked (to compare the promoter region of Ef-cd with late-maturing genotype There are one the missings of 36 bases).Research show International Rice institute's later stage cultivate early-maturing variety (IR30, IR36, IR50, IR58, IR64 and IR74) as survey 64 containing Ef-cd sites (such as Figure 28, early-maturing variety Ce64, IR30, IR36, IR50, IR58, IR64 and IR74 are containing Ef-cd gene locis, but late variety IR24 and IR29 are free of Ef-cd gene positions Point)；Simultaneously containing Ef-cd earliness genes in the main Poa linn of China, for example 64-7 (the 1980s) is surveyed, Bright extensive 77 (the 1990s), R402 (at the beginning of 21 century) and To463 (modern times), but late-maturing restorer it is such as bright extensive 63, 9311st, but there is no (such as Figure 29, Ef-cd gene loci are in the main Poa linn product of China in another name for Sichuan Province extensive 881 and good fortune extensive 838 There are presence, such as Ce64-7, bright extensive 77 (MH77), R402 and To463 in kind.But in the main late-maturing restorer kind of China In be not present, such as bright extensive 63 (MH63), 9311, another name for Sichuan Province extensive 881 (SH881) and good fortune extensive 838 (FH838)).

It is found by the genome analysis to 1495 kinds of Fine Quality Hybrid Rice kinds, in the variation at Sanya and Hangzhou heading stage Relevant main QTL is Ef-cd-OsSOC1 gene locis.Therefore, using 122500indel-1 labeled analysis in Sanya and The heading stage of 1439 kinds of Hangzhou Fine Quality Hybrid Rice kind (long-grained nonglutinous rice-indica hybrid).As a result as shown in figs. 30 and 31, in Sanya (a) with Hangzhou (b), the precocious D248 homozygous genotypes (AA) and heterozygous genotypes that SNP marker vf0301285586 is identified (AT) Hybrid generally heading more early than wild SH881 genotype (TT) kind.* conspicuousnesses p<0.01, NS refers to not significantly.Institute Have and all ear relatively early with homozygous or heterozygosis Ef-cd genotype Hybrid.These results also illustrate Ef-cd gene pairs water The precocious close linkage of rice.

Embodiment 5：The Effect on growth period of Ef-cd genes is applied with quantitative effect experiment

(1) Ef-cd near isogenic lines heading stage is obviously shortened heading stage

It is planted within 2015 paddy fields and is planted by the way of random district's groups and ground in Chinese Academy of Sciences's heredity with development Study carefully institute (39 ° of 54 ' N, Beijing), Jiaxing Academy of Agricultural Sciences (30 ° of 75 ' N, Zhejiang Jiaxing), Sichuan Agricultural University (30 ° of 42 ' N, four River Chengdu), the Fujian agriculture academy of sciences (26 ° of 08 ' N, Fujian Foochow), Hainan Lingshui (18 ° of 22 ' N).Rice Cropping line-spacing 26cm, Spacing 17cm.Field management follows following normal agricultural fertilizer experience (per hectare)：50kg nitrogen, 60kg phosphorus and 95kg potassium are made For basic fertilizer；Tillering period nitrogen 90kg；Heading stage nitrogen 30kg.All rice materials are sowed in identical condition of culture It plants.District's groups test 3 repetitions.Heading stage is from the number of days for being seeded into first inflorescence of appearance above sword-like leave, daily record heading Phase.

As a result as shown in Figure 18,19 and 20, near isogenic lines (table 3) phenotype of advanced lines is similar to recurrent parent, but takes out Ear period shifts to an earlier date.When being planted in, Hainan Lingshui (18 ° of 22 ' N), Sichuan Chengdu (30 ° of 42 ' N) and Beijing (39 ° of 54 ' N) etc. 3 is different During photoperiod paddy fields, the heading stage of near isogenic lines shortens 8.9-19.7 days than wild type.

Table 3

Recurrent parents	NILs	Combination	Generation
				Shuhui881	D248	6442S-7/SH881	BC₅F₁₁
Minghui63	D330	6442S-7/MH63	BC₉F₁₂
				Shuhui527	D488	6442S-7/SH527	BC₉F₁₂
Mianhui725	D439	6442S-7/MH725	BC₁₃F₁₀
				Yihui577	D374	6442S-7/YH1577	BC₁₀F₉
II-32B	B3115	6442S-7/II-32B	BC₆F₁₄

(2) Ef-cd mutated-genotypes precocity is without the underproduction

Rice cultivation in 2015 is in Chinese Academy of Sciences's heredity and development research institute (39 ° of 54 ' N, Beijing), Jiaxing agricultural sciences Institute's (30 ° of 75 ' N, Zhejiang Jiaxing), Sichuan Agricultural University (30 ° of 42 ' N, Sichuan Province Chengdu), the Fujian agriculture academy of sciences (26 ° of 08 ' N, Fujian Foochow), planting patterns is the same as (1) in embodiment 5 Suo Shi.

Planting material is precocity near isogenic lines D248 and D330 and corresponding late-maturing parent SH881 and MH63；Southern product Kind hybrid paddy rice Shanyou 63 (ZS97AXMH63), II-You838 (II-32AXFH838)；Ef-cd is imported using near isogenic lines In above-mentioned hybrid paddy rice：ZS97A/D330, wherein D330 are that MH63 imports the near isogenic lines of Ef-cd genes, E-II-You838 (E-II-32A/FH838), wherein E-II-32A is the near isogenic lines that II-32A imports Ef-cd genes.

Compare the correlation with yield of precocious near isogenic lines (D248, D330) and corresponding late-maturing parent (SH881, MH63) Shape is displayed without significant difference including spike number, grain number, setting percentage and mass of 1000 kernel, illustrates that Ef-cd genes have no effect on yield.

Whether further to detect Ef-cd can be not influence come contracting heading stage and yield in introductive crossing rice, investigation be found The hybrid paddy rice heading of four all near isogenic lines containing Ef-cd in place is with the maturity period earlier than corresponding hybrid paddy rice 6.9-15.9 My god (Figure 21 and 22), spike number, plant height, grain number, setting percentage and mass of 1000 kernel are displayed without significant difference.Compare Ef-cd genotype Hybrid paddy rice finds that the hybrid paddy rice yield of two, Beijing Ef-cd genotype hybridizes than control with the corresponding yield for compareing hybrid paddy rice Rice wants high (Figure 23), and in Jiaxing, only there are one hybrid paddy rice E-II-You838 (E-II-32A/FH838) yield of Ef-cd genotype Than compareing high (Figure 24), other place yield are all identical (Figure 25 and 26).These results it is strong illustrate Ef-cd- OsSOC1 sites can be applied in hybrid paddy rice sterile line or restorer promote hybrid paddy rice precocious and not influence yield.(Figure 23- In Figure 26, * * represent p<0.01；NS represents not notable).

Sequence table

<110>Northeast Institute of Geography and Agricultural Ecology, Chinese Academy of Sc

<120>Rice Dominant negative mutants Ef-cd and its application

<160> 18

<210> 1

<211>2327

<212> DNA

<213>Rice（Oryza sativa）

<220>

<223>Rice Dominant negative mutants Ef-cd

<400> 1

attggttgtg aagagaatgt tagtggagaa gttgttatat tttagaacag agggagtaat 60

aattaaaata ctcaacttta tgaaatatcg atacagttat tcttttcttt gtatacttcc 120

aatgcattta tatcctaatt tcacaaactt cgatataata attgtttgag aaagaactta 180

ttttagaaca agtcgtataa gggttaaaaa caaacttatt taagatggag gtatttaggt 240

ggtgtttggt tggagggact aaagtggggc taaactttag tccctctcac aaaaacataa 300

gtctctagtt atatttttgt gagagggact aaatttagtc cctagttacc aaacaccccc 360

ttaagatgga gctagtaccc tgcatgacgg ccatgtgcta gtttatctct cgcctcggga 420

gtttgtgggc catatgctac gtagatgttg ggccatcttc gtgggctcag gctccaccag 480

cacgtccgct cacggcgcat gggatgcacg cggcgccatt tgttccccac actcccgtgg 540

gccgcatact acccaggacg gtggcgcgtt gggccgtatc gtccccaagc ccatcacggc 600

ttttgcccta catcgcctcg atcgcgtttg ggcccgctag tccatgctag tatacgcgac 660

agcgacgtgt acgtacgtct ccaacgcgaa tacaacacct ccacgtggcg tacttgcatg 720

tgaggcagtc ttcaacgtat gtttccctgt caaaattcta ccgattcctg cggtttcctg 780

catccttttt gcctctaatt gcgtgtgacc actagctttt cgcctatgtt cccttccgtg 840

cctccgcgtg acacgagctg tttgagcagc agtgtatatc aactgaacat cgaataaata 900

ctacttcatc cgtttcataa tataagttat tctagcattt ttcacattca tattgattat 960

atatatatat atatatatat atgagaaacg gagggagtaa tgatttatta ctaccatgta 1020

tactcgctgt aaagagaaat atactactac ttcagactcc agagttggaa aaatcacatc 1080

gcggctttgc aagcaaaaac cgttatcacg caccgaatcg agggatacta ctagaagcga 1140

tggggattag ggtccagaaa tctcctcggc ttccactgcc caatcgacac gtcgaccaca 1200

ggtagcattc aagagatcga aacgtcgacc aataccagca tagcgcgtca tatacaacag 1260

gggtacaggt caaagcgatc tgtcacctga atgtactccc tccctcttta aaactcctcc 1320

tttcgtcttc ctcccgttcc ggccttgtgc agagaatctc aacagagata tgttcagcat 1380

atgtttgctc caacatcaca tcaaatacca agccacaata aagcaggagt acaagtacgc 1440

acttgagatc tctcgattta gatttagatg ggtagtggag tctgccgatc gaagggtttc 1500

tgcctggtgg tgctgaatgg gtgggtgcat gcgtctagca tttcgcatcc atggacgaca 1560

atacgggtaa gtaaaatata cagcgtggtc tgtttttagg atggtttggt gtcattgcat 1620

gcctagctat catctccttt ctcaacgcag agatccagct tattcctggc ctgttactta 1680

agaatggggc atcgcttggc tatcttctgc agcaccgccg ctggagcgac ttctgccagg 1740

cagccctatg aatagctcag tttcgacatc catgttgtcg ttggtggtgt tgatgttacg 1800

gtccgggttc tcatcttcag cccggacagt caaaggagca gacaagggag gctgattctt 1860

acactgcaca aagttcatga tcaagggaag caaatcaggt gagttgatct ttcagtgtcg 1920

cagaaaattc tcggcattta tattggtgtg cacataagat atcgcacgaa ttagctcaaa 1980

taatcatatg ctaggctggg cgatcgactt aagctggctg ctggtgtctt ctcggcagta 2040

aaagtttcac tcgtcttacc ttttcgcgta actcttcatt gtccttgcgc agcttcatct 2100

cctggtcgat gttaatgcaa aaaagagaca aacacgacat taggaacaga tgattggtcc 2160

tgcgtttcag gttatatata tatttattta tttatagtaa gtacgaagtt ggtgaggtac 2220

cttctctctc agtttggcaa cctgctcctc aagcagcttt gtctagtatg cagagaattg 2280

gttgtaagtt tgtgcacatc atatatatag ataaattaat tgctgat 2327

<210> 2

<211>2633

<212> DNA

<213>Rice（Oryza sativa）

<220>

<223>The corresponding Nipponbare allele of Ef-cd

<400> 2

attggttgtg aagagaatgt tagtggagaa gttgttatat tttaggacaa atcctgagag 60

ttaaaagttg ttatatttta gaacagaggg agtaataatt aaaatactca actttatgaa 120

attttgatac agttattctt ttctttgtat acttccaatg catttatatc ctaatttcac 180

aaacttcgat ataataattg tttgagaaag aacttatttt agaacaaatc gtaaaagggt 240

taaaaacaaa cttatttaag atggaggtat ttaggtggtg tttggttcga gggactaaag 300

tggggctaaa ctttagtccc tctcataaaa acataaatcc ctagttatat ttttgtgaga 360

gagactaaat ttagtcccta gttaccaaac acccccttaa gatggagcta gtactctgca 420

tgacggccat gtgctagttt atctctcgcc acgggagttt gtgggccata tgctacgtag 480

atgttgggcc atcttcgtgg gctcaggctc caccagcacg tccgctcacg gcgcatggga 540

tgcacgcggc gccgtttgtt cccccacact cccgtggccg catactaccc aggacggtgg 600

cgcgttgggc cgtatcgtcc ccaagcccat cacggctttt gccctacatc gcctcgatcg 660

cgtttgggcc cgctagtcca tgctagtata cgcgacagcg acgtgtacgt acatctccaa 720

cgcgaataca acacctccac gtggcgtact tgcatgtgag gcagtcttca acgtatgttt 780

ccctgtcaaa attctaccga ttcctgcggt ttcctgcatc ctttttgcct ctaattgcgt 840

gtgaccacta gcttttcgcc tatgttccct tccgtgcctc cgcgtgacac gagctgtttg 900

agcagcagtg tgtatcaact gaacatcgaa taaatactac ttcatccatt tcataatata 960

agttattcta acatttttca cattcatatt gatcatatat atatatatat atgtatatgt 1020

atatatgtat gtatatatgt atgtatgtat atatgtatat atgtatatat atgtatatat 1080

atgtatatat gtatatatat gtatatatat gtatatgtat atatatatat atatgtatat 1140

atatatatat atatgtatat atatatatat gtatatatat atatgtatat atatatatat 1200

gtatgtatat atatatatgt atatatatat atatatatat atatatatat atatatatat 1260

atatatatat atatatatat atatatatga aacggagtga gtaatgattt attactacca 1320

tatatactcg ctgtaaagag aaatatactg ctactacttc agactccaga gttggaaaaa 1380

tcacatcgcg gctttgcaag caaaaaccgt tatcacgcac cgaatcgagg gatactacta 1440

gaagcgatgg ggattagggt ccagaagtct cctcggcttc cactgcccaa tcgacacgtc 1500

gaccacaggt agcattcaag agatcgaaac gtcgaccaat accagcatag cgcgccatat 1560

acaacagggg tacaggtcaa agcgatctgt cacctgaatg tactccctcc ctctttaaaa 1620

ctcctccttt cgtcttcctc ccgttccggc cttgtgcaga gaatctcaac agagatatgt 1680

tcagcatatg tttgctccaa catcacatca aataccaagc cacaataaag caggagtaca 1740

agtacgcact tgagatctct cgatttagat ttagatgggt agtggagtct gccgatcgaa 1800

gggtttctgc ctggtggtgc tgaatgggtg ggtgcatgcg tctagcattt cgcatccatg 1860

gacgacaata cgggtaagta aaatatacag cgtggtctgt ttttaggatg gtttggtgtc 1920

attgcatgcc tagcatcatc tcctttctca acgcagagat ccagcttatt cctggcctgt 1980

tacttaagaa tggggcatcg cttggctatc ttctgcagca ccgccgctgg agcgacttct 2040

gccaggcagc cctatgaata gctcagtttc gacatccatg ttgtcgttgg tggtgttgat 2100

gttacggtcc gggttctcat cttcagcccg gacagtcaaa ggagcagaca agggaggctg 2160

attcttacac tgcacaaagt tcatgatcaa gggaagcaaa tcaggtgagt tgatctttca 2220

gtgtcgcaga aaattctcgg catttatatt ggtgtgcaca taagatatcg cacgaattag 2280

ctcaaataat catatgctag gctgggcgat cgacttaagc tggctgctgg tgtcttctcg 2340

gcagtaaaag tttcactcat cttacctttt cgcgtaactc ttcattgtcc ttgcgcagct 2400

tcatctcctg gtcgatgtta atgcaaaaaa gagacaaaca cgacattagg aacagatgat 2460

tggtcctgcg tttcaggtta tatatatatt tatttattta tagtaagtac gaagttggtg 2520

aggtaccttc tctctcagtt tggcaacctg ctcctcaagc agctttgtct agtatgcaga 2580

gaattggttg taagtttgtg cacatcatat atatagataa attaattgct gat 2633

<210> 3

<211> 28

<212> DNA

<213>Artificial sequence

<220>

<223> RNAiP forward primer

<400> 3

gtcgactcaaagcgatctgtcacctgaa 28

<210> 4

<211> 24

<212> DNA

<213>Artificial sequence

<220>

<223> RNAiP reverse primer

<400> 4

ggatcctacttgtactcctgcttg 24

<210> 5

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> sg1213

<400> 5

gtcgattcggtgcgtgataa 20

<210> 6

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> sg1214

<400> 6

gtcatatacaacaggggtac 20

<210> 7

<211> 21

<212> DNA

<213>Artificial sequence

<220>

<223> Efcd-cas9 forward primer

<400> 7

tcagactccagagttggaaaa 21

<210> 8

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> Efcd-cas9 reverse primer

<400> 8

tgcgaaatgctagacgcata 20

<210> 9

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> Ef-cdQ forward primer

<400> 9

actcctcctttcgtcttcct 20

<210> 10

<211> 22

<212> DNA

<213>Artificial sequence

<220>

<223> Ef-cdQ reverse primer

<400> 10

gcttgattgtggcttggtattt 22

<210> 11

<211> 22

<212> DNA

<213>Artificial sequence

<220>

<223> OsSOC1Q forward primer

<400> 11

ctgcgttgagaaaggagatgat 22

<210> 12

<211> 22

<212> DNA

<213>Artificial sequence

<220>

<223> OsSOC1Q reverse primer

<400> 12

catggacgacaatacgggtaag 22

<210> 13

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> OsActin1Q forward primer

<400> 13

accattggtgctgagcgttt 20

<210> 14

<211> 21

<212> DNA

<213>Artificial sequence

<220>

<223> OsActin1Q reverse primer

<400> 14

cgcagcttccattcctatgaa 21

<210> 15

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> Hd3aQ forward primer

<400> 15

tcgaggtcggcggcaatgac 20

<210> 16

<211> 25

<212> DNA

<213>Artificial sequence

<220>

<223> Hd3aQ reverse primer

<400> 16

cctcgcgctggcagttgaagtagac 25

<210> 17

<211> 25

<212> DNA

<213>Artificial sequence

<220>

<223> RFT1Q forward primer

<400> 17

ccattcgtccggatcactaacctca 25

<210> 18

<211> 22

<212> DNA

<213>Artificial sequence

<220>

<223> RFT1Q reverse primer

<400> 18

cgcgctggcagttgaagtagac 22

Claims

1. rice Dominant negative mutants Ef-cd, it is characterised in that rice Dominant negative mutants Ef-cd, from long-grained nonglutinous rice sterility It is 6442S-7, SEQ ID NO in the nucleotide sequence of the gene such as sequence table:Shown in 1.

2. the nucleotide sequence such as sequence of the corresponding Nipponbare allele of rice Dominant negative mutants Ef-cd described in claim 1 SEQ ID NO in list:Shown in 2.

3. applications of the rice Dominant negative mutants Ef-cd described in claim 1 in plant precocity is promoted.

4. application according to claim 3, it is characterised in that：The plant is rice.

5. application according to claim 3, it is characterised in that：The precocious promotion plant is specially to promote Rice Heading.