CN105985954A - Application of paddy rice miR160b gene in regulation and control on tillering angle - Google Patents

Abstract

The invention belongs to the field of plant gene engineering and particularly relates to an application of a paddy rice miR160b gene in regulation and control on tillering angle. Through over-expression of the miR160b gene, a normal paddy rice plant has a phenotypic change of increased tillering angle, and through inhibition of the miR160b gene, the mutant phenotype of the over-expressed miR160b plant is recovered. The miR160b gene has the function of regulating and controlling plant type of paddy rice, wherein a stem-loop structure DNA sequence of the gene is represented as the SEQ ID No.1, a stem-loop structure precursor RNA sequence formed after transcription is represented as the SEQ ID No.2, and the sequence of a core region in mature miR160b formed after transcription and cutting is represented as the SEQ ID No.3. The invention discloses an expression mode of changing the paddy rice miR160b gene or the genes in other members of the family through gene engineering technology, so that the tillering angle of the paddy rice is regulated and controlled to form an ideal plant type, thereby increasing yield.

Description

The application in regulation and control tillering angle of the Oryza sativa L. miR160b gene

Technical field

The invention belongs to plant genetic engineering field.It is specifically related to functional verification and the application of a kind of adjusting and controlling rice tillering angle gene miR160b.This Invention, by overexpression miR160b gene, makes normal water rice plants the character mutation that tillering angle increases occur, and suppression miR160b gene is permissible The mutant phenotype making overexpression miR160b plant is recovered, and further relates to by this miRNA of the experimental verification suppression to target gene, and then adjusting and controlling rice Tillering angle.The invention still further relates to utilize the plant type of this gene pairs Oryza sativa L. to carry out on purpose genetic improvement by technique for gene engineering.

Background technology

Oryza sativa L. is one of main cereal crops, exceedes the staple food of half population as the whole world.Oryza sativa L. is with its less genome (about 430Mbp), fine physics and genetic map, and on genome, have preferable conllinear with other grasses such as Semen Maydis, Fructus Hordei Vulgaris, Semen Tritici aestivis Property, relative maturity and maneuverable genetic conversion system, thus it is considered the model plant of monocot genes functional study.Along with whole world people Constantly increasing and the minimizing year by year of cultivated area of mouth, Food Security is increasingly paid close attention to by people, wherein improves the product of Oryza sativa L. unit are Amount is one of important measures solving this problem.The fifties, the cultivation with the hybrid rice seventies of applying of Dwarfing Gene made China's rice yield occur Twice revolutionary leap, but a very long time China's rice yield has been at the situation fluctuated afterwards.In recent years, breeding man and something lost Passing scholars, to be expected that by selection-breeding new, to realize rice yield to cultivate high-yield rice with ideotype the means that combine with heterosis utilization Promote further.

Plant height, tiller number, tillering angle and fringe form are to affect the important factor of plant type of rice, and wherein tiller is the weight of Ideal Rice Plant Type and high yield Want one of economical character.Tiller is the fundamental characteristics of paddy growth, and it includes tillering angle and two aspects of tillering ability, the former reflect stem with The collecting and distributing degree of tiller；The latter is demonstrated by the number of tiller number.Tillering angle refers to tiller and the angle of stem before plant heading, and it directly affects With plant interior focusing, temperature, the competition of oxygen between rice plant, finally affect the yield of Oryza sativa L..Although loose plant type can avoid invading of disease pest Evil, but occupy too many space, make photosynthetic efficiency reduction, lodging tolerance decline, and compact plant limits the infiltration of light and air, The humidity of adjacent plant increases, and makes plant be easier to infect pest and disease damage (Wang etc., Rice rising.Nat Genet, 2008,40:1273-1275). Therefore, tillering angle is excessive or the too small raising being all unfavorable for rice yield of tillering angle.

At present, the important gene closely-related with tillering angle cloned has: TAC1, PRGO1, LAZY1 and LPA1.TAC1 is 2007 The major gene resistance controlling this quantitative trait of tillering angle being cloned in year, this gene is specific expressed at tiller base portion and eustipes part.Its 4th In intron, 3 ' hold the sudden change at shearing sites so that TAC1 expression declines, and its lateral bud base portion is in the most asymmetric compared with wild type Growth pattern, thus produce mutant phenotype (Baisheng etc., TAC1, a major quantitative trait locus that tillering angle is close to 0 ° controlling tiller angle in rice.Plant J,2007,52:891-898).One C2H2 type of PRGO1 gene code of clone in 2008 Zinc finger protein, expressing in lateral meristem and apical meristem of this gene specific.In its open reading frame, the transversion of a base causes The change of coding protein sequence, thus produce the phenotype that tillering angle increases.Tissue slice observes display, in this mutant, lateral bud base portion near Ground and privately face symmetrically growth pattern so that lateral bud cannot bend and grow up and present growth pattern of throwing oneself on the ground (Lubin etc., Control of a key transition from prostrate to erect growth in rice domestication.Nat Genet,2008,40:1360-1364；Jian etc., Genetic control of rice plant architecture under domestication.Nat Genet,2008,40:1356-1369).The LAZY1 of clone in 2007 Gene is specifically expressing in stem and plumule, and at its depletion mutant lateral bud, the polar translocation of auxin is destroyed, and causes endogenous auxins to divide Cloth is abnormal, thus the mutant phenotype that tillering angle increases occurs.Current research shows that only angle gold lactone synthesis mutant can make lazy1 tillering angle The mutant phenotype increased is replied, and only angle gold lactone, by the synthesis of Developing restraint element, reduces the content of IAA in lateral bud and then affects the tiller angle of Oryza sativa L. Degree (Sang etc., Strigolactones regulate rice tiller angle by attenuating shoot gravitropism through inhibiting auxin Biosynthesis.Proc Natl Acad Sci U S A, 2014,111:11199-11204), show that only angle gold lactone may participate in the tune of tillering angle in Oryza sativa L. Control.LPA1 is the transcription inhibitory factor of the distinctive domain of coded plant uncertain (ID) the most just found, its mutant lpa1 has Loose plant type, tiller angle and leaf angle increase, and internode shortens, thickens, stalk cell wall thickening, and grain and blade face shorten and broaden, overground part to weight Property weakens.LPA1 has special expression pattern, and it has relatively strongly expressed at the Leaf angle of tender tissue and internode and older tiller base portion.LPA1 Regulate and control gravity sensing and signal transduction by reconciling the rate of settling of amyloplaste in cell, finally affect the gravitropism of Oryza sativa L. aerial parts react from And regulate and control size Wu etc. of tiller angle and Leaf angle, Loose plant architecture1, an INDETERMINATE DOMAIN protein involved in shoot gravitropism,regulates plant architecture in rice.Plant Physiol,2013,161:317-329).Although at present It is cloned into some genes controlling rice tillering angle, but the molecular mechanism about rice tillering angle is the most unclear.

ARF is the transcription factor family with B3DNA binding domain that in plant, a class is special, and they are by being combined in specific gene promoter region AuxRE element (TGTCTC) on, activate or the expression of Developing restraint element response gene.Typical ARF is generally of three domains It is respectively N-terminal than more conservative DNA binding domain (DBD), not conservative zone line (MR), C-terminal than more conservative dimer district Territory (CTD).The AuxRE of DBD domain mediation ARF and originally auxin-responsive gene (GH3, SAUR, Aux/IAA) promoter region Specific bond, MR domain then determines the type of ARF transcription factor, and CTD domain is similar to III and the IV knot of Aux/IAA albumen Structure territory, the CTD domain of ARF can be combined with III and the IV domain of Aux/IAA albumen.Up-to-date research shows, to arabidopsis ARF The crystal structure analysis of ARF5 and ARF7 in family finds, the CTD region of ARF5 and ARF7 has PB1 region, and they can be formed The form of oligomer respectively with IAA17 and IAA21 interactions between protein, and then regulation and control auxin response suppression (Korasick etc., Molecular basis for AUXIN RESPONSE FACTOR protein interaction and the control of auxin response repression.Proc Natl Acad Sci USA,2014,111:5427-5432；Nanao etc., Structural basis for oligomerization of auxin transcriptional regulators.Nat Commun,2014,5:3617).There are 25 members in Oryza sativa L. ARF family, reported have OsARF1, OsARF12, OsARF16, OsARF19, OsARF23, OsARF24 and OsARF25, each of which participates in the different growth and development processes of Oryza sativa L..Up-to-date research display OsARF12 Participate in affect the balance of phosphorus in Oryza sativa L. body, in osarf12 and osarf12osarf25 mutant, Pi sufficient/lack under conditions of, with phosphorus response The expression of relevant gene is all increased (Wang etc., Auxin response factor (OsARF12), a novel regulator for phosphate homeostasis in rice(Oryza sativa).New Phytologist,2014,201:91-103).OsARF23 and OsARF24 can form heterodimeric Body protein, jointly acts on the promoter region of RMD gene, directly regulates the expression of RMD gene, the growth of adjusting and controlling rice cell and form There is (Li etc., Rice actin-binding protein RMD is a key link in the auxin-actin regulatory loop that controls cell growth. Proc Natl Acad Sci U S A,2014,111:10377-10382).OsARF19 is by regulating expression and then the regulation and control water of OsGH3.5 and OsBRI1 The size of rice Leaf angle.But Oryza sativa L. there is no the research report about OsARF Yu tillering angle relation.

A kind of special small RNA molecular miRNA160 is formed after miR160b genetic transcription involved in the present invention.MiRNAs is a class organism Endogenic, the small RNA molecular of about 22-24nt length, not coded protein, it be widely present in each species and evolve on high conservative, Participate in the multiple bioprocesss such as the spatiotemporal database of organismal development, cell differentiation, signal transduction, disease, Stress response.MiR-96 gene and its The gene of its encoding proteins is the same, is all made up of promoter, transcriptional domain and terminator.In plant, miRNA joins with downstream target gene To almost mate completely, its regulatory mechanism belongs to the regulation and control of post-transcriptional level mostly, and the AGO complex i.e. containing ripe miRNA can be direct The mRNA of target gene is cut, and then whole mRNA molecule is degraded.Result of study in arabidopsis shows, the target base of miRNA160 Because being ARF6, ARF10 and ARF17 of ARF gene family.MiRNA160 direct regulation and control ARF17 thus regulate and control GH3 further, have impact on Auxin distribution in each histoorgan of arabidopsis.As ARF17 gene (mARF17) that overexpression one is not regulated and controled by miRNA160, plant There is various mutations phenotype in strain, including leaf rolling, development of floral organs is abnormal, bloom in advance, sterile etc., and make auxin immediate early genes There is abnormal (Mallory etc., MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE in the expression of GH3 gene family FACTOR 17is essential for proper development and modulates expression of early auxin response genes.Plant Cell,2005,17:1360-1375).Arf10arf16 double-mutant and Pro35S:MIR160 plant show identical mutant phenotype, long including main root Degree minimizing, the increase of lateral root number, lose gravity sensing.Although ARF10 and ARF16 exists functional redundancy phenomenon, but they are to arabidopsis The growth of root cap is indispensable Wang etc., Control of root cap formation by microRNA-targeted auxin response factors in Arabidopsis.Plant Cell,2005,17:2204-2216).When the ARF10 gene that overexpression one is not regulated and controled by miRNA160 (mARF10), time, plant occurs in that again the mutant phenotypes such as the flower of zigzag leaf, the angle fruit of curling, distortion, the miR160 regulation and control to ARF10 Have impact on the seedling sensitivity to ABA after sprouting, show IAA and ABA have in the germination process of seed potential contact (Liu etc., Repression of AUXIN RESPONSE FACTOR 10by microRNA160is critical for seed germination and post-germination stages.Plant J,2007,52:133-146).Mutant foc has one in 3 ' regulation and control regions of arabidopsis MIR160a gene Individual Ds transposon inserts, and this mutant shows the mutant phenotypes such as jagged leaf, irregular flower, abnormal seed, viviparous seedling, enters one Step is analyzed and is found that this mutant phenotype is owing to the abnormal cell division during embry ogenesis causes, ripe miR160 in foc mutant Accumulation reduce and the expression pattern of target gene ARF10, ARF16 and ARF17 changes, it can thus be appreciated that 3 ' regulation and control regions for miR160a The expression of gene is necessary ((Liu etc., The role of floral organs in carpels, an Arabidopsis loss-of-function mutation in MicroRNA160a,in organogenesis and the mechanism regulating its expression.Plant J,2010,62:416-428).Up-to-date Data show, miRNA160 also can affect regeneration (Qiao etc., the Proper of bud in arabidopsis Process of in vitro to the regulating and controlling effect of ARF10 regeneration fromin vitro cultured Arabidopsis thaliana requires the microRNA-directed action of an auxin response factor.Plant J,2012).But in Oryza sativa L., also not about the report of miR160b gene regulation plant type of rice.

Summary of the invention

It is an object of the invention to clone and identify the gene miR160b of a kind of adjusting and controlling rice tillering angle, by controlling miR160b gene in Oryza sativa L. In expression pattern mould Ideal Rice Plant Type, reach volume increase purpose.

The miR160b gene loop-stem structure DNA sequence that the present invention identifies is as shown in SEQ ID NO:1, before its loop-stem structure formed after transcribing Body RNA sequence as shown in SEQ ID NO:2, after transcribing and cutting formed ripe miR160b nucleus sequence such as SEQ ID NO:3 Shown in.

The miR160b gene overexpression transfer-gen plant that the present invention identifies mainly shows as the plant type (see embodiment 4) that tillering angle increases.Excess The transgenic positive expressing miR160b makes plant all show the mutant phenotype that tillering angle increases from seedling stage to period of maturation, and the period of maturation is brighter Aobvious, in addition have also appeared available tillering and slightly reduce, setting percentage declines.

The present invention is by the expression pattern analysis (see embodiment 5) in transfer-gen plant of ripe miR160b, and the target of miR160b gene Mimicry overexpression vector the mutant phenotype of the transfer-gen plant of overexpression miR160b can be made to recover normal (see embodiment 6) it was confirmed MiR160b gene has the function of controlling plant type of rice.

The present invention be experimentally confirmed target gene that OsARF13, OsARF18 and OsARF22 of Oryza sativa L. ARF family is ripe miR160 (see Embodiment 6).The mRNA of target gene is sheared by the way of base complementrity by miR160b, thus the tillering angle of adjusting and controlling rice.

The concrete technical step realizing the present invention is as follows:

1. by building overexpression vector, it is achieved that the overexpression of Oryza sativa L. miR160b gene, result occurs in that the sudden change that rice tillering angle increases Phenotype (see embodiment 3,4).

2. OsARF13, OsARF18 and OsARF22 are the target genes of ripe miR160b to utilize RLM-5 ' RACE method to confirm, miR160b Gene is by the mRNA of degrade OsARF13, OsARF18 and OsARF22, and then the tillering angle (embodiment 6.1) of adjusting and controlling rice.

3. utilize quantitative RT-PCR technology analyze ripe miR160b and target gene OsARF13 in the positive and negative plant of miR160b overexpression, The expression of OsARF18 and OsARF22, finds that ripe miR160b is contrary table with target gene OsARF13, OsARF18 and OsARF22 Expression patterns (embodiment 5).

4. utilize the expression of target mimicry technology suppression Oryza sativa L. miR160b gene, the sudden change that the tillering angle of overexpression miR160b increases can be made Phenotype returns to wild-type status (embodiment 6.2).

More detailed technological invention details will be given by following embodiment.

Advantages of the present invention

1. in arabidopsis miR160 gene family, most gene is cloned, wherein few members be proved participate in thaliana flower organ growth, The regeneration etc. of bud in the growth of root cap and arabidopsis Process of in vitro.At present, Oryza sativa L. there is no the report about miR160 gene function.This The miR160b Gene Handling rice tillering angle that invention is identified, has resolved the biological function of miR160b gene regulation plant type of rice.

2. although Oryza sativa L. having been cloned some genes controlling plant tillering angle, but the molecule mechanism of rice tillering angle regulation and control being the most unclear.And The miR160b gene that the present invention identifies can control the tillering angle of Oryza sativa L., is conducive to resolving further the molecule mechanism of rice tillering angle regulation and control, It is easy to cultivate the rice varieties of reasonable plant type.

3. the present invention demonstrates the target gene that ARF family is miR160b, miR160b suppression target gene (OsARF13, OsARF18 and OsARF22) Expression, and miR160b gene overexpression makes rice tillering angle improve, and illustrates that OsARF13, OsARF18 and OsARF22 are to Oryza sativa L. Tillering angle has certain regulating and controlling effect, is conducive to the relation further appreciating that ARF family with tillering angle.

4. the present invention passes through overexpression miR160b gene, adds the size of rice tillering angle.Improved by technique for gene engineering or weaken The expression of miR160b gene can change the tillering angle of Oryza sativa L., thus improves plant type of rice, reaches to increase the purpose of rice yield.

The present invention is described in further detail below in conjunction with the accompanying drawings, but not limitation of the invention.

Accompanying drawing explanation

Sequence table SEQ .ID.NO:1 is the Oryza sativa L. miR160b gene loop-stem structure DNA sequence that the present invention identifies.

Sequence table SEQ .ID.NO:2 is the RNA loop-stem structure sequence that the Oryza sativa L. miR160b genetic transcription that the present invention identifies is formed.

Sequence table SEQ .ID.NO:3 is that the ripe miR160b core sequence formed is sheared in the Oryza sativa L. miR160b genetic transcription that the present invention identifies.

Fig. 1: be that Oryza sativa L. miR160b gene spends the expression pattern analysis in 11 in japonica rice variety.Description of reference numerals: abscissa represents root (R), stem (C), leaf (L), sheath (LS), lateral bud (B) and children's fringe (P) six tissue sites.

Fig. 2.: it is the detection of Oryza sativa L. miR160b overexpression plant copy number.Use λ-EcoT14 I digest DNA Marker, description of reference numerals: black Color arrow represents the follow-up study bill of materials copy plant 4-7 of selection.

Fig. 3: be the phenotype analytical of Oryza sativa L. miR160b overexpression plant.Description of reference numerals: Fig. 3 A: seedling stage plant photo.Fig. 3 B: the period of maturation Plant photo.Fig. 3 C: the photo of right-angle view tillering angle.Fig. 3 D: available tillering.Fig. 3 E: tillering angle.Fig. 3 F: setting percentage. The left side in above-mentioned each figure represents miR160b gene overexpression feminine gender plant (WT)；The right represents the miR160b gene overexpression positive and plants Strain (Ubi::miR160b).

Fig. 4: be the expression analysis of miR160b and target gene.Description of reference numerals: Fig. 4 A: be that ripe miR160b turns at overexpression miR160b Expression in the gene plant positive and feminine gender.Fig. 4 B: be that OsARF13 is in the overexpression Oryza sativa L. miR160b transfer-gen plant positive and feminine gender Expression.Fig. 4 C: be OsARF18 expression in the overexpression Oryza sativa L. miR160b transfer-gen plant positive and feminine gender.Fig. 4 D: be OsARF22 expression in the overexpression Oryza sativa L. miR160b transfer-gen plant positive and feminine gender.In figure: black post represents miR160b gene Overexpression negative control plant (WT), Lycoperdon polymorphum Vitt post represents miR160b gene overexpression positive plant (Ubi::miR160b).Abscissa Represent root (R), stem (S), leaf (L), sheath (LS), lateral bud (B) and children's fringe (P) six tissue sites.

Fig. 5: be the position of RLM-5 ' RACE analysis miR160b shearing target gene.Description of reference numerals: Fig. 5 A: be miR160 family in Oryza sativa L. The Multiple Sequence Alignment of mature sequence.Fig. 5 B: be that RLM-RACE analyzes miR160b cut-out target gene OsARF13, OsARF18 and OsARF22 The position of mRNA molecule.Fig. 5 C: be the position view that is sheared of the mRNA of OsARF13, OsARF18 and OsARF22, arrow in figure Head sum word table shows the ratio terminating RNA molecule at arrow correspondence position.

Fig. 6: be that Ubi::MIM160b makes Ubi::miR160b mutant phenotype recover.Description of reference numerals: Fig. 6 A: be MIM160b vector construction Schematic diagram.Fig. 6 B: be to spend 11 (ZH11) phenotype photo during Ubi::MIM160b turns.Fig. 6 C: be that Ubi::MIM160b turns Ubi::miR160b Phenotype photo.Fig. 6 D: be utilize QRT-PCR detection tri-target genes of OsARF13, OsARF18 and OsARF22 ZH11, Expression analysis in Ubi::MIM160b, Ubi::miR160b, Ubi::MIM160b/Ubi::miR160b.

Fig. 7: be utilize QRT-PCR methods analyst tillering angle gene (LAZY1, TAC1, PROG1) and auxin related gene (GH3.1, GH3.4, GH3.8, PIN1, IAA4, IAA21) at overexpression miR160b positive plant and the expression in negative plant.With Oryza sativa L. UBQ Gene is as internal reference, and meansigma methods is taken from 3 secondary pollutants and repeated and 3 technology repetitions.

Fig. 8: be to build the pU1301 empty carrier collection of illustrative plates used by Oryza sativa L. miR160b overexpression vector.

Fig. 9: be the plasmid figure building the MT375 plasmid used by Oryza sativa L. MIM160b overexpression vector.

Figure 10: be the collection of illustrative plates of pU1301-miR160b overexpression carrier.

Figure 11: be pU2301-MIM160b (that is: Ubi::MIM160b) carrier figure.

Figure 12: be pEASY-T1 carrier schematic diagram (i.e. carrier T pEASY-T 1cloning vector；Beijing Quanshijin Biotechnology Co., Ltd).

Detailed description of the invention

Embodiment 1 Oryza sativa L. miR160 gene family member analyzes

According to miRBase (Release 21) (http://www.mirbase.org/index.shtml) the up-to-date display of data base, Oryza sativa L. miR160 gene family One has 6 members, the most named miR160a to miR160f.The each member of this gene family is the most separately distributed in Oryza sativa L. coloured differently body, miR160 Gene family each member loop-stem structure length and sequence are different, only have 3 kinds through shearing the core miR160 sequence formed.

The table 1 Oryza sativa L. each member of miR160 gene family distribution on chromosome and stem ring size are analyzed

Embodiment 2 miR160b gene expression spectrum analysis

In order to analyze the expression pattern of miR160b gene, we are i.e. in the Oryza sativa L. japonica rice variety of tiller animated period after extracting rice transplanting and spend 11 for 30 days (ZH11) root (R), stem (C), leaf (L), sheath (LS), lateral bud (B), the RNA sample of children's fringe (P) these six tissue sites, By miRNA stem-loop RT-PCR (Chen etc., Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res, 2005,33:e179) expression of method detection miR160b gene, arranges 3 secondary pollutants repetitions, 2 technology weights Multiple.The primer is as follows:

MIR160b ST-RT primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTGGCAT, MIR160b Forward primer CAAATGCCTGGCTCCCTGTA, ST-R:5 ' GTGCAGGGTCCGAGGT3 '.With U6 as internal reference, corresponding primer is U6-L: 5 ' TACAGATAAGATTAGCATGGCCCC3 ', U6-R:5 ' GGACCATTTCTCGATTTGTACGTG3 '.Reverse transcription uses TransScript First-Strand cDNA Synthesis SuperMix test kit, system is 5.3 μ l:20ng/ μ l RNA template 2 μ l, 2 × TS Reaction Mix 2.5 μ l, Trans Enzyme 0.25 μ l, MIR160b ST-RT primer/U6-R 0.25 μ l, DEPC 0.3 μ l.In ABI 9700 On react, program is: 16 DEG C of 15min, 42 DEG C of 50min, 72 DEG C of 15min.Real-time PCR is directly carried out in above-mentioned reverse transcription product, Real-time PCR uses SYBR Premix Ex Taq (Perfect Real Time) test kit, and reaction system is 25.3 μ l: reverse transcription product 5.3 μ l, SYBR Premix Ex Taq 12.5 μ l, ROX Reference Dye II 0.5 μ l, MIR160b Forward/U6-L 1 μ l, ST-R/U6-R 1 μ l, dd H₂O 7μl.Reacting on ABI 7500, program is: 95 DEG C of 10s；95 DEG C of 5s, 60 DEG C of 34s, coreaction 40 circulation.Result As it is shown in figure 1, spend in No. 11 in japonica rice variety, miR160b expression in children's fringe is the highest, next to that lateral bud, the expression in leaf Minimum.

The structure of embodiment 3 Oryza sativa L. miR160b gene overexpression carrier and the acquisition of transfer-gen plant

1. the structure of Oryza sativa L. miR160b gene overexpression vector

Used carrier is the pU1301 (see Fig. 8) of the State Key Laboratory of Crop Genetic Improvent structure at the applicant place. PU1301 is Genetic Transformation in Higher Plants carrier pCAMBIA1301 (Sun etc., 2004, Xa26, a gene conferring commonly used in the world resistance to Xanthomonas oryzae pv.oryzae in rice,encoding a LRR receptor kinase-like protein.Plant Journal. On the basis of 37:517-527), transformation, carries the Agrobacterium-mediated genetic transformation of the maize ubiquitin promoter with composing type and overexpression feature Carrier.PCAMBIA1301 carrier is by Australia CAMBIA laboratory (Center for the Application of Molecular Biology to International Agriculture) give.Use PCR method, directly from rice genome, miR160b loop-stem structure sequence expanded, As follows with primer

miR160b-L 5'AGAGGTACCTCCGGTCATGGATATGGATAG(KpnI)

miR160b-R 5'CAAGGATCCGGCACAATATGATGGGTGCT(BamHI)

The cumulative volume of PCR reaction system is 20 μ l, oryza sativa genomic dna template 1 μ l (about 50ng), 10 × PCR Buffer 2 μ l, 2mmol/L dNTP 1.5 μ l, each 0.3 μ l of 10 μm primers, rTaq (purchased from precious biological engineering Dalian company limited) 0.3 μ l, dd H₂O 14.6μl.Response procedures is: 94 DEG C 5min, 94 DEG C of 45s, 55 DEG C of 45s, 72 DEG C of 1min, 35cycles, 72 DEG C of 5min, amplification 10 pipe, collect PCR primer in 1.5ml Centrifuge tube purification, adds equal-volume 24:1 chloroform isoamyl alcohol, jog 5 minutes, and 12000rpm is centrifuged 15 minutes, sucts clear, adds 2 times of volumes 95 % ethanol, 1/10 volume 3M sodium acetate (PH5.2), place 30 minutes for-20 DEG C, 12000rpm is centrifuged 20 minutes, abandons supernatant, adds 500 μ l 75% Ethanol is placed 5min, 12000rpm and is centrifuged 5 minutes, abandons supernatant, dries, adds 75 μ l ddH₂O dissolves.PCR primer and the pU1301 of purification Carrier enzyme action, system: cumulative volume 100 μ l, PCR primer or vector plasmid 75 μ l, restricted enzyme BamHI 30U, restricted enzyme KpnI 30U, 10 × K Buffer 5 μ l, ddH₂O 16 μ l, 37 DEG C of enzyme action 4 hours.Purification digestion products, method ibid, finally adds 10 μ l ddH₂O dissolves. Coupled reaction: 10 μ l PCR primer are completely used for coupled reaction, carrier 0.5 μ l, 2U T4Ligase, 5 × Ligase Buffer 3 μ l, total 15 μ l bodies Long-pending, connect 24 hours.Taking 1 μ l and connect product, voltage 18000V, it is (biological purchased from Pu Luomaige (Beijing) that electricity forwards escherichia coli DH10 β to Technology Co., Ltd., i.e. U.S. Promega company), add 800 μ l LB culture medium, recover 45 minutes, take 200 μ l and be applied to containing kanamycin LA flat board, 37 DEG C, overnight incubation.Choosing monoclonal, amplification culture takes out plasmid, digestion verification, and enzymatic cleavage methods is ibid.Choose positive colony, structure The carrier electricity built up convert Agrobacterium (Agrobacterium tumefaciens) EHA105 (purchased from MBIA company, Http:// www.cambia.org/daisy/cambia/materials/overview.html) take out plasmid, PCR verifies, takes 750 μ l containing building carrier Agrobacterium bacterium solution adds isopyknic 50% glycerol mixing ,-70 DEG C of preservations.By the carrier containing Oryza sativa L. miR160b gene overexpression The named EHA105-pU1301-miR160b of agrobacterium strains of pU1301-miR160b (see Figure 10).

2. genetic transformation

Use agrobcterium-mediated transformation (Hiei etc., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, 1994, Plant J, 6:271-282) by overexpression Bacterial strain EHA105-pU1301-miR160b Introduced into Rice japonica rice variety is spent in No. 11 wound healing, through preculture, infect, co-culture, screen tool Have hygromycin resistance wound healing, break up, take root, seedling exercising and transplant land for growing field crops and obtain transfer-gen plant (Agrobacterium-mediated genetic transformation reagent and formula See the application for a patent for invention prospectus of applicant, publication number: CN1995346, patent name: paddy lignin synthesis gene FC 1 and should With, application number: 200610018105.5).

3. transfer-gen plant copy number detection

By in the wound healing spending No. 11 in overexpression bacterial strain EHA105-pU1301-miR160b Introduced into Rice japonica rice variety, obtain transformation seedlings 40 altogether Strain, utilizes Southern to hybridize (concrete operations reference: Wu etc., Development of enhancer trap lines for functional analysis of The rice genome.2003, Plant J, 35:418-427) analyze the copy number of these overexpression transfer-gen plants.Result is as in figure 2 it is shown, obtaining In the 40 strain transfer-gen plants obtained, positive rate is 90%, and single copy plant has 10 strains.Choose No. 4-7 single copy plant and separate offspring for follow-up Research material.

The phenotype analytical of embodiment 4 overexpression miR160b transfer-gen plant

Obtain 40 strain transfer-gen plants by genetic transformation, and the method utilizing southern to hybridize determines single copy plant 4-7, to overexpression MiR160b mono-copy transgenic progeny carries out phenotype investigation, finds that overexpression miR160b transgenic positive makes plant all table from seedling stage to period of maturation Reveal the mutant phenotype that tillering angle increases, and the period of maturation becomes apparent from.Respectively to 30 strains miR160b overexpression negative control plant (WT) Statistical analysis is carried out with tillering angle, available tillering and the setting percentage of positive plant (Ubi::miR160b) plant.The tillering angle of the present invention It is defined as rice maturity stem and angle between edge available tillering.Result as it is shown on figure 3, with overexpression feminine gender plant (WT) phase Ratio, the tillering angle of overexpression positive plant increases to 37.9 ° ± 6.2 from 22.0 ° ± 5.3, and available tillering is reduced to 9.2 ± 2.7 from 14.2 ± 5.5, Setting percentage is reduced to 35.99% ± 0.05 from 75.11% ± 0.1, and the left side in each figure of Fig. 3 is that miR160b overexpression feminine gender plant is (with WT Labelling), the right in each figure of Fig. 3 is miR160b overexpression positive plant (with Ubi::miR160b labelling).

Gene expression analysis in embodiment 5 overexpression miR160b transfer-gen plant

In order to verify in overexpression miR160b transgenic positive plant whether the expression of ripe miR160b is improved, we are respectively at extracting In the overexpression miR160b transgenic positive of tiller animated period and the negative root (R) of plant, stem (C), leaf (L), sheath (LS), lateral bud (B), The RNA sample of children's fringe (P) these six tissue sites, by using the expression of method detection miR160b same as in Example 2, arranges 3 Secondary pollutant repeats, 2 technology repeat.We are by same RNA sample afterwards, and reverse transcription becomes cDNA, use QRT-PCR detection OsARF13, The expression (the QRT-PCR primer sequence of target gene is shown in Table 2) of OsARF18 and OsARF22 these three target gene.Specifically comprise the following steps that 1. Utilizing TransZol (Beijing Quanshijin Biotechnology Co., Ltd) extracting RNA sample, concrete operation step sees TransZol description.2. Reverse transcription uses M-MLV Reverse Transcription box (purchased from invitrogen company), specifically comprises the following steps that following system is placed in 37 DEG C of water-baths by (1) 15min:toal RNA 5 μ g, 10 × DNase buffer 1 μ l, DNase I 1 μ l, Add DEPC Water to 10 μ l；(2) in system, add 1 μ l 25mM EDTA, room temperature 2min；(3) 65 DEG C of water-bath 5min, are immediately placed in 5min on ice；(4) 1 μ l oli (dT), 65 DEG C of water-bath 10min are added, fast Speed is placed in 5min on ice；(5) in the above-mentioned RNA sample handled well, following system is added: 5 × First strand buffer (purchased from promega company) 4 μ l, 0.1M DTT 1 μ l, 10mM dNTP 1 μ l, RRI 1 μ l, DEPC Water 1 μ l, preheats 2min at 37 DEG C；(6) add in preheated system Entering 1 μ l M-MLV (200U/ μ l), careful mixing is placed on 37 DEG C of reaction 50min；(7) 70 DEG C of inactivation 15min ,-20 DEG C of preservations.3. quantitative RT-PCR Use 384 hole PCR plate to carry out on ViiA7, use the method for 2-△ CT relative quantification to carry out the comparison of expression.10 μ l reaction systems comprise: 3 μ l reverse transcription products, 5 μ lFastStart Universal SYBR Green Master (ROX) (purchased from Products Co., Ltd of Roche Diagnistics), left and right primer Each 0.25 μ l, Water 1.5 μ l.Response parameter is provided that 95 DEG C/10min；95 DEG C/15sec, 60 DEG C/60sec, 40cycles.Result such as Fig. 4 Shown in, ripe miR160b expression in overexpression positive plant 6 is organized has rising in various degree compared with overexpression feminine gender plant, It can thus be appreciated that Oryza sativa L. miR160b gene overexpression vector (construction method of this carrier is shown in the detailed description in embodiment 3) improves maturation really MiR160b expression in plant body, and the expression of these 3 target genes is organized than overexpression feminine gender plant overexpression positive plant 6 6 tissues have reduction in various degree it was confirmed miR160b is inhibited to the expression of target gene.

The functional analysis of embodiment 6 miR160b gene

1..RLM--5 ' RACE analyzes the shearing site of target gene

Think that the target gene of miR160 belongs to Oryza sativa L. ARF family by bio information prediction and document report.The target gene quilt of microRNA Therefore miRNA-RISC can analyze miRNA-RISC by the method for RACR cut RNA with stable existence a period of time after cutting off The position of molecule.Then 3 genes (OsARF13, OsARF18 and OsARF22) during applicant have chosen ARF family, use RLM-5 ' RACE technology detects the position that these 3 genes are sheared by ripe miR160b, and the primer of 3 target genes of design is as follows:

GeneRacer^TM5’Primer:CGACTGGAGCACGAGGACACTGA

GeneRacer^TM5’Nested Primer:GGACACTGACATGGACTGAAGGAGTA

OsARF13-RACE:AACACCACATCCCGTCAAACTGCCG

OsARF13-RACE-Nested:CATCATCTACACCCTCTGGAACCTG

OsARF18-RACE:CCAGCCGCATCACGGTAGAAAACA

OsARF18-RACE-Nested:GTGGTGCCATTCTGATTGCTTCTC

OsARF22-RACE:GAACTATCACGCCTGCTCAACTAAG

OsARF22-RACE-Nested:CGTATGACCCAAAGACAGACAAATC

MiRNA target gene shearing site uses GeneRacer^TMTest kit (purchased from invitrogen company), specifically comprises the following steps that

1, RNA oligo:(1 is connected) containing 0.25 μ g GeneRacer^TMThe centrifuge tube of RNA oligo adds the total serum IgE of 5 μ g children's fringes, Inhale up and down with rifle and beat mixing, suspension RNA oligo；(2) 65 DEG C of dry bath 5min, the secondary structure of release RNA；(3) 2min is placed on ice, Of short duration centrifugal；(4) in centrifuge tube, following reaction system is added: 10 × Ligase Buffer 1 μ l, 10mM ATP 1 μ l, RNase OUT^TM(40U/μl) 1 μ l, T4RNA Ligase (5U/ μ l), inhale gently and play mixing, of short duration centrifugal；(5) 37 DEG C of of short duration being centrifuged of dry bath 2h (6) are placed on ice afterwards.

2, purification and precipitation RNA:(1) add 90 μ lDEPC water, 100 μ l phenol: chloroform, reverse mixing 30s；(2) room temperature 12000rpm from Heart 5min (3) draws supernatant in new centrifuge tube；(4) adding 2 μ l10mg/ml mussel glycogen 10 μ l, 3M sodium acetate (pH5.2) mixing continues Continuous 220 μ l 95% ethanol that add mix；(5) 10min is placed on ice；(6) 4 DEG C of 12000rpm are centrifuged 20min and precipitate RNA；(7) rifle head is used Absorb supernatant, note the direction of precipitation；(8) 500 μ l 70% ethanol are added, reverse mixing；(9) 4 DEG C, 12000rpm is centrifuged 2min；(10) Remove supernatant with rifle head, of short duration centrifugal after remove remaining ethanol；(11) superclean bench dries up 1-2min；(12) 10 μ l DEPC water are added.

3, reverse transcription (uses SuperScript^TMIII RT reaction): (1) adds following system: primer 1 μ l in 10 μ l RNA sample, DNTP mix 1 μ l, sterile distilled water 1 μ l；(2) 65 DEG C of dry bath 5min, discharge RNA secondary structure；(3) 1min is placed on ice, Of short duration centrifugal；(4) in above centrifuge tube, serial response system is continuously added: 5 × First Strand Buffer 4 μ l, 0.1M DTT 1 μ l, RNaseOUT^TM(40U/ μ l) 1 μ l, SuperScript^TMIII RT(200u/μl)1μl；(5) dozen mixing is inhaled up and down with rifle, of short duration centrifugal；(6)50 DEG C dry bath 1h；(7) 70 DEG C of inactivation 15min, place 2min on ice, of short duration centrifugal；(8) add 1 μ l RNaseH (2U) (9) 37 DEG C and react 20min； (10) of short duration centrifugal ,-20 DEG C of preservations.

4, first round PCR, reaction system: take cDNA 2 μ l, GeneRacer that above-mentioned steps obtains^TM5 ' Primer 1 μ l, Gene specific primer 1 μ l, 10mM dNTP 1 μ l, 2 × GC buffer I 25 μ l, LA Taq 0.5 μ l, benefit sterilizing ultra-pure water to 50 μ l.Response procedures: 94 DEG C of 3min； 94 DEG C of 30s, 68 DEG C of 2min repetitive cycling 5 times；94 DEG C of 30s, 65 DEG C of 30s, 68 DEG C of 2min repetitive cycling 5 times；94 DEG C of 30s, 60 DEG C of 30s, 68 DEG C of 2min repetitive cycling 10 times；94 DEG C of 30s, 55 DEG C of 30s, 68 DEG C of 2min repetitive cycling 15 times；68℃7min；25℃1min.

Second takes turns PCR, reaction system: first round PCR primer 2 μ l, GeneRACE nest5 ' Primer 1 μ l, Gene specific nest primer 1 μ l, 10mM dNTP 1 μ l, 2 × GC buffer I 25 μ l, LA Taq 0.5 μ l, benefit sterilizing ultra-pure water to 50 μ l.Response procedures is with first round PCR. The PCR primer that second takes turns is dug glue recovery rear clone (limited purchased from Beijing full formula gold biotechnology in carrier T pEASY-T1cloning vector Company, Figure 12 is shown in by the collection of illustrative plates of this carrier) on, then sequence verification.Result as it is shown in figure 5, OsARF13, OsARF18 and OsARF22 this The site that 3 genes are sheared by miR160-RISC is positioned at the position of the 10th and the 11st nucleotide of miR160.Therefore applicant thinks These 3 genes of OsARF13, OsARF18 and OsARF22 are the target genes of miR160b.

2, expressing Ubi::MIM160b makes the mutant phenotype of Ubi::miR160b recover

Research before applicant finds, increases the expression of miR160b gene, the tillering angle of plant can be made to increase, and then, we think Know if suppressing whether the expression of miR160b gene there will be the phenotype contrary with the overexpression mutant phenotype of miR160b gene.The application Utilize the Oryza sativa L. natural target mimic supplying to carry on plasmid MT375 (see Fig. 9) that Germany horse general biological development institute Detlef professor Weigel carries Gene OsIPS1 (with the sequence that 24nt and Oryza sativa L. miRNA osa-MIR399 is complementary on the mRNA of OsIPS1 genetic transcription), with reference to target Mimicry technology (Franco-Zorrilla etc., Target mimicry provides a new mechanism for regulation of microRNA Activity.Nat Genet, 2007,39:1033-1037) primer of design MIM160b, i.e. in the 10th and the 11st base of ripe miR160b Between increase the prominent ring of 3 bases so that ripe miR160b can not be combined with target gene, and primer sequence is as follows:

MIM-III:CCCTGCCTTCATACGCTATT

MIM-IV:ATTGCCAAATGTTTGAACGA

MIM160b I:ATTGCCTGGCTCTAGACTGTATGCCACCTTAGTAGAGGTAAAAGTC

MIM160b II:GGTGGCATACAGTCTAGAGCCAGGCAATTATTCGGTGGATGTCTGT

Applicant uses the method for Overlap extension PCR sequence complementary with osa-MIR399 in OsIPS1 on MT375 plasmid to be replaced with afterwards The sequence that MIM160b is complementary, forms artificial target mimic gene.Specifically comprise the following steps that use primer combination MIM160b-I+MIM-III Carrying out the reaction of first round PCR with MIM160b-II+MIM-IV, reaction system is: 5 × phusion HF Buffer 10 μ l, 10mM dNTPs 1 μ l, 50ng/ μ l MT375 plasmid 1 μ l, 10 μMs of each 0.3 μ l of left and right primer, phusion DNA polymerase (finnzymes) 0.5 μ l, Mend sterilizing ultra-pure water to 20 μ l.Reaction condition is: 98 DEG C of 30s；98 DEG C of 10s, 58 DEG C of 30s, 72 DEG C of 15s repetitive cycling 35 times；72℃ 7 Min, 25 DEG C of 10s.By PCR primer in 0.8% agarose gel electrophoresis, (Shanghai is raw to reclaim test kit with SanPrep pillar DNA glue after digging glue Work biological engineering company limited) reclaim PCR primer, finally it is dissolved in 20 μ l ultra-pure waters.Enter by two kinds of PCR primer of the first round PCR reclaimed Row second takes turns PCR.Reaction system is: 10x ExTaqE Buffer 2 μ l, 10mM dNTPs 0.3 μ l, two kinds of each 0.5 μ l of first round PCR primer, The each 0.3 μ l of primer MIMIII and MIMIV, Ex Taq (precious biological engineering Dalian company limited) the 0.2 μ l of 10 μMs, mends sterilizing ultra-pure water to 20 μl.Reaction condition is: 94 DEG C of 5min；94 DEG C of 45s, 60 DEG C of 45s, 72 DEG C of 1min repetitive cycling 30 times；72 DEG C of 7min, 25 DEG C of 10s. Final PCR primer is cloned on carrier T pEASY-T1cloning vector (Beijing Quanshijin Biotechnology Co., Ltd), and then order-checking is tested Card.It is connected to pU2301 after utilizing BamH I and Kpn I enzyme action and (is changed the Hygromycin marker on carrier into G418 labelling by pU1301 empty carrier Transformation) on carrier, build and obtain pU2301-MIM160b carrier (i.e. Ubi::MIM160b, the two carrier is named different, substantially It is same carrier, sees Figure 11).The pU2301-MIM160b carrier electricity built is proceeded to Agrobacterium (Agrobacterium tumefaciens) bacterial strain EHA105, spends 11 (i.e. ZH11) and miR160b gene overexpression positive respectively in rice transformation japonica rice variety.Result respectively obtains 36 and 35 Strain transfer-gen plant, conversion ratio is respectively 80.6% and 77.8%, but Ubi::MIM160b spends in turning in 11 (ZH11) positive plant and do not sends out Change in existing plant type, and Ubi::MIM160b turns and occurs in that, in the positive plant of Ubi::miR160b, the plant that mutant phenotype is recovered, utilization QRT-PCR detection tri-target genes of OsARF13, OsARF18 and OsARF22 wild type (WT) ZH1, Ubi::MIM160b, Expression in Ubi::miR160b, Ubi::MIM160b/Ubi::miR160b finds, the expression of Ubi::MIM160b target gene is higher than WT, The expression of Ubi::MIM160b/Ubi::miR160b target gene is lower than WT, but higher than Ubi::miR160b (as shown in Figure 6).According to this Result understands MIM160b and inhibits the expression of miR160b gene, and the expression ultimately resulting in target gene rises, so that miR160b overexpression Mutant phenotype be restored.

2.. the expression detection of tillering angle and auxin related gene

The result of study display overexpression miR160b gene of the application shows the mutant phenotype that tillering angle increases, and the target base of miR160b Because belonging to Oryza sativa L. ARF family, ARF is a class auxin response factor in plant, and they are by being combined in the AuxRE of specific gene promoter region On element (TGTCTC), activate or the expression of Developing restraint element response gene.Therefore to further appreciate that Oryza sativa L. mi160b and tiller angle Degree and the relation of auxin, the application have selected Exemplary gene (such as LAZY1 (LOC_Os11g29840), the LPA1 of some regulation and control tillering angles (LOC_Os03g13400), TAC1 (LOC_Os09g35980), and gene (the such as GH3.1 relevant to auxin (LOC_Os01g57610), GH3.4 (LOC_Os05g42150, GH3.8 (LOC_Os07g40290), PIN1 (LOC_Os02g50960), IAA4 (LOC_Os01g08320), IAA21 (LOC_Os06g22870)).QRT-PCR is used to detect these genes at overexpression miR160b Expression (the quantitation RT-PCR primer sequence of design is shown in Table 2) in positive and negative plant lateral bud.

The QRT-PCR primer of the OsARF, tillering angle and the auxin related gene that use in table 2 present invention

The concrete steps of QRT-PCR experiment are carried out with reference to the step of embodiment 5, and result is as shown in Figure 7.Test shows, tillering angle gene LAZY1 Expression in overexpression miR160b positive plant is less than negative plant, and the expression of LPA1 and TAC1 gene is at overexpression miR160b Positive and negative plant change inconspicuous.GH3.4, IAA4 are decreased obviously compared with negative plant with the expression of IAA21 gene, other genes Change inconspicuous.It follows that the expression improving miR160b gene changes tillering angle and the expression of auxin related gene, explanation The suppression of the OsARF of miR160b mediation may take part in the response process of auxin and the regulation and control of rice tillering angle.

Claims (4)

1. The application in adjusting and controlling rice tillering angle of the 1.miR160b gene, it is characterised in that: the nucleoside of described miR160b gene Acid sequence is as shown in sequence table SEQ ID NO:1.
2. The application in adjusting and controlling rice tillering angle of the 2.miR160b gene, it is characterised in that: the ripe core formed after transcribing shearing Heart sequence is as shown in SEQ ID NO:3.
3. Apply the most as claimed in claim 1 or 2, it is characterised in that: express miR160b gene by overexpression or suppression, Change the regulation and control model of miR160b gene pairs plant type of rice.
4. Apply the most as claimed in claim 3, it is characterised in that described plant type of rice includes tillering angle.