CN103589734B - 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

The DTH2 gene haplotype of one group of control rice ear sprouting period and application

Division explanation

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

Technical field

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

Technical background

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

The heading stage of rice varieties determines (Chang T T, Li C C, Vergara B S. (1969) Euphytica, 18:79 ~ 91 primarily of its photosensitivity, temperature sensibility and basic nutrition growth property; 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 basic nutrition growth property combination, make the Genetic Performance complex at heading stage, classical genetics and molecular genetics all prove that heading stage is controlled by minority qualitative trait gene and multiple Quantitative Trait Genes (QTL Quantitative Trait Locus).Up to the present, there have been 20 rice ear sprouting period genes to be cloned, wherein had 6 to be cloned 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 the allelic natural variation of Hd1 with Ghd7 is relevant with the range of distribution of paddy rice, but the allelic variation of Hd1 and Ghd7 is too violent for the change at kind heading stage, also there is very strong epistatic action to other gene, therefore, the improvement at kind heading stage is difficult to application.And the slow change that polygenic change more easily conforms, have more adaptability, significant in breeding.Judge 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 after domestication in cultivated rice is favourable to the mankind in addition.Namely mankind's domestication have selected the proterties favourable to the mankind, but not all beneficial traits is all domestication.

Utilize RIL (RILs) colony coming from japonica rice variety Asominori and rice variety IR24, research before us detects QTL(Wei XJ at multiple 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 second chromosome long arm end RM318 and RM240 is named as DTH2 in the present invention), under natural long-day conditions, comprise Asominori allelic plant ratio and comprise the allelic plant of IR24 and ear in advance.Up to the present, also do not find that the gene of being cloned 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 conditions.Fine Mapping rice ear sprouting period QTL best bet builds CSSL population (CSSL) or near isogenic line (NIL) exactly, most backgrounds outside target QTL site are consistent, this site is made to 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 the homozygous individual plant of recessiveness is less, on the basis building NIL, also need F ₂f multiplies in colony _2:3family colony, makes phenotype be easy to qualification.Afterwards QTL be decomposed into individual gene and carry out map based cloning, the linkage relationship namely marked by analysis mutational site and known molecular determines goal gene physical location on chromosome.

Summary of the invention

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

Object of the present invention realizes by following technical scheme:

One controls rice ear sprouting period gene DTH2, 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 is classified as SEQ ID NO.1, 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 an International Rice 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, belong to haplotype 1(Haplotype1, the Hap1 of DTH2 gene).

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

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

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

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

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

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

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

According to the technological line of Fig. 1, the present invention is Fine Mapping QTL DTH2, utilizing with Asominori is background parent, CSSL population (CSSLs) colony (Agricultural University Of Nanjing's rice research is preserved) that IR24 is donor parents, identifies the family CSSL18 carrying DTH2-i Insert Fragment of late heading.From the secondary F that CSSL18 and Asominori hybridization builds ₂in colony, DTH2(LOD value 24.70 again detected, contribution rate 26.22%).Utilize the method (MAS) of molecular marker assisted selection further, construct near isogenic line--NIL (DTH2) of only carrying DTH2 and utilize the secondary F of 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.Sequential analysis finds the 5th gene, coding CCT/B-box zinc finger protein, and with Arabidopis thaliana COL9 homology, imply this gene may be goal gene.By building the transgenosis group total length complementing vector that comprises the 7867bp genomic segment of this gene, proceeding in NIL (DTH2), finding T ₂for transgenic lines heading in advance.Found by Real-time RTPCR, DTH2 is positioned at the upstream of Hd3a and RFT1 in rice ear sprouting period approach.Subsequently by the order-checking to 79 parts of cultivated rices and 48 parts of wild-rices, find to there are 5 base differences in DTH2 coding region, cause 3 amino acid changes, produce 5 kinds of haplotypes.And prove that DTH2 is selected in domestication, and originate from China.

Beneficial effect

1. the present invention is by building 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 is the gene promoting Rice Heading under long day condition that first method by map based cloning is cloned, new genetic resources is provided in range of distribution northwards for expanding cultivated rice, also for other crop utilization electronic cloning methods clone genes involved provides gene order, further analysis finds that this gene is a domestication gene, and the Study on Evolution for paddy rice provides new thinking.

2. being imported by DTH2-a comprises in the breeding material of DTH2-i, and 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 gene of the present invention clone is relevant with domestication, and its domestication pattern can be the photoperiodic reaction of the farm crop such as paddy rice, corn and soybean and molecular evolution research provides reference.

Accompanying drawing explanation

Fig. 1. general technical schema of the present invention

The Fine Mapping of Fig. 2 .DTH2 and the complementary T of 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 ₂the phenotype (C) of generation and two parent Asominori and NIL (DTH2) and data at heading stage (D), P ¹and P ²refer to the complementary transfer-gen plant of total length and NIL (DTH2) and the P value between interference plant and Asominori respectively, employing 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 detected in experiment, mean value ± s.e.m..

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

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

Figure A is the result of the DTH2 coding region order-checking to 79 cultivated rices and 48 wild-rices, shows 5 SNP and 5 kind of haplotypes; Figure B shows the regional distribution of 5 kinds of haplotypes; Figure C shows the transgene result of 5 kinds of haplotypes, and mean value ± s.e.m., P value adopts two tail t to detect and draws; Figure D shows 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 selection

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

Table 1. controls 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 see J. Pehanorm Brooker etc., 2002, Molecular Cloning: A Laboratory 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 μM, dNTP100 μM, 1 × buffer (Mg ²⁺and 1U rTaq archaeal dna polymerase (purchased from Japanese Takara company) plus).Pcr amplification condition is: 98 ° of denaturations 3 minutes; 94 ° 30 seconds, 55 ° 30 seconds, 72 ° 1 minute, 35 circulations; 72 ° extend 7 minutes.PCR primer carries out silver dye (Basam, et al. (1991) Anal.Biochem.196:80-83) after being separated on the polyacrylamide gel of 6%.

The Fine Mapping of embodiment 2DTH2 and map based cloning

1. the phenotypic evaluation at heading stage of liang parent

Refer to the date that first tassel of individual plant is extracted out heading stage; Heading stage, number of days referred to that individual plant is from the number of days being 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 is later than the family of parent NIL (DTH2).Under the NLD condition of Beijing (east gate solarium of Institute of Crop Science, Chinese Academy of Agricultural Science), NIL (DTH2) heading more late than Asominori 7.4 days (table 1, Fig. 2 C).And under nature short day (natural short-day, the NSD) condition of Hainan (Agricultural University Of Nanjing Nan Fan base, Lingshui, Hainan), two parent's heading do not have significant difference.Process with short day (SD, 10 h light/14 h dark) through illumination box strict long day (LD, 14 h light/10 h dark), result respectively with consistent (table 2) under NLD and NSD condition.

The heading stage performance of table 2.Asominori and NIL (DTH2) under different illumination conditions

^atwo tail t detects

2.DTH2 Fine Mapping and candidate gene approach

For reducing the interval of DTH2 further, we constructed NIL (DTH2) × Asominori F in 2006 ₂colony, obtains 2712 F ₂individual plant.Be easy to for making the phenotype of extreme individual plant observe, we are F ₂f multiplies in colony _2:3family colony.I.e. each F ₂seed two row that individual plant is received, often row 10 strain.We planted 2712 F in 2007 _2:3family is totally 54240 strains, wherein selects 652 extreme heading in evening familys, meets the 3:1 segregation ratio (χ of single Mendelian factor ²=1.33, P=0.249), show in this colony, heading stage sooner or later by a Dominant gene, and early takes out allelotrope and shows as dominant to taking out allelotrope evening.Because 652 extreme familys are not enough to Fine Mapping, therefore we by molecule marker at remaining F _2:3have selected the individual plant of 10 strains in DTH2 site heterozygosis in family, whole sowing, through winter at South of Hainan numerous added-generation, plant 1600 F in 2008 _3:4family, therefrom have 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 21 and 190 to exchange individual plant respectively.6 SSR marker RM13896 of recycling exploitation, RM6290, RM13904, RM13924, RM13929, RM530, mark ind6 with 1 InDel to screen these 211 exchange individual plants, find that between RM13896 and RM530, have 129 exchanges individual plant, between RM6290 and RM13929, there are 18 exchange individual plant, between RM13904 and ind6, have 13 exchange individual plant.Continual exploitation marks, and DTH2 is positioned in the scope of the 37.6Kb between dCAPS mark dcaps3 and dcaps5 the most at last, has 4 and exchanges 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

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

The transgenic function complementation experiment of embodiment 3DTH2

1. the structure of full-length genome complementing vector

According to the candidate gene sequence of prediction, be template with Japan's fine (English name Nipponbare, rice varieties completing genome sequencing) sequence, design primer, the DTH2 genome be derived from Asominori is checked order, finds in their sequence of this section consistent.So clone OsJNBa33B21(purchased from Chinese Academy of Sciences's National Gene research centre with the fine BAC of Japan) for template, amplification DTH2 full-length genome (primer is in table 4) obtains the DNA fragmentation of a 7867bp.Use homologous recombination test kit (http://bioinfo.clontech.com/infusion/, purchased from Japanese Takara company) the restructuring of this fragment to pCAMBIA1305.1(from one of Australia open report and the plasmid that uses, Fig. 3) Sma I (purchased from Canadian Fermentas company) site.

2. agriculture bacillus mediated rice transformation

By recombinant plasmid correct for the order-checking obtained, agrobacterium strains by this bacterial strain of agrobacterium strains EHA105(openly uses from CAMBIA company) the rice transformation system that mediates, proceed in the callus of recessive genotype parent NIL (DTH2).Through callus of induce, subculture, preculture, infect, callus that Dual culture, screening have hygromycin resistance, break up, take root, hardening transfer, obtain transfer-gen plant.Agriculture bacillus mediated japonica rice genetic conversion system mainly applies 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 NIL (DTH2) seed of maturation is shelled, then use the Ethanol Treatment 1 minute of 70% successively, 0.15% mercury chloride (HgCl ₂) seed-coat is sterilized 15 minutes;

2.1.2 seed is washed 4-5 time with sterilizing;

2.1.3 seed is placed on inducing culture;

2.1.4 postvaccinal substratum is placed in dark place's cultivation 4 weeks, temperature 25 ± 1 °.

2.2 callus subcultures

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

2.3 preculture

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

2.4 Agrobacteriums are cultivated

Preculture Agrobacterium EHA105 two days on the LB substratum selected with corresponding resistance, temperature 28 °.

2.5 Agrobacteriums are infected

2.5.1 pre-incubated callus being transferred to has gone out in the triangular flask of bacterium;

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

2.5.3 callus is soaked 30 minutes in agrobacterium suspension;

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

2.6 callus washings and selection are cultivated

2.6.1 callus is washed to cannot see Agrobacterium with aqua sterilisa;

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

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

2.6.4 shift in callus to Selective agar medium and select to cultivate 2-3 time, each 2 weeks.

2.7 differentiation

2.7.1 kanamycin-resistant callus tissue is transferred on pre-division culture medium and is placed in dark place's cultivation 5-7 days;

2.7.2 the callus shifting pre-differentiation culture, on division culture medium, is cultivated under illumination, temperature 26 °.

2.8 take root

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

2.9 transplant

Wash the remaining medium on root off, the seedling replanting with good root system is entered land for growing field crops.

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

Invention has been twice independently to transform, obtain the complementary transgenosis T of total length altogether ₀for plant 85 strain, within 2009, plant in east gate solarium of crop science institute of 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 strain, 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 ₀for the seed that positive plant is received, find T ₁present good single-gene segregation ratio for family, positive plant significantly shifts to an earlier date (table 5) than negative plant heading stage.Within 2011, continue plantation T in land for growing field crops, Beijing ₂for family, find the heading of genetically modified proterties stability ratio N IL (DTH2) in advance (Fig. 2 C-D).Demonstrate the goal gene that this candidate gene is exactly DTH2QTL.Also demonstrate this gene simultaneously and can improve rice varieties by genetic transformation.

4.DTH2 gene structure and function prediction

Obtained the total length CDS of DTH2-a by RT-PCR technology, altogether 1224bp, order-checking finds that the fine sequence of itself and Japan is completely the same, and sequence is as shown in SEQ ID NO.2.Obtain the structure of 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).

Rice genome annotation plan (http://rice.plantbiology.msu.edu/index.shtml) predicts this genes encoding CCT/B-box zinc finger protein, belongs to CONSTANS-Like transcription factor gene family, is made up of 407 amino acid.According to InterProScan(http: //www.ebi.ac.uk/InterProScan/) DTH2 protein structure is predicted, find 5-47,48-90,350-392 amino acid is encoded a B-box structural domain, a Zinc finger domain and a CCT structural domain respectively.Plant transcription factor database (http://plntfdb.bio.uni-potsdam.de/v3.0/) is utilized to find that DTH2 exists 17 homologous genes respectively in Arabidopis thaliana and paddy rice.In them multiple reported and flowering of plant regulation and control relevant (Yano, M.et al. (2000) Plant Cell12,2473 – 2483; 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).

The primer that table 4. builds for DTH2 complementing vector 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

Method (the Warthmann N.et al. of the reports such as Warthmann N is used by primer (table 6), (2008) increase one section and comprise the 261bp fragment of DTH2 artificial mi RNA sequence PLoS One3 (3): e1289), by the method for homologous recombination the restructuring of this fragment to pCAMBIA2300(from an open report of Australia and the plasmid that uses) Sma I (purchased from Canadian Fermentas company) site.

2. agriculture bacillus mediated rice transformation

The rice transformation system that recombinant plasmid correct for the sequence obtained is mediated by agrobacterium strains EHA105 is proceeded in the callus of dominant genotypes parent Asominori.A group conversion for total length complementing vector that concrete method for transformation is homogenic.

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

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

The application of embodiment 5DTH2 in early days in mankind's rice modification

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

For determining the variation type of DTH2 further, we have carried out the total length order-checking of this gene to 127 parts of representational cultivations and wild-rice, and order-checking sample comprises cultivated rice two subspecies 79 parts (33 parts of indica and 46 part japonica) and the covering wild-rice 47 parts (34 parts of O.rufipogon and 13 part O.nivara) in whole natural distributed district and an O.barthii as outgroup.By carrying out sequencing analysis to the coding region sequence in genome (2275bp) of DTH2, finding 5 variant sites altogether, laying respectively at the 25th, 169,461,1645 and 1721 base (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 is to this gene function change not contribution.According to residue 3 variant sites, 127 parts of cultivations and wild-rice are divided into 5 kinds of haplotypes, Hap1(and DTH2-i), Hap2, Hap3, Hap4 and Hap5(and DTH2-a) (Fig. 4 A).

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

We carry out transgenic experiments to study their function difference to the equipotential type that above-mentioned 5 kinds of occurring in natures exist subsequently, construct 5 corresponding rite-directed mutagenesis carriers according to different haplotype design primer (table 8).Concrete construction process is: first use NCO I (purchased from Canadian Fermentas company) enzyme to cut DTH2 total length transgenosis complementing vector, then T4DNA ligase enzyme (purchased from American NEB company) is used to make carrier from connecting, on primer, introduce mutational site is afterwards that template carries out pcr amplification with DTH2-i, after producing corresponding mutant fragments, homologous recombination is to BstE II (purchased from Canadian Fermentas company) site, then makes carrier from connecting generation relative configurations with T4DNA ligase enzyme.Recombinant plasmid correct for the sequence obtained is proceeded in the callus of recessive genotype parent NIL (DTH2) by the rice transformation system that agrobacterium strains EHA105 mediates.A group conversion for total length complementing vector that concrete method for transformation is homogenic.

Obtained the transgenic positive plant (detection GMOs primer is in table 8) of 36,28,27,24 and 22 strain Hap1-Hap5 by transgenosis respectively, within 2011, plant in Yuan Nei solarium of crop science institute of 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).Prove that Hap2 does not have function, Hap3, Hap4 and Hap5 all have function.

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

We have carried out analyzing finding to the distribution frequency of these 3 haplotypes in cultivated rice and wild-rice: Hap3 and Hap4 all exists 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.We are to the regional distribution analysis of Hap5, the all samples of further discovery Hap5 is all distributed in (nature long day) (Fig. 4 B) to the north of north latitude 35 degree, and we also prove that Hap5 has the very strong ability promoting Rice Flowering under long day condition by above-mentioned function test, therefore infer that Hap5 is that early stage mankind screening is fixed up, adapt to the ability of high latitude to strengthening cultivated rice thus expand the northern range of distribution of cultivated rice there is important effect.

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

Claims (1)

1. The gene of 1.CDS sequence as shown in SEQ ID NO.7 dTH2haplotype 4 obtaining the application in the rice varieties that shifts to an earlier date at heading stage.