CN1594564A - Method for discovering wild rice beneficial gene using convertible large fragment genome library - Google Patents
Method for discovering wild rice beneficial gene using convertible large fragment genome library Download PDFInfo
- Publication number
- CN1594564A CN1594564A CN 200410023348 CN200410023348A CN1594564A CN 1594564 A CN1594564 A CN 1594564A CN 200410023348 CN200410023348 CN 200410023348 CN 200410023348 A CN200410023348 A CN 200410023348A CN 1594564 A CN1594564 A CN 1594564A
- Authority
- CN
- China
- Prior art keywords
- rice
- wild
- transform
- library
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for exploring wild rice advantageous gene using transformable large fragment genome library, the steps including: constructing wild rice transformable large fragment genome library, inducing large fragment clone into cultivated rice through transgene technology, establishing full genome gene knock in mutant library, after field screening about character and genetic model controlled by large fragment clone in mutant, obtaining gene transferred mutant rice dramatically improved in yield ,resistance and quality etc. Once large fragment clone containing advantageous gene is authenticated, it can be utilized in other cultivated rice variety, which modifies the existing rice varieties and can be the same with exploring other plant gene resource.
Description
Technical field
The invention belongs to rice varieties improvement and breeding technical field.Relate to extensive excavation and utilize the Protocols in Molecular Biology and the gene engineering technology field of wild-rice beneficial gene resource.
Background technology
Contain abundant beneficial gene in the wild-rice.The successful excavation of wild-rice genetic resources, utilize the first time once brought the world and the Green Revolution for the second time.The transformation dwarf gene has brought the Green Revolution for the first time to cultivated rice from wild-rice; Hybrid rice is described as the fifth-largest invention of China, and before three more than ten years, Yuan Longping academician finds from wild-rice that at first the open country loses sterile gene and just successfully cultivated hybrid rice, has started the Green Revolution for the second time.Wild-rice is owing to be in wild situation for a long time, stand the selection of various disasters and poor environment, have the resistance of various disease and pests and the adaptability of various adverse circumstances, as disease-resistant, pest-resistant, cold-resistant, drought-enduring, salt tolerant alkali, anti-ly flood, proterties such as anti-shade and high yield, high-quality, male sterile, efficient utilizations of phosphorus potassium all are the important gene resources that rice genetic is improved.
The wild-rice beneficial gene can be cultivated rice and utilizes.Oryza (Oryza) has 20～25 differentiation well to plant, the various karyotypes of Oryza are divided into AA, BB, CC, BBCC, CCDD, EE, FF, GG and HHJJ, having only two kinds is that common cultivated rice (Oryza sativa) and naked body rice (O.glaberrina) belong to cultivated rice, other title wild-rice.Can directly utilize for AA karyotype wild-rice, promptly in conjunction with selecting, backcross to get final product success with the conventional hybridization method.As far back as the '20s in last century, China paddy rice expert Ding Ying utilizes cultivated rice and common wild-rice hybridization, has selected " No. 1, middle mountain " and series derivatives kind " it is short to embrace tire ", " bag selects No. 2 " etc.The cultivation that sterile line, long stigma sterile line, red lotus sterile line are lost in the open country successfully makes the hybrid rice big area promote and produce enormous benefits; Some resistant genes, for example anti-thick grass stunt disease, resisting bacterial leaf-blight etc. also realized transformation with crossing the conventional hybridization method, but these only limit to AA genome wild-rice is hybridized transformation.Non-AA group type wild-rice and cultivated rice hybridization is difficulty comparatively, main at present by technology such as embryo rescue, group training, protoplastis fusions, change some beneficial genes of non-AA karyotype wild-rice over to cultivated rice, comprise disease resistance (bacterial leaf-blight, rice blast etc.), insect-resistance (brown paddy plant hopper, yellow rice borer, white backed planthopper etc.) and the beneficial gene of aspect such as resistance, cultivated a series of introgression line.
Simultaneously, the existing technology of utilizing of wild-rice beneficial gene also exists many limitation.Separate with the long-term madness of hybridization transformation offspring of cultivated rice as wild-rice, stable slow, breeding cycle is long; Methods such as embryo rescue, tissue culture, protoplastis fusion are primarily aimed at non-AA karyotype wild-rice, and there is difficulty in these methods to some genotypic wild-rice, can not avoid offspring's madness to separate simultaneously; Easy, importing in a plurality of cultivated rice kinds, reproduction fast that the wild-rice beneficial gene is difficult to; The gene that proterties is shifted in control is difficult to follow the tracks of, and easily causes the unstable of offspring's objective trait, perhaps causes the importing of the portion gene of controlled target proterties.
Therefore, excavate the new technical tactic of wild-rice gene treasure-house expectation, press for and seek the wild-rice beneficial gene that a kind of easy, effective high-throughput techniques and method are excavated important economic worth and had broad application prospects.
Summary of the invention
First purpose of the present invention is to provide a kind of utilization can transform the method that wild-rice beneficial gene resource fast, is effectively excavated in big fragment gene group library, to overcome the defective that exists in the prior art, and the genetically modified mutant plant that provides proterties to obtain improveing.
For realizing above-mentioned purpose, the inventive method comprises the steps:
A. the donor wild-rice can transform the foundation of big segment genomic library;
B. can transform big segment genomic library by transgenic technology and import the acceptor cultivated rice, obtain full genome mutated body storehouse;
C. mutant library is screened, obtain the mutant plant of economical character improvement and the wild-rice beneficial gene of control good character.
Among the above-mentioned steps a, the foundation that the donor wild-rice can transform big segment genomic library is the big fragment paddy DNA for preparing the Megebase level by the agar entrapping method, utilizes to transform artificial chromosome carrier (TAC) or the big fragment gene group of double base bacterial artificial chromosome carrier (BAC) structure library again.Transgenic technology among the step b is any in agriculture bacillus mediated, particle gun, pollen tube channel, the injection of fringe neck, directly the host bacterium when perhaps building the storehouse with the Agrobacterium, full genomic clone and/or candidate clone are imported the cultivated rice kind, rice material to be transformed is good conventional rice and a hybrid rice parents of purity, makes up full genomic gene and knocks in mutant library.Among the step c, the mutant library that is produced is screened, its process is: use antibiotic-screening during the transgenic paddy rice seed seed soaking, the PCR repetition measurement, field management method is planted positive transfer-gen plant routinely, positive transfer-gen plant is moved to the land for growing field crops investigate economical character, the variation individual plant that screening has breeding to be worth.Use Southern blot equimolecular biology techniques and detect, identify mutant, determine to insert segmental size and copy number, and the large fragment DNA of correspondence is cloned.Screen (the preferably single copy) mutant plant that isozygotys, proceed variety test; Simultaneously hybridisation rice then being carried out parent's survey joins.Obtain the significantly genetically modified mutant paddy rice novel material of improvement at last, and find the beneficial gene of the wild-rice that may in cultivated rice, lose.
Advantage of the present invention:
1. utilization of the present invention can transform the large dna fragment cloning carrier technique and carry out a kind of new gene and knock in, genetic transformation by kind TAC library, the nearly source of allos, create mutant, select the clonal mutation character gene of simple inheritance then, the characteristics that this gene clone technology has is simple to operate, the cycle is short.And in clone gene, also can obtain the transgenic plant of genetic modification.
2. the mutant library of the present invention's structure can provide the proterties of large dna fragment cloning control in the genomic library and the new rice variety of hereditary pattern and improvement.
3. the character improvement gene of the present invention's screening and acquisition can be used for the improvement of other rice varieties, makes existing rice varieties obtain whole upgrading
4. the present invention takes the lead in utilizing on a large scale the wild-rice genetic resources to excavate the significantly beneficial gene of improvement of proterties such as rice yield, resistance, quality, will widen the hereditary basis of cultivated rice.
Description of drawings
Fig. 1 is the BIBAC2 cleavage map.Foliage filter screening is labeled as: Totomycin, bacteria screening is labeled as: kantlex, the used mono-clonal of library construction site is: BamH I.
Fig. 2 is that the GUS of rotaring gene plant blade detects, and goes up positive transgenic rice plant, is contrast down.The positive control plant leaf has been dyed blueness, and the adjoining tree blade is not colored.
Fig. 3 is the pcr analysis of large dna fragment cloning transgenic rice plant.1: the large dna fragment cloning plasmid; 2: paddy DNA (contrast); 3-10: large dna fragment cloning transgenic rice plant; 11:1Kb Ladder.Plasmid and transgenic paddy rice have all amplified the dna segment of about 620bp, and contrast does not have.
The economical character variation of Fig. 4 large dna fragment cloning transgenic rice plant.The right side is a transfer-gen plant, and a left side is an adjoining tree.The whole proterties of variant is good, ripe slightly late, and plant height is the high approximately 5cm of contrast, and fringe portion proterties has clear improvement.
Embodiment
Embodiment 1:
Utilize oryza officinalis can transform big fragment gene group library transformation cultivated rice 9311, R207, golden 23B
(1) with the oryza officinalis is the donor plant, at first prepares the big fragment paddy DNA of Megebase level by the agar entrapping method.Promptly utilize and to transform artificial chromosome carrier (TAC, Transformedartificial chromosome) or double base bacterial artificial chromosome carrier (BIBAC, binarybacterial artificial chromosome), by cloning about 100Kb, make up the big fragment gene group of oryza officinalis library.Building process is as follows:
A. the preparation of macromolecule nuclear DNA. water intaking rice etiolated seedling 20g, liquid nitrogen grinding, add 200mL 1 * HB solution (0.01mol/L Tris-HCl, 0.08mol/L KCl, 0.01mol/L EDTA, the smart ammonia of 1mmol/L, the 1mmol/L spermidine, 0.5mol/L sucrose, 0.15% beta-mercaptoethanol, pH=9.4～9.5), stir, 2 layers of gauze, 1 layer of Mirocloth filters, the centrifugal 20min of 1800 * g, precipitation suspends with 1 * HB solution, with isopyknic 1% low melting-point agarose in 45 ℃ of mixings, adding can be made in the mould of blob of viscose, and 4 ℃ solidify 1h, takes out blob of viscose, place lysate (0.5mol/L EDTA pH=9.0～9.3 of 5 times of volumes, 1% sodium lauryl sarcosine, 1mg/mL Proteinase K) 50 ℃ of cracking 24～48h in wash blob of viscose 1 time with 0.5mol/L EDTA (pH=9.0～9.3) solution then, place 4 ℃ 0.05mol/L EDTA (pH=8.0) solution 1h again, carry out pulse electrophoresis at last, the low melting-point agarose with 1% reclaims the above dna fragmentation of 2Mb, is stored in 0.5mol/L EDTA (pH=8.0) solution.
The b.BIBAC preparing carriers.With a large amount of plasmid DNA of extracting of csCl-ethidium bromide gradient equilibrium centrifugation method purifying, the closed-circular DNA of acquisition digests with BAM I, and (Epicenter USA) carries out dephosphorization by process specifications to digest completely BIBAC DNA usefulness HK Phosphoric acid esterase.
The structure in c.BAC library.Place 40 times of volumes to contain the TE of 1mmol/L PMSF the macromolecule dna gel piece of purifying, 50 ℃ of incubation 1h repeat 1 time; Rinsing 2 times in the TE of 40 times of volumes then places 4 ℃ of incubation 30min of Digestive system (1 * EcoR I damping fluid+4mmol/L spermidine) of 10 times of volumes again, and every then clotting glue adds 200 μ L Digestive systems, 1UBAM I, incubation 2h on ice; At last, 37 ℃ of digestion 10min, the digestion back is washed 2 times with termination reaction with ice-cold TE. by pulse electrophoresis (CHEF, BIO-RAD: agarose 1%, damping fluid 0.5 * TBE, burst length 60～90s, pulsed voltage 4.5V/cm, 120 ° of pulse angles, electrophoresis time 18h, 11 ℃ of temperature) the inspection enzyme is cut effect, downcut the low melting-point agarose gel piece at 200～600kb place, by controlling suitable pulse electrophoresis condition (CHEF, BIO-RAD: agarose 1%, damping fluid 0.5 * TBE, burst length 5s, pulsed voltage 4.5V/cm, 120 ° of pulse angles, electrophoresis time 15h, 11 ℃ of temperature) make dna fragmentation be compressed into 1 band greater than 200kb, downcut herein low melting-point agarose gel piece place 4 ℃ of TE solution balance 2h. gel pieces through gelase (Epicentre, USA) digestion mixes with the dephosphorization carrier by a certain percentage for behind the liquid, with T4DNA ligase enzyme (Boehringer, Germany) connect, and usefulness Cell-Porator (Gibco, BRL) the sharp conversion instrument of electricity transforms, picking transformant direct inoculation is to containing 80 μ L nutrient solution (LB, 6mmol/L K
2HPO
4, 13.2mmol/L KH
2PO
4, 1.7mmol/L citrate sodium, 12.5 μ g/mL paraxin, 0.4mmol/L MgSO
4, 6.8mmol/L (NH
4)
2SO
4, on 384 hole culture plates 4.4%glyceryl) (Nunc, 384 Well Plate), after 37 ℃ of shaken over night, be stored in-70 ℃ of refrigerators.
(2) with three class cultivated rice kinds: 9311, R207, gold 23B is a recipient plant, adopting agriculture bacillus mediated transgenic technology that oryza officinalis can be transformed big fragment gene group library is that full genomic clone imports three class cultivated rices (9311 respectively, R207, gold 23B), (β-glucuronidase) the transgenic positive plant (Fig. 2) that obtains each large dna fragment cloning detects in histological chemistry by GUS then, the GUS detection method is carried out as follows: blade to be determined is immersed an amount of X-Gluc solution, after 37 ℃ of incubated overnight, under stereoscopic microscope, observe Taking Pictures recording.Vacuumize if will be added with the material of X-Gluc solution, Color will be better.Have the pigment interference problem as material, dyeing back is with the alcohol decolouring, the color of pigment faded away and the blueness of dying is preserved.X-Gluc staining fluid: 0.2mol/L NaPO
4Damping fluid, pH7.0 (0.2mol/L Na
2HPO
4, 62ml; 0.2mol/L NaH
2PO
4, 38ml); 0.1mol/L K
3[Fe (CN)
6]; 0.1Mol/LK
4[Fe (CN)
6] .3H
2O; 1.0Mol/L Na
2EPTA; 0.1% X-Gluc.
Use antibiotic-screening, PCR repetition measurement to carry out the evaluation (Fig. 3) of positive transfer-gen plant when (3) subsequently transgenic paddy rice seed being soaked seed.
The antibiotic-screening method is: seed was sterilized 2-3 minute with 75% alcohol, and filter paper blots on the filter paper that is placed on culture dish, and the Totomycin that adds 50mg/L screens, normally the positive transfer-gen plant of germination person.
PCR repetition measurement method is: the primer sequence of amplification hygromycin gene is: Forword:5 '-GCTGTT ATG CGG CCA TTA TC; Revise:5 '-GAC GTC TGT CGA GAA GTTTC, the PCR product is 620bp.PCR reaction system: DNA 30-90ng, 10 * Buffer 2.0ul, 1mM dNTP 1.8ul, 25mM MgCl
21.5ul, two kinds of each 0.5ul of 10uM primer, Tag enzyme 1.5U adds dd h then
2O to 20ul reaction volume.The PCR cycling condition: 94 ℃, sex change 3 minutes; 94 ℃, 1 minute, 55 ℃ (were detected P
SAG12) or 68 ℃ (detecting NPTII), 1.5 minutes, 72 ℃, 1.5 minutes, 40 circulations; 72 ℃, extended 5 minutes.Electrophoresis detection: use 1.4% agarose gel electrophoresis, the photographic recording electrophoresis result.
(4) field management method is planted positive transfer-gen plant routinely, investigates economical character, the variation individual plant that screening has breeding to be worth.Continue to screen (the preferably single copy) mutant plant that isozygotys, carry out variety test; Simultaneously hybridisation rice then being carried out parent's survey joins.In the field proterties is investigated, find that the part transfer-gen plant has character variation, wherein the whole proterties of 1 strain variant is good, ripe slightly late, and plant height is the high approximately 5cm of contrast, fringe portion proterties have clear improvement (Fig. 4, table 1).This shows and can the wild-rice beneficial gene be changed in the cultivated rice by this approach that the initiative breeding material is cultivated new variety, and will help to clone the gene of control mutant character.
Table 1: the economical character of large dna fragment cloning transgenic paddy rice is investigated
The real grain of material plant height (cm) spike length (cm) number of productive ear grain number per spike number setting percentage (%) thousand seed weight (g) single-strain grain weight (g)
Variant 74.9 13.65 9.1 112.67 95.60 84.85 24.12 20.89
Gold 23B contrast strain 65.3 12.41 8.9 102.22 87.98 86.07 23.89 18.71
Claims (5)
1. utilize and to transform the method that the wild-rice beneficial gene is excavated in big fragment gene group library, it is characterized in that, comprise the steps:
A. the donor wild-rice can transform the foundation of big segment genomic library;
B. can transform big segment genomic library by transgenic technology and import the acceptor cultivated rice, obtain full genome mutated body storehouse;
C. mutant library is screened, obtain the mutant plant of economical character improvement and the wild-rice beneficial gene of control good character.
2. utilization according to claim 1 can transform the method that the wild-rice beneficial gene is excavated in big fragment gene group library, it is characterized in that it is that double base bacterial artificial chromosome storehouse maybe can transform the artificial chromosome storehouse that the donor plant can transform big segment genomic library.
3. utilization according to claim 1 can transform the method that the wild-rice beneficial gene is excavated in big fragment gene group library, it is characterized in that, the structure that the donor wild-rice can transform big segment genomic library is the big fragment paddy DNA for preparing the Megebase level by the agar entrapping method, utilizes to transform artificial chromosome carrier or the big fragment gene group of double base bacterial artificial chromosome vector construction library again.
4. utilization according to claim 1 can transform the method that the wild-rice beneficial gene is excavated in big fragment gene group library, it is characterized in that, described transgenic technology is a kind of in agriculture bacillus mediated, particle gun, pollen tube channel, the injection of fringe neck.
5. utilization according to claim 1 can transform the method that the wild-rice beneficial gene is excavated in big fragment gene group library, it is characterized in that the donor plant is a wild-rice, and recipient plant is a cultivated rice.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410023348 CN1277924C (en) | 2004-06-25 | 2004-06-25 | Method for discovering wild rice beneficial gene using convertible large fragment genome library |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410023348 CN1277924C (en) | 2004-06-25 | 2004-06-25 | Method for discovering wild rice beneficial gene using convertible large fragment genome library |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1594564A true CN1594564A (en) | 2005-03-16 |
| CN1277924C CN1277924C (en) | 2006-10-04 |
Family
ID=34663411
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410023348 Expired - Fee Related CN1277924C (en) | 2004-06-25 | 2004-06-25 | Method for discovering wild rice beneficial gene using convertible large fragment genome library |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1277924C (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104937598A (en) * | 2012-11-29 | 2015-09-23 | 霍夫曼-拉罗奇有限公司 | Accurate and fast mapping of targeted sequencing reads |
| CN106416681A (en) * | 2016-09-09 | 2017-02-22 | 山东省烟台市农业科学研究院 | Methods for dyeing and culturing butterfly orchids |
| CN114651686A (en) * | 2022-04-11 | 2022-06-24 | 海南省农业科学院粮食作物研究所 | Method for breeding new red rice variety by utilizing medicinal wild rice |
-
2004
- 2004-06-25 CN CN 200410023348 patent/CN1277924C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104937598A (en) * | 2012-11-29 | 2015-09-23 | 霍夫曼-拉罗奇有限公司 | Accurate and fast mapping of targeted sequencing reads |
| CN104937598B (en) * | 2012-11-29 | 2017-11-07 | 霍夫曼-拉罗奇有限公司 | The accurate and quick positioning of the sequencing reading value of targeting |
| US10127351B2 (en) | 2012-11-29 | 2018-11-13 | Roche Molecular Systems, Inc. | Accurate and fast mapping of reads to genome |
| CN106416681A (en) * | 2016-09-09 | 2017-02-22 | 山东省烟台市农业科学研究院 | Methods for dyeing and culturing butterfly orchids |
| CN114651686A (en) * | 2022-04-11 | 2022-06-24 | 海南省农业科学院粮食作物研究所 | Method for breeding new red rice variety by utilizing medicinal wild rice |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1277924C (en) | 2006-10-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107988229A (en) | A kind of method for obtaining the rice that tiller changes using CRISPR-Cas modification OsTAC1 genes | |
| CN108239647A (en) | A kind of gene, molecular labeling and application for controlling rape plant type | |
| CN108948170B (en) | Plant type growth and development related protein and coding gene and application thereof | |
| CN104004781A (en) | Preparation method of glyphosate resistant transgenic rice | |
| CN106480163B (en) | A method of joint apple callus cell culture and genetic transformation identify apple disease-resistant gene | |
| CN101186926B (en) | Modified method for transforming gramineous crop by agrobacterium mediated flower-dipping method | |
| CN110358772A (en) | The OsEBP89 gene and preparation method of raising rice abiotic stress resistance and application | |
| CN103088059A (en) | Efficient genetic transformation method of hybridized tulip tree | |
| CN101358193B (en) | Identification of specificity promoter for rice leaf senescence and uses | |
| CN108948169A (en) | It is a kind of promote cotton fiber green pigment synthesis protein, gene and its coded sequence and application | |
| CN1277924C (en) | Method for discovering wild rice beneficial gene using convertible large fragment genome library | |
| CN1298021A (en) | One-purpose expression promoter in rice endosperm tissue and its application | |
| CN113913440B (en) | Application of GhD1119 gene in regulating and controlling cotton flowering of upland cotton | |
| CN114350673B (en) | Rice KOB1 gene for regulating and controlling seed vigor and regulating and controlling method thereof | |
| CN102154337A (en) | Gossypium hirsutum mitogen-activated protein kinas 6 (GhMAPK6) gene and application thereof | |
| CN105039389B (en) | Sugarcane carrier-free frame transgenic method | |
| CN103172717A (en) | Plant low potassium stress resistant related protein GmWRKY50 as well as encoding gene and application thereof | |
| CN102382852A (en) | Method for obtaining tomatoes with high lycopene content and resistance to bacterial wilt through recombining genes | |
| CN114164208A (en) | Gene editing sequence and method for creating soybean cell nucleus male sterile line | |
| Sederoff et al. | Application of recombinant DNA techniques to pines: A molecular approach to genetic engineering in forestry | |
| CN105968178A (en) | Application of rice OsRAD1 protein or encoding gene thereof in regulating pollen fertility | |
| CN105219784A (en) | A kind of hybridized Chinese tuliptree LhRGL1 gene and application thereof | |
| CN103773801A (en) | Application for breeding transgenic water-saving and drought-resistance plant by using aspen ABA (Abscisic Acid) receptor PtPYRL gene | |
| CN115894643B (en) | Sorghum salt and alkali tolerance related gene AT1 and application thereof in crop salt and alkali tolerance | |
| CN109022479A (en) | A kind of screening technique of callus phase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20061004 Termination date: 20130625 |