CN105543276A - Method for cultivating transgenic plant changed from reproductive growth to vegetative growth - Google Patents
Method for cultivating transgenic plant changed from reproductive growth to vegetative growth Download PDFInfo
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Abstract
The invention discloses a method for cultivating a transgenic plant changed from reproductive growth to vegetative growth and a specific RNA fragment. The method for cultivating the transgenic plant changed from the reproductive growth to the vegetative growth comprises the following steps: inhibiting the expression of type-A genes in a germinated plant to obtain the transgenic plant changed from the reproductive growth to the vegetative growth. The way for implementing the inhibition of the expression of the type-A genes is to introduce a specific RNA into the germinated plant; and the specific RNA is the double-chain RNA shown as the sequence 6 in a sequence table. An experiment proves that the method can cause the paddy rice to change from the reproductive growth to the vegetative growth, so that the probability of the survival is guaranteed, and an important effect in the plant multiplying and agricultural production can be realized.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of cultivation and reverse the method for the transgenic plant for nourishing and growing and special RNA fragment thereof by reproductive growth.
Background technology
To experience angiospermous all one's life and nourish and grow and two periods of reproductive growth: nourish and grow and refer to that plant is formed and the growth of the vegetative organ such as root, stem, leaf from seed germination to seedling, for growing of fringe grain provides reliable material guarantee; Reproductive growth refers to plant heading, to bloom and solid.When angiosperm nourishes and grows to certain phase, under the induction of the factors such as extraneous illumination, temperature and autologous hormones, plant changes by nourishing and growing to reproductive growth.Blooming is plant from the important growth event transformed to reproductive growth of nourishing and growing, and being a qualitative change process on higher plant life history, is also the key link of Ontogeny of plant process.Blossom and bear fruit under appropriate conditions for producing offspring and ensureing that output is most important.
Flower is angiospermous important reproductive organ, is also one of distinctive innovation proterties of angiosperm.Typically spend for one and comprise four-wheel floral organ, be followed successively by from outside to inside: sepal, petal, stamen and carpel.Wherein stamen and carpel mainly produce female and male gametophytes, complete fertilization, finally produce offspring.Early 1990s, scientist, by the research to the colored genetic mutant of the dicotyledonous model plant Arabidopis thaliana in angiosperm, Common Snapdragon, proposes the ABC model of flower development.A, B, C represent the three classes characterizing gene relevant to development of floral organs respectively, and category-A gene controls separately the growth of sepal, the growth of A and category-B Gene Handling petal, B and C genoid controls the growth of stamen, and C genoid controls the growth of gynoecium.In addition, A and C genoid suppresses mutually, if i.e. A functional gene sudden change, C genoid is all expressed in four-wheel floral organ, and sepal is replaced by carpel, and petal staminody, vice versa.Along with the deep discovery of research, ABC model B, C functional gene in different plant species is guarded relatively, but causes A functional gene except having report in Arabidopis thaliana due to the shortage of mutant material, also indefinite in other species.
Paddy rice is one of important food crop, and can be used as monocotyledonous model plant, the normal development of its foreign steamer floral organ plays a very good protection to seed, but also little to the research of the category-A gene regulating and controlling foreign steamer development of floral organs in paddy rice at present.In paddy rice, category-A gene has four members, OsMADS14 gene, OsMADS15 gene, OsMADS18 gene and OsMADS20 gene respectively, but due to the function more complicated of gene in monocotyledons, often functional redundancy is there is between different members in same gene family, therefore, be necessary to do further in-depth analysis, to explain the adjusting function of category-A gene pairs paddy rice foreign steamer development of floral organs in paddy rice comprehensively to the functional relationship between the function of several member and these members.
Summary of the invention
Technical problem to be solved by this invention how to be reversed as nourishing and growing by reproductive growth by plant.
For solving the problems of the technologies described above, the present invention provide firstly a kind of cultivation and reverses the method for transgenic plant for nourishing and growing by reproductive growth.
Cultivation provided by the present invention reverses the method for the transgenic plant nourished and grown by reproductive growth, can comprise the steps:, in the expression and/or activity that suppress category-A gene in plant of setting out, to obtain transgenic plant; Described transgenic plant are reversed as nourishing and growing by reproductive growth at reproductive growth period; Described category-A gene can be following b1) or b2) or b3) b4) or b5) or b6) shown in gene:
B1) OsMADS14 gene, OsMADS15 gene, OsMADS18 gene and OsMADS20 gene;
B2) described OsMADS14 gene;
B3) described OsMADS15 gene;
B4) described OsMADS18 gene;
B5) described OsMADS20 gene;
B6) nucleotide sequence limited with described OsMADS14 gene, described OsMADS15 gene, described OsMADS18 gene or described OsMADS20 gene has gene or the assortment of genes of more than 75% or 75% identity.
Described reproductive growth can be heading stage and/or bloom early stage and/or boot stage and/or flowering period and/or productive phase period.
The nucleotide sequence of described OsMADS14 gene can as shown in the 1-741 position of sequence 1 in sequence in sequence table 1 or sequence table.
The nucleotide sequence of described OsMADS15 gene can as shown in the 1-807 position of sequence 2 in sequence in sequence table 2 or sequence table.
The nucleotide sequence of described OsMADS18 gene can as shown in the 1-750 position of sequence 3 in sequence in sequence table 3 or sequence table.
The nucleotide sequence of described OsMADS20 gene can as shown in sequence in sequence table 4.
In aforesaid method, the implementation of described " suppressing the expression of category-A gene and/or activity " can be and import the expression of suppression category-A gene and/or the material of activity in the plant that sets out.
In aforesaid method, described " suppressing the expression of category-A gene and/or the material of activity " can be special RNA; Described special RNA specifically can be the double-stranded RNA shown in sequence 6 in sequence table.
In aforesaid method, the implementation method of described " import in the plant that sets out and suppress the expression of category-A gene and/or the material of activity " can be as follows: specific DNA molecular is imported the plant that sets out; Described specific DNA molecular can comprise DNA fragmentation one, intervening sequence and DNA fragmentation two; Described DNA fragmentation one can be sequence 1 sequence shown in the 127 to 483 from 5 ' end in sequence table; Described DNA fragmentation two can be the reverse complementary sequence of the 127 to 483 from 5 ' end of sequence 1 in sequence table.
The nucleotide sequence of described specific DNA molecular specifically can as shown in the sequence 5 in sequence table.
In aforesaid method, set out described in described specific DNA molecular imports by recombinant expression vector plant.
Described recombinant expression vector specifically can be the recombinant expression vector will obtained between KpnI and the SacI recognition site of the DNA molecular insertion vector pU1301 shown in the sequence 5 in sequence table.
Described carrier pU1301 specifically can be and the DNA small segment between Hind III recognition sequence of carrier pCAMBIA1301 and BamHI recognition sequence is replaced with the DNA molecular shown in sequence 7 that nucleotide sequence is sequence table.
In aforesaid method, described in the Phenotypic Expression of nourishing and growing be: inside take turns floral organ phenotypic alternation and/or axil place and reappear send out roots bract that shape structure and/or base portion degenerate of tiller bud and/or eustipes part and once more to grow and/or send out roots shape thing and/or lepicena and small wooden raft sheet of little Hua base portion all extends and become foliation structure.Described interior floral organ phenotypic alternation of taking turns is mainly reflected in lodicule and becomes film like and/or glumelle carpellody and/or stamen and become slurry sheet structure.It is that the set out axil place tiller bud of plant is degenerated that described axil place reappears tiller bud, and the axil place tiller bud of transgenic plant occurs again.
In aforesaid method, described in the plant that sets out can be following a1) to a5) and in any one:
A1) dicotyledons; A2) monocotyledons; A3) grass; A4) paddy rice; A5) 11 are spent in rice varieties.
For solving the problem, present invention also offers special RNA, specific DNA molecular or special recombinant plasmid.
Special RNA provided by the present invention, is specially the double-stranded RNA shown in sequence 6 in sequence table.
Specific DNA molecular provided by the present invention, can comprise DNA fragmentation one, intervening sequence and DNA fragmentation two; Described DNA fragmentation one can be sequence 1 sequence shown in the 127 to 483 from 5 ' end in sequence table; Described DNA fragmentation two can be the reverse complementary sequence of the 127 to 483 from 5 ' end of sequence 1 in sequence table.
The nucleotide sequence of described specific DNA molecular specifically can as shown in the sequence 5 in sequence table.
Special recombinant plasmid provided by the present invention can be the recombinant plasmid containing described specific DNA molecular.
Special recombinant plasmid provided by the present invention specifically can be the special recombinant plasmid will obtained between KpnI and the SacI recognition site of the DNA molecular insertion vector pU1301 shown in the sequence 5 in sequence table.
Described carrier pU1301 specifically can be and the DNA small segment between Hind III recognition sequence of carrier pCAMBIA1301 and BamHI recognition sequence is replaced with the DNA molecular shown in sequence 7 that nucleotide sequence is sequence table.
The application that described special RNA, described specific DNA molecular or described special recombinant plasmid are reversed in the transgenic plant for nourishing and growing by reproductive growth in cultivation also belongs to protection scope of the present invention.
In above-mentioned application, described plant can be following a1) to a5) in any one:
A1) dicotyledons; A2) monocotyledons; A3) grass; A4) paddy rice; A5) 11 are spent in rice varieties.
In above-mentioned application, described in the Phenotypic Expression of nourishing and growing be: inside take turns floral organ phenotypic alternation and/or axil place and reappear send out roots bract that shape structure and/or base portion degenerate of tiller bud and/or eustipes part and once more to grow and/or send out roots shape thing and/or lepicena and small wooden raft sheet of little Hua base portion all extends and become foliation structure.Described interior floral organ phenotypic alternation of taking turns is mainly reflected in lodicule and becomes film like and/or glumelle carpellody and/or stamen and become slurry sheet structure.It is that the set out axil place tiller bud of plant is degenerated that described axil place reappears tiller bud, and the axil place tiller bud of transgenic plant occurs again.
Experiment proves, in paddy rice, category-A gene is played an important role by reproductive growth reverse plant in nourishing and growing.In paddy rice the expression of category-A gene four members all suppressed after, causing plant to be reversed as nourishing and growing by reproductive growth, ensureing the possibility of chances of survival, in plant procreation and agriculture production, there is important effect.
Accompanying drawing explanation
Fig. 1 is GUS coloration result.
Fig. 2 is the relative expression quantity of real-time quantitative PCR augmentation detection gene.
Fig. 3 is the plant and the tassel phenotype that are in transgenic plant at heading stage.
Fig. 4 is in the interior of transgenic plant in earlier stage of blooming to take turns floral organ phenotype.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Spend 11 in rice varieties purchased from crop science institute of the Chinese Academy of Agricultural Sciences, spend in rice varieties 11 hereinafter referred to as in spend 11.
Alternately, condition is: 28 DEG C for illumination alternate culture and light culture and dark culture; 14 h light are cultivated/10 h dark and are cultivated; Intensity of illumination during illumination cultivation is 90 μ E/m
2/ s.
YEB liquid nutrient medium: by beef extract 5g, yeast extract paste 1g, peptone 5g, sucrose 5g, MgSO
47H
2o0.04g is dissolved in 1L deionized water, regulates pH to 7.2, autoclave sterilization 20min with the 10MNaOH aqueous solution.
Inducing culture: by a large amount of for 50 × N6 mother liquor 20mL, 100 × B5 trace mother liquor 10mL, the organic mother liquor 1mL of 200 × MS mother liquid of iron salt 5mL, 1000 × B5, caseinhydrolysate 300mg, glutamine 500mg, proline(Pro) 2.8g, 2,4-D2.0mg, sucrose 30.0g and plant gel 4.5g are dissolved in 1L distilled water, regulate pH to 5.8,121 DEG C of sterilizing 15min.
Dual culture substratum: by a large amount of for 50 × N6 mother liquor 20mL, 100 × B5 trace mother liquor 10mL, 200 × MS mother liquid of iron salt 5mL, the organic mother liquor 1mL of 1000 × B5, caseinhydrolysate 300mg, glutamine 500mg, proline(Pro) 2.8g, 2, 4-D2.0mg, sucrose 30.0g, plant gel 4.5g, Pyocianil 100mg and cephamycin 400mg is dissolved in 1L distilled water, regulate pH to 5.2, 121 DEG C of sterilizing 15min, glucose 10g to adding when 50 ~ 60 DEG C through 0.22 μM of filtration sterilization to be cooled, Pyocianil 100mg and cephamycin 400mg and Syringylethanone, the final concentration of Syringylethanone in system is 100 μMs.
Primary screening substratum: by a large amount of for 50 × N6 mother liquor 20mL, 100 × B5 trace mother liquor 10mL, the organic mother liquor 1mL of 200 × MS mother liquid of iron salt 5mL, 1000 × B5, caseinhydrolysate 300mg, glutamine 500mg, proline(Pro) 2.8g, 2,4-D2.0mg, sucrose 30.0g, plant gel 4.5g are dissolved in 1L distilled water, regulate pH to 5.8,121 DEG C of sterilizing 15min, Totomycin 30mg, Pyocianil 100mg to adding when 50 ~ 60 DEG C through 0.22 μM of filtration sterilization to be cooled and cephamycin 400mg.
Postsearch screening substratum: except the Totomycin 30mg in primary screening substratum is replaced with Totomycin 50mg, other composition and content are all constant.
Division culture medium: a large amount of for 20 × MS mother liquor 50mL, 200 × MS trace mother liquor 5mL, the organic mother liquor 5mL of 200 × MS mother liquid of iron salt 5mL, 200 × MS, caseinhydrolysate 1000mg, glutamine 500mg, sucrose 30.0g, sorbyl alcohol 20g, NAA0.4mg, 6-BA2.0mg, KT1mg and plant gel 4.5g are dissolved in 1L distilled water, regulate pH to 5.8,121 DEG C of sterilizing 15min.
Root media: a large amount of for 20 × MS mother liquor 25mL, 200 × MS trace mother liquor 2.5mL, the organic mother liquor 2.5mL of 200 × MS mother liquid of iron salt 2.5mL, 200 × MS, sucrose 15g and plant gel 4.5g are dissolved in 1L distilled water, regulate pH to 5.8,121 DEG C of sterilizing 15min, the Totomycin 30mg to adding when 50 ~ 60 DEG C through 0.22 μM of filtration sterilization to be cooled.
1/2MS nutrient solution: a large amount of for 20 × MS mother liquor 25mL, 200 × MS trace mother liquor 2.5mL, the organic mother liquor 2.5mL of 200 × MS mother liquid of iron salt 2.5mL, 200 × MS are dissolved in 1L distilled water, regulate pH to 5.8.
Solute and the concentration thereof of a large amount of mother liquor of 50 × N6 are: 141.50g/LKNO
3, 20g/LKH
2pO
4, 23.15g/L (NH
4)
2sO
4, 9.25g/LMgSO
47H
2o, 8.30g/LCaCl
22H
2o, solvent is water, pH nature.
Solute and the concentration thereof of 100 × B5 trace mother liquor are: 0.3g/LH
3bO
3, 1g/LMnSO
44H
2o, 0.0025g/LCoCl
26H
2o, 0.0025g/LCuSO
45H
2o, 0.2g/LZnSO
47H
2o, 0.025g/LNa
2moO
42H
2o, 0.075g/LKI, solvent is water, pH nature.
Solute and the concentration thereof of the organic mother liquor of 1000 × B5 are: 2g/L glycine, 100g/L inositol, 1g/L nicotinic acid, 1g/L pyridoxine hydrochloride, 10g/L vitamin, and solvent is water, pH nature.
Solute and the concentration thereof of a large amount of mother liquor of 20 × MS are: 38.00g/LKNO
3, 8.80g/LCaCl
22H
2o, 7.40g/LMgSO
47H
2o, 3.40g/LKH
2pO
4, 33.00g/LNH
4nO
3, solvent is water, pH nature.
Solute and the concentration thereof of 200 × MS trace mother liquor are: 4.46g/LMnSO
44H
2o, 0.166g/LKI, 1.24g/LH
3bO
3, 1.72g/LZnSO
47H
2o, 0.050g/LNa
2moO
42H
2o, 0.005g/LCuSO
45H
2o, 0.005g/LCoCl
26H
2o, solvent is water, pH nature.
The solute of 200 × MS mother liquid of iron salt and concentration thereof are: 5.56g/LFeSO
47H
2o, 7.46g/LNa
2eDTA2H
2o, solvent is water, pH nature.
Solute and the concentration thereof of the organic mother liquor of 200 × MS are: 20g/L inositol, 100mg/L nicotinic acid, 100mg/L pyridoxine hydrochloride, 100mg/L vitamin, 400mg/L glycine, and solvent is water, pH nature.
Carrier pBIntron in following embodiment is for replacing with the DNA molecular shown in sequence 8 that nucleotide sequence is sequence table by the DNA small segment between Hind III recognition sequence of carrier pBluescriptII (SK) (Clotech Products) and NotI recognition sequence.
In following embodiment, the building process of carrier pU1301 is as follows: (1) take corn gene group DNA as template, with the primer U-1:5 ' of synthetic-
aAGCTTcTGCAGTGCAGCGTGACCC-3 ' (underscore part is the recognition sequence of Hind III) and primer U-2:5 '-
gGATCCfor primer carries out pcr amplification, (response procedures is 94 DEG C of denaturation 4min to CTGCAGAAGTAACACCAA-3 ' (underscore part is the recognition sequence of BamHI); 94 DEG C of sex change 1min, 57 DEG C of annealing 1min, 72 DEG C extend 2min40s, 30 circulations; Last 72 DEG C extend 10min), reclaim the pcr amplification product of about 2Kb; (2) with restriction enzyme Hind III and this pcr amplification product of BamHI double digestion, digestion products is reclaimed; (3) with restriction enzyme Hind III and BamHI double digestion carrier pCAMBIA1301, (product of CenterfortheApplicationofMolecularBiologytoInternational Agriclture, network address is
www.cambia.org), reclaim carrier framework; (4) digestion products is connected with carrier framework, obtains recombinant plasmid, be i.e. carrier pU1301.According to sequencing result, it is as follows that the carrier pU1301 obtained step (4) carries out structrual description: the DNA small segment between Hind III recognition sequence of carrier pCAMBIA1301 and BamHI recognition sequence is replaced with the DNA molecular shown in the sequence 7 that nucleotide sequence is sequence table.
The acquisition of embodiment 1, transgenic paddy rice and qualification
One, the structure of recombinant plasmid Ai
According to the nucleotide sequence comparison result of four members of category-A gene in paddy rice (OsMADS14 gene, OsMADS15 gene, OsMADS18 gene and OsMADS20 gene), carry out a large amount of preliminary experiment, sequence 1 the 127 to 483 target area section as RNAi from 5 ' end in Selective sequence table.
OsMADS14 gene nucleotide series as shown in sequence in sequence table 1, its encoding sequence as sequence in sequence table 1 from shown in 5 ' end 1-741 position Nucleotide.
OsMADS15 gene nucleotide series as shown in sequence in sequence table 2, its encoding sequence as sequence in sequence table 2 from shown in 5 ' end 1-807 position Nucleotide.
OsMADS18 gene nucleotide series as shown in sequence in sequence table 3, its encoding sequence as sequence in sequence table 3 from shown in 5 ' end 1-750 position Nucleotide.
OsMADS20 gene nucleotide series is as shown in sequence in sequence table 4.
1, extract in rice varieties and spend the genomic dna of 11 and using it as template, with P1-SmaI and P2-HpaI of synthetic for primer carries out pcr amplification, reclaim pcr amplification product 1.
P1-SmaI:5 '-GG
cCCGGGgTCGCGCTCATCATCTTCTC-3 ' (underscore part is the recognition sequence of SmaI, and sequence is thereafter the 127-146 position of sequence 1);
P2-HpaI:5 '-GC
gTTAACaGTGACTTTTCCTTCCGTTG-3 ' (underscore part is the recognition sequence of HpaI, and sequence is thereafter the reverse complementary sequence of the 463-482 position of sequence 1).
2, with Restriction enzyme Sma I and HpaI double digestion pcr amplification product 1, digestion products 1 is reclaimed.
3, with Restriction enzyme Sma I and HpaI double digestion carrier pBIntron, the carrier framework 1 of about 3500bp is reclaimed.
4, digestion products 1 is connected with carrier framework 1, obtains recombinant plasmid 1.
5, to spend the genomic dna of 11 for template in the rice varieties of step 1 extraction, with the P3-Hind III of synthetic and P4-KpnI for primer carries out pcr amplification, pcr amplification product 2 is reclaimed.
P3-Hind III: 5 '-GG
aAGCTTgTCGCGCTCATCATCTTCTC-3 ' (underscore part is the recognition sequence of Hind III, and sequence is thereafter the 127-146 position of sequence 1);
P4-KpnI:5'-GC
gGTACCaGTGACTTTTCCTTCCGTTG-3 ' (underscore part is the recognition sequence of KpnI, and sequence is thereafter the reverse complementary sequence of the 463-482 position of sequence 1).
6, with restriction enzyme Hind III and KpnI double digestion pcr amplification product 2, digestion products 2 is reclaimed.
7, with restriction enzyme Hind III and KpnI double digestion recombinant plasmid 1, the carrier framework 2 of about 3.7kb is reclaimed.
8, digestion products 2 is connected with carrier framework 2, obtains recombinant plasmid 2.
9, with restriction enzyme KpnI and SacI double digestion recombinant plasmid 2, the digestion products 3 of about 360bp is reclaimed.
10, with restriction enzyme KpnI and SacI double digestion carrier pU1301, the carrier framework 3 of about 3.9kb is reclaimed.
11, digestion products 3 is connected with carrier framework 3, obtains recombinant plasmid Ai.
According to sequencing result, it is as follows that the recombinant plasmid Ai obtained step 11 carries out structrual description: the DNA small segment between the KpnI recognition sequence of carrier pU1301 and SacI recognition sequence is replaced with the DNA molecular shown in the sequence 5 that nucleotide sequence is sequence table.Recombinant plasmid Ai just can form the double-stranded RNA as shown in sequence 6 on rna level, causes RNAi, thus can be used for the expression suppressing OsMADS14 gene, OsMADS15 gene, OsMADS18 gene and OsMADS20 gene.
Two, the genetic transformation of paddy rice
1, recombinant plasmid Ai step one built imports agrobacterium tumefaciens EHA105 by electric shock, obtains recombinational agrobacterium, called after EHA105-Ai.
2, EHA105-Ai mono-clonal is inoculated in the YEB liquid nutrient medium of 20mL containing kantlex 50mg/L and Rifampin 50mg/L, 28 DEG C, after 220rpm shakes cultivation 12 ~ 16h, then the YEB liquid nutrient medium containing Syringylethanone 100 μMs is inoculated in the ratio of 2%-4% (volumn concentration), 28 DEG C, 220rpm shaking culture is to OD
600value reaches about 0.5, obtains Agrobacterium and infects liquid.
3, middle 11 seeds of spending shell threshing, be placed in 100mL triangular flask, add 70% (volume percent) aqueous ethanolic solution and soak 30sec, be placed in 25% (volume percent) aqueous sodium hypochlorite solution again, 120rpm shakes sterilizing 30min, aseptic water washing 3 times, use filter paper suck dry moisture, then seed embryo is placed in callus induction on inducing culture down, 30 DEG C of full exposures induce 7 days, and the callus grown is pinched after bud for rice conversion.
4, after completing steps 3, get the good embryo callus of growth conditions, be soaked in the Agrobacterium that step 2 obtains and infect liquid, 28 DEG C, 80rpm shaking table jog callus, infect 30min, then, be placed on be covered with one deck sterilizing filter paper Dual culture substratum on, 25 DEG C of light culture 4 days.
5, the callus that step 4 obtains is got, be placed in primary screening substratum, illumination alternate culture is after 2 weeks, the primary screening substratum more renewed, callus continues illumination alternate culture 2 weeks, the resistant calli that former callus grows is transferred on postsearch screening substratum, illumination alternate culture 2 weeks.
6, after completing steps 5, get eugonic resistant calli, be placed in division culture medium, illumination alternate culture 3 weeks, resistant calli differentiates resistance budlet.
7, after completing steps 6, get resistance budlet, be placed in root media, illumination alternate culture, obtains resistant plant.When resistant plant grows to 6 ~ 10cm, carry out open Aquaponic, grow intermediate house after new root, obtain T
0in generation, intends transgenic rice plant.
The T that extraction step 7 obtains
0in generation, intends the genomic dna of transgenic rice plant and as template, adopts primers F: the 127-146 position of the sequence 1 of 5'-GTCGCGCTCATCATCTTCTC-3'(sequence table) and the reverse complementary sequence of 463-484 position of sequence 1 of primer R:5'-GCAGTGACTTTTCCTTCCGTTG-3'(sequence table) primer pair that forms carries out pcr amplification.
PCR reaction conditions: 95 DEG C, 5 minutes; 95 DEG C 40 seconds, 56 DEG C 40 seconds, 72 DEG C 40 seconds, 35 circulations; 72 DEG C extend 10 minutes.
Result shows, can increase can not obtain size be about 360bp band for positive T
0for transgenic rice plant.Each plant is a strain.Stochastic choice three strains, by its called after L1, L2 and L3.
Three, the acquisition of empty carrier plant is turned
Replace recombinant plasmid Ai to carry out step 2 with carrier pU1301, obtain turning empty carrier plant.
Four, T
0for the qualification of transgenic rice plant
1, GUS dyeing qualification
After getting group training seedling 30 days L1 seedling, L2 seedling, L3 seedling or turn the blade of empty carrier plant shoots, carry out GUS dyeing.
Fig. 1 is the coloration result of the blade of L3.Experimental result is as follows: the blade GUS of L1, L2 and L3 dyes rear in blue, after turning the blade GUS dyeing of empty carrier plant, does not produce blue material.Visible, L1, L2 and L3 are transgenic rice plant.
2, real-time quantitative PCR amplification
(1) when the tassel length of paddy rice (in spend 11, turn empty carrier plant, L1, L2 or L3) is 5 ~ 10 centimetres, get sword-like leave blade, extract with RNA the RNA that test kit (Invitrogen Products) extracts, obtain the RNA of leaf tissue.
(2) with the RNA of leaf tissue for template, detected the relative expression quantity (with ACTIN1 gene for reference gene) of OsMADS14 gene, OsMADS15 gene, OsMADS18 gene or OsMADS20 gene by real-time quantitative PCR.
Identify that the primer of OsMADS14 gene is 5 '-ACGGAAGGAAAAGTCACTGC-3 ' and 5 '-TCCTCTATCCTTTCGCCAGC-3 ', object fragment is if sequence in sequence table 1 is from shown in 5 ' end the 465th to the 668th.
Identify that the primer of OsMADS15 gene is 5 '-TGAGTCCATTTCCGAGCTG-3 ' and 5 '-TCTTCTGCCTCTCCACCAGT-3 ', object fragment is if sequence in sequence table 2 is from shown in 5 ' end the 444th to the 532nd.
Identify that the primer of OsMADS18 gene is 5 '-CTGCAAAAGCTCATGGAGAC-3 ' and 5 '-CTGACTCCCCTGATCCTCTT-3 ', object fragment is if sequence in sequence table 3 is from shown in 5 ' end the 502nd to the 676th.
Identify that the primer of OsMADS20 gene is 5 '-CCACTAAAGCTGCTGCTCCT-3 ' and 5 '-GGCAAGCCATTGTTGCTGCT-3 ', object fragment is if sequence in sequence table 4 is from shown in 5 ' end the 566th to the 704th.
Internal reference is ACTIN1 gene, and the primer of internal reference is 5 '-TGCTATGTACGTCGCCATCCAG-3 ' and 5 '-AATGAGTAACCACGCTCCGTCA-3 '.
The middle relative expression quantity of OsMADS14 gene in 11 of spending is shown in Fig. 2 as the relative expression quantity of OsMADS14 gene in 1, L1, L2 and L3.Result shows, in L1, L2 and L3, the expression amount of OsMADS14 gene is respectively middle 0.05 times, 0.055 times and 0.06 times of spending the expression amount of OsMADS14 gene in 11; The expression amount and the middle expression amount of OsMADS14 gene in 11 of spending that turn OsMADS14 gene in empty carrier plant are basically identical.
The middle relative expression quantity of OsMADS15 gene in 11 of spending is shown in Fig. 2 as the relative expression quantity of OsMADS15 gene in 1, L1, L2 and L3.Result shows, in L1, L2 and L3, the expression amount of OsMADS15 gene is respectively middle 0.22 times, 0.25 times and 0.24 times of spending the expression amount of OsMADS15 gene in 11; The expression amount and the middle expression amount of OsMADS15 gene in 11 of spending that turn OsMADS15 gene in empty carrier plant are basically identical.
The middle relative expression quantity of OsMADS18 gene in 11 of spending is shown in Fig. 2 as the relative expression quantity of OsMADS18 gene in 1, L1, L2 and L3.Result shows, in L1, L2 and L3, the expression amount of OsMADS18 gene is respectively middle 0.11 times, 0.09 times and 0.03 times of spending the expression amount of OsMADS18 gene in 11; The expression amount and the middle expression amount of OsMADS18 gene in 11 of spending that turn OsMADS18 gene in empty carrier plant are basically identical.
The middle relative expression quantity of OsMADS20 gene in 11 of spending is shown in Fig. 2 as the relative expression quantity of OsMADS20 gene in 1, L1, L2 and L3.Result shows, in L1, L2 and L3, the expression amount of OsMADS20 gene is respectively middle 0.009 times, 0.105 times and 0.008 times of spending the expression amount of OsMADS20 gene in 11; The expression amount and the middle expression amount of OsMADS20 gene in 11 of spending that turn OsMADS20 gene in empty carrier plant are basically identical.
Result shows, in L1, L2 and L3, the expression of category-A gene four members is all suppressed.
3, the phenotypic evaluation of transgenic rice plant
Centering spends 11, turn empty carrier plant, L1, L2 and L3 compares rice plant phenotype and adds up in growth and development process.Experiment in triplicate, repeats 30 strains at every turn.
Result shows, though the expression amount of OsMADS14 gene, OsMADS15 gene, OsMADS18 gene or OsMADS20 gene is inconsistent in L1, L2 and L3, but the expression due to each gene all drops to lower level (being less than 25%), therefore, L1, L2 and L3 phenotype is similar.
Experimental result is shown in Fig. 3 and Fig. 4.
Fig. 3 is the plant and the tassel phenotype that are in heading stage.Result shows, with in spend compared with in the of 11, the tassel being in L1, L2 and the L3 at heading stage can not normally be extracted out (in Fig. 3 A, B, C and D), axil place produces more tiller bud (in Fig. 3 E), eustipes part sends out roots shape structure (in Fig. 3 F), the bract that base portion is degenerated grows (in Fig. 3 G) once more, and little Hua base portion sends out roots shape thing, and lepicena and small wooden raft sheet all extend and become foliation structure (in Fig. 3 G, H and I) simultaneously.
(wherein A is wild-type little floral anatomy figure, B is the little floral anatomy figure of mutant to Fig. 4, and wherein le is lemma, pa is glumelle, and lo is lodicule, and st is stamen, pi is gynoecium, and eg is lepicena, and rg is the lepicena of degenerating) take turns floral organ phenotype for being in the plant in earlier stage that blooms.Result shows, with in spend compared with in the of 11, L1, L2 and L3 interior takes turns floral organ also obvious change, and lodicule becomes film like (in Fig. 4 C and D), glumelle carpellody (in Fig. 4 E and F), stamen becomes slurry sheet structure (in Fig. 4 G and H).
Turn empty carrier plant and the middle phenotype of 11 of spending all without significant difference.
Result shows, in paddy rice the expression of category-A gene four members all suppressed after, plant is caused to be reversed as nourishing and growing by reproductive growth, take turns floral organ phenotypic alternation and/or axil place to reappear send out roots bract that shape structure and/or base portion degenerate of tiller bud and/or eustipes part once more to grow and/or send out roots shape thing and/or lepicena and small wooden raft sheet of little Hua base portion all extends and become foliation structure as interior, wherein, wheel floral organ phenotypic alternation is mainly reflected in lodicule and becomes film like and/or glumelle carpellody and/or stamen and become slurry sheet structure, it is that the middle axil place tiller bud of 11 of spending is degenerated that axil place reappears tiller bud, the axil place tiller bud of transgenic plant occurs again.Visible, in paddy rice, category-A gene is played an important role by reproductive growth reverse plant in nourishing and growing.
Claims (10)
1. cultivate and reverse the method for transgenic plant for nourishing and growing by reproductive growth, comprise the steps:, in the expression and/or activity that suppress category-A gene in plant of setting out, to obtain transgenic plant; Described transgenic plant are reversed as nourishing and growing by reproductive growth at reproductive growth period;
Described category-A gene is following b1) or b2) or b3) b4) or b5) or b6) shown in gene:
B1) OsMADS14 gene, OsMADS15 gene, OsMADS18 gene and OsMADS20 gene;
B2) described OsMADS14 gene;
B3) described OsMADS15 gene;
B4) described OsMADS18 gene;
B5) described OsMADS20 gene;
B6) nucleotide sequence limited with described OsMADS14 gene, described OsMADS15 gene, described OsMADS18 gene or described OsMADS20 gene has gene or the assortment of genes of more than 75% or 75% identity.
2. the method for claim 1, is characterized in that: the implementation of described " suppressing the expression of category-A gene and/or activity " is import to suppress the expression of category-A gene and/or the material of activity in the plant that sets out.
3. method as claimed in claim 2, is characterized in that: described " suppressing the expression of category-A gene and/or the material of activity " is special RNA;
Described special RNA is the double-stranded RNA shown in the sequence 6 in sequence table.
4. method as claimed in claim 2 or claim 3, is characterized in that:
The implementation method of described " import in the plant that sets out and suppress the expression of category-A gene and/or the material of activity " is as follows: specific DNA molecular is imported the plant that sets out;
Described specific DNA molecular comprises DNA fragmentation one, intervening sequence and DNA fragmentation two; Described DNA fragmentation one is sequence in sequence table 1 sequence shown in the 127 to 483 from 5 ' end; Described DNA fragmentation two is sequence 1 reverse complementary sequence of the 127 to 483 from 5 ' end in sequence table.
5. method as claimed in claim 4, is characterized in that: the nucleotide sequence of described specific DNA molecular is as shown in the sequence 5 in sequence table.
6. the method as described in as arbitrary in claim 1 to 5, is characterized in that: set out described in described specific DNA molecular is imported by recombinant expression vector plant; The recombinant expression vector of described recombinant expression vector for obtaining between KpnI and the SacI recognition site of the DNA molecular insertion vector pU1301 shown in the sequence 5 in sequence table; Described carrier pU1301 is for replacing with the DNA molecular shown in sequence 7 that nucleotide sequence is sequence table by the DNA small segment between Hind III recognition sequence of carrier pCAMBIA1301 and BamHI recognition sequence.
7. the method as described in claim 1 to 6, is characterized in that: described in the plant that sets out be following a1) to a5) and in any one:
A1) dicotyledons; A2) monocotyledons; A3) grass; A4) paddy rice; A5) 11 are spent in rice varieties.
8. special RNA, specific DNA molecular or special recombinant plasmid;
Described special RNA is the double-stranded RNA shown in the sequence 6 in sequence table;
Described specific DNA molecular, comprises DNA fragmentation one, intervening sequence and DNA fragmentation two; Described DNA fragmentation one is sequence in sequence table 1 sequence shown in the 127 to 483 from 5 ' end; Described DNA fragmentation two is sequence 1 reverse complementary sequence of the 127 to 483 from 5 ' end in sequence table;
Described special recombinant plasmid is the recombinant plasmid containing described specific DNA molecular.
The nucleotide sequence of described specific DNA molecular specifically can as shown in the sequence 5 in sequence table.
9. special RNA, described specific DNA molecular or described special recombinant plasmid described in claim 8 reverse the application in the transgenic plant for nourishing and growing in cultivation by reproductive growth.
10. apply as claimed in claim 9, it is characterized in that: described plant is following a1) to a5) in any one:
A1) dicotyledons; A2) monocotyledons; A3) grass; A4) paddy rice; A5) 11 are spent in rice varieties.
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Non-Patent Citations (3)
| Title |
|---|
| KAORU KOBAYASHI等: "Inflorescence meristem identity in rice is specified by overlapping functions of three AP1/FUL-like MADS box genes and PAP2,a sepallata mads box gene", 《THE PLANT CELL》 * |
| 于新等: "水稻花器官发育的分子机理", 《分子植物育种》 * |
| 王旻: "《生物工程》", 31 August 2015 * |
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