CN101948870B - Method for reducing branch quantity of plant and improving chlorophyll and anthocyanin contents of plant - Google Patents
Method for reducing branch quantity of plant and improving chlorophyll and anthocyanin contents of plant Download PDFInfo
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Abstract
The invention discloses a method for reducing branch quantity of a plant and improving chlorophyll and anthocyanin contents of the plant by using an miR171 gene and target genes SCL6 thereof, which relates to the technical field of agricultural science. The invention comprises (1) a method for reducing the branch of a plant, improving the chlorophyll and anthocyanin contents of the plant and enhancing anti-aging and drought resistances of the plant through the function over-expression of the miR171 gene and the function loss or reduction of SCL6-II, SCL6-III and SCL6-IV genes; and (2) a method for reducing the branch quantity of the plant, improving the chlorophyll and anthocyanin contents of the plant and enhancing the anti-aging and drought resistances of the plant through the function over-expression of the miR171 gene and the function loss or reduction of the SCL6-II, SCL6-III and SCL6-IV genes. The invention has application of the miR171 gene, the SCL6-II gene, the SCL6-III gene and the SCL-IV 6 gene in reducing the branch quantity of the plant and improving the chlorophyll and anthocyanin contents of the plant, the function of the miR171 gene in the anti-aging and drought resistances and the application thereof in enhancing the anti-aging and drought resistances. The obtained effect of the invention is obvious.
Description
Technical field
What the present invention relates to is regulation and control plant branching quantity and the chlorophyll in a kind of agricultural cience and farming techniques field, the method for anthocyanin content; In particular to be to utilize miR171 and target gene SCL6 thereof to reduce plant branching quantity and the method that improves chlorophyll and anthocyanin content.
Background technology
In the evolutionary process of whole plants, different plants, desirable branch requires different.If can have important effect through the branch of each kind of plant of technique means regulation and control.Plant branching is by the meristematic tissue of axil at first, and this process receives complicated networking regulation and control.Secondary meristematic tissue produces a series of axil meristematic tissue with identical developmental potentiality, these meristematic tissue or selection develops into side shoot, or select dormancy.And this process receives the etap, the tight regulation and control of environmental factors and plant hormone.Growth hormone (indolylacetic acid; IAA) be considered to growth of higher plant branch and top and build up most important hormone in the process.And phytokinin (cytokinin) can promote branched generation, even under the tangible situation of apical dominance under the growth hormone control.The only angle of another newfound hormonal substance gold lactone (strigolactone) can suppress pea, petunia, the tillering of the branch of Arabidopis thaliana and paddy rice.In the branched signal network of regulation and control, growth hormone, phytokinin and only angle these three kinds of hormones of gold lactone exist complex interactions.Found the gene that some regulation and control side shoots form and grow equally through genetics and molecular biological method, as: LATERAL SUPPRESSOR (LAS), MONOCULM1 (MOC1), REGULATOR ofAXILLARY MERISTEMS REVOLUTA (RAX).Some that whether also have other are grown with plant branching and the relevant gene of morphogenesis is also treated further searching.
The branch regulation and control of plant have important effect in whole plants circle.For example: various farm crop, through reducing sterile and the relatively poor branch of fertility, can improve the quality of crop preferably, and can improve output through rational dense planting; For forest, particularly some tall and big arbors can be improved wood quality through reducing branch, obtain the plant of Gao Erzhi; For gardening plant, some cut-flower plants particularly for example will obtain the cut-flower of superior in quality feverfew, need great amount of manpower and material resources to remove side shoot usually.If can obtain the cut-flower new variety of plant of no side shoot or the minimizing of side shoot quantity through means such as transgenic or screening mutants, will save great amount of manpower and material resources.
Chlorophyll is one type of most important pigment relevant with photosynthesis (photosynthesis).Photosynthesis is the process that luminous energy is changed into chemical energy through more synthetic organic cpds.In fact chlorophyll be shown in all can carry out photosynthetic organism, comprises the blue-green algae (blue bacterium) of green plants, protokaryon and the algae of eucaryon.Chlorophyll absorbs energy from light, energy is used to change carbonic acid gas into glucide then.Chlorophyll also is one type of very beneficial material to human body.For example: rich trace elements iron in the chlorophyll is natural hematopoiesis raw material; Rich trace elements iron in the chlorophyll is natural hematopoiesis raw material; Chlorophyll can be kept the activity of ferment, makes it give play to extremely strong antioxygenation, and the opposing radical delays senility; Chlorophyll is best natural toxinicide, the toxin that sanitas, additive and the essence etc. that contain in the various junk foods that can neutralize accumulate in vivo; Chlorophyll can also preventing infection, prevents the diffusion of inflammation, has the effect of bactericidal antiphlogistic.Except these physiological functions, it can be that vegetables have better appearance that chlorophyll content raises, the existing better article phase of plant of sight.
Anthocyanin mainly is present in the ghost of vegetable cell, plays the injury effect of defence radical pair cell.Owing on the structure of anthocyanin an electronics shortage is arranged; Highly active so seem; But its effect is indirect: the hydrogen peroxide through forming in electron transfer system (chloroplast(id) oxygenate system/mitochondrial respiratory system electronic transfer chain) penetrates ghost, under the catalysis of flavonoid px, borrows flavonoid elimination hydrogen peroxide such as cyanidin(e).According to the anthocyanin that added up in the last few years the HUMAN HEALTH role is had: strong receptivity, anti-oxidative damage the oxygen negative radical; Improve capillary vessel impedance and perviousness, microcirculation improvement; Inhibition/activated to some key enzyme activity is regulated, and changes metabolic condition.
Number of patent application: CN00813062.0 is found in retrieval through to the prior art document; Title: regulate the gene of plant branching, contain the carrier of this gene; By this carrier microorganism transformed, and utilize this mikrobe to regulate the method for plant branching, this technology is regulated and control the branch of plant through the MADS gene of justice and antisense expression paddy rice; Employed miR171 gene among the MADS gene that uses and the present invention (open source literature: Sunkar and Zhu; 2004, Plant Cell, 16:2001-2019; Llave etc., 2002, Science, 297:2053-2056; Gene number is respectively: At3G51375, At1G11735 and At1G62035) and SCL6 gene (open source literature: Llave etc., 2002, Science, 297:2053-2056; Gene number is respectively: At2G45160, At3G60630 and At4G00150) have no the homology on the sequence.Overexpression MADS gene has been described in this patented claim can increase the branch of buckwheat, and utilizes this gene of technological expression of sense-rna can reduce the branch of buckwheat.But in its whole case description, do not have the phenotype that data presentation buckwheat branch quantity changes.This patented claim transfer-gen plant that only the demonstration antisense transforms in the 4th secondary figure of Fig. 5 A is grown obviously slowly, but does not show branched phenotype.
Summary of the invention
The present invention is through obtaining the overexpression plant miR171 gene (gene that contains any one sequence in 5 ' tgattgagcc gcgccaaata t 3 ' or 5 ' ttgagccgtg ccaatatcac g, 3 ' these two nucleotide sequences; With reference to Sunkar etc.; 2005, Plant Cell, transfer-gen plant 17:1397-1411); Realized that the overexpression of miR171 gene can significantly reduce the branch quantity of plant, and the content that increases chlorophyll and anthocyanin; Target gene SCL6 through making up miR171 (comprises SCL6-II, SCL6-III and SCL6-IV; Be meant the gene that contains 5 ' gggatattgg cgcggctcaa t, 3 ' nucleotide sequence; With reference to Llave etc., 2002, Science; Two mutants 297:2053-2056) has realized that scl6-IIscl6-III double-mutant and scl6-II scl6-III scl6-IV Trimutant plant present the phenotype that the obviously minimizing of branch quantity, chlorophyll and anthocyanin content increase.Therefore can be through making up the transfer-gen plant of overexpression miR171; Perhaps formulate scl6-II scl6-III double-mutant or scl6-II scl6-III scl6-IV Trimutant; Reduce the branch quantity of plant, and the content that improves chlorophyll and anthocyanin.The present invention is applicable to and cultivates no branch or few branched ornamental plant of viewing and admiring and afforesting and farm crop and cash crop, and the vegetables and the ornamental plants in garden of chlorophyll and the increase of anthocyanin content.
Therefore; One aspect of the present invention relates to two kinds and reduces plant branching, increase chlorophyll and anthocyanin content; Can also improve simultaneously the method for plants antisenescence and drought resistance; These two kinds of methods comprise makes miR171 gene function overexpression, perhaps makes SCL6-II, SCL6-III and forfeiture of SCL6-IV gene function or reduction.
The present invention relates to two kinds on the other hand and obtains to reduce branch, increase chlorophyll and anthocyanin content; Simultaneously can also improve the method for the plant of plants antisenescence and drought resistance; These two kinds of methods comprise makes miR171 gene function overexpression, perhaps makes SCL6-II, SCL6-III and forfeiture of SCL6-IV gene function or reduction.
The invention still further relates to miR171 and express the plant of increase and the plant of SCL6-II, SCL6-III and SCL6-IV gene function disappearance or reduction.
The invention still further relates to miR171, SCL6-II, SCL6-III and the SCL6-IV gene purposes in influencing plant branching and chlorophyll and anthocyanin content.
The invention still further relates to the effect of miR171 gene in anti-ageing and drought-resistant process, and the purposes in influencing these processes.
The present invention is through overexpression miR171 and reject said SCL6-II, SCL6-III and SCL6-IV gene and realize said inactivation.The present invention is applicable to: cultivate the ornamental plant that confession that branch quantity reduces is viewed and admired and afforested, and various arbors, the cut-flower of various types of viewing and admiring, various farm crop and cash crop, and to utilize blade be the various vegetables and the food crop of leading.Described plant is including, but not limited to each kind of plant of Cruciferae Btassica, Solanaceae tomato genus, Solanaceae Nicotiana, Papilionaceae, composite family, Gramineae Oryza, Gramineae Hordeum.
The present invention can adopt the whole bag of tricks to make the miR171 overexpression, and SCL6-II, SCL6-III and forfeiture of SCL6-IV gene function or reduction, and these methods comprise:
1. pass through the transfer-gen plant of the gene constructed overexpression of overexpression miR171;
2. adopt fast neutron, gamma-ray physical method; The method that chemical mutagens such as ethylmethane sulfonate, T-DNA or transposon insert; Through making up mutant library and screening mutant, obtain scl6-II, scl6-III and scl6-IV two mutants plant that SCL6-II, SCL6-III and SCL6-IV gene are undergone mutation respectively; With scl6-II, scl6-III and the sexual hybridization of scl6-IV single mutant, in the offspring, select to obtain scl6-II scl6-III double-mutant and scl6-II scl6-IIIscl6-IV Trimutant plant then;
3. utilize the cDNA sequence of SCL6-II, SCL6-III and SCL6-IV gene, make up the plant expression vector that RNA disturbs (RNAi), import plant and obtain transfer-gen plant; Screen the transfer-gen plant that each genetic expression descends respectively, can obtain the function total loss of 3 genes such as SCL6-II, SCL6-III and SCL6-IV or the plant of reduction.
The present invention is vegetable material with the Arabidopis thaliana; Make up overexpression plant, scl6-II scl6-III double-mutant and the scl6-II scl6-III scl6-IV Trimutant of miR171; Obtained the increase of branch minimizing, chlorophyll and anthocyanin content; Use gene to be: the gene of Arabidopis thaliana miR171c number is At1G62035, and the gene of Arabidopis thaliana SCL6-II number is At2G45160, and the gene of the gene of Arabidopis thaliana SCL6-III number is At3G60630; The gene of Arabidopis thaliana SCL6-IV number is At4G00150, and the gene of rice Os-miR171a number is Os06G13106.
All there are miR171 and SCL6-II, SCL6-III and SCL6-IV gene in all higher plants; Can set up the plant that branch minimizing, chlorophyll and anthocyanin content increase through overexpression miR171, equally can be through knocking out scl6-II scl6-III double-mutant and the scl6-II scl6-III scl6-IV Trimutant that the gene constructed branch minimizing of SCL6-II, SCL6-III and SCL6-IV, chlorophyll and anthocyanin content increase.These make up the method that reduces branch and the plant that increases chlorophyll and anthocyanin and are applicable to all dicotyledonous and monocotyledonss.At present the plant of the known miR171 of having and SCL6-II, SCL6-III and SCL6-IV gene comprises Arabidopis thaliana (Cruciferae Btassica) in the dicotyledons, tomato (Solanaceae tomato genus), tobacco (Solanaceae Nicotiana), pea (Papilionaceae) etc., the paddy rice in the monocotyledons (Gramineae Oryza) etc.
Utilize promotors such as 35S, pKY71 (open source literature source: Schardl etc., 1987, the Gene of cauliflower mosaic virus; 61:1-11) or pHB (open source literature source: Mao etc.; PNAS, 2005, the 102:12270-12275) gene order of plant expression vector such as grade and miR171; Come the plant expression vector of construction expression miR171, import plant and obtain transfer-gen plant.Such plant is the transfer-gen plant of overexpression miR171 gene.
The present invention also can adopt following method to make SCL6-II, SCL6-III and the forfeiture of SCL6-IV gene function or reduce, and comprises that the method that physics (fast neutron, gamma-rays), chemical mutagen (ethylmethane sulfonate), T-DNA or transposon insert obtains mutant library.Through round pcr and determined dna sequence; Identify SCL6-II, SCL6-III and the SCL6-IV gene order of two mutants; Scl6-II, scl6-III and the scl6-IV single mutant that can obtain SCL6-II, SCL6-III and SCL6-IV afunction or reduce;, in the offspring separable acquisition scl6-II scl6-III double-mutant and scl6-II scl6-III scl6-IV Trimutant further through sexual hybridization.
The cDNA sequence of the present invention SCL6-II also capable of using, SCL6-III and SCL6-IV gene makes up RNA interferential plant expression vector, and the importing plant obtains SCL6-II, SCL6-III and the SCL6-IV gene is not expressed or the transfer-gen plant of down-regulated expression.Such plant is the transfer-gen plant of SCL6-II, SCL6-III and SCL6-IV afunction or reduction.
In the present invention, " the gene function overexpression of miR171 " is meant that the gene function of miR171 is higher than wild-type or expression level is higher than wild-type.On phenotype, the phenotype that the overexpression of miR171 gene function causes the obviously minimizing of branch quantity, chlorophyll and the anthocyanin content of plant obviously to increase.The expression amount of miR171 is higher, and the phenotype that quantity minimizing of transfer-gen plant branch and chlorophyll and anthocyanin content increase is stronger.
In the present invention, " SCL6-II, SCL6-III and the forfeiture of SCL6-IV gene function perhaps reduce " is meant the whole or forfeiture partly of function of SCL6-II, SCL6-III and SCL6-IV gene.On phenotype; SCL6-II, SCL6-III and the forfeiture of SCL6-IV gene function or reduction cause plant faint branch phenotype may occur; But scl6-II scl6-III double-mutant similar branch quantity can occur with scl6-II scl6-III scl6-IV Trimutant significantly to be reduced, and chlorophyll and the remarkable phenotype that increases of anthocyanin content.
The present invention utilizes overexpression miRNA171 gene and makes SCL6 gene function inactivation or down-regulated expression, reduces the branch quantity of plant; The construction process of the transfer-gen plant of overexpression miRNA171 of the present invention is simple, and in the branch quantity that reduces dicotyledons, and the while is remarkable in the effect on aspect increase plant chlorophyll and the anthocyanin content; The present invention is easy to be obtained the obvious minimizing of branch quantity and not to have branched plant fully, and the chlorophyll of these plant leafs and all significantly increases of cyanidin(e) content.
Description of drawings
The phenotype of the 35Spro-miR171c transfer-gen plant of Figure 1A overexpression Arabidopis thaliana miR171c;
Lotus throne leaf branch amount quantitative analysis in Figure 1B 35Spro-miR171c overexpression plant;
Stem leaf branch amount quantitative analysis in Fig. 1 C 35Spro-miR171c overexpression plant;
Wherein: the Y axle representes that relevant branch number plant accounts for the per-cent of sum; Black: produce 0 percentage ratio that branched plant is shared; Grey: produce 1 percentage ratio that branched plant is shared, white: produce more than or equal to 2 percentage ratios that branched plant is shared.The sample number of each genotype statistics is more than or equal to 30.
Wherein: the genotype of digitized representation is respectively: wild-type WT (1), No. 2 plant of 35Spro-miR171c transgenic (2), No. 3 plant of 35Spro-miR171c transgenic (3) and No. 4 plant of 35Spro-miR171c transgenic (4), scale is 3 centimetres.
The structure synoptic diagram of the overexpression carrier of Fig. 2 A rice Os miR171a.
The phenotype of the 35Spro-OsmiR171a transfer-gen plant of Fig. 2 B overexpression rice Os miR171a
Fig. 3 A long day is 53 days plant phenotype analytical of growth down;
Wherein: the genotype of digitized representation is respectively: WT (1), scl6-II (2), scl6-III (3), scl6-IV (4), scl6-II scl6-III (5), scl6-II scl6-IV (6), scl6-III scl6-IV (7), scl6-II scl6-IIIscl6-IV (8) and 35Spro-MIR171#2 (9), scale is 3 centimetres.
Wild-type that Fig. 3 B and Fig. 3 C long day grew 44 to 53 days down and the analysis of two mutants plant branch quantity B and plant height C;
Wherein: represent that corresponding plant among the A respectively in the numeral of B and C base portion, relatively there is significant difference in relative genotype plant and the wild-type of asterisk representative, Student ' s t-test, and * * P＜0.01, n >=30, error line is represented ± SD.
Fig. 4 A long day is 25 days plant of growth down;
Wherein: wild-type (1), 35Spro-MIR171c#2 (2) and scl6-II scl6-III scl6-IV Trimutant (3), scale is 3 centimetres.
Fig. 4 B long day is 25 days blade of growth down;
Wherein: wild-type (1), 35Spro-MIR171c#2 (2) and scl6-II scl6-III scl6-IV Trimutant (3), scale is 1 centimetre.
Fig. 4 C long day is 7 days cotyledon of growth down;
Wherein: wild-type (1), 35Spro-MIR171c#2 (2) and scl6-II scl6-III scl6-IV Trimutant (3).Scale is 5 millimeters.
Fig. 4 D and Fig. 4 E long day be the chlorophyllous accumulation of plant of growth 10 days (D) and 25 days (E) down;
Wherein: wild-type (white), 35Spro-MIR171c#2 (black) and scl6-II scl6-III scl6-IV Trimutant (grey).Relatively there is significant difference in relative genotype plant and the wild-type of asterisk representative, Student ' s t-test, and * * P＜0.01, n >=30, error line representes ± SD that experiment repeats through three times, all shows similar situation at every turn.
Fig. 5 utilizes 5 days seedling to measure the content of anthocyanin;
Wherein: 1 is wild-type, and 2 is the 35Spro-MIR171c transfer-gen plant, and 3 is scl6-II scl6-III scl6-IV Trimutant.
The dark held of the excised leaf of wild-type that Fig. 6 long day grew 21 days and 35Spro-MIR171c transfer-gen plant two days;
Wherein: 1 is wild-type, and 2 is the 35Spro-MIR171c transfer-gen plant.
The dehydration experiment that Fig. 7 utilizes about 21 days wild-type of growth and 35Spro-MIR171c transfer-gen plant plant excised leaf to carry out; Water conservation be the 35Spro-MIR171c transfer-gen plant; Percentage of water loss is the lost moisture and the per-cent of initial fresh weight; Illustrated value is three MV and the standard deviations measured, and sample size each time is a 5-8 sheet excised leaf;
Wherein: 1 is wild-type, and 2 is the 35Spro-MIR171c transfer-gen plant.
The siccocolous experiment that Fig. 8 utilizes about 21 days wild-type of growth and 35Spro-MIR171c transfer-gen plant excised leaf to carry out, drought-resistant is the 35Spro-MIR171c transfer-gen plant;
Wherein: 1 is wild-type, and 2 is the 35Spro-MIR171c transfer-gen plant.
Embodiment
Provide embodiment below in conjunction with summary of the invention and accompanying drawing, further set forth the present invention.The present invention makes the method for the gene function overexpression of miR171 be not limited to aforesaid method, and any method that can make the gene function overexpression of miR171 may be used to the technical scheme of embodiment of the present invention.The present invention makes the method for SCL6-II, SCL6-III and forfeiture of SCL6-IV gene function or reduction be not limited to aforesaid method, and any method that can make SCL6-II, SCL6-III and the forfeiture of SCL6-IV gene function or reduce may be used to the technical scheme of embodiment of the present invention.Following examples only be used to the present invention is described and be not used in the restriction scope of the present invention.
The experimental technique of unreceipted actual conditions among the following embodiment is usually according to normal condition, like molecular cloning laboratory manual (Molecular cloning:A laboratory manual; 3rd ed.; Sambrook etc., Cold Spring HarborLaboratory, 2001) and molecular biology of plants laboratory manual (Plant Molecular Biology-A LaboratoryManual; Clark etc.; Springer-Verlag, 1997) condition described in, or the condition of advising according to manufacturer.
The transfer-gen plant that in Arabidopis thaliana, makes up overexpression Arabidopis thaliana miR171 gene reduces the branch quantity of plant
With Arabidopis thaliana miR171c is example, and the building process of the transfer-gen plant of miR171 overexpression is described.With Arabidopis thaliana (Arabidopsis thaliana) the miR171c gene clone of pcr amplification BamHI and XbaI site to the pBS carrier, after the order-checking with the segment of miR171c with BamHI and XbaI cutting-out, insert pKY71 (Schardl etc. respectively; 1987, Gene, 61:1-11) carrier or pHB (Mao etc.; PNAS; 2005,102:12270-12275) back of the 35S promoter of carrier obtains the plant expression vector of overexpression miR171, transforms Agrobacterium GV3101 bacterial strain with the plasmid that obtains.With the bacterial strain that obtains, through flower-dipping method (Clough and Bent, 1998; Plant J; 16:735-743) again this carrier is transformed plant, obtain the 35Spro-MIR171c transfer-gen plant of overexpression miR171c gene, these transfer-gen plant branch numbers obviously reduce (Figure 1A).Find through statistical analysis, No. 2 of the 35Spro-MIR171c plant with No. 3 transfer-gen plants in do not produce any lotus throne leaf branch above 70% plant, No. 4 transfer-gen plant does not almost all produce lotus throne leaf branch (Figure 1A, 1B).Same, in these transfer-gen plants, surpass 80% and all do not produce stem leaf branch (Fig. 1 C).These presentation of results obtain the method for the transfer-gen plant of this gene of overexpression through the miR171 gene that utilizes Arabidopis thaliana, can significantly reduce the branch quantity of plant.
The transfer-gen plant that in Arabidopis thaliana, makes up overexpression paddy rice miR171 gene reduces the branch quantity of plant
With paddy rice miR171a gene (Os-miR171a, Sunkar etc., 2005, Plant Cell, 17:1397-1411.Gene number: Os06G13106) be example, the building process of the transfer-gen plant of Os-miR171a overexpression is described.The segment of the Os-miR171a of pcr amplification is cloned into the BamHI and the XbaI site of pBS carrier; After the order-checking segment of Os-miR171a is downcut with BamHI and XbaI; The back of inserting the 35S promoter of pKY71 carrier or PHB carrier respectively obtains the plant expression vector (Fig. 2 A) of overexpression Os-miR171a, transforms Agrobacterium GV3101 bacterial strain with the plasmid that obtains.With the bacterial strain that obtains, again this carrier is transformed plant through flower-dipping method, obtain transfer-gen plant.Experiment through northern bolt detects the expression amount of Os-miR171a, and the transfer-gen plant of acquisition is the transfer-gen plant of overexpression miR171.These transfer-gen plant branch numbers obviously reduce, and many transfer-gen plants almost all do not produce lotus throne leaf branch (Fig. 2 B).These presentation of results obtain the method for the transfer-gen plant of this gene of overexpression through the Os-miR171 gene that utilizes paddy rice, can significantly reduce the branch quantity of plant.
In Arabidopis thaliana, make up the branch quantity that scl6-II scl6-III double-mutant and scl6-II scl6-III scl6-IV Trimutant reduce plant
With the Arabidopis thaliana is example, and the procurement process of SCL6-II, SCL6-III and the forfeiture of SCL6-IV gene function or reduction two mutants is described.Method with T-DNA inserts is formulated dicotyledons seed mutant library.With the M1 seedling in generation,, and confirm the mutational site through order-checking with PGR method increase respectively SCL6-II, SCL6-III and the SCL6-IV site of above two mutants.After obtaining each single mutant of scl6-II, scl6-III and scl6-IV of confirming, can obtain F1 for seed earlier with scl6-II and the hybridization of scl6-III two mutants.F2 is carried out the PCR screening for seedling, and the scl6-II scl6-III double-mutant plant of selecting SCL6-II, SCL6-III mutational site all to isozygoty hybridizes with the scl6-IV two mutants then.F1 for selfing after, obtain F2 for seed.After the F2 seed sterilization in generation, evenly plant on Murashige and Skoog (MS) solid medium, under light, cultivate after 6 days, select the seedling that main root shortens, the cotyledon green is obviously deepened, transplant to soil.Wait to grow up to bolting when blooming, carry out PCR and detect, obtain 3 genes such as SCL6-II, SCL6-III and the SCL6-IV scl6-IIscl6-III scl6-IV of body Trimutant that isozygotys of all undergoing mutation.Shown in Fig. 3 A, scl6-II scl6-III double-mutant and scl6-IIscl6-III scl6-IV Trimutant branch number obviously reduce (Fig. 3 A, No. 5 and No. 8 plant).Statistical analysis shows that the branch numbers of poles of scl6-II scl6-III double-mutant and scl6-II scl6-III scl6-IV Trimutant is markedly inferior to wild-type WT (Fig. 3 B), and their the plant plant height utmost point is higher than wild-type WT (Fig. 3 C) significantly.These presentation of results, SCL6-II, SCL6-III and SCL6-IV gene function are lost or the method for the two mutants of reduction through making up, and can significantly reduce the branch quantity of plant.
The transfer-gen plant and the scl6-II scl6-III scl6-IV Trimutant that in Arabidopis thaliana, make up overexpression Arabidopis thaliana miR171 gene increase chlorophyll content
Find through observing the 35Spro-MIR171c transfer-gen plant and the scl6-II scl6-III scl6-IV Trimutant that obtain; The blade of 35Spro-MIR171c transfer-gen plant and scl6-II scl6-III scl6-IV Trimutant Cheng Miao is obviously longer and narrower than the wild-type, and is deep green (Fig. 4 A and B).The cotyledon of same 35Spro-MIR171c transfer-gen plant and scl6-IIscl6-III scl6-IV Trimutant is obvious greener than the wild-type (Fig. 4 C) also.The chlorophyll content (method is with reference to 2009Nature 457:327-331 such as Ni) of scl6-IIscl6-III scl6-IV Trimutant is significantly higher than wild-type WT (Fig. 4 D and E) in Cheng Miao and seedling.These test-results explanations through obtaining the method for overexpression MIR171c transfer-gen plant or scl6-II scl6-III scl6-IV Trimutant, can significantly improve the content of plant chlorophyll.
Embodiment 5
The transfer-gen plant and the scl6-II scl6-III scl6-IV Trimutant that in Arabidopis thaliana, make up overexpression Arabidopis thaliana miR171 gene increase anthocyanin content
At 5 days seedling of normal growth, the cotyledon of 35Spro-MIR171c transfer-gen plant and scl6-II scl6-III scl6-IV Trimutant is obviously red in wild-type WT.Content through measuring anthocyanin (method is with reference to Yang etc., and 2000, Cell finds that 103:815-827) content of anthocyanin is minimum in the wild-type, in other two kinds of plant, anthocyanin content is about 2 times (Fig. 5) of wild-type.These test-results explanations through obtaining the method for overexpression MIR171c transfer-gen plant or scl6-IIscl6-III scl6-IV Trimutant, can significantly improve the content of plant anthocyanin.
Embodiment 6
The transfer-gen plant that in Arabidopis thaliana, makes up overexpression Arabidopis thaliana miR171 gene comes the anti-ageing ability of enhancement of plant
Blade isolated culture through the wild-type that will grow under normal operation 21 days and 35Spro-MIR171c transfer-gen plant is found; After dark held two days; The blade that can find wild-type is obviously old and feeble, and 35Spro-MIR171c transfer-gen plant aging rate obviously slow down (Fig. 6).These presentation of results through obtaining the method for overexpression MIR171c transfer-gen plant, can significantly improve the anti-ageing ability of plant.
Embodiment 7
The transfer-gen plant that in Arabidopis thaliana, makes up overexpression Arabidopis thaliana miR171 gene comes the drought-resistant ability of enhancement of plant
Plant through growth under normal operation carries out dehydration experiment, cuts the plant leaf of cultivating 21 days, is placed on temperature and is 22 ℃, relative humidity and keep 45% and intensity of illumination 140 μ mol.m
-2.s
-1The incandescent and fluorescent lamp handle down, measure its moisture loss, represent with the weight percent of initial fresh leaf, as (Leung etc., 1997, Plant Cell, 9:759-771) said.Dehydration through the excised leaf with wild-type WT and 35Spro-MIR171c transfer-gen plant carries out is tested.The result shows that the rate-of-loss of coolant of miR171 overexpression plant significantly is lower than wild-type WT (Fig. 7).In siccocolous experiment, find that the overexpression plant survival time of miR171 obviously prolongs in the plant of not watering; Under the identical time, the overexpression plant strain growth state of miR171 obviously is better than wild-type (Fig. 8).These presentation of results through obtaining the method for overexpression MIR171c transfer-gen plant, can significantly improve the drought-resistant ability of plant.
Claims (4)
- One kind reduce plant branching, increase chlorophyll and anthocyanin content, enhancement of plant is anti-ageing and the method for drought-resistant ability; It is characterized in that; Described plant is an Arabidopis thaliana, and the miR171c gene that this method is used to come from Arabidopis thaliana obtains the transgenic arabidopsis of this gene of overexpression.
- 2. method that reduces plant branching, increases chlorophyll and anthocyanin content; It is characterized in that; Described plant is an Arabidopis thaliana; This method makes SCL6-II, SCL6-III and forfeiture of SCL6-IV gene function or reduction in the Arabidopis thaliana through in Arabidopis thaliana, making up SCL-6II SCL6-III SCL6-IV Trimutant.
- 3. a method that obtains to reduce branch, increases the plant of chlorophyll and anthocyanin content is characterized in that described plant is an Arabidopis thaliana, and the miR171c gene that this method is used to come from Arabidopis thaliana obtains the transgenic arabidopsis of this gene of overexpression.
- 4. method that obtains to reduce branch, increases the plant of chlorophyll and anthocyanin content; It is characterized in that; Described plant is an Arabidopis thaliana; This method makes SCL6-II, SCL6-III and forfeiture of SCL6-IV gene function or reduction in the Arabidopis thaliana through in Arabidopis thaliana, making up SCL6-II SCL6-III SCL6-IV Trimutant.
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