CN104630233A - Application of OsChls2 gene in regulation and control of chlorophyll content in paddy - Google Patents

Abstract

The invention belongs to the field of plant gene engineering, and relates to an application of OsChls2 gene in regulation and control of chlorophyll content in paddy. The systematical investigation is carried out on the chlorophyll content by paddy core germplasm sequencing; the sites with a significant effect are detected through genome-wide association study; a major gene OsChls2 for controlling natural variation of the chlorophyll is obtained; a nucleotide sequence of the major gene OsChls2 is as shown in SEQ ID NO:2; a sequence of protein is as shown in SEQ ID NO:6; the nucleotide sequence of an allelic gene of the major gene OsChls2 is as shown in SEQ ID NO:1; the sequence of the protein is as shown in SEQ ID NO:4. Non-synonymous mutations of three amino acids exist in CDS of hap2 in the major gene OsChls2 by sequencing; frameshift mutation and translation are terminated in advance caused by deficiency of 26 basic groups; a transformation experiment proves that the family chlorophyll content in over-expression is reduced; and the gene has important value in high-photosynthetic efficiency breeding of the paddy.

Description

The application of OsChls2 gene in adjusting and controlling rice chlorophyll content

Technical field

The present invention relates to field of plant genetic, be specifically related to one and be positioned at the clone and the application that paddy rice the 2nd karyomit(e) control the gene OsChls2 of chlorophyll content.

Background technology

Chlorenchyma is that plant carries out photosynthetic main place, and the raising of its photosynthetic efficiency is significant to crop yield.Crop yield potentiality in theory can calculate (Monteith, 1977) according to formula Y=0.487*St* ε i* ε c* ε p.Research finds, after modern rice varieties rational close planting to the intercepting efficiency ε i of photosynthetically active radiation up to 80-90%, harvest index ε p also reaches 50-60%, and the space that both promote further is all very limited.Because Globalradiation energy St is fixing in certain geographical position and the season of growth, what uniquely can be used in above formula significantly to improve output is optical energy utilization efficiency ε c (Horton, 2000; Zhu et al2008; 2010).The maximum optical energy utilization efficiency of C3 plant can reach 4.6% in theory, but the maximum efficiencies of light energy utilization of crop such as the paddy rice of Field observation are often only 1/3 of theoretical value, and the average efficiency of light energy utilization is only 0.5%.Chlorophyll has the effect of collecting and transmitting luminous energy, and light energy conversion can also be chemical energy by the special chlorophyll a molecule of reactive center.Research shows, the optimization by antenna chlorophyll content in crop breeding practice improves photosynthetic efficiency.

The hereditary basis more complicated of chlorophyll content, by multiple gene regulating, is typical quantitative character.Utilize molecular markers linkage map spectrum and segregating population can navigate to the QTLs controlling this proterties, meanwhile, also by association analysis, the gene controlling this proterties is positioned.Association analysis is a kind of based on linkage disequilibrium and prerequisite, is tested and appraised the analytical procedure of each proterties and molecule marker or candidate gene dependency in natural population, can identifies constant gene segment C (QTL) (the Gupta et al2005 controlling biological specific trait; Rafalski2010).Compared with traditional Q TL localization method, the advantage of association analysis is mainly reflected in: (1) association analysis is more direct and convenient in Fine Mapping, its mapping precision higher (Buckler et al 2002); (2) association analysis can detect multiple allelotrope simultaneously, and likely identifies optimum allelotrope; (3) due to the germ plasm resource colony that association analysis uses nature to exist, therefore do not need to utilize the time for many years to build mapping population (Meuwissen et al2000; Flint-Garcia et al2003; Whitt et al2003; Flint-Garcia et al2005).

Usual association analysis has two kinds of strategies, comprises whole-genome association (Genome-Wide Association Study, GWAS) and candidate gene association analysis (Candidate Gene Association Study) (Aranzana et al2005; Cockram et al2010; Huang et al2010).But no matter be which kind of strategy, substantially comprise following 4 steps: (1) selects to have the multifarious germplasm materials of high genetic; (2) group structure analysis avoids false positive to associate; (3) to countless specific objective trait carry out multiple spot, for many years, multiple multiple accurate investigation (Flint-Garcia et al2003; Wilson et al2004); (4) after the phenotypic data analyzing LD level and objective trait between the group structure of germplasm materials, mark, association analysis software (as TASSEL or ANOVA) can be used to carry out association analysis (Wu et al2001).

The present invention adopts new-generation sequencing technology to complete to 533 parts of Rice Core Germplasms sequence of resurveying, establish whole-genome association platform, and system thinking has been carried out to kind of a matter chlorophyll content, the site of tool pole active effects is detected further by whole-genome association (GWAS).The present invention adopts the strategy of association analysis to excavate and controls chlorophyll content natural variation major gene OsChls2, then in natural population, select portion of material carry out heavy sequence verification, find to there are 5 kinds of haplotypes in natural population, select the allelotrope of one of them haplotype as conversion fragment, overexpression is carried out to it, finds that this allelotrope significantly can reduce chlorophyll content of rice.

Summary of the invention

The object of the invention is to the defect overcoming prior art, from paddy rice, separating clone one controls the complete coding region segment DNA fragment of chlorophyll content gene, and overexpression research has been carried out to this gene, find that this gene extremely significantly can reduce chlorophyll content of rice, obviously this gene has important Breeding value in adjusting and controlling rice chlorophyll content.This unnamed gene is OsChls2 by applicant.

Object of the present invention also comprises the application of OsChls2 gene in adjusting and controlling rice chlorophyll content.

Do not achieve the above object, in the present invention, applicant is checked order to 533 parts of Core Germplasms by high throughput sequencing technologies, according to sequencing result, material is divided into different subgroups.Then by carrying out the degree of depth order-checking of OsChls2 full-length genome to ind and aus subgroup, and analyze chlorophyll content proterties, conversion test shows that decline has appearred in the family chlorophyll content of overexpression.

The OsChls2(of separating clone of the present invention is sometimes expressed as hap2 in this manual) nucleotide sequence of major gene is as shown in sequence table SEQ ID NO:2, and the nucleotide sequence of its allelotrope hap1 is as shown in SEQ ID NO:1.

According to sequencing result, this gene is divided into 5 haplotypes, the translation albumen of hap1 is consistent with the protein sequence of " Japan is fine ", and wherein there are 3 amino acid whose nonsynonymous mutations in hap2 in coding region, and have the disappearance of 26 bases, hap3 existence causes an amino acid whose disappearance by phase shift mutation, cause its translation albumen there is an amino acid whose disappearance, be wherein numbered hap1 and hap2 haplotype protein sequence as shown in sequence table SEQ ID NO:4 and SEQ ID NO:6.

In Some Species matter, expression analysis is done to the different haplotype of this gene, has found that hap2 and hap1 exists pole significant difference (see figure 3) on expression amount.

Overexpression has been carried out to obtained gene, has found that the fragment of hap2 gene proceeds to flower 11(hap1 in paddy rice) afterwards transgenic positive strain occur that chlorophyll content declines (see Fig. 7 A-Fig. 7 D).

The invention has the advantages that: the method that the present invention applies whole-genome association first in paddy rice have found major gene and its allelotrope that one controls chlorophyll content, and these genes have important Breeding value in the paddy rice breeding of high photosynthetic efficiency.The present invention simultaneously also provides technological borrowing for cloning genes involved in other crop.

More detailed technical scheme is shown in described in " embodiment ".

Accompanying drawing explanation

Sequence table SEQ ID NO:1 is the nucleotide sequence of the allelotrope (numbering hap1) of the OsChls2 major gene of separating clone, and sequence is 1455bp; Wherein: 1-64 position and 1112-1455 position are exon regions, intron region is 65-1111 position, its 135 amino acid of encoding.Wherein CDS district is 1-64 position and 1112-1455 position, and TGA is terminator.

Sequence table SEQ ID NO:2 is the nucleotide sequence of the OsChls2 major gene (numbering hap2) of separating clone, and sequence is 1378bp; To encode 109 amino acid.Wherein: CDS district is 1-64 position and 1113-1378 position, and TAA is terminator.In this major gene, 1-64 position and 1113-1378 position are exon region; Intron region is region, 65-1112 position.

Sequence table SEQ ID NO:3 is the CDS district of the allelotrope (hap1) of the OsChls2 major gene of splicing, and sequence is 408bp, 135 aminoacid sequences of encoding.

Sequence table SEQ ID NO:4 is the sequence of the protein of the allelotrope (hap1) of OsChls2 major gene.

Sequence table SEQ ID NO:5 is the CDS district of the OsChls2 major gene (numbering hap2) of splicing, and sequence is 330bp, 109 aminoacid sequences of encoding.

Sequence table SEQ ID NO:6 is the sequence of the protein of OsChls2 major gene.

Fig. 1. for the phenotype analytical of material in aus of degree of depth order-checking.Find that the revision test result of 2 years shows: two kinds single times shape material in aus exists significant difference in SPAD, chlb, chla/chlb phenotype, and chla and total chl also detected significant difference (seeing Figure 1A-1D respectively) in 1 year.

Fig. 2. for the phenotype analytical of material in ind of degree of depth order-checking.Find that the revision test result of 2 years shows, in ind there is significant difference (seeing Fig. 2 A-2E respectively) in hap2 and hap5 and hap1 haplotype material in SPAD, chla, chlb, chla/chlb and total chl phenotype.

Fig. 3 .OsChls2 gene expression level schematic diagram in paddy rice hap2 and hap1.Get four kinds of haplotype materials of ind, do expression amount and detect, find that the expression amount of hap2 and hap5 is extremely remarkable in hap1.

Fig. 4. original plasmid pCAMBIA1301.

Fig. 5. the overexpression plasmid pCAMBIA1301s that the present invention builds, compared with original plasmid pCAMBIA1301, adds 35S strong promoter on multiple clone site side.

Fig. 6. transgenic lines positive detection figure, wherein totally 16 transfer-gen plants, detect and obtain the positive individual plant (see swimming lane 1-8,10-12,14-15) of 13 strains, the negative individual plant (see swimming lane 9,13,16) of 3 strains.

Fig. 7. transgenic lines phenotype statistical study figure.Wherein transgenic positive plant compares the decline (see Fig. 7 A-7D) having occurred chlorophyll content with negative plant.

Fig. 8. the comparison chart of two kinds of protein types.H1 is that the exon of the nucleotide sequence of hap1 translates into amino acid whose sequence, H2 is that the exon of the nucleotide sequence of hap2 translates into amino acid whose sequence, great difference is there is in H1 and H2 in amino acid quantity and on putting in order, black represents that, by the identical aminoacid sequence of comparison, grey represents by the different aminoacid sequence of comparison.

Embodiment

Following examples define the present invention further, those skilled in the art can determine essential characteristic of the present invention, and when not departing from the present invention's essence and scope, various change and amendment can be made to the present invention, to make its applicable various uses and condition.

First, collect Core Germplasms, the method extracting DNA in its full-page proof DNA(extraction rice tissue is common method), adopt new-generation sequencing technology to check order to it.In conjunction with field material, it is carried out to the system examination of chlorophyll content, sequencing data and phenotypic data are found the gene OsChls2 being positioned at a control chlorophyll content on No. 2 karyomit(e)s by the method for whole-genome association.Then in differing materials, there is natural variation in conjunction with this gene, degree of depth order-checking has been carried out to portion of material, according to sequencing result, Haplotyping A has been carried out to this gene, find that hap2 exists the disappearance of aminoacid sequence, and translation premature termination.Real-Time PCR expression analysis finds, lower expression is there is in the hap2 of this gene in Core Germplasms, there is significant difference in the expression amount of hap2 and hap1, the chlorophyllous data that coupling system is investigated simultaneously find that the phenotype between two haplotypes also exists pole significant difference.The fragment that this gene discovery of overexpression contains hap2 gene proceeds to middle colored 11(hap1) after, transgenic positive strain occurs that chlorophyll content declines.

Experiment material comes from Core Germplasms from all parts of the world, and its field leaf look exists abundant phenotypic variation.Extract its DNA, adopt new-generation sequencing technology to check order to it, form the sequencing data of low cover degree.

The clone of embodiment 1:OsChls2 gene

(1) system thinking of Core Germplasms chlorophyll content

After Rice Core Germplasm heading about 3 days, get the foam box that healthy blade puts into dress ice immediately, wrap with black cloth, then take back in laboratory immediately.Accurately weigh with analytical balance, shred, add mixing extract, 4 DEG C of lucifuge extractings, period shakes up sample at set intervals, on spectrophotometer, its absorbance is measured after sample decolours completely, thereafter (method is with reference to Jiang G to calculate chlorophyll content according to Arnon method correction formula, He Y, Xu C, Li X, Zhang Q.The genetic basis of stay-green in rice analyzed in a population of doubled haploid lines derived from an indica by japonica cross.Theor Appl Genet, 2004, 108:688-698).

(2) association analysis of chlorophyll content candidate gene is controlled

The blade of sampling 533 parts of Core Germplasms, extract its DNA, (method is with reference to Huang X to obtain SNP marker after adopting s-generation sequencing technologies to be checked order, Zhao Y, Wei X, Li J, Wang A, Zhao Q, Li W, Guo Y, Deng L, Zhu C, Fan D, Lu Y, Weng Q, Liu K, Zhou T, Jing Y, Si L, Dong G, Huang T, Lu T, Feng Q, Qian Q, Li J, Han B.Genome-wide association study of flowering time and grain yield traits in a worldwide collection of rice germplasm.Nat Genet, 2012, 44:32-39), investigate Core Germplasms chlorophyll content simultaneously, whole-genome association detects the significant site of P value.By LMM model at No. 2 chromosomal physical location 10831243 places, P value is found to be the remarkable site of 3.7e-06, at this remarkable site upstream 58kb place, find that there is an annotate genes relevant with chlorophyll metabolism approach, determine that this gene is target candidate gene OsChls2.

(3) function prediction of OsChls2 major gene

According to InterProScan (http://www.ebi.ac.uk/InterProScan/), protein structure is predicted, the protein that OsChls2 gene (Hap1) is encoded is made up of (see sequence table SEQ ID NO:4) 135 amino acid, and it comprises a conservative PF12740 structural domain.Utilize BLASTp to search for the protein structure that OsChls2 gene coding amino acid forms, find this genes encoding one serine dehydrogenase.Research in the past shows that this chlorphyllase is the key enzyme in chlorophyll degradation process, and methyl jasmonate can induced lipase die body, and this enzyme can catalyzing glycerol three ester lipase as serine dehydrogenase.

Embodiment 2: compare the natural variation of checking order and determining between OsChls2 allelotrope

(1) sequencing

Part Core Germplasms is increased this gene, Hi-Fi LA-Taq and rTag (purchased from Japanese TakaRa company) is adopted to carry out pcr amplification from the genome of these materials, according to common PCR reaction program, 30 circulations are set, annealing temperature 58 DEG C, the extension time, 1min30s was according to common PCR reaction program, 30 circulations are set, annealing temperature 58 DEG C, extension time 1min30s, then PCR order-checking (PCR order-checking is undertaken by Wuhan Hua Da genome company) is carried out, for sequencing primer in table 1.Sequencher4.5 software (U.S. Gene Codes Corporation) is used to splice sequence.The nucleotide sequence of this gene (hap2) is as shown in SEQ ID NO:2, and its coding region sequence is shown in SEQ ID NO:5, and the sequence of its protein is shown in SEQ ID NO:6.

(2) sequencing analysis of this gene in natural population

Sequence comparing analysis discovery existence 40 SNP and InDel differences in Core Germplasms two subgroups (aus and ind) that target area is intersegmental, wherein 29 sudden changes are positioned at and include subarea, and 11 sudden changes are positioned at the exon of coding region.There are 7 same sense mutations coding region, the change of the aminoacid sequence that the change of 1 nonsynonymous mutation and 3 indel causes, and therefore the colony obtained that checks order can be divided into 5 haplotypes, the protein types that wherein hap1 is fine with Japan is consistent.

Table 1 compares the primer of order-checking for the present invention

The phenotype of embodiment 3:OsChls2 gene between different haplotype and expression analysis

The SPAD of aus and ind subgroup, chlorophyll a (chla), chlorophyll b (chlb), Chlorophyll content (total chl), chlorophyll a/b isophenous are analyzed, find that two haplotypes in aus subgroup all exist significant difference in five kinds of phenotypes that 2012 detect, within 2013, between SPAD, chlb, chlorophyll a/b isophenous, there is significant difference (see figure 1).In ind subgroup, different haplotype material is all had to there is significant difference in above-mentioned five proterties, especially all there is pole significant difference (see figure 2) in hap1 and hap2 in each phenotype, extract each haplotype material RNA simultaneously and do expression amount detection (extraction of RNA is see Trans Zol test kit), analyze and show between hap1 and hap2, to there is pole significant difference (see figure 3).

The reproducible results display of 2 years, this gene pairs controls chlorophyll content and has pole active effects.

The application of embodiment 4:OsChls2 gene in genetic transformation (transgenosis)

1, OsChls2 transgenic technology route:

Select a hap2 material Taishan glutinous (China Agricultural University professor Li Zichao present), design a pair PCR specific primers amplify genomic dna with restriction endonuclease BamHI and SalI joint, conveniently PCR response procedures, 30 circulations are set, annealing temperature 58 DEG C, extension time 1min30s, being connected to TA clones in carrier T (purchased from Promega company), pick out the correct clone without sudden change containing candidate gene, restriction endonuclease BamHI and SalI enzyme is utilized to cut positive colony, carry out subclone, be connected on double base overexpression vector pCAMBIA1301S, multiple clone site is other adds a 35S strong promoter (see Fig. 4, Fig. 5), adopt general transgenic method, the plasmid of the correct clone obtained is imported in rice varieties " in spend 11 " by agriculture bacillus mediated rice transformation system, through induction, subculture, infect, Dual culture, screening has the callus of hygromycin resistance, differentiation, take root, acclimatization and transplants, obtain genetically modified rice plant.Agriculture bacillus mediated paddy rice (japonica rice subspecies) genetic conversion system mainly applies the method for the people such as Hiei report (see Efficient transformation of rice, Oryza sativa L., mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, 1994, Plant Journal6:271-282) basis is optimized.

The present invention obtains separate transgenic (Over-expression) T altogether ₀for rice plant 16 strain, comprise the positive individual plant of 13 strains and the negative individual plant of 3 strains.Show after tested, the chlorophyll content (see figure 6) significant difference of the transgenic paddy rice positive and negative plant.Transgenosis T ₀all show the phenotype of chlorophyll content decline for positive overexpression individual plant, demonstrating this candidate gene OsChls2 is exactly the gene controlling chlorophyll content.

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 plant hormone in the present invention used by substratum is expressed as follows: 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(Casein Enzymatic Hydrolysate, caseinhydrolysate); HN(Hygromycin B, Totomycin); DMSO(Dimethyl Sulfoxide, dimethyl sulfoxide (DMSO)); N6max(N6 macroelement ingredient solution); N6mix(N6 Trace Elements solution); MSmax(MS macroelement ingredient solution); MSmix(MS Trace Elements solution)

(2) main solution formula

1) N _6maxsubstratum macroelement mother liquor (according to 10 times of concentrated solution (10X) preparations):

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

2) N _6minsubstratum trace element mother liquor (according to 100 times of concentrated solution (100X) preparations):

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

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

By 3.73 grams of b diammonium disodium edta (Na ₂eDTA2H ₂o) and 2.78 grams of FeSO ₄7H ₂o dissolves respectively, and mixing is also settled to 1000 milliliters with distilled water, and to 70 DEG C of temperature baths 2 hours, 4 DEG C saved backup.

4) vitamins stock liquid (according to the preparation of 100X concentrated solution):

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

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

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

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

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

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

Weigh 2,4-D100 milligram, dissolve 5 minutes with 1 milliliter of 1N potassium hydroxide, be settled to 100 milliliters after then adding 10 ml distilled waters dissolvings completely, preserve under room temperature.

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

Weigh 6-BA100 milligram, dissolve 5 minutes with 1 milliliter of 1N potassium hydroxide, after then adding 10 ml distilled waters dissolvings completely, be settled to 100 milliliters, room temperature preservation.

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

Weigh NAA100 milligram, dissolve 5 minutes with 1 milliliter of 1N potassium hydroxide, be settled to 100 milliliters after then adding 10 ml distilled waters dissolvings completely, 4 DEG C keep in Dark Place.

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

Weigh IAA100 milligram, dissolve 5 minutes with 1 milliliter of 1N potassium hydroxide, be settled to 100 milliliters after then adding 10 ml distilled waters dissolvings completely, 4 DEG C keep in Dark Place.

11) preparation of glucose storage liquid (0.5 grams per milliliter):

Weigh glucose 125 grams, then dissolve with distilled water and be settled to 250 milliliters, after sterilizing, 4 DEG C save backup.

12) preparation of AS stock solution:

Weigh AS0.392 gram, add DMSO10 milliliter and dissolve, divide and be filled in 1.5 milliliters of centrifuge tubes ,-20 DEG C save backup.

13) 1N potassium hydroxide stock solution preparation:

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

(3) for the culture medium prescription of rice transformation:

1) inducing culture

Adding distil water to 900 milliliter, 1N potassium hydroxide adjust ph is to 5.9, boil and be settled to 1000 milliliters, be dispensed into 50 milliliters of triangular flasks (30 milliliters/bottle), sterilizing according to a conventional method after sealing (sterilizing 15 minutes at such as 121 DEG C, following medium sterilization method is identical with the sterilising method of basal culture medium).

2) subculture medium:

Adding distil water to 900 milliliter, 1N potassium hydroxide adjust ph, to 5.9, is boiled and is settled to 1000 milliliters, is dispensed into 50 milliliters of triangular flasks (30 milliliters/bottle), sealing, sterilizing as stated above.

3) pre-culture medium (japonica rice can not be cooked this step):

Adding distil water to 250 milliliter, 1N potassium hydroxide adjust ph, to 5.6, is sealed, sterilizing as stated above.

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

4) suspension medium:

Adding distil water to 100 milliliter, adjust ph, to 5.4, is dispensed in the triangular flask of two 100 milliliters, sealing, sterilizing as stated above.

1 milliliter of sterile dextrose stock solution and 100 microlitre AS stock solutions are added before using.

Adding distil water to 250 milliliter, 1N potassium hydroxide adjust ph, to 5.6, is sealed, sterilizing as stated above.

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

6) screening culture medium:

Adding distil water to 250 milliliter, adjust ph, to 6.0, is sealed, sterilizing as stated above.

Dissolve substratum before using, add 250 microlitre HN(50 mg/ml) and 400 microlitre CN(10 gram CN/36 ml water) packing pours (25 milliliters/ware) in culture dish into.(note: Selective agar medium Pyocianil concentration is 400 mg/litre for the first time, second time and later Selective agar medium Pyocianil concentration are 250 mg/litre).

7) pre-division culture medium (japonica rice can not be cooked this step):

Adding distil water to 250 milliliter, 1N potassium hydroxide adjust ph, to 5.9, is sealed, sterilizing as stated above.

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

8) division culture medium:

Adding distil water to 900 milliliter, 1N potassium hydroxide adjust ph is to 6.0.

Boil and be settled to 1000 milliliters with distilled water, being dispensed into 100 milliliters of triangular flasks (50 milliliters/bottle), sealing, sterilizing as stated above.

9) root media

Adding distil water to 900 milliliter, by 1N potassium hydroxide adjust ph to 5.8.

Boil and be settled to 1000 milliliters with distilled water, being dispensed into and taking root (25 milliliters/pipe) in pipe, sealing, sterilizing as stated above.

(4) Agrobacterium-mediated genetic transformation step:

3.1 callus of induce

1) will in maturation, spend 11 rice paddy seeds to shell, then use the Ethanol Treatment 1 minute of 70% successively, 0.15% mercury chloride (HgCl ₂) seed-coat sterilizes 15 minutes;

2) seed is washed 4-5 time with sterilizing;

3) 8-10 grain seed is placed on inducing culture;

4) postvaccinal substratum is placed in dark place and cultivates 4-5 week, temperature 26 ± 1 DEG C.

3.2 callus subcultures:

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

3.3 precultures:

Select consolidation and the embryo callus subculture of relatively dry, be put in the lower cultivation of dark on pre-culture medium 2 weeks, temperature 26 ± 1 DEG C.

3.4 Agrobacteriums are cultivated:

1) at the LA substratum selected with corresponding resistance, (preparation of LA substratum is with reference to J. Pehanorm Brooker etc., Molecular Cloning: A Laboratory guide, the third edition, Jin Dongyan etc. (translating), Science Press, 2002, Beijing) agrobacterium strains that openly uses from CAMBIA company of upper this bacterial strain of line preculture Agrobacterium EHA105() two days, culture temperature 28 DEG C;

2) Agrobacterium is transferred in suspension medium, 2-3 hour cultivated by 28 DEG C of shaking tables.

3.5 Agrobacteriums are infected:

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

2) regulate the suspension of Agrobacterium to OD ₆₀₀=0.8-1.0;

3) callus is soaked 30 minutes in agrobacterium suspension;

4) shift callus to blot to the good filter paper of sterilizing; Then be placed on Dual culture base and cultivate 3 days, temperature 19-20 DEG C.

3.6 callus washings and selection are cultivated:

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

2) be immersed in the aqua sterilisa containing 400 milligrams/L Pyocianil (CN) and shake 30 minutes;

3) shift callus to blot to the good filter paper of sterilizing;

4) shift in callus to Selective agar medium and select to cultivate 2-3 time, each 2 thoughtful kanamycin-resistant callus tissues grown.

3.7 differentiation:

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

2) shift the callus of pre-differentiation culture on division culture medium, every bottle is evenly distributed three independently callus, cultivates and thoughtfully grow large young plant, culture temperature 26 DEG C in 5 weeks-6 under illumination.

3.8 take root and hardening:

1) the old root that differentiation phase produces is cut;

Then transfer them in root media and to remove sealed membrane after cultivating under illumination that 2-3 is thoughtful and growing large young plant and add part of running water hardening and transplant again for one week, temperature 26 DEG C.

3.9 transplant

Wash the remaining medium on root off, the seedling with good root system is proceeded to greenhouse, kept moisture moistening at initial several days simultaneously, transplant again to land for growing field crops etc. after growing way stalwartness.

Claims (4)

1. The application of 1.OsChls2 major gene in adjusting and controlling rice chlorophyll content, is characterized in that the nucleotide sequence of this gene is as shown in SEQ ID NO:2.
2. The application of 2.OsChls2 major gene in adjusting and controlling rice chlorophyll content, is characterized in that the sequence of the protein of this gene is as shown in SEQ ID NO:6.
3. The application of allelotrope hap1 in adjusting and controlling rice chlorophyll content of 3.OsChls2 major gene, is characterized in that the nucleotide sequence of this gene is as shown in SEQ ID NO:1.
4. The application of allelotrope hap1 in adjusting and controlling rice chlorophyll content of 4.OsChls2 major gene, is characterized in that the sequence of the protein of this gene is as shown in SEQ ID NO:4.