CN103589734A - Haplotype of group of genes DTH2 for controlling rice heading period and application of genes DTH2 - Google Patents

Abstract

The invention belongs to the technical field of gene engineering and relates to a group of genes DTH2 for controlling the rice heading period, and a haplotype and application of the genes DTH2. A CDS sequence of the haplotype 4 of the genes DTH2 is presented by SEQ ID NO.7. A transgenosis experiment shows that the genes have a function of allowing the rice heading period to be advanced and proves that the genes are selected during domestication and are from China.

Description

One group of DTH2 gene haplotype and application of controlling rice ear sprouting period

Division explanation

This case is that the application number that 2012-03-05 submits is 2012100546596, and name is called the dividing an application of Chinese invention patent application of DTH2 gene and haplotype and the application of rice ear sprouting period " a group control ".

Technical field

The invention belongs to gene engineering technology field, be specifically related to one group of DTH2 gene haplotype and application of controlling rice ear sprouting period.

Technical background

Be an important economical character heading stage, and may in the domestication process of crop, be selected (Izawa T.Journal of Experimental Botany58:3091 – 3097 (2007)).Cultivated rice is distributed in worldwide from 53 ° of north latitude to the wide geographic area (Chang T.T.Euphytica25,425 – 441 (1976)) 40 °, south latitude at present.Yet ancestors' common wild-rice of Asian Cultivated Rice, is mainly distributed in South Asia, South East Asia and Australia are northern, the most northern Dongxiang Wild Rice that is limited in 28 ° of north latitude of its distribution.A free-revving engine of paddy rice domestication is to produce the rice varieties that adapts to each department plantation, thereby expands cultivated area.Affecting the key constraints that cultivated rice expands is northwards exactly heading stage.For example, in high latitude area (northeast, Asia), early heading and to insensitive characteristic of photoperiod, guaranteed paddy rice can be before cold snap arrives seed development ripe.Therefore, for meeting the demand of growing population to grain, searching can expand the key gene of cultivated rice cultivated area, and clones corresponding gene, illustrates its hereditary basis and molecule mechanism, to cultivating eurytropy rice varieties, has important theory and practice meaning.

Mainly determine (Chang T T, Li C C, Vergara B S. (1969) Euphytica, 18:79～91 by its photosensitivity, temperature sensibility and basic nutrition growth property the heading stage of rice varieties; Tsai K H. (1985) Rice Genet Newslett, 2:77～78; Tsai K H. (1986) In:Rice Genetics.International Rice Research Institute, 339～349).The diversity of rice varieties photosensitivity, temperature sensibility and the combination of basic nutrition growth property, make the Genetic Performance complex at heading stage, classical genetics and molecular genetics all prove to be controlled by minority qualitative trait gene and a plurality of Quantitative Trait Genes (QTL Quantitative Trait Locus) heading stage.Up to the present, there have been 20 rice ear sprouting period genes to be cloned, wherein had 6 to be to clone by the approach of Fine Mapping map based cloning.As Hd1, Hd6, Hd3a, Ehd1, Ghd7 and DTH8(Yano, M.et al. (2000) Plant Cell12,2473 – 2483; Takahashi, Y., Shomura, A., Sasaki, T. & Yano, M. (2001) Proc Natl Acad Sci USA98,7922 – 7927; Kojima, S,, Takahashi, Y. & Kobayashi, Y. (2002) Plant Cell Physiol43,1096 – 1105; Doi, K.et al. (2004) Genes.Dev.18,926-936; Xue, W.Y.et al. (2007) Nat.Genet.40,761 – 767; Wei, X.J.et al. (2010) Plant.Physiol.153,1747 – 1758).For Heading date gene, due to the photoperiodic difference in various places, the people of different areas may select dissimilar light sensitive material to adapt to local illumination condition.Research finds that Hd1 is relevant with the range of distribution of paddy rice with the allelic natural variation of Ghd7, but the allelic variation of Hd1 and Ghd7 is too violent for the change at kind heading stage, other gene is also had to very strong epistatic action, therefore, in the kind improvement at heading stage, be difficult to application.And the slow variation that polygenic change more easily conforms has more adaptability, significant in breeding.Judge that in addition whether a gene is domestication gene, mainly see the allelic gene type of this gene in wild-rice to the mankind without profit, and the allelic gene type in cultivated rice is favourable to the mankind after taming.Be that the mankind tame and selected the proterties favourable to the mankind, but not all favourable proterties is all tamed.

Utilization comes from RIL (RILs) colony of japonica rice variety Asominori and rice variety IR24, research before us detects QTL(Wei XJ at a plurality of heading stage, et al. (2010) Mol Breeding25:287 – 298), this QTL of minor effect QTL(at heading stage that wherein we pay close attention between the long-armed end RM318 of the second karyomit(e) and RM240 is named as DTH2 in the present invention), under nature long day condition, comprise the allelic plant ratio of Asominori and comprise the allelic plant of IR24 and ear in advance.Up to the present, also there is no to find that the gene of cloning by QTL belongs to the type (Tsuji H, Taoka KI and Shimamoto K (2010) Current Opinion in Plant Biology14:1 – 8) of Accelerate bloom under long day condition.Fine Mapping rice ear sprouting period QTL best bet builds chromosome segment substitution line (CSSL) or near isogenic line (NIL) exactly, most backgrounds outside target QTL site are consistent, make this site show as typical Mendelian inheritance, i.e. quantitative character quality.But for minor effect QTL, at F ₂in colony, because the phenotypic difference of heterozygous individual plant and recessive homozygous individual plant is less, building on the basis of NIL, also need F ₂f multiplies in colony _2:3family colony, makes phenotype be easy to identify.Afterwards QTL is decomposed into individual gene and carries out map based cloning, by analyzing the linkage relationship of mutational site and known molecular mark, determine the physical location of goal gene on karyomit(e).

Summary of the invention

The object of the invention is to the above-mentioned deficiency for prior art, one group of DTH2 gene and haplotype and application of controlling rice ear sprouting period is provided.

Object of the present invention can be achieved through the following technical solutions:

Control rice ear sprouting period gene DTH2 for one, it is from paddy rice, length is 7867bp, 407 amino acid of corresponding encoded CCT/B-box zinc finger protein, dominant allele DTH2-a(DTH2 allelotrope is from a Japanese japonica rice variety Asominori) total order classifies SEQ ID NO.1 as, its CDS sequence, as shown in SEQ ID NO.2, belongs to haplotype 5(Haplotype5, the Hap5 of DTH2 gene).

The application of described control rice ear sprouting period gene DTH2 in rice varieties improvement.

The Recessive alleles DTH2-i(DTH2 allelotrope of described gene DTH2 from the international paddy rice of Yi Ge rice variety IR24), complete nucleotide sequence is as shown in SEQ ID NO.3, its CDS sequence is as shown in SEQ ID NO.4, the haplotype 1(Haplotype1, the Hap1 that belong to DTH2 gene).

The application of the Recessive alleles DTH2-i of described gene DTH2 in rice varieties improvement.

The haplotype 2(Hap2 of described gene DTH2), its CDS sequence is as shown in sequence table SEQ ID NO.5.

The application of the haplotype 2 of described gene DTH2 in rice varieties improvement.

The haplotype 3(Hap3 of described gene DTH2), its CDS sequence is as shown in sequence table SEQ ID NO.6.

The application of the haplotype 3 of described gene DTH2 in rice varieties improvement.

The haplotype 4(Hap4 of described gene DTH2), its CDS sequence is as shown in sequence table SEQ ID NO.7.

The application of the haplotype 4 of described gene DTH2 in rice varieties improvement.

According to the technological line of Fig. 1, the present invention is Fine Mapping QTL DTH2, utilization be take Asominori as background parent, chromosome segment substitution line (CSSLs) colony (Agricultural University Of Nanjing's rice research is preserved) that IR24 is donor parents, identifies the family CSSL18 that carries DTH2-i Insert Fragment of late heading.Secondary F from CSSL18 and Asominori hybridization structure ₂in colony, DTH2(LOD value 24.70 again detected, contribution rate 26.22%).Further utilize the method (MAS) of molecular marker assisted selection, built the near isogenic line--NIL (DTH2) that only carries DTH2 the secondary F that utilizes NIL (DTH2) * Asominori ₂, F _2:3and F _3:4colony, DTH2 is limited in the interval range of about 37.6kb between mark dCAPS3 and dCAPS5 the most at last, has 9 candidate genes.The 5th gene found in sequential analysis, coding CCT/B-box zinc finger protein, and with Arabidopis thaliana COL9 homology, implying that this gene may be goal gene.By building the complementary carrier of transgenosis group total length of a 7867bp genome section that comprises this gene, proceed in NIL (DTH2), find T ₂for the heading of transgenosis family in advance.By Real-time RT PCR, find, DTH2 is positioned at the upstream of Hd3a and RFT1 in rice ear sprouting period approach.By the order-checking to 79 parts of cultivated rices and 48 parts of wild-rices, find in DTH2 coding region, to have 5 base differences subsequently, cause 3 amino acid to change, produce 5 kinds of haplotypes.And prove that DTH2 is selected in domestication, and originate from China.

Beneficial effect

1. the present invention passes through to build near isogenic line NIL (DTH2), and utilizes the secondary F of NIL (DTH2) * Asominori ₂, F _2:3and F _3:4colony, by the method for map based cloning, finally clone one and expanded cultivated rice range of distribution and control the minor gene DTH2 of rice ear sprouting period northwards, this gene be first method clone who passes through map based cloning under long day condition, promote the gene of Rice Heading, for expanding cultivated rice, provide new genetic resources in range of distribution northwards, also for other crop utilization electronic cloning methods clone genes involved provides gene order, further analyze and find that this gene is a domestication gene, for the Study on Evolution of paddy rice provides new thinking.

2. DTH2-a is imported in the breeding material that comprises DTH2-i, can make the heading stage of this material in advance, planting range is northwards moved by its country of origin, expands the range of distribution northwards of cultivated rice.

3. the present invention clone's gene is relevant to domestication, and photoperiodic reaction and molecular evolution research that its domestication pattern can be the farm crop such as paddy rice, corn and soybean provide reference.

Accompanying drawing explanation

Fig. 1. general technical schema of the present invention

The complementary T of the Fine Mapping of Fig. 2 .DTH2 and transgenosis total length ₂generation and interference T ₂the result in generation.

A figure is DTH2 Fine Mapping figure; B figure represents the gene structure of DTH2; C-D figure represents the complementary T of transgenosis total length ₂generation and interference T ₂phenotype (C) and the data at heading stage (D) of generation and two parent Asominori and NIL (DTH2), P ¹and P ²refer to respectively the complementary transfer-gen plant of total length and NIL (DTH2) and disturb the P value between plant and Asominori, adopting Wilcoxen signed rank test, P ³refer to the P value between two parents,

Adopt two tail t to detect, n refers to the plant number detecting in experiment, mean value ± s.e.m..

Fig. 3. the carrier figure of the binary vector pCAMBIA1305.1 using in the present invention.

The natural variation of Fig. 4 .DTH2 coding region and regional distribution analysis.

Figure A is the result to the DTH2 coding region order-checking of 79 cultivated rices and 48 wild-rices, has shown 5 SNP and 5 kinds of haplotypes; Figure B has shown the regional distribution of 5 kinds of haplotypes; Figure C has shown the transgene result of 5 kinds of haplotypes, mean value ± s.e.m., and P value adopts two tail t to detect and draws; Figure D has shown 5 kinds of haplotype distributions between different subspecies and the evolutionary relationship between them.

Embodiment

The structure of embodiment 1DTH2 near isogenic line

The QTL of 1.DTH2 detects, and backcrosses and selects

Utilize the RILs colony of Asominori and IR24, research before us detects a plurality of gene locuss of controlling heading stage, one of them site DTH2, at (natural long-day of natural long day, NLD), under condition, the plant that comprises DTH2-a ears in advance than the plant that comprises DTH2-i.Utilization be take Asominori as background parent, IR24 is the CSSLs colony of donor parents, we find that CSSL18 has ripe and expression extremely significantly late than background parent Asominori and stablizes, because CSSL18 compares with background parent Asominori, only in some fragment, inserted IR24 fragment, the long-grained nonglutinous rice fragment IR24 therefore inserting causes CSSL18 to have ripe basic reason extremely significantly late.We hybridize CSSL18 and Asominori once, continuous backcross 4 times, and then selfing builds a CSSL18/Asominori BC who comprises 374 individual plants ₄f ₂colony, adopts IciMapping v2.1 software, DTH2 therefrom again detected.Target section is determined at two SSR(Simple Sequence Repeat) between mark RM318 and RM240 (RM series primer is public, sees website www.gramene.org), genetic distance 9.6cM(centimorgan).As can be seen from Table 1 at QTL who controls heading stage of this interval existence, contribution rate 26.22%, use MAS technology, be chosen in DTH2 section for the IR24 genotype of isozygotying, and be that the isozygoty individual plant of Asominori background is NIL (DTH2) at other sections.

Table 1. is controlled the QTL at heading stage between RM318 and RM240

%PVE: interpret table form variation rate; A: additive effect; D: dominant effect.

2. microsatellite marker genotype identification method (SSR technology)

PCR standard program is referring to J. Pehanorm Brooker etc., 2002, molecular cloning experiment guide, the third edition, Jin Dongyan etc. (translating), Science Press.PCR adopts 20 μ l reaction systems as follows: DNA masterplate 20-50ng, primer 0.5 μ M, dNTP100 μ M, 1 * buffer (Mg ²⁺plus) and 1U rTaq archaeal dna polymerase (purchased from Japanese Takara company).Pcr amplification condition is: 98 ° of denaturations 3 minutes; 94 ° 30 seconds, 55 ° 30 seconds, 72 ° 1 minute, 35 circulations; 72 ° are extended 7 minutes.PCR product carries out silver and dyes (Basam, et al. (1991) Anal.Biochem.196:80-83) on 6% polyacrylamide gel after separation.

The Fine Mapping of embodiment 2DTH2 and map based cloning

1. liang parent's phenotypic evaluation at heading stage

The date that first tassel of heading index futures individual plant is extracted out; Heading stage, number of days referred to that individual plant is from the number of days that is seeded into first tassel extraction; Extreme heading in evening individual plant refers to the individual plant that heading stage is identical or more late with parent NIL (DTH2); Extreme heading in evening man means that most of individual plant heading in a random family is all identical or be later than the family of parent NIL (DTH2).Under the NLD condition of Beijing (solarium, Institute of Crop Science, Chinese Academy of Agricultural Science east gate), NIL (DTH2) is than the late heading of Asominori 7.4 days (table 1, Fig. 2 C).And under nature short day (natural short-day, the NSD) condition of Hainan (Nan Fan of Agricultural University Of Nanjing base, Lingshui, Hainan), two parent's heading do not have significant difference.Through strict long day (LD, 14 hours illumination/10 hour dark) of illumination box and short day (SD, 10 hours illumination/14 hour dark), process, result respectively with consistent (table 2) under NLD and NSD condition.

Table 2.Asominori and NIL (DTH2) performance at heading stage under different illumination conditions

^atwo tail t detect

2.DTH2 Fine Mapping and candidate gene approach

For further dwindling the interval of DTH2, we built NIL (DTH2) * Asominori F in 2006 ₂colony, obtains 2712 F ₂individual plant.For making the phenotype of extreme individual plant be easy to observe, we are F ₂f multiplies in colony _2:3family colony.Be each F ₂seed two row that individual plant is received, every row 10 strains.We have planted 2712 F in 2007 _2:3family is totally 54240 strains, wherein selects 652 extreme heading in evening familys, meets the 3:1 separation of single Mendelian factor than (χ ²=1.33, P=0.249), show in Ci colony, heading stage is sooner or later by a Dominant gene, and early takes out allelotrope and show as dominant to taking out allelotrope evening.Because 652 extreme familys are not enough to Fine Mapping, thus we by molecule marker at remaining F _2:3in family, selected the individual plant of 10 strains in DTH2 site heterozygosis, all sowing, at South of Hainan numerous added-generation, planted 1600 F through winter in 2008 _3:4family, has therefrom selected again 447 extreme heading in evening familys.Subsequently, we utilize 1099 extreme heading in evening familys to carry out Fine Mapping to DTH2, utilize RM318 and RM240 to find respectively 21 and 190 exchange individual plants.6 SSR mark RM13896, RM6290, RM13904, RM13924, RM13929, the RM530 of recycling exploitation, with 1 InDel mark ind6, these 211 exchange individual plants are screened, between RM13896 and RM530, there are 129 exchange individual plants in discovery, between RM6290 and RM13929, there are 18 exchange individual plants, between RM13904 and ind6, have 13 exchange individual plants.Continual exploitation mark, DTH2 is positioned in the scope of the 37.6Kb between dCAPS mark dcaps3 and dcaps5 the most at last, have 4 exchange individual plants, and CAPS mark caps1 is divided into from (Fig. 2 A).The sequence information of these marks is in Table 3.

Table 3. is for the primer of Fine Mapping of the present invention

Utilize Japanese Rice annotation plan database (http://rapdb.dna.affrc.go.jp/) and Michigan State Usa vertical university rice genome annotation plan (http://rice.plantbiology.msu.edu/index.shtml), in the interval of 37.6Kb, there are 9 candidate genes in discovery, a CCT/B-box zinc finger protein of ORF5 coding wherein, reach 50.95% with Arabidopis thaliana floral genes COL9 amino acids homologous rate, all belong to CONSTANS-Like transcription factor gene family, the path of blooming of this gene family member wide participation Plant Light cycle regulating (Griffiths S.et al. (2003) Plant Physiol131:1855 – 1867).So ORF5(LOC_Os02g49230) by first-selection, be the candidate gene of DTH2.

The transgenic function complementation experiment of embodiment 3DTH2

1. the structure of the complementary carrier of genome total length

According to the candidate gene sequence of prediction, with Japan's fine (English name Nipponbare, rice varieties that completes genome sequencing) sequence, be template, design primer, the DTH2 genome being derived from Asominori is checked order, find consistent in their sequence of this section.So take Japan fine BAC clone OsJNBa33B21(purchased from country of Chinese Academy of Sciences cara gene) be template, the amplification full genome of DTH2 (primer is in Table 4) has obtained the DNA fragmentation of a 7867bp.Use homologous recombination test kit (http://bioinfo.clontech.com/infusion/, purchased from Japanese Takara company) this fragment is recombinated to pCAMBIA1305.1(from an open report of Australia and the plasmid using, Sma I Fig. 3) (purchased from Canadian Fermentas company) site.

2. agriculture bacillus mediated rice transformation

By the correct recombinant plasmid of the order-checking obtaining, the agrobacterium strains openly using from CAMBIA company by this bacterial strain of agrobacterium strains EHA105() the rice transformation system of mediation, proceeds in the callus of recessive gene type parent NIL (DTH2).Through callus of induce, subculture, preculture, infect, cultivate altogether, callus that screening has hygromycin resistance, break up, take root, hardening transfer, obtain transfer-gen plant.Agriculture bacillus mediated japonica rice genetic conversion system is mainly applied the method (Hiei Y.et al., 1996) of people's reports such as Hiei, and is optimized on this basis.Concrete steps are as follows:

2.1 callus of induce

2.1.1 ripe NIL (DTH2) seed is shelled, then use successively 70% Ethanol Treatment 1 minute, 0.15% mercury chloride (HgCl ₂) to seed-coat sterilization 15 minutes;

2.1.2 with sterilizing, wash seed 4-5 time;

2.1.3 seed is placed on inducing culture;

2.1.4 postvaccinal substratum is placed in to dark place and cultivates 4 weeks, 25 ± 1 ° of temperature.

2.2 callus subcultures

Select the embryo callus subculture of glassy yellow, consolidation and relatively dry, be placed in dark lower cultivation 2 weeks on subculture medium, 25 ± 1 ° of temperature.

2.3 preculture

Select the embryo callus subculture of consolidation and relatively dry, be placed in dark lower cultivation 2 weeks on pre-culture medium, 25 ± 1 ° of temperature.

2.4 Agrobacteriums are cultivated

On the LB substratum of selecting with corresponding resistance, preculture Agrobacterium EHA105 is two days, 28 ° of temperature.

2.5 Agrobacteriums are infected

2.5.1 pre-incubated callus is transferred in the triangular flask of the bacterium of having gone out;

2.5.2 regulate the suspension of Agrobacterium to OD6000.8-1.0;

2.5.3 callus is soaked in agrobacterium suspension 30 minutes;

2.5.4 shift callus blots to the filter paper of the bacterium of having gone out; Then be placed on common substratum and cultivate 3 days, temperature 19-20 °.

2.6 callus washings and selection are cultivated

2.6.1 with aqua sterilisa, wash callus to cannot see Agrobacterium;

2.6.2 callus is immersed in containing in the aqua sterilisa of 400 milligrams/L carboxylic benzyl sistomycocin 30 minutes;

2.6.3 shift callus blots to the filter paper of the bacterium of having gone out;

2.6.4 shift callus to selecting selection on substratum to cultivate 2-3 time, each 2 weeks.

2.7 differentiation

2.7.1 kanamycin-resistant callus tissue is transferred to and on pre-division culture medium, is placed in dark place and cultivates 5-7 days;

2.7.2 shift the callus of pre-differentiation culture to division culture medium, under illumination, cultivate, 26 ° of temperature.

2.8 take root

Cut the root that differentiation phase produces; Then transfer them in root media and cultivate 2-3 week under illumination, 26 ° of temperature.

2.9 transplant

Wash the residual substratum on root off, the seedling replanting with good root system is entered to land for growing field crops.

3. the detection of transfer-gen plant and gene function checking

The present invention has carried out twice and has independently transformed, and has obtained altogether the complementary transgenosis T of total length ₀for plant 85 strains, within 2009, plant in solarium, crop science institute east gate, Beijing Chinese Academy Of Agricultural Sciences, by PCR Molecular Detection, (front primer designs on Insert Fragment, rear primer designs on carrier, in Table 4) obtain positive plant 62 strains, ratio turned empty carrier plant and significantly shifted to an earlier date (table 5) heading stage.T at Beijing field planting list copy in 2010 ₀the seed of receiving for positive plant, finds T ₁for family, present the separated ratio of good single-gene, positive plant significantly shifts to an earlier date (table 5) heading stage than negative plant.Within 2011, in land for growing field crops, Beijing, continue plantation T ₂for family, find genetically modified proterties stability ratio N IL (DTH2) heading (Fig. 2 C-D) in advance.Proved that this candidate gene is exactly the goal gene of DTH2QTL.Also proved that this gene can improve rice varieties by genetic transformation simultaneously.

4.DTH2 gene structure and function prediction

By RT-PCR technology, obtained the total length CDS of DTH2-a, 1224bp, checks order and finds that the fine sequence of itself and Japan is in full accord altogether, and sequence is as shown in SEQ ID NO.2.The structure that has obtained DTH2 gene by comparing total length CDS and 7867bp genome sequence, the sequence of coding region in genome is 2775bp, comprises 4 exons and 3 introns (Fig. 2 B).

CCT/B-box zinc finger protein of this genes encoding is predicted in rice genome annotation plan (http://rice.plantbiology.msu.edu/index.shtml), belongs to CONSTANS-Like transcription factor gene family, 407 amino acid, consists of.According to InterProScan(http: //www.ebi.ac.uk/InterProScan/) DTH2 protein structure is predicted, find 5-47,48-90,350-392 the amino acid B-box structural domain of encoding respectively, a Zinc finger domain and a CCT structural domain.Utilize plant transcription factor database (http://plntfdb.bio.uni-potsdam.de/v3.0/) to find that DTH2 exists respectively 17 homologous genes in Arabidopis thaliana and paddy rice.A plurality of report and flowering of plant regulation and control relevant (Yano, M.et al. (2000) Plant Cell12,2473 – 2483 in them; Lee YS, et al. (2010) Plant J63:18-30; Datta S.et al. (2006) The Plant Cell18:70 – 84; Kim SK, et al. (2008) Planta228:355 – 365; Cheng XF.et al. (2006) Plant J43:758 – 768).

Table 4. is for the primer of the complementary vector construction of DTH2 of the present invention

Table 5. transgenosis group of the present invention total length T ₀generation and T ₁for transgene result

^atwo tail t between transfer-gen plant and empty carrier detect

(+) and (-) represent transgenic positive and negative plant, ^bwilcoxen signed rank test

The transgenosis interference experiment of embodiment 4DTH2

1. the structure of transgenosis interference carrier

By primer (table 6), use method (the Warthmann N.et al. of the reports such as Warthmann N, (2008) Sma I PLoS One3 (3): the 261bp fragment that e1289) increases a section and comprise DTH2 artificial mi RNA sequence, the method by homologous recombination recombinates to pCAMBIA2300(this fragment from an open report of Australia and the plasmid using) (purchased from Canadian Fermentas company) site.

2. agriculture bacillus mediated rice transformation

The rice transformation system that the correct recombinant plasmid of the sequence obtaining is mediated by agrobacterium strains EHA105 proceeds in the callus of dominant gene type parent Asominori.A group conversion for total length complementation carrier that concrete method for transformation is homogenic.

3. the detection of transfer-gen plant and gene function checking

By transgenosis, 4 strain T have been obtained ₀for transgenic positive plant (transgenosis detects front primer and designs on carrier, and rear primer designs on Insert Fragment, in Table 6), within 2009, plant in the Nan Bin of Sanya, Hainan Institute of Crop Science, Chinese Academy of Agricultural Science farm and carry out breeding.Gather in the crops latter 2010 plantation T ₁for transfer-gen plant, under illumination box LD condition, find T ₁for family, present the separated ratio of good single-gene, positive plant significantly postpones (table 7) heading stage than negative plant.2011 solarium, crop science institute east gate, Nian Beijing Chinese Academy Of Agricultural Sciences plantation T ₂for family, find that genetically modified proterties stabilization ratio Asominori heading postpones (Fig. 2 C-D).Further proved that this gene is exactly goal gene.

The primer that table 6. builds for DTH2 interference carrier of the present invention

Table 7. the present invention turns DTH2 and disturbs T ₁for transgene result

(+) and (-) represent transgenic positive and negative plant

^awilcoxen signed rank test

Embodiment 5DTH2 is the application in mankind's rice modification in early days

1. the equipotential type of DTH2 in cultivated rice and wild-rice Core Germplasms

For further determining the variation type of DTH2, we have carried out the total length order-checking of this gene to 127 parts of representational cultivations and wild-rice, order-checking sample comprises that the wild-rice 47 parts (34 parts of O.rufipogon and 13 parts of O.nivara) in 79 parts of two subspecies of cultivated rice (33 parts of indica and 46 parts of japonica) and covering whole natural distributed district and an O.barthii are as outgroup.By to the coding region of DTH2 the sequence in genome (2275bp) carry out sequencing analysis, find altogether 5 variant sites, lay respectively at the 25th, 169,461,1645 and 1721 bases (Fig. 4 A).Wherein 169 variant sites are only present in (frequency is 4%) in 5 parts of wild-rices, and 1645 variant sites do not cause amino acid variation (for same sense mutation), so the variation on these 2 sites changes not contribution to this gene function.According to 3 variant sites of residue, 127 parts of cultivations and wild-rice are divided into 5 kinds of haplotypes, Hap1(is DTH2-i), Hap2, Hap3, Hap4 and Hap5(are DTH2-a) (Fig. 4 A).

2. the functional analysis of the existing DTH2 equipotential of occurring in nature type

The equipotential type that we exist above-mentioned 5 kinds of occurring in natures is subsequently carried out the function difference that transgenic experiments is studied them, according to different haplotype design primers (table 8), has built 5 corresponding rite-directed mutagenesis carriers.Concrete construction process is: first use NCO I (purchased from Canadian Fermentas company) enzyme to cut the complementary carrier of DTH2 total length transgenosis, then use T4DNA ligase enzyme (purchased from U.S. NEB company) to make carrier from connecting, on primer, introducing afterwards mutational site take DTH2-i and carries out pcr amplification as template, produce after corresponding sudden change fragment homologous recombination to BstE II (purchased from Canadian Fermentas company) site, oneself connects generation relative configurations then with T4DNA ligase enzyme, to make carrier.The rice transformation system that the correct recombinant plasmid of the sequence obtaining is mediated by agrobacterium strains EHA105 proceeds in the callus of recessive gene type parent NIL (DTH2).A group conversion for total length complementation carrier that concrete method for transformation is homogenic.

By transgenosis, obtained respectively the transgenic positive plant (transgenosis detects primer in Table 8) of 36,28,27,24 and 22 strain Hap1-Hap5, within 2011, planted in the Yuan Nei of crop science institute solarium, Beijing Chinese Academy Of Agricultural Sciences.We find that the heading stage of Hap2 and Hap1 is close; The heading stage of Hap3 and Hap4 is close, early than Hap1 and Hap2; The heading stage of Hap5 is than front 4 all Zao (Fig. 4 C).Proof Hap2 does not have function, and Hap3, Hap4 and Hap5 all have function.

The different equipotential types of 3.DTH2 are the application in mankind's rice modification in early days

We have carried out analyzing discovery to the distribution frequency of these 3 haplotypes in cultivated rice and wild-rice: Hap3 and Hap4 all exist in wild rice in China and cultivated rice, and Hap5 only exists in temperate zone cultivated rice (temperate japonica), and there is a sequence change (Fig. 4 D) by Hap4 in Hap5.Our regional distribution analysis to Hap5, the all samples of further finding Hap5 is all distributed in to the north of north latitude 35 degree (nature long day) (Fig. 4 B), and we also prove that Hap5 has the very strong ability that promotes Rice Flowering under long day condition by above-mentioned function test, therefore infer that Hap5 is that early stage mankind's screening is fixed up, thereby there is important effect to strengthening the northern range of distribution of ability expansion cultivated rice of cultivated rice adaptation high latitude.

The primer that table 8. is analyzed for DTH2 domestication of the present invention

Claims (2)

1. The haplotype 4 of gene DTH2 shown in 1.SEQ ID NO.1, its CDS sequence is as shown in sequence table SEQ ID NO.7.
2. 2. the application of the haplotype 4 of described gene DTH2 claimed in claim 1 in obtaining the rice varieties shifting to an earlier date heading stage.

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