CN105316346A - Application of gene OsPIL16 in reducing rice plant height and improving tiller number - Google Patents

Abstract

The invention discloses application of a gene OsPIL16 in reducing rice plant height and improving tiller number. A cDNA fragment of the gene OsPIL16 serves as an application gene and the gene is transferred to rice forwards, so that the expression level of the OsPIL16 gene is improved, and a transgenic rice plant with improved rice OsPIL16 gene expression is obtained. The transgenic positive rice plant is manifested in reduced plant height and increased tiller number.

Description

Gene OsPIL16 is reducing the application in Plant Height of Rice, raising tiller number

Technical field

The present invention relates to field of agricultural sciences, specifically, the expression that the present invention relates to by improving rice Os PIL16 gene improves Tillering Ability in Rice and reduces Plant Height of Rice.

Background technology

Paddy rice is global topmost food crop, and the whole world has the population of 50% to take rice as staple food.Rice Production is to guarantee world food safety, and reduce poverty population and rural area employment play an important role.For a long time, paddy rice is the grain variety that China's sown area is maximum, ultimate production maximum, per unit area yield is the highest, is the staple food of China about 65% population, in grain-production and consumption, be in dominant position.We know that the three elements determining rice yield are spike number, grain number per spike and thousand seed weight, and spike number is determined by tiller number, so tiller number is one of proterties determining rice yield.In addition, Plant Height of Rice refers to the height of rice plant basal part of stem to fringe point (not comprising awn length).Traditional high stalk rice varieties property resistant to lodging is poor, and under nature adverse environmental factor, (as wind and rain) easily lodges and causes production loss in various degree.Although therefore plant height proterties does not directly form the output of paddy rice, be basic premise (Xu Xue etc., Nuclear Physics comment, 2008, the 25:171-175 of rice high yield stable yields; Wei Lingzhu etc., biotechnology journal, 2012,28:144-153).In recent years, increasing genetic evidence shows, plant height and Plant hormones regulators,gibberellins (Gibberellin, the GA) approach of many plants are closely related.In addition, other plant hormone comprises brassinolide (Brassinosteriod, BR) and growth hormone (Indole-3-aceticacid, IAA) and associated transcription factor of blooming, cell walls forms the plant height that gene also can affect plant.That studies along with rice tillering and plant height regulatory mechanism deepens continuously, and utilizes genetically engineered improvement Plant Height of Rice and tiller number to be the effective ways improving rice yield.

Growing by the strict regulation and control of inherited genetic factors of rice tillering.Axil place on the internode do not extended at base portion the tillering of paddy rice is initial, then independent of trunk diameter growth.The gene that the regulation and control of cloning the earliest are tillered is that MOC1 (MONOCULM1), MOC1 encode a GRAS family transcription factor.MOC1 deletion mutant body is not tillered, and the tiller number of the process LAN plant of this gene significantly increases, and infers that MOC1 is the Main Factors (Lietal.Nature2003,422:618 – 21) of adjusting and controlling rice tillering accordingly.The nucleoprotein that LAXPANICLE2 (LAX2) genes encoding one is new, the deletion mutant of this gene is similar to moc1 mutation type surface.The phenotype of Lax2moc1 double-mutant is even more serious, infers that LAX2 and MOC1 is worked by different approaches, or work in same protein complexes (Tabuchietal.PlantCell2011,23:3276 – 87.).How does is the activity of MOC1 conditioned? how does MOC1 activate downstream gene? the phenotype of tilleringanddwarf1/tillerenhancer (tad1/te) mutant is contrary with moc1 mutant, and tiller number increases.Encode anaphase of cell division of TAD1/TETAD1 starts co-activation of mixture (anaphase-promotingcomplex is called for short APC/C).APC/C is the E3 ubiquitin ligase of a function high conservative in eukaryote.TAD1 and MOC1 does mutually, forms a complex body with OsAPC10, and as co-activation of APC/C, the mode relied on by the cell cycle is degraded target MOC1 (Xuetal.2012NatureCommunications, 2012,3:750).Lower the expression that meristematic tissue determines gene homeobox1 subsequently, thus suppress the initial sum of lateral shoot meristem to be formed.MOC1 regulates the expression of two 2 transcription factor gene OSH1 and OsTB1 in downstream, thus participates in regulating merismatic initial sum axillary bud growth (Lietal.Nature2003; 422:618 – 621).Consistent with this, OsTB1 antagonism is tillered the activity (Guoetal.NatCommun2013 of negative regulator OsMADS57; 4:1566 – 1577).OsMADS57 can directly act on DWARF14 (D14, also known as making D88 or HTD2-HIGHTILLERINGANDDWARF2); D14/D88/HTD2 regulates tiller number (Gao, etal.PlantMolBiol2009 by regulating plant hormone only angle gold lactone signal approach; 71:265 – 76.; Liuetal.Planta2009; 230:649 – 58.).The check activity of OsMADS57 to D14/D88/HTD2 depends on interaction between OsTB1.The expression of OsMADS57 is also by the regulation and control (Guoetal.NatCommun2013 of miR444a; 4:1566 – 77.).Therefore, may realize by regulating only angle gold lactone (strigolactone, SL) signal pathway tillering of MOC1 regulation and control.

Tillering is a kind of proterties of complexity, and by multiple approach or gene regulating, wherein plant hormone plays key effect, except typical plant hormone auxin and phytokinin, and the formation that the only angle gold lactone SL negative regulation found recently is tillered.SL synthesizes at plant root, is upwards transported to stem by root, thus suppresses rice tillering.Therefore the gene participating in SL synthesis and signal pathway all have impact on rice tillering number, as D27 (DWARF27), D17, D10 and OsMAX1 (MOREAXILLARYGROWTH) mediate SL synthesis, D3, D14 and D53 have regulated and controled SL signal (Li Shujia etc., Botany Gazette, 2015,50:539-548).IDEALPLANTARCHITECTURE1/WEALTHYFARMER ' SPANICLE (IPA1/WFP) encoding transcription factors OsSPL14, its expression regulates by OsmiR156.IPA1 is by just regulating and controlling OsTB1 and suppress to tiller (Lu, etal.PlantCell2013; 25:3743 – 3759.).

In recent years, increasing genetic evidence shows, plant height and Plant hormones regulators,gibberellins (Gibberellin, the GA) approach of many plants are closely related.In addition, other plant hormone comprises brassinolide (Brassinosteriod, BR) and growth hormone (Indole-3-aceticacid, IAA) and associated transcription factor of blooming, cell walls forms the plant height that gene also can affect plant.GA is the important hormone (KazanandManners, JExpBot, 2011,62:4087-4100) of regulating plant plant height.3 the GA defective type half Dwarf Mutant d35, the sd1 (semidwarf1) and d18 that find in paddy rice cause (Itohetal. by gene OsKO2, OsGA20ox2 and OsGA3ox2 Mutation on GA route of synthesis respectively, PlantCell, 2002,14:57-70; Sasakietal., Nature, 2002,416:701-702; Itohetal., PlantMolBiol, 2004,533-547).GA signal pathway GID1 (GA-INSENSITIVEDWARF1) genes encoding GA receptor protein, this gene lacks functionality Mutant Rice gid1 plant significantly downgrades (Ueguchi-Tanakaetal., Nature, 2005,437:693-698).DELLA albumen is the negative regulatory factor of GA signal transduction path, and in paddy rice, two genes of encoding D ELLA albumen are SLR1 (SLENDERRICE-1) and SLRL1 (SLR1-LIKE1).The plant height of SLR1 and SLRL1 gene overexpression paddy rice significantly reduces (Itohetal., PlantCell, 2002,14:57-70).Recently, the people such as Kovi (TheorApplGenet, 2011,123:705-714) navigate to the gene that regulates Plant Height of Rice, the Plant hormones regulators,gibberellins response protein of this genes encoding chitin induction.The gene of other plant hormone as brassinolide and growth hormone approach all affects plant height.

The present invention is isolated and cloned into the gene of a coding bHLH structural domain from paddy rice Japan is fine.The people such as Nakamura (2006) predict this gene order in rice genome, by its called after OsPIL16.But the Unknown Function of this gene in adjusting and controlling rice economical character at present.

Summary of the invention

In the rice mutant that present invention applicant improves at a Drought and salt stress tolerance, a sequence number is AK287958, the OsPIL16 gene of coding bHLH structural domain to utilize gene expression atlas to identify.Rice Os PIL16 gene ORF region sequence is 1515 bases, encode 504 amino acid and a terminator codon.The present invention passes through PCR method, amplify the full length coding region (cDNA comprises 1515 bases) of rice Os PIL16 gene, this gene forward is proceeded in paddy rice, improve the expression level of OsPIL16 gene, obtain the transgenic rice plant that rice Os PIL16 genetic expression improves.Find in genetically modified T3 is for plant homozygous lines, the plant height of positive plant reduces, and tiller number improves.

The present invention for build overexpression OsPIL16 gene cDNA plant expression vector, strengthen the cDNA sequence of gene OsPIL16 of OsPIL16 genetic expression as shown in SEQIDNO:1, aminoacid sequence is as shown in SEQIDNO:2.

The invention still further relates to gene OsPIL16 and reduce the application method in Plant Height of Rice, raising tiller number, concrete grammar is as follows: it is first by the full length coding region cDNA of PCR method amplifying rice OsPIL16 gene, then add A at PCR primer end, be connected to pMD18-T carrier; Then utilize BamHI and SpeI enzyme to cut the OsPIL16 gene cDNA fragment be cloned on pMD18-T carrier, between BamHI and SpeI of insertion pCAMBIA1390-Ubi plant expression vector, obtain plant expression vector pCAMBIA1390-Ubi-PIL16; Recycling agrobcterium-mediated transformation plant over-express vector is converted into rice varieties Japan fine in, improve the expression of OsPIL16 gene, obtain the transgenic rice plant that rice Os PIL16 genetic expression improves.

Wherein, the maize ubiquitin promoter Ubi be cloned on pMD18-T carrier is utilized HindIII and BamHI double digestion by plant expression vector pCAMBIA1390-Ubi, insert on pCAMBIA1390 plant expression vector between HindIII and BamHI, transform the plant expression vector obtained.

This gene forward, as applying gene, proceeds in paddy rice, improves the expression level of OsPIL16 gene by the cDNA fragment of the OsPIL16 gene that the present invention utilizes rice varieties Japan fine, and transgenic positive rice plant performance tiller number increases.Under field condition, control material plant height is 102.1cm, and transgenosis plant height is then 89.0-95.4cm, is starkly lower than contrast.The available tillering of control material is 9.9/strain, and the available tillering of transgenic rice plant is 13.5-14.2/strain, apparently higher than contrast.This provides new genetic resources and new way for cultivating high-yield crop kind, has certain economic implications.

The invention has the advantages that:

(1) the invention provides a kind of gene OsPIL16 reducing plant height and improve tiller number.Applicant is in paddy rice after overexpression OsPIL16 gene, and find that transgenic paddy rice plant height reduces, tiller number improves.

(3) expression method of involved in the present invention raising OsPIL16 gene, except with except constitutive promoter Ubi, can also by other constitutive promoter or organ specific promoters.Therefore the expression by improving OsPIL16 gene has application space comparatively widely at reduction plant height, raising tiller number New Crop Varieties.

(4) gene applied in the present invention can provide support for the cereal crops such as paddy rice and the research of other economical character such as crop plant height and tiller number.

Accompanying drawing explanation

Fig. 1 is the structure schematic diagram of OsPIL16 expression vector of the present invention.BamHI and SpeI enzyme is utilized to cut the OsPIL16 gene cDNA fragment be cloned on pMD18-T carrier, on insertion pCAMBIA1390-Ubi (1390-Ubi) plant expression vector between BamHI and SpeI, after the cDNA of such OsPIL16 gene inserts maize ubiquitin promoter (Ubi) with regard to forward, be namely built into pCAMBIA1390-Ubi-PIL16 plant expression vector (1390-Ubi-PIL16);

Fig. 2 is the Southern results of hybridization figure of OsPIL16 transfer-gen plant in the present invention.Wherein OsPIL16-OX represents the cDNA transgenic paddy rice strain turning OsPIL16 gene, and #1, #13, #27 represent different positive transgenic strain; WT represents wild rice plant;

Fig. 3 is for detecting T3 for OsPIL16 gene transcript levels result figure in transgenic paddy rice plant.OsPIL16-OX represents the cDNA transgenic paddy rice strain turning OsPIL16 gene, and #1, #13, #27 represent different positive transgenic strain; WT represents wild rice plant;

Fig. 4 turns the rice plant of OsPIL16 gene and the wild rice plant phenotypic map large Tanaka.#1, #13 and #27 represent different transgenic paddy rice strain; WT represents wild rice plant;

Fig. 5 turns the rice plant of OsPIL16 gene and the plant height of Wild plant and available tillering comparison diagram.A is OsPIL16-OX transgenic paddy rice strain and wild-type (WT) plant height comparison diagram; B is OsPIL16-OX transgenic paddy rice strain and wild-type (WT) available tillering comparison diagram; #1, #13 and #27 represent different transgenic paddy rice strain; WT represents wild rice plant.

Embodiment

Following examples define the present invention, and describe the present invention at separating clone for building the DNA fragmentation of OsPIL16 gene plant expression vector, and the method for authentication function.According to following description and these embodiments, those skilled in the art can determine essential characteristic of the present invention, and when not departing from spirit and scope of the invention, various change and amendment can be made to the present invention, being applicable to different purposes and condition to make it.

Embodiment 1: separating clone is for building the cDNA fragment of OsPIL16 gene plant expression vector

In order to analyze the function of OsPIL16 gene, we construct the over-express vector of OsPIL16 gene cDNA, and concrete steps are as follows.

TRIZOL reagent (Invitrogen) is adopted to extract total serum IgE from the blade of rice varieties Japan fine (kind of open report).Concrete steps are as follows: put 20 milligrams of blades in the mortar of Liquid nitrogen precooler, add liquid nitrogen and clay into power fast, loaded by powder in 1.5ml centrifuge tube, add rapidly 1mlTrizol (Invitrogen) and put upside down mixing, and room temperature leaves standstill 5 minutes.At 4 DEG C, centrifugal 10 minutes of 12000rpm, gets supernatant liquor and moves in new 1.5ml centrifuge tube.Add 200 μ l chloroforms, acutely shook for 15 seconds with hand, room temperature leaves standstill 2-3 minute.4 DEG C, centrifugal 15 minutes of 12000rpm.Get in the new 1.5ml centrifuge tube of colourless aqueous phase to, add 250 μ l Virahols, 250 μ l high level salt solutions, put upside down mixing, and room temperature leaves standstill 10 minutes.4 DEG C, centrifugal 10 minutes of 12000rpm, absorbs supernatant liquor.Add 75% ethanol that 1ml is ice-cold, several times, then 4 DEG C, centrifugal 5 minutes of 7500rpm, abandons supernatant, in drying at room temperature to precipitating bleach to turned upside down.Add appropriate DEPC water (being generally 60 μ l) dissolution precipitation, utilize ultraviolet spectrophotometer to measure the concentration of RNA.

Utilize ThermoScript II SuperScriptII (Invitrogen) that its reverse transcription is become cDNA, concrete steps are as follows: add 1 μ l500 μ g/mloligo (dT) 12-18,2 μ g total serum IgE, 1 μ l10mMdNTP mixture and DEPC water to 12 μ l successively, in 65 DEG C of water-baths 5 minutes, rapid ice bath 5 minutes, collected by centrifugation sample is at the bottom of pipe a little.Then 4 μ l5 × the first chain damping fluids, 2 μ l0.1MDTT and 1 μ lRNaseOUT (40U/ μ l) are added successively, 42 DEG C, 2 minutes.Then add 1 μ lSuperScriptII, slightly mix, 42 DEG C are reacted 50 minutes, and then 70 DEG C of water-baths make enzyme deactivation in 15 minutes, have so just synthesized the first chain cDNA.

With with restriction enzyme site upstream primer OsPIL16F1 (5 '-AGGATCCATGCTACGCGGGAACGACACCG-3 '; SEQIDNO:3; the additional BamHI site of sequence specific primers and a protection base) and downstream primer OsPIL16R1 (5 '-AACTAGTCACGCCTGCTTCACGGCGGGG-3 '; SEQIDNO:4, the additional SpeI site of sequence specific primers and a protection base).Utilize PrimerSTARHSDNApolymerasewithGCbuffer (TaKaRa) to carry out amplification object fragment, PCR reaction conditions is 94 DEG C of denaturations 1 minute; 98 DEG C 10 seconds, 68 DEG C 4 minutes, 30 circulations.TArgetCloneTM-Plus test kit (TOYOBO) is utilized to add A at PCR primer end.Then pMD18-T carrier (TaKaRa) is connected to.Screening positive clone also checks order, and obtaining required DNA fragmentation (sequence is as shown in SEQIDNO:1), is pMD18-OsPIL16 by this clone designation.

The structure of embodiment 2:OsPIL16 gene plant expression vector and genetic transformation

In order to analyze the function of OsPIL16 gene better, applicant makes the cDNA sequence of OsPIL16 gene improve at the fine middle expression level of rice varieties Japan by overexpression technology.The function of this gene is studied according to the phenotype of transfer-gen plant and physiological characteristic.

In order to build OsPIL16 gene plant expression vector, first the application constructs the plant expression vector of pCAMBIA1390-Ubi.First utilize following method to extract corn gene group DNA, from the blade of single 981 (kinds of open report) in corn variety Shandong, extract STb gene.Concrete steps are as follows: the blade that takes a morsel (2-3cm is long), put into 1.5ml centrifuge tube, liquid nitrogen flash freezer, grind fast.Add 200 μ lDNAExtractionbuffer (20mMTris-HClpH=7.5; 250mMNaCl; 25mMEDTA; 0.5%SDS; 0.2mg/ml Proteinase K), vortice mixes, puts into 65 DEG C of water-baths and be incubated 2h.The centrifugal 10min of 12000rpm subsequently, moves to supernatant liquor in new 1.5ml centrifuge tube, and add the mixing of equal-volume chloroform, turn upside down for several times, static several minutes, to liquid level layering, repeat twice.Get upper phase to move in new 1.5ml centrifuge tube, add equal-volume Virahol, turn upside down after mixing for several times, the centrifugal 10min of 12000rpm, evacuation supernatant liquor, adds 70% ethanol of 200 μ l, the centrifugal 5min of 12000rpm.Dry DNA under room temperature, with the resuspended genomic dna of 25 μ lTE-RNaseAbuffer.

Utilize corn gene group DNA as template; utilize upstream primer UbiHindF1 (5 '-ATAAGCTTGCATGCCTGCAGTGCAGCG-3 '; SEQIDNO:5; the additional HindIII site of sequence specific primers and two protection bases) and downstream primer UbiBamHR1 (5 '-AAGGATCCTCTAGAGTCGACCTGCAGAAG-3 '; SEQIDNO:6, the additional BamHI site of sequence specific primers and two protection bases).Extension amplification outcome is at pMD18-T carrier, and screening positive clone also checks order, and obtain required DNA fragmentation, being Ubi promoter region, is pMD18-Ubi by this clone designation.By above-mentioned clone HindIII and BamHI double digestion, reclaim Insert Fragment; Equally, the enzyme that uses the same method cuts pCAMBIA1390 carrier (international conventional Genetic Transformation in Higher Plants carrier), reclaims carrier segments.To make with carrier segments of the Insert Fragment reclaiming and be connected, transformation of E. coli XL1-Blue, cuts screening positive clone by enzyme, namely obtains pCAMBIA1390-Ubi plant expression vector (see Fig. 1).

By the positive colony pMD18-OsPIL16 BamHI that obtains in embodiment 1 and SpeI double digestion, reclaim Insert Fragment; Equally, the enzyme that uses the same method cuts the plant expression vector of pCAMBIA1390-Ubi (1390-Ubi), reclaims carrier segments.Ligation is done, transformation of E. coli XL1-Blue with the Insert Fragment reclaimed and carrier segments.Cut screening positive clone by enzyme, obtain plant expression vector, respectively called after pCAMBIA1390-Ubi-PIL16 (see Fig. 1).PCAMBIA1390-Ubi-PIL16 is converted into EHA105 Host Strains.

By agriculture bacillus mediated rice transformation system imported to rice varieties Japan fine in, through preculture, infect, callus that Dual culture, screening have hygromycin resistance, break up, take root, transplanting seedlings obtains transfer-gen plant.Agriculture bacillus mediated rice transformation system improves on the method basis of people's reports such as Hiei carries out (Hieietal., 1994, PlantJ, 6:271-282).Transform and obtain 15 strains independently transgenic rice plant respectively.

Concrete steps are as follows:

(1) callus of induce: the fine rice paddy seed of wild-type Japan shelled, with 70% ethanol surface sterilization 1 minute; 5% (active chlorine content) NaClO solution surface is sterilized 20 minutes; Aseptic water washing 4-5 time; Be seeded in the upper evoked callus of calli induction media (composition sees below), after 25-26 DEG C of light culture 4-7 days, nascent callus will be induced from mature embryo scultellum, remove the plumule that embryo grows with tweezers simultaneously, subculture continues cultivation 2 weeks in calli induction media, until it is yellowish to grow color and luster, quality is hard in granular embryo callus.

(2) preculture of callus: callus is gone to fresh calli induction media and cultivate, in 25-26 DEG C of light culture 4 days.

(3) Agrobacterium is cultivated: picking Agrobacterium mono-clonal is inoculated into (kantlex containing 50mg/L) in 5mLYEP liquid nutrient medium, 28 DEG C, 220rpm, is cultured to logarithmic growth late period (approximately cultivating 18-24 hour).By the bacterium liquid of acquisition by 1% inoculum size be transferred to 50mL fresh, in AB liquid nutrient medium containing 50mg/L kantlex (composition sees below); 28 DEG C, 220rpm, being cultured to OD600 value is about 0.5 (cultivating 5-6 hour).

(4) Agrobacterium is infected: 50mL bacterium liquid is proceeded to centrifuge tube, 4 DEG C, and centrifugal 10 minutes of 4000g, abandons supernatant, adds the resuspended thalline of isopyknic AAM substratum.Fine for the Japan of (2) embryo callus is immersed above-mentioned AAM bacterium liquid, infects 2 minutes, slowly shake.With aseptic thieving paper, callus is blotted, be placed in (substratum upper berth one deck aseptic filter paper) on Dual culture substratum, 26 DEG C, dark Dual culture 2-3 days.

(5) callus washing and select to cultivate: the aseptic washing 4 times of the callus after Dual culture, and then with containing the aseptic washing 2 times of 500mg/L Pyocianil Cb, then blot with aseptic thieving paper and be placed on worktable and blow 30 minutes.Callus is placed in solid screening culture medium (containing 25mg/L Totomycin, 400mg/L Pyocianil), 26 DEG C of light culture 2 weeks.Then transfer in solid screening culture medium (containing 30mg/L Totomycin, 300mg/L Pyocianil), 26 DEG C of light culture, every 2 weeks subcultures once, screen 4 weeks.

(6) differentiation culture: resistant calli is transferred on division culture medium, 28 DEG C of illumination cultivation 7 days, after switching once, are cultured to generation regrowth.

(7) strong sprout, transplanting: the plantlet of regeneration is gone on fresh 1/2MS substratum, strong plantlets and rootage in culturing bottle.Treat that seedling grows to about 10cm, open sealed membrane, hardening 2-3 days, regrowth is moved in soil and cultivates.

Agent prescription:

(1) reagent and solution abbreviation: the abbreviation of the plant hormone be applied in the present invention is expressed as follows: Cb (Cabenicillin, Pyocianil); KT (Kinetin, kinetin); NAA (Napthaleneaceticacid, naphthylacetic acid); 2,4-D (2,4-Dichlorophenoxyaceticacid, 2,4 dichlorophenoxyacetic acid); AS (Acetosyringone, Syringylethanone); DMSO (Dimethylsulfoxide, dimethyl sulfoxide (DMSO)).

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

1) YEP liquid nutrient medium: 2gBacto-peptone, 2g yeast powder, 1gNaCl, adds water and is settled to 200mL, adjusts pH to 7.0 with 5NNaOH.

2) calli induction media: N6 is a large amount of, N6 trace, molysite, N6 VITAMIN, 0.5g/L acid hydrolyzed casein, 30g/L sucrose, 2mg/L2,4-D, Gelrite (Sigma) 4g/L, pH5.8.

3) AB liquid nutrient medium: 3g/LK ₂hPO ₄, 1g/LNaH ₂pO ₄, 1g/LNH ₄cl, 300mg/LMgSO ₄7H ₂o, 150mg/LKCl, 10mg/LCaCl ₂2H ₂o, 2.5mg/LFeSO ₄7H ₂o, 5g/L glucose, pH7.0.

4) AAM substratum: AA is a large amount of, AA trace, 0.9g/LL-glutamine, 0.3g aspartic acid, MS VITAMIN, 0.5g/L acid hydrolyzed casein, 36g/L glucose, 68.5g/L sucrose, 20mg/LAS, pH5.2.

5) Dual culture substratum: N6 is a large amount of, N6 trace, molysite, N6 VITAMIN, 30g/L sucrose, 10g/L glucose, 0.5g/L acid hydrolyzed casein, 2mg/L2,4-D, 20mg/LAS, Gelrite (Sigma) 4g/L, pH5.8.

6) solid screening culture medium: N6 is a large amount of, N6 is micro-and N6 VITAMIN, 0.5g/L acid hydrolyzed casein, 30g/L sucrose, 2mg/L2,4-D, Gelrite (Sigma) 4g/L, pH5.8, the Totomycin of suitable concn and Pyocianil.

7) division culture medium: MS is a large amount of, MS trace, molysite and MS VITAMIN, 2g/L acid hydrolyzed casein, 30g/L sucrose, 30g/L sorbyl alcohol, 2mg/LKT, 0.2mg/LNAA, pH5.8,30mg/L hygromycin B, 200mg/L Pyocianil.

8) 1/2MS substratum: 1/2MS is a large amount of, 1/2MS trace, MS VITAMIN, 30g/L sucrose, 4g/LGelrite, 30mg/L hygromycin B, 200mg/L Pyocianil, pH5.8.

(3) main solution formula:

1) N6 macroelement (10 ×)

2) N6 trace (1000 ×):

3) N6 VITAMIN (1000 ×)

4) MS macroelement (10 ×)

5) MS trace (1000 ×):

6) MS VITAMIN (1000 ×)

7) molysite (200 ×)

9) AA trace (1000 ×)

10) 2,4-D storage liquid (2mg/ml)

Take 2,4-D100mg, be dissolved in 1mlDMSO, add distilled water and be dissolved to 49ml, then add 0.5NNaOH, to dissolving completely, in-20 DEG C of preservations.

11) Kinetin storage liquid (0.2mg/ml)

Take Kinetin10mg, be dissolved in 1ml1NKOH, adding distil water is dissolved to 50ml, in 4 DEG C of preservations.

12) NAA storage liquid (0.2mg/ml)

Take NAA10mg, be dissolved in 0.5ml1NKOH, adding distil water is dissolved to 50ml, in 4 DEG C of preservations.

13) Syringylethanone (100mg/ml)

Take Syringylethanone 100mg, be dissolved in 1mlDMSO, in-20 DEG C of preservations.

14) kantlex (50mg/ml)

Take kantlex 500mg, be dissolved in 8ml distilled water, adding distil water is dissolved to 10ml, then uses 0.22 μm of frit degerming, in-20 DEG C of preservations.

Embodiment 3: the insertion point detecting OsPIL16 gene in transgenic rice plant

With warm and fine 3 the OsPIL16 transgenic paddy rice strains of rice varieties Japan for material, carry out Southern hybridization, concrete steps are as follows: extract genomic dna by CTAB method, concrete steps are with reference to MurrayandThompson, Nucl.AcidsRes, 1980,8:4321-4325.Get 100 μ g genomic dnas to utilize Hind Ш to carry out enzyme to cut, use 0.8% agarose gel electrophoresis DNA isolation subsequently.DNA is transferred to nylon membrane Hybond-N+; AmershamCo.Ltd., NJ, USA.Because foreign gene OsPIL16 and resistance selective marker gene HP TII is in same T-DNA region, therefore we use HPTII gene as the insertion point of probe identification OsPIL16 gene.With pCAMBIA1390-Ubi plasmid for template, with the upstream primer HPTIIF1 of HPTII gene (5 '-TTCAGCTTCGATGTAGGAGG-3 ', and downstream primer HPTIIR1 (5 '-TACACAGCCATCGGTCCAGA-3 ', SEQIDNO:8) SEQIDNO:7).Utilize PrimerSTARHSDNApolymerasewithGCbuffer (TaKaRa) to carry out amplification object fragment, PCR reaction conditions is 98 DEG C of denaturations 2 minutes; 94 DEG C 10 seconds, 53 DEG C of 30s, 68 DEG C 1 minute, 35 circulations.PCR primer (expanding fragment length is 858bp) utilizes WizardSVgel and PCRclean-upsystem (Promega, USA) to carry out purifying (implementing according to test kit specification sheets).According to DIGHighPrimeDNALabelingandDetectionStarterKitI test kit specification sheets, the PCR fragment after purifying is carried out DIG mark, hybridization.As shown in Figure 2, in three transgenic lines #1, #13 and #27 of OsPIL16-OX, foreign gene is incorporated in rice genome result all, and insertion point is different, is three independently transgenic lines.

Embodiment 3: the transcript degree detecting OsPIL16 gene in transgenic rice plant and wild rice

With the T3 of the paddy rice wild-type fine kind of Japan and 3 independently OsPIL16-OX for transgenic rice plant for material, extract the RNA of 2 leaf phase rice leafs, utilize fluorescence quantitative RT-RCR to detect the transcript degree of OsPIL16 gene in rice leaf.Concrete grammar is as follows: extract total serum IgE according to embodiment 1, and synthesis cDNA first chain, utilizes SYBRPremixExTaq ^tMpCR kit (TaKaRa) carries out quantitative fluorescent PCR reaction.Concrete steps are as follows: add 10 μ l2 × SYBRPremixExTaq successively ^tM, 2.0 μ lcDNA templates, 0.2 μM of gene-specific primer pair, use RNase-freeH ₂it is 20 μ l that O supplies reaction system.Auele Specific Primer used to upstream primer OsPIL16qF1 (5 '-CGACTACCTCTTCATCAAGAATC-3 ', and downstream primer OsPIL16qR1 (5 '-GACAGGGTGGTAGATGCAACCA-3 ', SEQIDNO:10) SEQIDNO:9).Use the reference gene that paddy rice endogenous translation elongation factor gene (OsEF-1a) are analyzed as quantitative fluorescent PCR simultaneously, its special upstream primer be OsEF-1aF (5 '-TTTCACTCTTGGTGTGAAGCAGAT-3 ', SEQIDNO:11), special downstream primer is OsEF-1aR (5 '-GACTTCCTTCACGATTTCATCGTAA-3 ', SEQIDNO:12).PCR reaction conditions is 95 DEG C of denaturations 30 seconds; 95 DEG C of sex change 30 seconds, 60 DEG C of annealing extensions 30 seconds, 40 circulations.After PCR reaction terminates, analyze the amplification curve of each sample and solubility curve to determine the confidence level of experimental result.Then the Ct value obtained by 2-Δ Δ Ct method process quantitative fluorescent PCR with Excel also calculates standard error, maps to final process result.As shown in Figure 3, in 3 transgenic lines (#1, #13 and #27), the transcript degree of OsPIL16 gene is apparently higher than wild-type, proves external source OsPIL16 gene great expression in transgenic paddy rice.

Embodiment 4: the rice plant plant height turning OsPIL16 gene reduces, tiller number increases

The present embodiment analyzes the OsPIL16 gene overexpression strain of large Tanaka growth and the plant height of wild-type and tiller number.Concrete steps are as follows: transfer-gen plant and WT lines 70% ethanol surface sterilization 1 minute; 5% (active chlorine content) NaClO solution surface is sterilized 20 minutes; Aseptic water washing 4-5 time, sprouts 3 days under a dark condition.Transfer to subsequently in soil and grew to for 4 leaf phases, transplant to large Tanaka (May 21, Jinan).Spacing in the rows between 2 rice plants and line-spacing are 15cm and 25cm respectively.After grouting terminates, take pictures (Fig. 4).The plant height on each plant of transfer-gen plant and wild-type and number of productive ear is analyzed during results.Result shows, the plant height of transgenic line is approximately 89.0-95.4cm, and wild-type is about 102.1cm, and the plant height of transgenic line is starkly lower than wild-type, analyzes and shows, significant difference (Fig. 5 A) through TTEST.Average every strain available tillering of transgenic line is 13.5-14.2, and wild-type is about 9.9, and the available tillering of transgenic line, apparently higher than wild-type, is analyzed through TTEST and shown, significant difference (Fig. 5 B).This result shows, the plant height of transfer-gen plant significantly reduces, and available tillering significantly increases.

Claims (4)

1. gene OsPIL16 is improving the application in rice tillering number.

2. gene OsPIL16 is improving the application method in rice tillering number, it is characterized in that, using the cDNA fragment of OsPIL16 gene as applying gene, this gene forward is proceeded in paddy rice, improve the expression level of OsPIL16 gene, obtain the transgenic rice plant that rice Os PIL16 genetic expression improves.

3. gene OsPIL16 is reducing the application in Plant Height of Rice.

4. gene OsPIL16 is reducing the application method in Plant Height of Rice, it is characterized in that, using the cDNA fragment of OsPIL16 gene as applying gene, this gene forward is proceeded in paddy rice, improve the expression level of OsPIL16 gene, obtain the transgenic rice plant that rice Os PIL16 genetic expression improves.