CN103923916A - Application of OsFLA19 protein in regulation of plant leaf included angle - Google Patents

Abstract

The present invention discloses an application of OsFLA19 protein in regulation of the plant leaf included angle. The present invention provides an application of a substance expressed by the coding gene of OsFLA19 protein in a plant with silencing or inactivating target in regulation of the plant leaf included angle, wherein the amino acid sequence of the OsFLA19 protein is a sequence 2 in the sequence table, and the regulated plant leaf included angle is the increased plant leaf included angle. According to the present invention, experiment results show that: the DNA molecules comprising the partial specific sequence of the coding gene of the OsFLA19 is constructed into the target vector pTCK303 to obtain a RNA interference vector, an agrobacterium-mediated method is adopted to transform the RNA interference vector into rice Kitaake callus to obtain the OsFLA19 RNAi line, and the line presents the significantly-increased plant leaf included angle characteristic compared with the rice with no coding gene transformation so as to show that the silence of the OsFLA19 expression is closely related to the improvement of the rice leaf included angle.

Description

The application of OsFLA19 albumen in regulating plant leaf angle

Technical field

The present invention relates to biological technical field, relate in particular to the application of a kind of OsFLA19 albumen in regulating plant leaf angle.

Background technology

Paddy rice is one of important food crop in the whole world, accounts for 1/3 of global cereal crop cultivated area.Along with population increases rapidly, global crisis in food is day by day serious.Urbanization in China, water resources restriction etc. cause Cultivated Land Area Decrease, add pattern of farming adjustment, the existing downtrending of the actual cultivated area of paddy rice of China, meet the demand of huge population growth to rice, solve Food Security, must realize the new breakthrough on rice breeding.The sixties in 20th century, China selects the of short stem anti-fall kind that Comprehensive Traits is good, has started new era of China's rice dwarf breeding, and the selection and popularization of Semidwarf rice kind becomes very important research topic in rice breeding practice.Therefore, excavate and qualification affect the gene of rice dwarf, carry out the research of the aspect such as location, clone and the mechanism of action of dwarf-related gene, realization, for the orderly improvement of Plant Height of Rice, has very important theory significance and using value.

Downgrade as a kind of good character of paddy rice, there is effect resistant to lodging and volume increase, and the excavation of dwarf gene in paddy rice and breeding utilization are for huge contribution has been made in mankind's grain-production.But, very poor, the dwarf gene simplification of China's resource of short stem and available short source are more rare, genetic research shows, in production, the of short stem and Semi-dwarf cultivar overwhelming majority of application is kind and the derived varieties thereof with sd1, but chain due to sd1 gene and bad economical character, make it on producing, extensively utilize the risk (Yu et al., 2005) single by heredity and that bring that lies dormant.Therefore create, screen the short source of non-sd1, to expand the hereditary basis of dwarf gene in paddy rice, increase genetic diversity or be very important and necessary as the deposit of dwarf gene, the research is subject to vast rice breeding worker and germ plasm resource worker's great attention; Up to the present, found multiple Dwarf Mutant, and most of mutant is all excessively downgraded or do not possess practical economical character.Therefore excavate new short bar resource, seek new short source breeding to using value by number of ways, in theory still on breeding practice, all will there is very important practical value.

Rice plant is downgraded the effect of being commonly considered as dwarf gene and is caused the variation on rice plant morphology or cytology, as internode shortens and cell number minimizing etc.; Meanwhile, the expression of gene is also subject to the impact of outside atmosphere and endogenous condition.In recent years, extensive and deep research has been carried out in the aspect such as clone and utilization of the genetics to rice dwarf genes involved, the hormone regulating and controlling of Dwarf Mutant and dwarf-related gene, has especially made significant headway utilizing aspect genetic engineering means control Plant Height of Rice.Plant hormone almost participates in the whole process of rice growth, and dwarfing plants sudden change is relevant with Brassinosteroids (Brassinosteroid, BR) with plant Plant hormones regulators,gibberellins (Gibberellin, GA); The sudden change of minority dwarfing plants is relevant with growth hormone (Auxin, IAA).Rice dwarf mutant dwarfl (d1) is GA non-sensitive type mutant, shows as dwarfing, leaf is wide and be blackish green, inflorescence is tight.Research shows, water f rice 1G base Adwar is in conjunction with albumen because of coding, and this albumen plays an important role f1GTP (Ashikari et al., 1999) in the growing of plant.The rice dwarf mutant gid2 of the separation such as Sasaki is also GA insensitiveness mutant, because suppressing the signal conduction of GA, causes plant to downgrade (Sasaki et al., 2003).Paddy rice brd1 mutant is a kind of BR defective type Dwarf Mutant, and external source applies BR can return to normal phenotype.This mutant leaf sheath is short, blade is short and bending, tiller less, sterile.Endogenous BR content analysis finds that the BR-6-oxydase in BR building-up process reduces (Mori et al., 2002).Hong etc. study discovery, and Dwarf Mutant d2 is BR responsive type mutant, and external source applies 10 ^-6the BL (a kind of activity form of BR) of M, can make the phenotype of mutant return to wild-type, the D2 gene that utilized d2 mutant clone, a newcomer in this genes encoding Cytochrome P450 family, belong to the CYP90D family (Hong etal., 2003) similar to BR synthetic enzyme height.Along with carrying out and widespread use of RFLP, RAPD, AFLP, SSR equimolecular labeling technique, the gene of many control plant height proterties has obtained location and clone, thereby utilize the biosynthesizing of these Gene Handling GA or BR or signal conduction to obtain applicable plant height, to be following important means (Mori et al., 2002 that produce; Hong et al., 2003; Hedden et al., 2003; Sakamoto et al., 2003).

Rice yield and its plant type are closely related, except plant height, also comprise tillering number, tillering angle, floral shape and leaf angle etc.It is the New Policy that obtains at present increasing production of rice that upright blade plant type and dense planting combine, and the plant that has upright blade can absorb more sunlight and promote photosynthesis and seed grouting, thereby improves the output of whole strain.Paddy rice is exactly the phenotype that produces upright blade to an important morphological feature of brassinolide (BR) response.Scientists research synthetic to BRs in paddy rice and signal transducers mechanism has for many years dropped into great enthusiasm, and increasing paddy rice BRs key element synthetic and signal transduction is cloned and confirms.The mutant of the synthetic mutant reducing of BR and signal weakening shows the features such as dwarfing, upright blade, seed diminish, for example brd2, d2, d11, brd1, d61, on the contrary, the positive regulatory factor of overexpression synthetic gene or signal transduction can increase the angle of leaf angle, for example overexpression BZR1.Therefore, separating rice brassinolide signal element, the molecular mechanism that not only perfect rice-rape element lactone signal transmits, and for improvement plant type of rice, to improve rice yield also significant.

There is Arabinogalactan-Protein (the Arabinogalactan proteins of class fasciclin structural domain (Fasciclin-like domain), AGP) be the class in Arabinogalactan-Protein family, it not only includes the glycosylation region of class AGP, but also there is l-2 fasciclin-like structural domain, this proteinoid is by many functions of exercising molecule adhesion that experimental results show that.The methods analyst by information biology such as Faik the FLA gene in paddy rice and wheat, found that, the FLA albumen structure identical with having of Arabidopis thaliana of these cereals, comprises fasciclin-like and AGP-like structural domain.In these genes 70% are also contained GPI-anchor series by supposition.RNAgel blot analyzes and finds most of FLA genes faint expression in seed and root, and the expression of most of wheat FLA genes is lowered (Faik et al., 2006) because of coercing of abiotic condition.Up to the present, in rice genome, existing 29 members of FLA gene family are out identified.The FLA genoid one of having reported in arabidopsis gene group has 24 members.That also knows about the function of plant FLA albumen at present is less, and the evidence that oneself has shows, FLA albumen may work in the process of cell elongation, cytoadherence and secondary wall maturation.When a sos5 of Arabidopis thaliana (fla4) mutant is grown on the substratum of high salt concentration, the elongation that shows tip of a root expansion, root has been subject to inhibition, sequencing result shows that mutational site is arranged in this intragenic fasciclin-like structural domain, therefore, FLA4 is cell proliferation necessary (shi et al., 2003).Another FLA Gene A of Arabidopis thaliana tFLA11, its mRNA is specifically expressing in the sclerenchyma (sclerenchyma) of inflorescence stem and pod, and expression intensity is along with the maturation of fruit constantly strengthens, infer that AtFLA11 may promote the lignifying of secondary cell wall, the maturation of secondary cell wall is had to important effect (Ito et al., 2005).

At present, although people make some progress the research of AGP genoid family protein, the research in paddy rice is also fewer, and the biological procedures that this genoid participates in is not clear.

Summary of the invention

An object of the present invention is to provide the purposes of the material that in silence or inactivation object plant, OsFLA19 protein coding gene is expressed.

The invention provides the application in regulating plant leaf angle of material that OsFLA19 protein coding gene in silence or inactivation object plant expresses; The aminoacid sequence of described OsFLA19 albumen is the sequence 2 in sequence table.

In above-mentioned application, the nucleotides sequence of described OsFLA19 protein coding gene is classified the sequence 1 in sequence table as.

In above-mentioned application, in described silence or inactivation object plant OsFLA19 protein coding gene express material be following 1)-3) and at least one:

1) RNA molecule, for following (a) or (b):

(a) the RNA molecule shown in the sequence 4 of sequence table (single stranded RNA or double-stranded RNA);

(b) with the RNA molecule (single stranded RNA or double-stranded RNA) of sequence 4 reverse complementals of sequence table;

2) DNA molecular of above-mentioned RNA molecule;

3) recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contain described DNA molecular.

In above-mentioned application, the nucleotides sequence of described DNA molecular is classified sequence 3 in sequence 3 or the sequence table in sequence table as from 5 ' end 16-1199 position Nucleotide;

Described recombinant vectors is that described DNA molecular is inserted in expression vector, the carrier obtaining, and in an embodiment of the present invention, expression vector is specially carrier pTCK303.

In above-mentioned application, described regulating plant leaf angle is to increase leaf angle.

Above-mentioned being applied as imports described DNA molecular in object plant, obtains transgenic plant, and the leaf angle of described transgenic plant is greater than described object plant;

Described leaf angle is specially the first leaf angle under sword-like leave angle or sword-like leave;

In above-mentioned application, described object plant is dicotyledons or monocotyledons; Described monocotyledons is specially paddy rice.

Another object of the present invention is to provide a kind of method of cultivating transgenic plant.

Method provided by the invention, comprises the steps: that in silence or inactivation object plant, OsFLA19 protein coding gene is expressed, and obtains transgenic plant, and the leaf angle of described transgenic plant is greater than described object plant; The aminoacid sequence of described OsFLA19 albumen is the sequence 2 in sequence table.

In aforesaid method, the nucleotides sequence of described OsFLA19 protein coding gene is classified the sequence 1 in sequence table as.

In aforesaid method, in described silence or inactivation object plant, OsFLA19 protein coding gene is expressed as the described DNA molecular in above-mentioned application is imported in object plant, obtains transgenic plant.

In aforesaid method, described described DNA molecular in above-mentioned application is imported to object plant by the described recombinant vectors in above-mentioned application.

Described leaf angle is the first leaf angle under sword-like leave angle or sword-like leave.

In aforesaid method, described object plant is dicotyledons or monocotyledons; Described monocotyledons is specially paddy rice.

Of the present invention experimental results show that, the OsFLA19 that the present invention finds, the DNA molecular of its encoding gene part distinguished sequence composition is building up in object carrier pTCK303, obtain rna interference vector, utilize agrobacterium-mediated transformation by rna interference vector rice transformation Kitaake callus, detect through hygromycin selection and PCR, obtain the RNAi strain of OsFLA19, this plant shows the characteristic that obvious leaf angle increases compared with not proceeding to the paddy rice of this gene, illustrates that reticent OsFLA19 expresses with raising rice leaf intersection angle closely related.

Brief description of the drawings

Fig. 1 is the RT-PCR method more special sequence in the full-length cDNA of OsFLA19 that increases

Fig. 2 is the part-structure schematic diagram of RNAi expression vector OsFLA19RNAi

Fig. 3 is the Real-time-PCR qualification of transgenic paddy rice

Fig. 4 is that OsFLA19RNAi transgenic paddy rice leaf angle becomes large Phenotypic Observation

Embodiment

The experimental technique using in following embodiment if no special instructions, is ordinary method.

Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.

The structure of embodiment 1, interference OsFLA19 encoding gene expression vector OsFLA19RNAi and the acquisition of interference DNA molecular

1, disturb the acquisition of the target fragment of OsFLA19 encoding gene expression

According to the paddy gene sequence of submitting at GenBank, find coding OsFLA19 gene, its complete reading frame sequence is for No. GenBank NM_001053180(sequence 1; The albumen called after OsFLA19 of coding, its aminoacid sequence is sequence 2 in sequence table), design accordingly forward and reverse primer, 5 ' end primer: 5 '-CGG gGTACC? aCTAGTcAATAAGGAGCACAAGAAG-3 ' (underscore sequence is Kpn I, Spe I site), 3 ' end primer: 5 '-CGC gGATCC? gAGCTCcTGAGTGAATCATCATCAACA-3 ' (underscore sequence is BamH I, Sac I site), with Kitaake paddy rice (Oryza sativaL.cv.Kitaake in tri-leaf period, Qu et al., J.Exp.Bot.2008,59:24172424, the public can obtain from Institute of Botany, Chinese Academy of Sciences, is designated hereinafter simply as wild-type paddy rice.) cDNA of the total RNA reverse transcription of seedling is template, adopts the increase distinguished sequence of 316bp in OsFLA19 full-length cDNA of RT-PCR method.Specific operation process is as follows:

The extraction of rice total RNA: choose Kitaake paddy rice in tri-leaf period.Seedling 100mg is material, in liquid nitrogen, grinds, and the lyophilized powder grinding in liquid nitrogen is transferred in the 1.5ml centrifuge tube containing 1ml Trizol reagent (Invitrogen), fully mixes; 25 DEG C of placements of room temperature 5 minutes; In every pipe, add the fresh chloroform of 0.2ml, violent jolting 15 seconds, 25 DEG C of incubations 2 ~ 3 minutes; 12,000rpm, 4 DEG C, centrifugal 15 minutes; The water 0.5ml of supernatant is transferred in a new 1.5ml centrifuge tube, add 0.5ml Virahol, room temperature is placed and within 10 minutes, is made RNA precipitation; 12,000rpm, 4 DEG C, centrifugal 10 minutes; Remove supernatant, RNA precipitation is cleaned 2 times with 1ml75% ethanol, super clean bench blows to half-dried; With 50 μ l DEPC-ddH ₂the resuspended precipitation of O, 60 DEG C of water-baths 10 minutes, to dissolve RNA precipitation.By-70 DEG C of preservations after this RNA solution packing, the standby template of doing reverse transcription.

RT-PCR: get the above-mentioned RNA sample of 1 μ l, use DEPC-ddH ₂100 times of O dilutions, by spectrophotometric determination RNA concentration.With reference to RT-PCR test kit (Promega) specification sheets, according to the quantitative result of RNA, get 2 these RNA of μ g, add 1.0 μ gOligo dT primers, use DEPC-ddH ₂o is supplemented to 15 μ l, mixes rear 70 DEG C of sex change 5 minutes, ice bath 5 minutes.Of short duration centrifugal after, add 25 μ l reverse transcription mixtures (5 μ l M-MLV5 × ReactionBuffer, 6 μ l dNTP Mixture (2.5mM), 1 μ l M-MLV Reverse Transcriptase, 0.5 μ l RNaseInhibitor, 12.5 μ l DEPC-ddH ₂o).After mixing, 42 DEG C of water-baths complete transcriptive process,reversed for 1 hour; 75 DEG C of water-baths make ThermoScript II inactivation in 10 minutes, obtain the mixture that contains the first chain cDNA.

Get the template of above-mentioned the first chain cDNA of 1 μ l as PCR, carry out PCR reaction by following system: 0.2 μ l LATaq(5U/ μ l), 10 μ l2 × GC buffer, 1.8 μ l dNTPs, 0.5 μ l5 ' end primer (10 μ M), 0.5 μ l3 ' end primer (10 μ M), adds ddH ₂o final volume 20 μ l.Primer sequence is as front, and PCR program is: 94 ° of C denaturations enter PCR circulation after 3 minutes, and loop parameter is that extend for ° C1 minute ° C30 renaturation → 72 second, 94 ° of C30 sex change → 58 second, after 30 circulations, continues synthetic 10 minutes at 72 ° of C.

The PCR product of amplification separates through 0.8% agarose gel electrophoresis, and result as shown in Figure 1, as can be seen from the figure, obtains the band of the about 0.3kb of molecular weight, reclaims this segment obtain 20 μ l recovery products with AxyPrep DNA gel recovery test kit.Carry out sequencing analysis, the PCR fragment of sequencing result 316bp has the Nucleotide of sequence 1 between 5 ' end 713-1028 in sequence table, by this PCR fragment called after A.

The structure of cloning vector and purifying: get the above-mentioned recovery fragment of 3.5 μ l, add T4-DNA ligase enzyme 1 μ l(3U/ μ l), 2 × ligase enzyme damping fluid, 5 μ l, pGEM-T Easy carrier (Promega) 0.5 μ l(50mg/ml) 4 DEG C of connections spend the night, to connect product and transform bacillus coli DH 5 alpha competent cell, obtain transformant through the resistant panel screening containing Pyocianil, obtain plasmid pTeasy-A.

Through order-checking, plasmid pTeasy-A is for by sequence in sequence table 1, the Nucleotide between 5 ' end 713-1028 inserts the plasmid obtaining in pGEM-T Easy carrier.

2, the structure of OsFLA19RNAi expression vector

PTCK303 carrier enzyme is cut: be documented in as in Publication about Document with restriction enzyme Spe I and Sac I double digestion carrier pTCK303(pTCK303: Wang Z, Chen C, Yunyuan Xu, et al.2004.A Practical Vectorfor Efficient Knockdown of Gene Expression in Rice (Oryza sativa) .PlantMolecularBiology reporter22:1-9.; The public can obtain from Institute of Botany, Chinese Academy of Sciences), the enzyme system of cutting is: plasmid 10 μ l, 10x enzyme cutting buffering liquid 5 μ l, Spe I 1 μ l(10U/ μ l), Sac I I0.8 μ l(10U/ μ l), add ddH ₂o postreaction system to 50 μ l, 37 DEG C of enzymes are cut 4 hours.With agarose gel electrophoresis, enzyme is cut to product and separate, reclaim the linearizing pTCK303 large fragment of 14621bp, be dissolved in 20 μ l ddH ₂in O.

The acquisition of forward fragment and oppositely fragment: with restriction enzyme Spe I and Sac I similarity condition double digestion pTeasy-A and reclaim 316bp enzyme and cut product, called after OsFLA19F(forward fragment).Equally, carrier pTeasy-A is carried out to double digestion and reclaim 316bp enzyme and cut product, the reverse fragment of called after OsFLA19R(with restriction enzyme BamHI and KpnI).

OsFLA19RNAi expression vector: fetch the OsFLA19F product 10 μ l of the 316bp of receipts, the carrier pTCK303 solution 6 μ l of recovery, (3U/ μ l), 10x ligase enzyme damping fluid 2 μ l are mixed with T4DNA ligase enzyme 2 μ l, 16 DEG C connect 16 hours, obtain connecting product, connection product is proceeded in intestinal bacteria, obtain transformant.Extract the plasmid of transformant, send to order-checking, by this plasmid called after OsFLA19F/pTCK303.Order-checking successfully this plasmid is carried out double digestion with restriction enzyme KpnI and BamH, and the enzyme system of cutting is: plasmid 10 μ l, 10x enzyme cutting buffering liquid 5 μ l, KpnI1 μ l(10U/ μ l), BamH I0.8 μ l(10U/ μ l), add ddH ₂o postreaction system to 50 μ l, 37 DEG C of enzymes are cut 4 hours.With agarose gel electrophoresis, enzyme is cut to product and separate, reclaim the linearizing OsFLA19F/pTCK303 large fragment of 14937bp, be dissolved in 20 μ l ddH ₂in O.Fetch the OsFLA19R product 10 μ l of the 316bp of receipts, the carrier OsFLA19F/pTCK303 solution 6 μ l of recovery, (3U/ μ l), 10x ligase enzyme damping fluid 2 μ l are mixed with T4DNA ligase enzyme 2 μ l, 16 DEG C connect 16 hours, obtain connecting product, connection product is proceeded in intestinal bacteria, obtain transformant.Extract the plasmid of transformant, send to order-checking.

This plasmid is the plasmid obtaining between the Spe I of the DNA molecular insertion pTCK303 carrier shown in the sequence in sequence table 3 and BamH, be the recombinant vectors that contains forward and reverse insertion object fragment OsFLA19F and OsFLA19R, called after OsFLA19RNAi(OsFLA19RNAi carrier structure collection of illustrative plates as shown in Figure 2).

DNA molecular shown in sequence 3 is made up of forward fragment, intron and reverse fragment, forward fragment is that in sequence table, sequence 3 is from 5 ' end 884-1199 position Nucleotide (be in sequence table sequence 1 from the Nucleotide of 5 ' end 713-1028), and in intron sequences table, sequence 3 is from 5 ' end 394-871 position Nucleotide; Oppositely fragment be in sequence table sequence 3 from 5 ' end 16-331 position Nucleotide.The RNA of the DNA molecule encode shown in sequence 3 is single stranded RNA or double-stranded RNA, the RNA molecule shown in the sequence 4 that this RNA is sequence table or with the RNA molecule of sequence 4 reverse complementals of sequence table.

Acquisition and the phenotype research of embodiment 2, interference OsFLA19 encoding gene express transgenic paddy rice

One, disturb the acquisition of OsFLA19 encoding gene express transgenic paddy rice

1, disturb the acquisition of OsFLA19 encoding gene express transgenic paddy rice

Genetic transformation: with reference to electric exciter (EasyJecT Plus electric exciter, EquiBio company of Britain) operational guidance, plasmid OsFLA19RNAi is transformed to Agrobacterium EHA105(Biovector Co. with electric shocking method, the catalog number (Cat.No.) Biovec-11 of LTD company), obtain the RNAi engineering bacteria of positive colony through the resistant panel screening containing kantlex, called after EHA105/OsFLA19RNAi.

The screening of the positive seedling of paddy rice: by EHA105/OsFLA19RNAi Introduced into Rice Kitaake(Oryza sativa L.cvKitaake, wild-type paddy rice) callus, by the sterilized water washing 4-5 time containing 300mg/L cephamycin for this callus, after blotting, aseptic filter paper goes to N ₆d ₂s ₁substratum (in subordinate list) is upper, a screening generation.After two weeks, be transferred to N ₆d ₂s ₂substratum (in table 1) two generations of upper screening (2 weeks/generation).Taking-up is screened eugonic resistant calli through 3 generations, is transferred to pre-division culture medium (in table 1) upper, in differentiation culture case (12 hour photoperiod, 28 DEG C of daytimes, 25 DEG C of nights), cultivates 7 days; Then be transferred on division culture medium, in differentiation culture case, be cultured to generation regrowth.The plant of regeneration is at the upper strong plantlets and rootage of Rooting and hardening-off culture base (in table 1).In the time that seedling grows to 10 centimetres of left and right, open container closure film, hardening 2-3 days, then moves into seedling phytotron cultivation, and in totally 21 T0 generations, turn OsFLA19RNAi rice strain to obtain 7.

The substratum and the composition thereof that in the cultivation of table 1 rice tissue and conversion process, use

2, the qualification of transgenic paddy rice

1) GUS histochemical stain (having gus gene on pTCK303 carrier): the long root segment of 2-3mm that is turned OsFLA19RNAi paddy rice by above-mentioned 1 21 T0 generations that obtain is put into respectively in GUS staining fluid, the several minutes of bleeding, then be placed in 37 DEG C and be incubated overnight, 70% ethanol decolorization of the tissue after dyeing.It is positive transgenic line that root is all blue plant.GUS staining fluid (pH7.0) component is: 100mM Na ₃pO ₄(pH7.0), 0.1%TritonX-100,10mM EDTA, 0.5mM yellow prussiate of potash, the 0.5mM Tripotassium iron hexacyanide, 1mg/ml X-Gluc.Result identifies altogether 5 strains and adds up to 15 positive T0 generations to turn OsFLA19RNAi paddy rice, this seedling is moved to greenhouse production, according to different strain sowings, obtain T1 for turning OsFLA19RNAi rice paddy seed, on inferior basis, obtain isozygotying T2 for turning OsFLA19RNAi rice paddy seed through breeding.In experiment afterwards, choosing T2 is material for turning OsFLA19RNAi paddy rice (R1), (R3) and homozyous seed T2 (R6).

2) quantitative PCR qualification: turn from being numbered the T2 generation of R1, R3 and R6 the seedling of OsFLA19RNAi paddy rice and extract mRNA, and transcribe respectively obtain cDNA(with reference to the method in embodiment 1), taking wild-type paddy rice (paddy rice kitaake) for contrasting.Utilize fluorescence real-time quantitative PCR method, taking cDNA as template, with 5 ' end primer (10 μ M) (5 '-ACGCTGCTCCGCCTCCTCAA-3 '), 3 ' end primer (10 μ M) (5 '-GCCGTCGTAGGTGGTCTTCA-3 ') is that the gene expression abundance of OsFLA19 in primer pair transfer-gen plant detects.Reagent for quantitative analysis is SYBR Green Real-time PCR Master Mix(TOYOBO).Instrument is the real-time fluorescence quantitative PCR instrument Mx3000P of Stratagene company of the U.S..Draw 1 μ l the first chain cDNA solution, dilute 50 times as template, carry out PCR reaction by following system: 10 μ l SYBR Green Realtime PCRMaster Mix, 4 μ l masterplates, 1 μ l5 ' end primer 1 (10 μ M), 1 μ l3 ' end primer 1 (10 μ M), adds ddH ₂o final volume 20 μ l.

With Ubiqutin as internal reference, its 5 ' end primer: 5 '-ACCACTTCGACCGCCACTACT-3 ', 3 ' end primer be: 5 '-ACGCCTAAGCCTGCTGGTT-3 '.PCR program is: denaturation 2 minutes, enter PCR circulation, and loop parameter is ° C15 second → 72,94 ° of C15 second → 57 ° C15 second, totally 40 circulations.

Result as shown in Figure 3, at Ubiqutin gene as internal reference in the situation that, compared with wild-type paddy rice (WT), the gene expression abundance that the T2 generation of R1, R3 and R6 turns OsFLA19 in OsFLA19RNAi rice seedling has had downward in various degree, the carrier that illustration purpose gene connects has successfully proceeded to paddy rice, and it has disturbed the expression of OsFLA19 gene in paddy rice.

Adopting uses the same method proceeds to empty carrier pTCK303 in wild-type paddy rice, obtains turning empty carrier paddy rice, and sowing sowing turns empty carrier paddy rice until obtain T2 generation, adopts above-mentioned quantitative PCR qualification, and result and wild-type are without significant difference.

Two, disturb the phenotype research of OsFLA19 encoding gene express transgenic paddy rice

In wild-type paddy rice, T2 generation of being numbered R1 and R6, turned to the seed that OsFLA19RNAi paddy rice and T2 generation turn empty carrier paddy rice and broadcast in the mixture of flower nutrition soil and vermiculite (both blending ratios are 9:1), after 30 DEG C of sproutings, be placed on (25 DEG C) in greenhouse and be cultured to tri-leaf period, after outdoor strong sprout, be transplanted to mid-May in outdoor paddy rice solarium and be cultured to heading stage.

Each strain 8 strains, test in triplicate results averaged.

Observe leaf angle (the first leaf angle under sword-like leave angle and sword-like leave) growth phenotype result as follows:

The observation of taking pictures, result is as Fig. 4 A, wherein wild-type paddy rice WT(Kitaake), T2 generation turns OsFLA19RNAi paddy rice (R1, R6), can see that T2 generation turns the obvious leaf angle of OsFLA19RNAi paddy rice and becomes large phenotype.

At after planting approximately 80 days statistics leaf angles, (sword-like leave angle was the angle of sword-like leave and stem stalk; Under sword-like leave, the first leaf angle is the angle of the first leaf and stem stalk under sword-like leave), under wild-type Flag Leaves in Rice angle, sword-like leave the first leaf angle be respectively 41.0,28.8(degree); The T2 generation that is numbered R1 turns under OsFLA19RNAi rice leaf intersection angle, sword-like leave that the first leaf angle is respectively 80.0,50.0(degree), the T2 generation that is numbered R6 turns under OsFLA19RNAi paddy rice R6 leaf angle, sword-like leave that the first leaf angle is respectively 56.0,34.0(degree).

Calculate relative leaf angle, leaf angle is the ratio that T2 generation turns OsFLA19RNAi paddy rice and wild-type leaf angle relatively, wild-type rice leaf intersection angle is denoted as to 1, result as shown in Figure 4 B, can find out, the relative leaf angle of sword-like leave of wild-type paddy rice, under sword-like leave, the relative leaf angle of the relative leaf angle of the first leaf and the second leaf under sword-like leave is 1, in the T2 generation that is numbered R1, turns the relative leaf angle of sword-like leave of OsFLA19RNAi paddy rice, under sword-like leave, the relative leaf angle of the first leaf is 1.95, 1.74, in the T2 generation that is numbered R6, turns the relative leaf angle of sword-like leave of OsFLA19RNAi paddy rice, under sword-like leave, the relative leaf angle of the first leaf is 1.37, 1.18.T2 is for turning empty carrier paddy rice and wild-type paddy rice result without significant difference.

Illustrate and disturb the expression of rice Os FLA19 gene can improve leaf angle.

Claims (10)

1. the application of the material that in silence or inactivation object plant, OsFLA19 protein coding gene is expressed in regulating plant leaf angle; The aminoacid sequence of described OsFLA19 albumen is the sequence 2 in sequence table.

2. application according to claim 1, is characterized in that:

The nucleotides sequence of described OsFLA19 protein coding gene is classified the sequence 1 in sequence table as.

3. application according to claim 1 and 2, is characterized in that:

In described silence or inactivation object plant OsFLA19 protein coding gene express material be following 1)-3) and at least one:

1) RNA molecule, for following (a) or (b):

(a) the RNA molecule shown in the sequence 4 of sequence table;

(b) with the RNA molecule of sequence 4 reverse complementals of sequence table;

2) the encode DNA molecular of described RNA molecule;

3) recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contain described DNA molecular.

4. application according to claim 3, is characterized in that:

The nucleotides sequence of described DNA molecular is classified sequence 3 in sequence 3 or the sequence table in sequence table as from 5 ' end 16-1199 position Nucleotide;

Described recombinant vectors is that described DNA molecular is inserted in expression vector, the carrier obtaining.

5. according to arbitrary described application in claim 1-4, it is characterized in that:

Described object plant is dicotyledons or monocotyledons.

6. cultivate a method for transgenic plant, comprise the steps: that in silence or inactivation object plant, OsFLA19 protein coding gene is expressed, obtain transgenic plant, the leaf angle of described transgenic plant is greater than described object plant; The aminoacid sequence of described OsFLA19 albumen is the sequence 2 in sequence table.

7. method according to claim 6, is characterized in that:

The nucleotides sequence of described OsFLA19 protein coding gene is classified the sequence 1 in sequence table as.

8. according to the method described in claim 6 or 7, it is characterized in that:

In described silence or inactivation object plant, OsFLA19 protein coding gene is expressed as the described DNA molecular in arbitrary described application in claim 3-5 is imported in object plant.

9. method according to claim 8, is characterized in that:

Described described DNA molecular in arbitrary described application in claim 3-5 is imported to object plant by the described recombinant vectors in arbitrary described application in claim 3-5.

10. according to arbitrary described method in claim 6-9, it is characterized in that:

Described object plant is dicotyledons or monocotyledons.