CN101892246B - Clone and application of pleiotropic gene Ghd8 for controlling yield, florescence and plant height of rice grain - Google Patents

Abstract

The invention relates to the plant genetic engineering field, in particular to clone of a pleiotropic gene Ghd8 which affects yield, florescence and plant height of rice grain. In the invention, the gene Ghd8 is separated and cloned by experiments such as establishment of a near-isogenic line of a target gene, preliminary location, comparative sequencing, genetic transformation and complementation and the like, wherein the sequence of the gene Ghd8 is shown as SEQ ID NO:2. Compared with a recessive allelic single plant, the yield of a dominant allelic single plant is increased by 58%, the plant height thereof is increased by 25.6%, but the heading stage thereof is only increased by 9 days. By means of results of Ghd8 main mutation site and trait correlation analysis and haploid cluster analysis of protein sequences of different varieties, the near-isogenic lines of different allelic types are established to obtain four favorable Ghd8 allelic types, wherein, the Ghd8-9311 and Ghd8-ruf allelic types can increase yield of the rice grain but not delay blossoming, thus being applicable to breeding the rice grain in areas with good light and temperature conditions; and Ghd8-MH63 and Ghd8-Nip allelic types are not photosensitive, thus being applicable to increasing yield and breeding the rice grain in areas under a short-day condition.

Description

Control rice grain output, clone and the application of the pleiotropic gene Ghd8 of flowering period and plant height

Technical field

The invention belongs to the plant gene engineering technology field.Being specifically related to one affects rice grain output, clone and the application of flowering period and plant height pleiotropic gene Ghd8.The present invention has carried out Fine Mapping to the quantitative trait locus gene that this has enormous benefits, utilizes agriculture bacillus mediated genetic transformation, and the function of this gene is verified; Made up simultaneously the near isogenic line of the different allelotypes of this gene, the function of the different allelotypes of this gene has been verified.

Background technology

At present, world population is approaching 7,000,000,000, wherein half population with paddy rice as staple food grain.Satisfy growing population to the demand of grain, the yield per unit that especially improves paddy rice in the situation that current Economic development arable area constantly reduces is significant.

Rice yield is that its Components mainly comprises plant height, grain number per spike, heading stage, setting percentage, tiller number, thousand seed weight etc. by the complicated economical character of many factors joint effect.The real grain of every fringe number is that every fringe grain husk spends number and setting percentage to determine, and every fringe grain husk spends number to refer to directly determine rice grain output by the number of grain husk flower on each spike of rice.The plant height of paddy rice is important Plant type indices, is determining lodging resistance and the biological yield of paddy rice, and acts on rice grain output, and the improvement of plant height also is the importance of Ideotype Breeding.Become in current molecular breeding and transgenic breeding in the situation of choosing of certainty that breaks through the rice yield bottleneck and sought the real number of the every fringe of control, the critical sites of heading stage and plant height reaches to cloning corresponding gene and illustrating its hereditary basis and molecule mechanism, has great theory and practice significance.

The grain number per spike of paddy rice, heading stage and plant height are the complex characters that a class is subjected to controlled by multiple genes, present the phenotypic variation of continuous normal distribution law in separating the offspring.These polygene are known as quantitative character gene locus therefor (quantitative trait loci, QTL).The typical amounts proterties such as the output controlled by multiple genes of originally experiencing is difficult to single-gene namely to occur at F2 and separates, and generally also needs to make up the Mendelian that the consistent near isogenic line of genetic background makes objective trait present Dominant gene and separates.The structure of near isogenic line is divided into Advanced backcross method (Ashikari et al.2005; Fan et al.2006) with based on two kinds of method of the Characters direct construction (Zhang et al., 2006).Near isogenic line namely is based on RIL F among the present invention ₇Dai Zhongyi proterties occurs to separate family and make up.

For the clone of the Quantitative Trait Genes of hereditary basis complexity, map based cloning still is effective method.Map based cloning (map-basedcloning) 1986 at first proposes (Coulson et al. by the Alan Coulson of Cambridge University, 1986), to carry out according to the position of goal gene on karyomit(e) with the method isolated genes, need not to know in advance the dna sequence dna of gene, also need not to know in advance its expression product for information about.It is the hereditary basis of determining mutant phenotype by the linkage relationship of analyzing mutational site and known molecular mark.

The employing map based cloning methods such as Yano have been cloned since the rice ear sprouting period genes involved Hd1, and rice ear sprouting period QTL clone work had also obtained huge achievement after especially the paddy rice whole genome sequence discharged.(Yano et al., 2000 such as the rear clone Heading date gene Hd1 of elder generation, Hd6, Hd3a and Ehd1; Takahashi et al., 2001; Kojima et al., 2002; Kazuyuki Doi et al., 2004).First control Rice Panicle grain base was cloned (AshikariM et al., 2005) because of Gn1a in 2005, utilized to comprise about 13,000 F ₂The segregating population of individual plant is limited to Gn1a zone and the most at last this gene clone of 6.3kb.The gene (cytokinin oxidase/dehydrogenase, OsCKX2) of genes encoding phytokinin oxydehydrogenation enzyme, the pyramiding breeding of this gene and Green Revolution gene sd1 has shown huge yield potential.Xue Weiya etc. (Xue et al., 2008) utilize bright extensive 63 to clone with the near isogenic line that precious Shan 97 is derived and to be positioned at paddy rice relevant pleiotropic gene Ghd7 at the 7th chromosomal heading stage, studies have shown that CO-LIKE gene of its coding.This gene is so that two kinds of allelotypes of its near isogenic line plant height under the long day condition differs 30cm, and single plant yield amplification reaches 50% (Xue et al., 2008), simultaneously also so that postponed 20 days heading stage, has reduced its production application and has been worth.

The RIL F that utilization of the present invention is derived from HR5/ZS97 based on the method for proterties difference ₇Choosing the phenotypic difference individual plant among the separation family RIL39 in generation carries out hybridizing the structure near isogenic line after background is selected.With this near isogene be the basis, utilize the map based cloning method, finally cloned this and controlled simultaneously rice anthesis, the pleiotropy QTL of plant height grain number per spike, Ghd8.This site so that between near isogenic line two allelotypes single plant yield increase to 23.8 grams by 15.1 grams, plant height increases to 98 centimetres by 78 centimetres, and only increased to 86 days by 77 days heading stage, production application has a high potential.

Summary of the invention

The object of the invention is to clone the pleiotropic gene Ghd8 of control rice grain output, heading stage and a plant height.Utilize Grain Yield, heading stage and the plant height proterties of this gene regulating paddy rice, have preferably application prospect.This unnamed gene that the applicant will clone is Ghd8.Full length gene 891bp (being SEQ ID NO:2), the albumen that contains the CCAAT binding domains of encoding, 296 amino acid of total length, the sequence of its protein is as described in the SEQ ID NO:3.

The present invention is achieved in that

RIL (hereinafter to be referred as the RIL) F7 that utilizes the precious Shan 97 of rice varieties (ZS97) and HR5 to make up separates family for colony, and (method is seen Zhang et al., 2006), therefrom select and obtain 2 kinds of extreme phenotype individual plant: N15 and (show as small ear, short strain, early blossoming; The N15 material is seen Zhanget al., 2006), N16 (spend for large fringe, high strain by evening; The N16 material is seen Zhang et al., 2006) hybridize, make up near isogenic line (hereinafter to be referred as NIL).Extreme early blossoming individual plant from the NIL-F2 segregating population, utilize molecule marker with the gene Fine Mapping in the fragment of a 70Kb.Candidate gene (LOC_08g07740) in this 70Kb section is checked order, the discovery coding region there are differences, (be purchased from Arizona Genomics Institute from the 9311BAC library, website, research centre http://www.genome.arizona.edu/orders/direct.html? library=OSI9Ba) be separated to a sequence (being SEQ ID NO:1) that comprises the long 10.26kb of this candidate gene in, be connected to binary vector plasmid pCAMBIA1301 (from the plasmid of open report of Australia and use), adopt the agrobacterium mediation converted method, complementary conversion NIL-ZS97 genotype plant (being the N15 individual plant).The performance of transgenosis individual plant is very similar to Ghd8NIL-HR5 genotype (being the N16 individual plant) phenotype.Ghd8 is successfully cloned.

Utilize 198 parts of Mini core collection kinds to carry out the main variant sites of Ghd8 (S1-S5) with rice grain output, the association analysis of flowering period and plant height.Simultaneously in conjunction with the cluster analysis of 94 parts of kind protein sequence haploidy, the near isogenic line that makes up different allelotypes verifies protein function, found to be beneficial to volume increase but not the allelotype Ghd8-9311 allelotype of late blooming (coding region sequence such as SEQID NO:2) and Ghd8-ruf allelotype (coding region sequence such as SEQ ID NO:8); The Ghd8-nip allelotype (coding region sequence such as SEQ ID NO:4) that can increase production and only slightly postpone breeding time; Affect hardly the Ghd8-MH63 allelotype (coding region sequence such as SEQ ID NO:6) of breeding time in the time of volume increase, for breeding improvement provides new approach.

The invention has the advantages that:

(1) the present invention utilizes a kind of novel method based on the Characters to make up near isogenic line, and the method is simple, and is efficient, avoided the method shortcoming consuming time that backcrosses of many generations.

(2) the present invention has cloned one affects rice grain output, heading stage and plant height one because of pleiotropic gene Ghd8.This gene is controlled rice anthesis simultaneously, plant height and grain number per spike, and for improveing simultaneously plant type of rice, output and regional adaptability provide new thinking and possibility.

(3) utilize association analysis method that proterties has been carried out subdivision and verify with the near isogene based material, found the approach that utilizes Ghd8 to carry out output and regional adaptability improvement breeding (volume increase but not late blooming).For different areas or season, the different allelotypes of reasonable selection Ghd8 breed high-yield variety.

Description of drawings

Sequence table SEQ ID NO:1 is the sequence that comprises candidate gene, is used for transforming the N15 individual plant.

Sequence table SEQ ID NO:2 is Ghd8-9311 allelotrope (deriving from rice varieties 93-11) encoding sequence.

Sequence table SEQ ID NO:3, SEQ ID NO:13 are Ghd8-9311 allelotrope encoding amino acid sequences.

Sequence table SEQ ID NO:4 is Ghd8-Nip (it is fine to derive from rice varieties Japan), Ghd8-HR5 (deriving from rice varieties HR5) allelotrope encoding sequence.

Sequence table SEQ ID NO:5, SEQ ID NO:10 are Ghd8-Nip, Ghd8-HR5 allelotrope encoding amino acid sequence.

Sequence table SEQ ID NO:6 is Ghd8-MH63 allelotrope (deriving from rice varieties bright extensive 63) encoding sequence.

Sequence table SEQ ID NO:7, SEQ ID NO:11 are Ghd8-MH63 allelotrope encoding amino acid sequences.

Sequence table SEQ IDNO:8 is Ghd8-ruf allelotrope (deriving from rice varieties O.rufipogon) encoding sequence.

Sequence table SEQ ID NO:9, SEQ ID NO:12 are Ghd8-ruf allelotrope encoding amino acid sequences.

Fig. 1: the general technical route map that is separating clone Ghd8 gene of the present invention.

Fig. 2: the Characters that is near isogenic line among the present invention

Fig. 3: be that the present invention utilizes extreme recessive individual plant Fine Mapping Ghd8 collection of illustrative plates and based on the structure diagram of the conversion carrier of binary vector pCAMBIA1301, karyomit(e) indicates with the overstriking black line, the black line top indicates the mark title, corresponding numeral reorganization time, and isozygotied chromosome segment for the HR5 background of black dotted lines box indicating.

Fig. 4: be the comparison of candidate gene aminoacid sequence among two kinds of parents of near isogenic line (precious Shan 97, HR5) among the present invention.

Fig. 5: be the performance of near isogenic line of the present invention and transgenosis individual plant.NIL (zs97), NILzs97-are respectively the recessive parent of near isogenic line and the negative individual plant of transgenosis, and NIL (Hr97), NILzs97+ then are the dominant parent of near isogenic line and transgenic positive individual plant.

Fig. 6: be for detection of the primer of genotype and gene sequencing among the present invention.

Embodiment

Following examples further define the present invention, and have described separating clone and the functional verification of Ghd8 gene.

The Fine Mapping of embodiment 1:Ghd8 gene

1, near isogenic line makes up and estimates

According to the technological line plantation ZS97 of Fig. 1 and the RIL F7 colony of HR5, find that plant height, the isophenous separation of grain number per spike (table 1) heading stage appears, in RIL39 family inside.Select wherein short strain, fringe early, the N15 individual plant of small ear and high strain, late fringe, the large N16 individual plant of fringe, rice genetic linkage map (Temnykh et al., 2000 of having delivered with reference to Temnykh etc.; Temnykh et al., 2001), chosen at N15, had 126 SSR marks of polymorphism between the N16 to carrying out the background screening, the F that simultaneously N15 and N16 individual plant is derived ₂Colony carries out QTL scanning (table 2), F ₂Phenotype presents separation in 3: 1, plant height, fringe size, be divided into flowering period from and the low value proterties be recessive (seeing accompanying drawing 2).This QTL is positioned at the 8th X chromosome centric territory, between molecule marker RM5432 and the RM547.The near isogenic line in this site (claiming Ghd8NIL) makes up to be finished.

Table 1 N15, N16 and original parent HR5 thereof, the Characters of ZS97

The various proterties QTL effects of table 2 near isogenic line colony

2.Ghd8 the Fine Mapping of gene

The season of growth in 2006 (May is to October) comprises the Ghd8NIL-F of 2260 individual plants in the Wuhan plantation ₂Segregating population, the screening of individual plant of recombinating of 340 individual plants choosing wherein extreme early blossoming, respectively with SSR mark RM547, RM310, RM5556, RM4085, RM5432 (sequence is seen Fig. 6) reaches from creating polymorphism mark SNP3, SEQ3-1, SEQ5-1 (sequence is seen Fig. 6) screening restructuring individual plant and the most at last the assignment of genes gene mapping in the fragment of a 70Kb, (see Fig. 3), with mark selfpro (sequence is seen Fig. 6) be divided into from.

3. candidate gene determines

70Kb candidate section comprises the gene (LOC_Os08g07740) of coding CCAAT-BOX Binding factor (CBF) albumen.According to Orna Ben-Naim's etc., the CBF genoid has participated in the Arabidopis thaliana regulation and control of blooming, further relatively order-checking discovery to candidate gene, HR5 has identical nucleotide sequence with Japanese fine this section gene, and two kinds of allelotype: Ghd8-ZS97 of Ghd8NIL, Ghd8-HR5 allelotrope are except existing at promoter region the difference of many places, have a phase shift mutation in the coding region from ATG the 322nd base place so that Ghd8-ZS97 allelotrope coded amino acid premature termination (Fig. 4), LOC_Os08g07740 is considered to the candidate gene of Ghd8.

Separation and the clone of embodiment 2:Ghd8 gene

Candidate gene sequence according to prediction, amplification PCR fragment screening 9311BAC library, pick out the clone 50N15 that contains candidate gene LOC_Os08g07740, utilize restriction endonuclease SacI enzyme to cut positive colony, carry out subclone, obtain the segment of a 10.26K b, this fragment comprises the sequence of transcripting start point upstream 3.5kb and termination site downstream 5.7Kb, only contains complete genome of candidate gene in this fragment.

This fragment is connected to binary vector pCAMBIA1301 upper (Fig. 3), and order-checking is adopted agrobcterium-mediated transformation after determining that conversion carrier is correct, obtains genetically modified paddy rice.Transgenosis concrete steps of the present invention are as follows:

Correct clone's plasmid imports in recessive gene type (Ghd8-ZS97) the plant callus by agriculture bacillus mediated rice transformation system, through callus of induce, subculture, preculture, infect, cultivate altogether, callus that screening has hygromycin resistance, break up, take root, practice transplantation of seedlings, obtain transfer-gen plant.Agriculture bacillus mediated paddy rice (japonica rice subspecies) genetic conversion system is mainly used on method (Hiei Y etal., the 1996) basis of the people such as Hiei report and is optimized.

The method of the key step of genetic transformation of the present invention, substratum and preparation thereof is as described below:

(1) reagent and solution abbreviation

The abbreviation of the used plant hormone of substratum is expressed as follows among the present invention: 6-BA (6-BenzylaminoPurine, 6-benzyladenine); CN (Carbenicillin, Pyocianil); KT (Kinetin, kinetin); NAA (Napthalene acetic acid, naphthylacetic acid); IAA (Indole-3-acetic acid, indolylacetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid, 2,4 dichlorophenoxyacetic acid); AS (Acetosringone, Syringylethanone); CH (CaseinEnzymaticHydrolysate, caseinhydrolysate); HN (Hygromycin B, Totomycin); DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide (DMSO)); N6max (N6 macroelement composition solution); N6mix (N6 Trace Elements solution); MSmax (MS macroelement composition solution); MSmix (MS Trace Elements solution)

(2) main solution formula

1) N6 substratum macroelement mother liquor (according to 10 times of concentrated solutions (10X) preparation):

Saltpetre (KNO ₃) 28.3 grams

Potassium primary phosphate (KH ₂PO ₄) 4.0 grams

Ammonium sulfate ((NH ₄) ₂SO ₄) 4.63 grams

Sal epsom (MgSO ₄7H ₂O) 1.85 grams

Calcium chloride (CaCl ₂2H ₂O) 1.66 grams

Mentioned reagent is dissolved one by one, then be settled to 1000 milliliters with distilled water under the room temperature.

2) N6 substratum trace element mother liquor (is prepared according to 100 times of concentrated solutions (100X)

Potassiumiodide (KI) 0.08 gram

Boric acid (H ₃BO ₃) 0.16 gram

Manganous sulfate (MnSO ₄4H ₂O) 0.44 gram

Zinc sulfate (ZnSO ₄7H ₂O) 0.15 gram

Mentioned reagent is at room temperature dissolved and be settled to 1000 milliliters with distilled water.

3) molysite (Fe ₂EDTA) stock solution (according to the preparation of 100X concentrated solution)

With 3.73 gram b diammonium disodium edta (Na ₂EDTA2H ₂O) and 2.78 the gram FeSO ₄7H ₂O dissolves respectively, mixes and is settled to 1000 milliliters with distilled water, bathes 2 hours to 70 ℃ of temperature, and 4 ℃ save backup.

4) VITAMIN stock solution (according to the preparation of 100X concentrated solution)

Nicotinic acid (Nicotinic acid) 0.1 gram

VITMAIN B1 (Thiamine HCl) 0.1 gram

Vitamin B6 (Pyridoxine HCl) 0.1 gram

Glycine (Glycine) 0.2 gram

Inositol (Inositol) 10 grams

Adding distil water is settled to 1000 milliliters, and 4 ℃ save backup.

5) MS substratum macroelement mother liquor (MSmax mother liquor) (according to the preparation of 10X concentrated solution)

Ammonium nitrate (NH ₄NO ₃) 16.5 grams

Saltpetre 19.0 grams

Potassium primary phosphate 1.7 grams

Sal epsom 3.7 grams

Calcium chloride 4.4 grams

Mentioned reagent is at room temperature dissolved, and be settled to 1000 milliliters with distilled water.

6) MS substratum trace element mother liquor (MSmin mother liquor) (according to the preparation of 100X concentrated solution)

Manganous sulfate (MnSO ₄4H ₂O) 2.23 grams

Zinc sulfate (ZnSO ₄7H ₂O) 0.86 gram

Boric acid (H ₃BO ₃) 0.62 gram

Potassiumiodide (KI) 0.083 gram

Sodium orthomolybdate (Na ₂MoO ₄2H ₂O) 0.025 gram

Copper sulfate (CuSO ₄5H ₂O) 0.0025 gram

Cobalt chloride (CoCl ₂6H ₂O) 0.0025 gram

Mentioned reagent is at room temperature dissolved, and be settled to 1000 milliliters with distilled water.

7) 2, the preparation of 4-D stock solution (1 mg/ml):

Weigh 100 milligrams of 2,4-D, with 1 milliliter of 1N potassium hydroxide dissolving 5 minutes, be settled to 100 milliliters after then adding 10 ml distilled water dissolve completes, under room temperature, preserve.

8) preparation of 6-BA stock solution (1 mg/ml):

Weigh 100 milligrams of 6-BA, with 1 milliliter of 1N potassium hydroxide dissolving 5 minutes, be settled to 100 milliliters, room temperature preservation after then adding 10 ml distilled water dissolve completes.

9) preparation of naphthylacetic acid (NAA) stock solution (1 mg/ml):

Take by weighing 100 milligrams of NAA, with 1 milliliter of 1N potassium hydroxide dissolving 5 minutes, be settled to 100 milliliters after then adding 10 ml distilled water dissolve completes, 4 ℃ save backup.

10) preparation of indolylacetic acid (IAA) stock solution (1 mg/ml):

Weigh 100 milligrams of IAA, with 1 milliliter of 1N potassium hydroxide dissolving 5 minutes, be settled to 100 milliliters after then adding 10 ml distilled water dissolve completes, 4 ℃ save backup.

11) preparation of glucose stock solution (0.5 grams per milliliter):

Weigh glucose 125 grams, then be settled to 250 milliliters with dissolved in distilled water, sterilizing rear 4 ℃ saves backup.

12) preparation of AS stock solution:

Weigh AS 0.392 gram, add 10 milliliters of dissolvings of DMSO, divide to be filled in 1.5 milliliters of centrifuge tubes, 4 ℃ save backup.

13) 1N potassium hydroxide stock solution

Weigh potassium hydroxide 5.6 grams, be settled to 100 milliliters with dissolved in distilled water, room temperature preservation is for subsequent use.

(3) be used for the culture medium prescription of rice transformation

1) inducing culture

100 milliliters in N6max mother liquor (getting the 10X concentrated solution that has prepared, lower same)

10 milliliters in N6mix mother liquor (getting the 100X concentrated solution that has prepared, lower same)

Fe ²⁺10 milliliters of EDTA stock solutions (getting the 100X concentrated solution that has prepared, lower same)

10 milliliters of VITAMIN stock solutions (getting the 100X concentrated solution that has prepared, lower same)

2.5 milliliters of 2,4-D stock solutions (get above-mentioned prepare)

Proline(Pro) (Proline) 0.3 gram

CH 0.6 gram

Sucrose 30 grams

Phytagel 3 grams

Adding distil water to 900 milliliter, 1N potassium hydroxide is regulated pH value to 5.9, boil and be settled to 1000 milliliters, divide and install to 50 milliliters of triangular flasks (25 milliliters/bottle), according to a conventional method sterilization after the sealing (for example sterilized 25 minutes down for 121 ℃, following medium sterilization method is identical with the sterilising method of basal culture medium).

2) subculture medium

100 milliliters in N6max mother liquor (10X)

10 milliliters in N6mix mother liquor (100X)

Fe ²⁺10 milliliters of EDTA stock solutions (100X)

10 milliliters of VITAMIN stock solutions (100X)

2.0 milliliters of 2,4-D stock solutions

Proline(Pro) 0.5 gram

CH 0.6 gram

Sucrose 30 grams

Phytagel 3 grams

Adding distil water to 900 milliliter, 1N potassium hydroxide is regulated pH value to 5.9, boils and is settled to 1000 milliliters, minute installs to 50 milliliters of triangular flasks (25 milliliters/bottle), sealing, as stated above sterilization.

3) pre-culture medium

12.5 milliliters in N6max mother liquor (10X)

1.25 milliliters in N6mix mother liquor (100X)

Fe ²⁺2.5 milliliters of EDTA stock solutions (100X)

2.5 milliliters of VITAMIN stock solutions (100X)

0.75 milliliter of 2,4-D stock solution

CH 0.15 gram

Sucrose 5 grams

Agar powder 1.75 grams

Adding distil water to 250 milliliter, 1N potassium hydroxide is regulated pH value to 5.6, sealing, as stated above sterilization.

Use front heating for dissolving substratum and add 5 milliliters of glucose stock solutions and 250 microlitre AS stock solutions, (25 milliliters/ware) in the culture dish are poured in packing into.

4) be total to substratum

12.5 milliliters in N6max mother liquor (10X)

1.25 milliliters in N6mix mother liquor (100X)

Fe ²⁺2.5 milliliters of EDTA stock solutions (100X)

2.5 milliliters of VITAMIN stock solutions (100X)

0.75 milliliter of 2,4-D stock solution

CH 0.2 gram

Sucrose 5 grams

Agar powder 1.75 grams

Adding distil water to 250 milliliter, 1N potassium hydroxide is regulated pH value to 5.6, sealing, as stated above sterilization.

Use front heating for dissolving substratum and add 5 milliliters of glucose stock solutions and 250 microlitre AS stock solutions, (25 milliliters/every ware) in the culture dish are poured in packing into.

5) suspension medium

5 milliliters in N6max mother liquor (10X)

0.5 milliliter in N6mix mother liquor (100X)

Fe ²⁺0.5 milliliter of EDTA stock solution (100X)

1 milliliter of VITAMIN stock solution (100X)

0.2 milliliter of 2,4-D stock solution

CH 0.08 gram

Sucrose 2 grams

Adding distil water to 100 milliliter is regulated pH value to 5.4, minute installs in two 100 milliliters the triangular flask sealing, as stated above sterilization.Add 1 milliliter of aseptic glucose stock solution and 100 microlitre AS stock solutions before using.

6) select substratum

25 milliliters in N6max mother liquor (10X)

2.5 milliliters in N6mix mother liquor (100X)

Fe ²⁺2.5 milliliters of EDTA stock solutions (100X)

2.5 milliliters of VITAMIN stock solutions (100X)

0.625 milliliter of 2,4-D stock solution

CH 0.15 gram

Sucrose 7.5 grams

Agar powder 1.75 grams

Adding distil water to 250 milliliter is regulated pH value to 6.0, sealing, as stated above sterilization.

Dissolving substratum before using adds 250 microlitre HN (50 mg/ml) and (25 milliliters/ware) in the culture dish are poured in 400 microlitre CN (250 mg/ml) packing into.(annotate: selecting substratum Pyocianil concentration for the first time is 400 mg/litre, and Totomycin concentration is 50 mg/litre) selects substratum Pyocianil concentration for the second time and later on is 250 mg/litre, and Totomycin concentration is 50 mg/litre).

7) pre-division culture medium

25 milliliters in N6max mother liquor (10X)

2.5 milliliters in N6mix mother liquor (100X)

Fe ²⁺2.5 milliliters of EDTA stock solutions (100X)

2.5 milliliters of VITAMIN stock solutions (100X)

0.5 milliliter of 6-BA stock solution

0.5 milliliter of KT stock solution

NAA stock solution 50 microlitres

IAA stock solution 50 microlitres

CH 0.15 gram

Sucrose 7.5 grams

Agar powder 1.75 grams

Adding distil water to 250 milliliter, 1N potassium hydroxide is regulated pH value to 5.9, sealing, as stated above sterilization.

Dissolving substratum before using, 250 microlitre HN (50 mg/ml), 250 microlitre CN (250 mg/ml), (25 milliliters/ware) in the culture dish are poured in packing into.

8) division culture medium

100 milliliters in N6max mother liquor (10X)

10 milliliters in N6mix mother liquor (100X)

Fe ²⁺10 milliliters of EDTA stock solutions (100X)

10 milliliters of VITAMIN stock solutions (100X)

2 milliliters of 6-BA stock solutions

2 milliliters of KT stock solutions

0.2 milliliter of NAA stock solution

0.2 milliliter of IAA stock solution

CH 1 gram

Sucrose 30 grams

Phytagel 3 grams

Adding distil water to 900 milliliter, 1N potassium hydroxide is regulated pH value to 6.0.

Boil and be settled to 1000 milliliters with distilled water, minute install to 50 milliliters of triangular flasks (50 milliliters/bottle), sealing, as stated above sterilization.

9) root media

50 milliliters in MSmax mother liquor (10X)

5 milliliters in MSmix mother liquor (100X)

Fe ²⁺5 milliliters of EDTA stock solutions (100X)

5 milliliters of VITAMIN stock solutions (100X)

Sucrose 20 grams

Phytagel 3 grams

Adding distil water to 900 milliliter is regulated pH value to 5.8 with 1N potassium hydroxide.

Boil and be settled to 1000 milliliters with distilled water, minute install to (25 milliliters/pipe) in the pipe of taking root, sealing, as stated above sterilization.

(4) agriculture bacillus mediated genetic transformation step

3.1 callus of induce

1) ripe Hejiang 19, No. 8, Mudanjiang, rice paddy seed shells, and then uses successively 70% Ethanol Treatment 1 minute, 0.15% mercury chloride (HgCl ₂) seed-coat sterilization 15 minutes;

2) wash seed 4-5 time with sterilization;

3) seed is placed on the inducing culture;

4) postvaccinal substratum is placed dark place cultivate 4 weeks, 25 ± 1 ℃ of temperature.

3.2 callus subculture

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

3.3 preculture

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

3.4 Agrobacterium is cultivated

1) (LA substratum compound method is with reference to J. Pehanorm Brooker etc. at the LA substratum of selecting with corresponding resistance, the molecular cloning experiment guide, the third edition, Jin Dongyan etc. (translating), Science Press, 2002, Beijing) upper preculture is carried the Agrobacterium EHA105 (agrobacterium strains that this bacterial strain openly uses from CAMBIA company) two days of SEQ ID NO:1 sequence fragment, 28 ℃ of temperature;

2) Agrobacterium is transferred in the suspension medium, cultivated 2-3 hour on 28 ℃ of shaking tables.

3.5 Agrobacterium is infected

1) pre-incubated callus is transferred in the bottle of the bacterium of having gone out;

2) regulate the suspension of Agrobacterium to OD ₆₀₀0.8-1.0;

3) callus was soaked in agrobacterium suspension 30 minutes;

4) shifting callus blots to the good filter paper of sterilization; Then be placed on the common substratum and cultivated temperature 19-20 ℃ 3 days.

3.6 callus washing and selection are cultivated

1) aqua sterilisa washing callus is to cannot see Agrobacterium;

2) be immersed in the aqua sterilisa that contains 400 mg/litre Pyocianils (CN) 30 minutes;

3) shifting callus blots to the good filter paper of sterilization;

4) shift callus to selecting to select on the substratum cultivation 2-3 time, each 2 weeks.

3.7 differentiation

1) kanamycin-resistant callus tissue is transferred on the pre-division culture medium in dark place cultivation 5-7 days;

2) callus that shifts pre-differentiation culture is cultivated 26 ℃ of temperature under the illumination to division culture medium.

3.8 take root

1) cuts the root that differentiation phase produces;

2) then transfer them in the root media and to cultivate 2-3 week, 26 ℃ of temperature under the illumination.

3.9 transplant

Wash the residual substratum on the root off, the seedling that will have good root system changes the land for growing field crops isolation environment over to, and field management is with common land for growing field crops.

After the process complementation was converted into the Ghd8-ZS97 Genotype, the transgenic positive individual plant occurred postponing the phenotype (Fig. 5) that plant height increases and grain number per spike increases flowering period.Four independent results that transform familys all confirm transgenosis individual plant phenotype and genotype be divided into from.Plant T1 generation, show that separation (26: 76) in 1: 3 appears in one of them family phenotype, with gene internal labeling Z9M-1 (sequence is seen Fig. 6) and gus dyeing all confirm phenotype and genotype be divided into from, after the species test result showed that Ghd8-9311 allelotrope changes Ghd8-ZS97 genotype (Recessive alleles) plant over to, phenotype had obtained answer (table 3).Thus, Ghd8 is successfully cloned, and the present invention confirms simultaneously, can utilize Ghd8 to serve Yield Breeding by the transgenosis means.

Table 3 Ghd8 near isogenic line and the complementary transgenosis individual plant species test table that transforms

Table 3 notes: NIL (zs97), NIL (Hr5) represent respectively Ghd8-ZS97 among the Ghd8NIL, Ghd8-HR5 allelotype plant, and NILzs97-, NILzs97+ represent respectively transgenosis feminine gender, positive plant

Embodiment 3: seek the Ghd8 allelotype of distinguishing the different effect that produces

With PCR product direct Sequencing method: with primer Ghd8-S1, Ghd8-S2 Ghd8-S3, Ghd8-S4, Ghd8-S5 Ghd8-S16Ghd8-S7, Ghd8-S8 is each section of totally 8 pairs of primers (primer sequence is referring to Fig. 6) amplification Ghd8 gene, the PCR product through SAP and EXOI after 37 ℃ of digestion 1 as a child (digestion reaction system 8 microlitres), 80 ℃ of sex change, get 2.5 microlitre digestion products with amplified reaction (the 96 ℃ of sex change in 2 minutes of the first step of checking order of ABI company sequencing kit, 96 ℃ of sex change in 10 seconds of second step, 50 ℃ of renaturation, 60 ℃ were extended 4 minutes, this step repeats 33 circulations, 10 minutes 4 ℃ of the 3rd steps), amplified production is with 95% ethanol: 3M sodium-acetate (pH4.8)=precipitate at 25: 1, the every hole reaction of 26 microlitres, precipitate after 15 minutes with 3000g revolution centrifugal collecting precipitation, add 75 microlitres, 70% washing with alcohol after getting supernatant, 3000g removes supernatant after centrifugal 10 minutes, dries, add 7 microlitre methane amides, carry out sequencing with the ABI3730 sequenator after the sex change in 3 minutes of 96 degree.Sequence is carried out the splicing of sequence through sequencher4.1.2.In conjunction with the phenotype data of all material and sequence with TASSEL software (Bradbury et al, .2007) MLM in carries out association analysis, the result shows and mainly contains 5 site (S1, S2, S3, S4, S5) and heading stage, Correlated Yield Characters is closely related, wherein S2 and S5 are 2005,2006 with experiment in 2,007 three in significant relevant to having heading stage, be positioned at Ghd8-MH63 (encoding amino acid sequence such as SEQ ID NO:7), Ghd8-9311 (encoding amino acid sequence such as SEQ ID NO:3), Ghd8-ruf (encoding amino acid sequence such as SEQ ID NO:9) coding the 86th amino acids replaces with the variation of L-Ala (Ala) by the Serine (Ser) of Ghd8-nip allelotrope (encoding amino acid sequence SEQ ID NO:5), and the variation of 174 glycine multiplicity is simultaneously to heading stage and output all existing significant correlation (table 4) in experiment in 3 years.

The not homoallelic Effect Evaluation of embodiment 4:Ghd8

By take rice varieties 93-11, bright extensive 63, Japanese warm and fine O.rufipogon as donor parents, ZS97 is recurrent parent, backcross 3-6 time, utilize simultaneously genetic marker Ghd8-S4 to determine the existence of goal gene Ghd8, pass through again 1-2 selfing, the final genetic background basically identical (being ZS97) that obtains, a series of near isogenic line (ZS97 that Ghd8 isozygotys and imports ⁹³¹¹, ZS97 ^Mh, ZS97 ^Nip, ZS97 ^Ruf).Carry out processing relatively length day and find: Ghd8-9311 allelotype (coding region sequence such as SEQ ID NO:2) and Ghd8-ruf allelotype (SEQ ID NO:8) are the powerful site, postpone heading stage and increase substantially simultaneously grain number per spike; And with Ghd8-nip allelotype (coding region sequence such as SEQ IDNO:4) for being represented as the functional medium site, can improve output and simultaneously under the long day condition, only postpone 1.5 days breeding times, under the short day condition 3.4 days in advance; And the class allelotype take Ghd8-MH63 allelotype (coding region sequence such as SEQ ID NO:6) as representative is as weak functional site, it is minimum on impact breeding time in volume increase, (table 5), developing during evolution in this presentation of results Ghd8-nip, the Ghd8-MH63 allelotype the favourable allelic variation that breeding improvement is had using value.

Table 4 nucleotide site difference and proterties correlation analysis

Table 5 Ghd8 is different, and the allelotype near isogene ties up to phenotype under the different sunshine conditions

(annotate: ZS97 ^Nip, ZS97 ^Mh, ZS97 ^Ruf, ZS97 ⁹³¹¹Be respectively the near isogenic line that imports Ghd8-nip, Ghd8-MH63, Ghd8-9311, Ghd8-ruf allelotrope structure under the ZS97 background." ^*" represent significant difference, " ^*" to represent difference extremely remarkable.)

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Claims (6)

1. a clone's Ghd8 gene increases rice grain output under the long day, postpones the application in heading stage and the increase plant height, it is characterized in that its nucleotide sequence is shown in sequence table SEQ ID NO:2.

2. a clone's Ghd8 gene postpones the application in rice ear sprouting period and the increase plant height under short day, it is characterized in that its nucleotide sequence is shown in sequence table SEQ ID NO:2.

3. the allelotrope Ghd8-ruf of the described gene of claim 1 increases rice grain output under the long day, postpones heading stage, increases the application in the plant height, it is characterized in that its nucleotide sequence is shown in sequence table SEQ ID NO:8.

4. the allelotrope Ghd8-ruf of the described gene of claim 1 increases rice grain output and the application that postpones in heading stage under short day, it is characterized in that its nucleotide sequence is shown in sequence table SEQ ID NO:8.

5. the allelotrope Ghd8-MH63 of the described gene of claim 1 increases rice grain output under the long day, does not postpone the interim application of Rice Heading, it is characterized in that its nucleotide sequence is shown in sequence table SEQ ID NO:6.

6. the allelotrope Ghd8-Nip of the described gene of claim 1 increases rice grain output under the long day, postpones the application in heading stage and the increase plant height, it is characterized in that its nucleotide sequence is shown in sequence table SEQ ID NO:4.

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