CN106701756A - Application of microRNA172 to regulation of plant height, ear type and leaf size of rice - Google Patents

Abstract

The invention relates to the technical field of plant genetic engineering, and particularly relates to application of microRNA172 to regulation of plant height, ear type and leaf size of rice. The invention further provides an application method of an MIM172 gene segment of an antagonist miR172 to the genetic improvement of the rice. The miR172b is a nucleotide sequence shown in SEQ ID NO:1; the pri-miR172b is a nucleotide sequence shown in SEQ ID NO:2. Overexpression of pri-miR172b leads to smaller leaf of the rice, branching of an ear part, and reducing of ear grains. The antagonist MIM172 is a nucleotide sequence shown in SEQ ID NO:3, and reducing the expression quantity of miR172 through the antagonist MIM172 leads to shorter plant height of the rice, larger leaf, branching of the ear part, and increasing of the ear grains. The results prove that the miR172 can regulate the plant type of the rice, and has an important application value.

Description

Applications of the microRNA172 in adjusting and controlling rice plant height, fringe type and leaf blade size

Technical field

The present invention relates to field of plant genetic.Specifically related to a kind of microRNA172b (hereinafter abbreviated as miR172b) and its precursor-gene Pri-microRNA172b (hereinafter abbreviated as pri-miR172b), microRNA172 (hereinafter abbreviated as miR172) antagonist MIM172 are in regulation and control water Application in rice plant height, fringe type and leaf blade size.Increase the expression quantity of miR172b by technique for gene engineering, Rice Panicle branch stalk can be caused to reduce, Grain number per spike is reduced, blade shortens, blade narrows；And using antagonist MIM172, (antagonist is one section of engineer long with the 24bp of synthesis Genetic fragment, can be used to reduce the expression quantity of miR172), then Plant Height of Rice can be caused to become, and short, fringe portion branch stalk increases, grain number per spike increases, leaf Piece is elongated, blade broadens, and these results show that the expression quantity by manipulating miR172 can provide a kind of method to improve the plant type of paddy rice.

Background technology

With the continuous growth and the change of human consumption's mode of population in the world, the crisis in food in the whole world is increasingly serious.Paddy rice is that the whole world is most important One of cereal crops, China has more than the population of half with paddy as staple food grain, therefore improves the yield of paddy rice to ensureing that it is heavy that the grain security of country has The strategic importance wanted.Eighties of last century, because the utilization of breeding wheat for semidwarfness and hybrid vigour, the yield of paddy rice there occurs leaping for matter twice.But it is near several Over 10 years, the yield of paddy rice but secular stagnation not before, the need for being increasingly difficult to meet socio-economic development, therefore need new technology and genetic resources badly The Main Agronomic Characters such as yield, resistance, quality for improveing paddy rice.

The yield of paddy rice is closely related with its plant type, and the Breeding Model with ideotype as Breeding objectives is paid attention in agricultural production by height.Water The plant type of rice mainly includes content (Wang and Li, the Molecular basis such as size and angle of plant height, tiller, fringe type, root morphology and organ of plant architecture.Annu Rev Plant Biol,2008,59:253-279).Wherein plant height, tiller, fringe type and grain weight are all for a long time to lose Pass the important goal proterties of improvement.Plant height be with rice yield, photosynthesis and the proterties closely related with lodging resistance, eighties of last century is reducing Plant height greatly improves the yield of global crops for the first time " green revolution " of breeding objective.With the development of science of heredity and molecular biology, Hormone particularly gibberellin is illustrated and rape element sterol has played effect (Wang the and Li, Molecular of key in the plant height of regulation plant basis of plant architecture.Annu Rev Plant Biol,2008,59:253-279)。

The fringe of paddy rice is the purpose organ harvested in agricultural production, and its form and composition directly determine the yield of paddy rice.The fringe of paddy rice is a kind of circular cone Inflorescence, is developed by inflorescence meristem, and it can typically produce Primary branch and Secondary branch, and higher level can also be produced in a few cases Branch stalk, branch stalk can further bud into small ear so that produce flower, rice will be formed after floral organ chasmogamy.With the plant such as arabidopsis Difference, the growth course of rice panicle type is determined by a series of transformation of separate living tissue destiny.Under the conditions of suitable internal and external environment, paddy rice is by nutrition Reproductive growth is grown into, at the same time, apical meristem produces Primary branch point also converted into inflorescence meristem by inflorescence meristem Raw tissue, after a number of Primary branch separate living tissue is produced, inflorescence meristem can degenerate.And Primary branch separate living tissue can be produced The Secondary branch separate living tissue of raw higher level, also can directly differentiate small ear separate living tissue, and Secondary branch separate living tissue can be repeated once branch and obstruct mitogenetic The development models of tissue, final branch stalk separate living tissue is all changed into small ear separate living tissue.And small ear separate living tissue can then produce floral meristem, and then Produce the differentiation of floral organ.The fringe time of different separate living tissues largely determines fringe type (Kyozuka et al., the Control of of paddy rice grass inflorescence form by the fine-tuning of meristem phase change.Curr Opin Plant Biol,2014,17:110-115)。 If the time that inflorescence meristem is maintained is more long, more Primary branch will be produced；And if the differentiation of small ear separate living tissue is postponed, just There may be even three times branch stalks of more Secondary branch, so as to produce more grain number per spikes.In view of significance of the fringe type in agricultural production, section Scholar has discovered that the gene of substantial amounts of control fringe type development, and some genes applied in the practices of breeding (Zhang and Yuan, Molecular control of grass inflorescence development.Annu Rev Plant Biol,2014,65:553-578)。

Growing for plant needs to consume substantial amounts of energy, and photosynthesis is the topmost energy source of plant.Leaf is that plant carries out photosynthesis Topmost organ, it grows and not only determines the plant type of plant, while also determine that plant carries out photosynthesis, assimilation carbohydrate Ability.The leaf of paddy rice includes blade and leaf sheath, and pulvinus can be also formed in the junction of the two, and pulvinus often differentiates auricle and the tip of a leaf.Leaf It is to be come by the apical meristem development of vegetative growth phase, because the polar translocation of auxin can cause to form one in the edge of separate living tissue The region of local auxin Cmax, separate living tissue specific expression gene OSH1 can be suppressed in the region, so that the region is thin Born of the same parents are finally divided into phyllogen basal cell, start the development program of leaf.With the development of phyllopodium, can gradually form top based polar, abdomen backplane and in Side polarity, so as to determine the spatial framework of leaf.The final size of blade then depends on the comprehensive function that cell division and cell extend.

MicroRNA (miRNA) be a class length about in the 20-24 RNA molecule of the endogenous non-coding of nucleotides, in nearly all research Have been found that the gene of substantial amounts of coding miRNA is present in the higher organism crossed.MiRNA often through regulating mRNA stability or tune Control protein translation process controls the activity of target gene mRNA, so as to adjust including each including growth, development, metabolism, response environment etc. Vital movement process.Because the regulatory mechanism of its powerful biological function and complexity, miRNA related research has become current life science One of popular domain, and it is possible to played an important role in the genetic improvement of crops.

Coding multiple miRNAs in paddy rice body, but the biological function of the overwhelming majority is still unknown, needs more work badly to illustrate these The effect of tiny RNA and the value in rice genetic improvement.In addition in view of same miRNA is often to be encoded by multiple precursor-genes, Therefore it is extremely difficult to obtain the loss of function mutant of specific miRNA.Studies have reported that can be with using a kind of technology for being referred to as target mimicry Effectively reduce expression quantity (Franco-Zorrilla et al., the Target mimicry provides a new mechanism for of miRNA in plant regulation of microRNA activity.Nat Genet,2007,39:1033-1037).The technology is by designing a kind of and target miRNA parts The simulation target RNA for matching somebody with somebody, it can be combined with miRNA, but can not be sheared by miRNA.This simulation target RNA of overexpression in plant Can just be combined with target miRNA, and prevent effects of the miRNA to target gene real in plant；Simulation target RNA can also lead to simultaneously Crossing a kind of unknown mechanism causes target miRNA to be degraded.Although the technology can be used for disturbing the activity of miRNA, it is also rarely used for The genetic improvement of paddy rice.

The means that the present invention passes through plant genetic engineering, study the function of miRNAs in paddy rice, and excavation is possibly used for changing water in agricultural production The miRNAs of rice plant type, to provide new genetic resources for rice breeding；And disturb these miRNA's using the technology of target mimicry Activity improves the economical character of paddy rice.

The content of the invention

It is to provide a kind of miR172b and precursor-gene pri-miR172b, miR172 antagonist MIM172 in adjusting and controlling rice that the purpose of the present invention is Application in plant type.Another object of the present invention is to provide a kind of carrier of overexpression miR172b and MIM172, and the carrier is turning Application in trans-genetic hybrid rice.The present invention also provides a kind of method for changing plant type of rice by adjusting the expression quantity of miR172 simultaneously.miR172b With such as SEQ ID NO:Nucleotide sequence shown in 1, also including with SEQ ID NO:Nucleotides sequence shown in 1 shows more than 90% homology Gene order, also mutant allele or derivative including being produced because inserting, substitute or lacking one or more bases.Present invention additionally comprises The precursor-gene pri-miR172b of miR172b is encoded, the pri-miR172b has such as SEQ ID NO:Nucleotide sequence shown in 2, Yi Jiyu SEQ ID NO:Nucleotides sequence shown in 2 shows the gene order of more than 90% homology, also including because inserting, substituting or lacking one or more alkali Base and allele of mutant for producing or derivatives thereof.Simultaneously present invention additionally comprises the antagonist MIM172, antagonist MIM172 of miR172 With such as SEQ ID NO:Nucleotide sequence shown in 3, and include the DNA fragmentation of MIM172, and/or the expression containing MIM172 is carried Body, and with SEQ ID NO:Nucleotides sequence shown in 3 shows not more than 6 derivatives of base difference.

The present invention can cause that Rice Panicle branch stalk is reduced, grain number per spike is reduced, blade shortens, blade narrows by increasing the expression quantity of miR172b； And the expression quantity of miR172 is reduced using antagonist MIM172, then can cause that Plant Height of Rice becomes short, fringe portion branch stalk increases, grain number per spike increases, Variable length blade, blade broaden, these results show by the expression quantity that manipulates miR172 can for improve the economical character of paddy rice provides it is a kind of newly Breeding method.MiR172b and its precursor-gene pri-miR172b can be used to be opened with other controlling elements, such as constitutive promoter or organ specificity Mover constructs expression vector；Also can by transgenic technology, antisense RNA, RNAi, Zinc finger nuclease (zinc-finger nucleases, ZFN), activating transcription factor sample effector nuclease (transcription activator-like effector nucleases, TALEN) and The technologies such as CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)/Cas9 manipulate to regulate and control the plant type of plant to it； And antagonist MIM172 then can be used for constructing gene expression load with other controlling elements, such as constitutive promoter or organ specific promoters Body, the means such that it is able to pass through genetic engineering change the plant type of plant.

Realize that concrete technical scheme of the invention is as follows：

1st, the result annotated according to rice genome, isolates the DNA for including coding miR172b precursor-genes pri-miR172b from paddy rice Fragment, specific separation method has a detailed description in embodiment 1.

2nd, the above-mentioned DNA fragmentation for including pri-miR172b is connected on pCAMBIA1301S carriers, builds the overexpression of miR172b Carrier, the carrier is named as 35S by us:miR172bOE；Specific construction method has a detailed description in example 2.

3rd, using method (Franco-Zorrilla et al., the Target mimicry provides a new mechanism for of target mimciry regulation of microRNA activity.Nat Genet,2007,39:1033-1037), design can disturb the artificial antagonist of miR172 activity MIM172, its nucleotide sequence such as SEQ ID NO：Shown in 3；And the method for passing through Overlap extension PCR, MIM172 is fused to and is come from In order to build the expression vector for genetic transformation in the Gene A tIPS1 of the non-coding protein of arabidopsis；Specific method has in detail in embodiment 3 Thin description.

4th, the AtIPS1 that will merge has antagonist MIM172 is connected on pCAMBIA1301S carriers, builds overexpression antagonist MIM172 Carrier 35S:MIM172.Specific method has a detailed description in example 4.

5th, using agriculture bacillus mediated transgenic method (Lin and Zhang, the Optimising the tissue culture conditions for for optimizing high efficiency transformation of indica rice.Plant Cell Rep,2005,23:540-548) by expression vector 35S:MiR172bOE and 35S:11 (Institute of Crop Science, Chinese Academy of Agricultural Science) are spent in MIM172 Introduced into Rice acceptors, transformed plant is obtained；Specific method is being implemented Had a detailed description in example 5.

6th, positive transgenic plant is analyzed and identified by the method for PCR；And in the genotype and phenotype of T1 generation identification transfer-gen plants, be total to Separation detection, and statistical analysis is carried out to Relevant phenotype；Specific method has a detailed description in embodiment 6.

7th, using method (Shen et al., the Global expression profiling of rice microRNAs by one-tube of stem-loop RT-PCR stem-loop reverse transcription quantitative PCR revealed important roles of microRNAs in abiotic stress responses.Mol Genet Genomics,2010,284:477-488) analyze the expression quantity of miR172 in transfer-gen plant；Specific method is in embodiment Had a detailed description again in 7.

8th, by hybridize method analyze MIM172 whether the effect of antagonism miR172 overexpressions；Specific method is in embodiment 8 by retouching in detail State.

Compared with prior art, advantages of the present invention is as follows：

1st, present invention discover that overexpression miR172b can reduce the length and width of blade；

2nd, the present invention adjusts the expression quantity of miR172 in paddy rice using antagonist MIM172 first；

3rd, the present invention has found using the expression quantity of antagonist MIM172 reductions miR172 Plant Height of Rice can be caused to become short, fringe portion branch stalk in paddy rice Rise with grain number per spike, blade becomes big, and the change of the change of these proterties, especially panicled characters, the need for just meeting rice genetic improvement；

4th, the present invention utilizes the activity for adjusting miR172 for the genetic improvement of plant type of rice and blade is provided a method that.

Brief description of the drawings

Sequence table SEQ ID NO:1 is the nucleotide sequence of miR172b.

Sequence table SEQ ID NO:2 is the nucleotide sequence of the precursor pri-miR172b of miR172b, and its 208-228 nucleotide sequence coded miR172b。

Sequence table SEQ ID NO:3 is the nucleotide sequence of the antagonist MIM172 of the miR172 of engineer.

Fig. 1：The RNA secondary structures of the precursor-gene pri-miR172b of miR172b.Description of reference numerals：

The secondary structure is using disclosed software kit ViennaRNA Package (http://www.tbi.univie.ac.at/RNA/) according to minimum free Can calculate, part is the sequence of miR172b shown in red letters.

Fig. 2：Expression vector 35S:MiR172bOE building process schematic diagrames.Description of reference numerals：

A figures in Fig. 2：Insertion expression vector 35S:The DNA fragmentation schematic diagram for including pri-miR172b of miR172bOE.

B figures in Fig. 2：For construction of expression vector 35S:The structural representation of the empty carrier pCAMBIA1301S of miR172bOE.The present invention Fragment shown in A figures in Fig. 2 is inserted into the B in Fig. 2 by KpnI and BamHI double digestions and schemes shown carrier pCAMBIA1301S's The expression vector 35S of conversion is formed in the middle of KpnI and BamHI restriction enzyme sites:miR172bOE.

Fig. 3：The expression vector 35S that the present invention builds:The collection of illustrative plates of miR172bOE.

Figure:4：Artificial antagonist MIM172 is with miR172b oligonucleotide ligands to schematic diagram.Description of reference numerals：

Red letters A represents the 10th nucleotides of miR172b；Blue letters U represents the 11st nucleotides of miR172b；The four of purple Individual letter represent in MIM172 with the unmatched nucleotides of miR172 and the small circular protrusion that is formed, the structure is that MIM172 plays a role Key point.

Fig. 5：Antagonist MIM172 fusions are entered the process schematic of arabidopsis AtIPS1 genes using Overlap extension PCR method.Reference Explanation：

Red, blue, green and purple fragment are represented for designing the section of primer in AtIPS1, and orange fragment represents antagonist MIM172. In synthetic primer MIM172F and MIM172R, directly the sequence of MIM172 is blended in the middle of the sequence of AtIIPS1.

Fig. 6：Expression vector 35S:MIM172 building process schematic diagrames.Description of reference numerals：

A figures in Fig. 6：Insertion expression vector 35S:The DNA fragmentation schematic diagram for including antagonist MIM172 of MIM172.

B figures in Fig. 6：For construction of expression vector 35S:The structural representation of the empty carrier pCAMBIA1301S of MIM172.

Fragment shown in A figures in Fig. 6 is inserted the B in Fig. 6 by KpnI and BamHI double digestions and schemes shown carrier by the present invention The expression vector 35S of conversion is formed in the middle of KpnI the and BamHI restriction enzyme sites of pCAMBIA1301S:MIM172.

Fig. 7：The expression vector 35S that the present invention builds:The collection of illustrative plates of MIM172.

Fig. 8：11 (WT) and 35S are spent in wild type:The phenotype of miR172bOE (miR172OE) transfer-gen plant compares.Description of reference numerals：

A figures in Fig. 8：It is to spend 11 (WT) and 35S in wild type:The plant height of miR172bOE (miR172OE) transfer-gen plant and tiller Form compares.

B figures in Fig. 8：It is to spend 11 (WT) and 35S in wild type:The fringe type of miR172bOE (miR172OE) transfer-gen plant compares.

C figures in Fig. 8：It is to spend 11 (WT) and 35S in wild type:The Primary branch form of miR172bOE (miR172OE) transfer-gen plant

Compare.

Fig. 9：11 (WT) and 35S are spent in wild type:The phenotype of MIM172 (MIM172) transfer-gen plant compares.Description of reference numerals：

A figures in Fig. 9：It is to spend 11 (WT) and 35S in wild type:The plant height of MIM172 (MIM172) transfer-gen plant and the form of tiller Compare.

B figures in Fig. 9：It is to spend 11 (WT) and 35S in wild type:The fringe type of MIM172 (MIM172) transfer-gen plant compares.

C figures in Fig. 9：It is to spend 11 (WT) and 35S in wild type:The Primary branch form of MIM172 (MIM172) transfer-gen plant compares.

Figure 10：11 (WT), 35S are spent in wild type:MiR172bOE (miR172OE) and 35S:MIM172 (MIM172) transfer-gen plant Sword-like leave form compare.

Figure 11：11 (WT) and 35S are spent in detecting wild type using real-time quantitative PCR:In miR172bOE (miR172OE) transfer-gen plant MiR172b expression quantity.Data display is 3 average value ± standard errors of repetition.

Figure 12：11 (WT) and 35S are spent in detecting wild type using real-time quantitative PCR:MiR172b in MIM172 (MIM172) transfer-gen plant Expression quantity.Data display is 3 average value ± standard errors of repetition.

Figure 13：Antagonisms of the MIM172 to miR172.Description of reference numerals：

A in Figure 13：It is to spend 11 (WT), 35S in wild type:MiR172bOE (miR172OE) transfer-gen plant, 35S:MIM172(MIM172) The fringe type of transfer-gen plant and miR172OE and MIM172 hybrids compares.

B in Figure 13：It is to spend 11 (WT), 35S in wild type:MiR172bOE (miR172OE) transfer-gen plant, 35S:MIM172(MIM172) The statistics of every fringe Primary branch of transfer-gen plant and miR172OE and MIM172 hybrids, every fringe Secondary branch and grains per panicle.Data show What is shown is from 10 average value ± standard errors of individual plant.

Specific embodiment

Embodiment 1：Separation includes the DNA fragmentation of the precursor-gene pri-miR172b of miR172b

Using Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/) and miRBase (http://www.mirbase.org/) two websites are retrieved, and (gene accession number of miRBase databases is to obtain miR172b MIMAT0001070) and its precursor-gene pri-miR172b (gene accession number of miRBase databases is MI0001140) nucleotide sequence. MiR172b maturation body sequences such as SEQ ID NO:Shown in 1, the sequence such as SEQ ID NO of its precursor-gene pri-miR172b:Shown in 2, secondly level Structure is as shown in Figure 1.

According to the analysis result of database sequence, to expand the genetic fragment of pri-miR172b, following primer is designed：

MiR172bOEF (forward primer):5'-GGTACCCAGTAGAGAGTGTGATGCCGCAGCT-3'(underlined sequences are known for KpnI Other site)；

MiR172bOER (reverse primer):5'-GGATCCGCGGCGTTGGTACAATTAAGCTGATG-3'(underlined sequences are BamHI Recognition site).

11 (Institute of Crop Science, Chinese Academy of Agricultural Science) blade STb genes are spent in extracting rice varieties.DNA method for extracting is CTAB methods (Zhang et al.,Genetic diversity and differentiation of indica an japonica rice detected by RFLP analysis.Theor Appl Genet,1992,83:495-499).It is template that 11 blade STb genes are spent in, is entered with primer MiR172bOEF and MiR172bOER Performing PCR is expanded.PCR reaction cumulative volumes are 20 μ l, comprising DNA profiling 100ng, the μ l of 2 μ l, 10mM dNTP of 10xPCR buffer 2, Each μ l of 0.3 μ l, rTaq enzyme 0.2 of 10mM primers MiR172bOEF and MiR172bOER, plus deionized water is to 20 μ l (used PCR Buffer, dNTP, rTaq enzyme etc. is purchased from precious bioengineering Dalian Co., Ltd).PCR reaction conditions are as follows：1. 94 DEG C of 4min, 2. 94 DEG C 40 S, 3. 58 DEG C of 40s, 4. 72 DEG C of 1min, 5. from 2. -4. circulate 38 times, 6. 72 DEG C of 7min, 7. 4 DEG C of preservations.PCR primer is in 1% (matter Amount/volume) TBE Ago-Gels on electrophoresis detection, recovery length is that (the target dna section of 305bp is added 317bp plus on primer Two restriction enzyme site 12bp) DNA fragmentation.The PCR primer of recovery is connected to T-A cloning vectors using T4DNA ligases PGEMT-vector it is upper (T4DNA ligases and carrier pGEMT-vector, are purchased from Pu Luomaige (Beijing) Bioisystech Co., Ltd, That is U.S. Promega companies).(electric conversion instrument is eppendorf Products to the method that connection product is converted by electricity, and applied voltage of the present invention is 1800V, operation instructions of the concrete operations with reference to the instrument) import in Escherichia coli DH10B (being purchased from Promega companies), plus 400 μ l LB Culture medium recovery 45min, is applied to and contains ampicillin, the chloro- 3- indoles-β-D- galactolipins of the bromo- 4- of 5- and isopropyl-β-D-thiogalactoside On LA culture medium flat plates, (LA and LB is formulated reference to 37 DEG C of incubator culture 14-16h：Pehanorm Brooker,《Molecular Cloning:A Laboratory guide》3rd Version, Science Press, 2002).Picking is presented blue monoclonal, and Amplification Culture simultaneously extracts plasmid, by KpnI and BamHI double digestions Screening positive clone, and carry out sequence verification using the PCR primer that T7 and SP6 primer pairs on carrier are inserted.The PCR primer include just like SEQ ID NO:The sequence of the pri-miR172b shown in 2.Recombinant plasmid comprising the PCR primer is named as plasmid TA-miR172b.

Embodiment 2：The structure of the overexpression vector of miR172b

With KpnI and BamHI double digestion plasmids TA-miR172b (described KpnI and BamHI are bought from precious bioengineering Dalian Co., Ltd, Detailed directions refer to the specification of the said firm's corresponding product with consumption, and plasmid TA-miR172b comes from embodiment 1, as shown in the A figures in Fig. 2), The DNA fragmentation of the 317bp comprising pri-miR172b is reclaimed, then with the vector plasmid by KpnI and BamHI double digestions [carrier is disclosed report to pCAMBIA1301S：Zhou et al.,Over-expression of aspartate aminotransferase genes in rice resulted in altered nitrogen metabolism and increased amino acid content in seeds.Theor Appl Genet,2009, 118:1381-1390；Its basic framework originates from Australian CAMBIA laboratories (http://www.cambia.org/daisy/cambia/materials/overview.html) pCAMBIA1301, by adding 35S promoter reality Now to the expression regulation of transformed gene；Its structure is as shown in the B figures in Fig. 2] (Promega companies are purchased from, specifically using T4DNA ligases Usage refers to the specification of the said firm's product with consumption) it is attached.(electric conversion instrument is eppendorf public to the method that connection product is converted by electricity Department product, applied voltage of the present invention be 1800V, concrete operations with reference to the instrument operation instructions) import Escherichia coli DH10B (be purchased from Promega companies) in, plus 400 μ l LB culture medium recovery 45min, it is applied on the LA culture medium flat plates of the kanamycins containing 50mg/L, 37 (LA and LB is formulated reference for DEG C incubator culture 14-16h：Pehanorm Brooker,《Molecular Cloning:A Laboratory guide》The third edition, Science Press, 2002 Year).Picking monoclonal, Amplification Culture simultaneously extracts plasmid, by KpnI and BamHI double digestion screening positive clones, and the expression of gained is carried Body is named as 35S:MiR172bOE, its structure is as shown in Figure 3.

Embodiment 3：The design of antagonist MIM172

According to document (Franco-Zorrilla et al., the Target mimicry provides a new mechanism for regulation of of open report microRNA activity.Nat Genet,2007,39:1033-1037), the activity of Mirnas of plant can be disturbed using the method for target mimicry, Thus designing can be with the antagonist MIM172 sequence of antagonism miR172 activity, the sequence signature such as SEQ ID NO:Shown in 3.MIM172 is 24 length of nucleotides, 3 nucleotides are had more than the miR172 of 21 nucleotides, the two can with complementary pairing, but in miR172 10 and the 11st bit base matches the centre of sequence, and MIM172 has more 3 nucleotides and forms a small circular protrusion.Because receiving miRNA in plant The mRNA of regulation and control is typically all (Schwab et al., the Specific being cut from the centre position matched with miRNA the 10th and the 11st bit base effects of microRNAs on the plant transcriptome.Dev Cell,2005,8:517-527), 3 cores therefore in antagonist MIM172 The small circular protrusion that thuja acid is formed destroys the shear action of miR172, is the key that MIM172 played a role.MiR172 in paddy rice body It is to encode (being respectively pri-miR172a, pri-miR172b, pri-miR172c and pri-miR172d) by 4 precursor-genes, four for being formed Individual miR172 maturations body sequence is highly consistent, is at most no more than 2 bases in head or tail difference each other, therefore designed by the present invention MIM172 can be the miR172 of form of ownership in antagonism paddy rice body, and it is as shown in Figure 4 with the match condition of miR172b.

Applicant further by the method for Overlap extension PCR by the sequence of MIM172 (Overlap extension PCR method refers to Pehanorm Brooker,《Point Sub- cloning experimentation guide》The third edition, Science Press, 2002；Basic procedure is as shown in Figure 5) it is connected to the base of arabidopsis non-coding protein Because of AtIPS1 (database TAIR addresses:http://www.arabidopsis.org；Gene accession number：At3g09922 gone so as to carrier construction in the middle of) Carry out transgenosis.To realize above-mentioned target, following primer is designed：

IPS1OEF (forward primer):

5'-ACGGGTACCTGGCCATCCCCTAGCTAGGT-3'(underlined sequences are KpnI recognition sites)；

IPS1OER (reverse primer):

5'-TACGGATCCCGGAAGCAAATTTACATGCACT-3'(underlined sequences are BamHI recognition sites)；

MIM172F (forward primer):

5'-TTATGCAGCATCGAGTTCAAGATTCCAGCTTCGGTTCCCCTCGGAATCA-3'(underlined sequences are coding The sequence of MIM172)；

MIM172R (reverse primer):

5'-CTGGAATCTTGAACTCGATGCTGCATAATTTCTAGAGGGAGATAAACA-3'(underlined sequences are coding The complementary series of MIM172)；

The blade STb gene of extracting Col types arabidopsis (deriving from Institute of Botany, Chinese Academy of Sciences), DNA method for extracting is with embodiment 1.With The DNA is template, respectively with primer I PS1OEF and MIM172R, MIM172F and IPS1OER for combination carries out two groups of PCR, PCR System and program are consistent with the PCR system and program described in embodiment 1.Make after respectively taking 0.5 μ l mixing in 2 groups of PCR product more than Be the template of next round Overlap extension PCR, with primer I PS1OEF and IPS1OER to combine into performing PCR amplification, PCR system and program with PCR system and program described in embodiment 1 is consistent.Finally giving middle fusion has the restructuring AtIPS1 fragments of MIM172.By PCR primer The electrophoresis detection on the TBE Ago-Gels of 1% (mass/volume), and the PCR primer of recovery is connected to T-A cloning vectors pGEMT-vector Upper (being purchased from Pu Luomaige (Beijing) Bioisystech Co., Ltd, i.e. U.S. Promega companies), should using T7 and SP6 primer pairs on carrier PCR primer carries out sequence verification.Recombinant plasmid comprising the PCR primer is named as plasmid TA-MIM172.

Embodiment 4：The structure of antagonist MIM172 overexpression vectors

With KpnI and BamHI double digestion plasmids TA-MIM172 (described KpnI and BamHI are bought from precious bioengineering Dalian Co., Ltd, Detailed directions refer to the specification of the said firm's corresponding product with consumption, and plasmid TA-MIM172 comes from embodiment 3, as shown in the A figures in Fig. 6), The DNA fragmentation of the 515bp comprising MIM172 is reclaimed, then with the vector plasmid pCAMBIA1301S by KpnI and BamHI double digestions [the disclosed report of the carrier：Zhou et al.,Over-expression of aspartate aminotransferase genes in rice resulted in altered nitrogen metabolism and increased amino acid content in seeds.Theor Appl Genet,2009,118:1381-1390；Its is basic Skeleton originates from purchase from CAMBIA companies of Australia (http://www.cambia.org/daisy/cambia/materials/overview.html) PCAMBIA1301, the expression regulation to transformed gene is realized by adding 35S promoter；Its structure is as shown in the B figures in Fig. 6] utilize T4DNA Ligase (being purchased from Promega companies, detailed directions refer to the specification of the said firm's product with consumption) is attached.Connection product is converted by electricity Method (electric conversion instrument be eppendorf Products, applied voltage of the present invention be 1800V, concrete operations with reference to the instrument operation instructions) Import in Escherichia coli DH10B (being purchased from Promega companies), plus 400 μ l LB culture medium recovery 45min, be applied to the card containing 50mg/L that On the LA culture medium flat plates of mycin, (LA and LB is formulated reference to 37 DEG C of incubator culture 14-16h：Pehanorm Brooker,《Molecular Cloning:A Laboratory guide》 The third edition, Science Press, 2002).Picking monoclonal, Amplification Culture simultaneously extracts plasmid, and sun is screened by KpnI and BamHI double digestions Property clone, and the expression vector of gained is named as 35S:MIM172, its structure is as shown in Figure 7.

Embodiment 5：The acquisition of transgenic paddy rice

By expression vector 35S:MiR172bOE (coming from embodiment 2) and 35S:MIM172 (coming from embodiment 4) is by Agrobacterium EHA105 11 callus is spent in the genetic transforming method Introduced into Rice kind of mediation, by preculture, infect, co-culture, screen with hygromycin (use In a kind of antibiotic of the positive transgenosis callus of screening, Roche Group Co., Ltd of the purchase from Denmark) callus of resistance, break up, take root And acclimatization and transplantses crop field, obtain transfer-gen plant.The method of Agrobacterium genetic transformation and reagent used and formula are excellent according to the report of Hiei et al. Change (Hiei et al., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.Plant J,1994,6:271-282；Lin and Zhang,Optimising the tissue culture conditions for high efficiency transformation of indica rice.Plant Cell Rep,2005,23:540-548)。

The Agrobacterium-mediated genetic transformation reagent and formula that the present invention relates to are as follows：

(1) reagent and solution are abridged

6-BA (6-BenzylaminoPurine, 6-benzyladenine)；KT (Kinetin, kinetin)；NAA (Napthalene acetic acid, Methyl α-naphthyl acetate)；IAA (Indole-3-acetic acid, heteroauxin)；2,4-D (2,4-Dichlorophenoxyacetic acid, 2,4- dichloro-benzenes Fluoroacetic acid)；AS (Acetosringone, acetosyringone)；CH (Casein Enzymatic Hydrolysate, caseinhydrolysate)；HN (Hygromycin B, hygromycin)；DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide (DMSO))；N6max (a large amount of ingredient solutions of N6)； N6min (N6 a small amount of ingredient solution)；MSmax (a large amount of ingredient solutions of MS)；MSmin (MS a small amount of ingredient solution)

(2) main solution formula

1) N6max mother liquors [10 times of concentrates (10X)]

Dissolve one by one, 1000ml is then settled at room temperature.

2) N6min mother liquors [100 times of concentrates (100X)]

1000ml is dissolved and is settled at room temperature.

3)Fe₂- EDTA stores liquid (100X)

300ml distilled water and ferric sulfate (FeSO are added in a big triangular flask₄·7H₂O)2.78g

300ml distilled water is added in another big triangular flask and 70 DEG C are heated to, b diammonium disodium edta is subsequently adding (Na₂EDTA·2H₂O)3.73g

Mixed after they all dissolve, 2h is kept in 70 DEG C of water-baths, be settled to 1000ml, 4 DEG C save backup.

4) vitamins stock liquid (100X)

Add water and be settled to 1000ml, 4 DEG C save backup.

5) MSmax mother liquors (10X)

1000ml is dissolved and is settled at room temperature.

6) MSmin mother liquors (100X)

1000ml is dissolved and is settled at room temperature.

7) 2,4-D stores liquid (1mg/ml)

2,4-D 100mg.

1ml 1N potassium hydroxide dissolves 5min, then plus after 10ml distillations water dissolves are complete is settled to 100ml, room temperature preservation.

8) 6-BA stores liquid (1mg/ml)

6-BA 100mg.

1ml 1N potassium hydroxide dissolves 5min, then plus after 10ml distillations water dissolves are complete is settled to 100ml, room temperature preservation.

9) NAA stores liquid (1mg/ml)

NAA 100mg.

1ml 1N potassium hydroxide dissolves 5min, then plus after 10ml distillations water dissolves are complete is settled to 100ml, and 4 DEG C save backup.

10) IAA stores liquid (1mg/ml)

IAA 100mg.

1ml 1N potassium hydroxide dissolves 5min, then plus after 10ml distillations water dissolves are complete is settled to 100ml, and 4 DEG C save backup.

11) glucose storage liquid (0.5g/ml)

Glucose 125g

Distillation water dissolves are settled to 250ml, and 4 DEG C save backup after sterilizing.

12) AS storages liquid

AS 0.392g

DMSO 10ml

In packing to 1.5ml centrifuge tubes, 4 DEG C save backup.

13) 1N potassium hydroxide storage liquid

Potassium hydroxide (KOH) 5.6g

Distillation water dissolves are settled to 100ml, and room temperature preservation is standby.

14) KT stores liquid (1mg/ml)

KT 100mg.

1ml 1N potassium hydroxide dissolves 5min, then plus after 10ml distillations water dissolves are complete is settled to 100ml, room temperature preservation.

(3) culture medium prescription

1) inducing culture

Plus distilled water, to 900ml, 1N potassium hydroxide adjusts pH value to 5.9, boils and be settled to 1000ml, is dispensed into 50ml triangular flasks (25ml / bottle), sealing sterilizing.

2) subculture medium

Plus distilled water, to 900ml, 1N potassium hydroxide adjusts pH value to 5.9, boils and be settled to 1000ml, is dispensed into 50ml triangular flasks (25ml / bottle), sealing sterilizing.

3) pre-culture medium

Plus distilled water is to 250ml, 1N potassium hydroxide adjusts pH value to 5.6, sealing sterilizing.

Using preceding heating for dissolving culture medium and 5ml glucose storages liquid and 250 μ l AS are added to store liquid, packing is poured into culture dish (25ml/ wares).

4) base is co-cultured

Plus distilled water is to 250ml, 1N potassium hydroxide adjusts pH value to 5.6, sealing sterilizing.

Using preceding heating for dissolving culture medium and 5ml glucose storages liquid and 250 μ l AS are added to store liquid, packing is poured into culture dish (25ml/ wares).

5) suspension medium

Plus distilled water, to 100ml, regulation pH value is dispensed into two triangular flasks of 100ml to 5.4, sealing sterilizing.

Liquid is stored using preceding addition 1ml glucose storages liquid and 100 μ lAS.

6) Selective agar medium

Plus distilled water, to 250ml, regulation pH value to 6.0, sealing sterilizes.

Using preceding dissolving culture medium, the hygromycin and 400ppm cephalosporins of 250 μ l 50mg/ml are added, (25ml/ is poured into culture dish in packing Ware).

7) pre- differential medium

Plus distilled water is to 250ml, 1N potassium hydroxide adjusts pH value to 5.9, sealing sterilizing.

Using preceding dissolving culture medium, the hygromycin and 400ppm cephalosporins of 250 μ l 50mg/ml are added, (25ml/ is poured into culture dish in packing Ware).

8) differential medium

Plus distilled water, to 900ml, 1N potassium hydroxide adjusts pH value to 6.0.

1000ml is boiled and be settled to, 100ml triangular flasks (50ml/ bottles), sealing sterilizing is dispensed into.

9) root media

Plus distilled water, to 900ml, 1N potassium hydroxide adjusts pH value to 5.8.

1000ml is boiled and be settled to, is dispensed into pipe of taking root (25ml/ pipes), sealing sterilizing.

10) LA culture mediums (LB culture mediums are free of agar powder)

Distillation water dissolves are settled to 250ml, and loaded on 500ml triangular flasks, room temperature is saved backup after sterilizing.

The step of Agrobacterium-mediated genetic transformation that the present invention relates to, is as follows：

(1) callus induction

1) ripe rice paddy seed is shelled, then successively with 70% Ethanol Treatment 1min, 0.15% mercury chloride (HgCl₂)15min

2) sterilizing washing seed 4-5 times；

3) seed is placed on inducing culture；

4) it is placed at dark and cultivates 5 weeks, 26 ± 1 DEG C of temperature.

(2) callus subculture

The embryo callus subculture of glassy yellow, consolidation and relatively dry is selected, dark lower culture 2 weeks, 26 ± 1 DEG C of temperature is put on subculture medium.

(3) preculture

The embryo callus subculture of consolidation and relatively dry is selected, dark lower culture 4d, 26 ± 1 DEG C of temperature is put on pre-culture medium.

(4) Agrobacterium culture

1) Agrobacterium EHA1052d of the preculture containing structure good vector, 28 DEG C of temperature on the LA culture mediums with kanamycins；

2) Agrobacterium is transferred in suspension medium, 2-3h is cultivated on 28 DEG C of shaking tables.

(5) Agrobacterium is infected

1) callus of preculture is transferred in the bottle for having sterilized；

2) suspension of regulation Agrobacterium is to OD₆₀₀0.8-1.0；

3) callus is soaked into 30min in agrobacterium suspension；

4) blotted on transfer callus to the filter paper for having sterilized；It is then placed within co-culturing on base and cultivates 2d, 19-20 DEG C of temperature.

(6) callus washing and selection culture

1) sterilize water washing callus to invisible Agrobacterium；

2) it is immersed in 30min in the aqua sterilisa containing 400ppm cephalosporins；

3) blotted on transfer callus to the filter paper for having sterilized；

4) selected 2-3 times, every time 2 weeks on transfer callus to Selective agar medium.(first time cephalosporin screening concentration is 400ppm, second It is later 250ppm)

(7) break up

1) kanamycin-resistant callus tissue is transferred to and cultivates 5-7d on pre- differential medium at dark；

2) shift on callus to the differential medium of pre- differentiation culture, cultivated under illumination, 26 DEG C, 5-7 weeks of temperature.

(8) take root

1) young plant for having broken up is extracted, the root produced during differentiation is cut；

2) it is then transferred to be cultivated 2-3 weeks under illumination in root media, 26 DEG C of temperature.

(9) transplant

Wash the remaining medium on root off, the seedling with good root system is transferred to greenhouse, while keeping moisture moistening at initial several days.In greenhouse After hardening about 2 weeks or so, then transfer load to crop field.

Embodiment 6：The identification of transgenic paddy rice and Phenotypic Observation

T0 is for 35S for extracting:MiR172bOE and 35S:The STb gene of MIM172 transformed plants (coming from embodiment 5) blade, then uses PCR Method carries out positive detection to T0 for transformed plant.DNA extracts the method system with PCR with embodiment 1.

Positive detection is carried out using beta-glucosiduronatase gene (GUS) primer, the sequence of GUS primers is as follows：

GUS-F (forward primer):5'-CCAGGCAGTTTTAACGATCAGTTCGC-3'

GUS-R (reverse primer):5'-GAGTGAAGATCCCTTTCTTGTTACC-3'

PCR testing results show, to expression vector 35S:MiR172bOE transgenosis and the GUS that comes positive plant for negative plant, The reduction of fringe portion branch stalk, grain number per spike reduction, width of blade reduction, length of blade reduction are all shown as, its character mutation is shown in Fig. 8 and Figure 10；And it is right Expression vector 35S:MIM172 transgenosis and the GUS that comes positive plant all shows as plant height for negative plant and becomes short, per fringe one Secondary branch stalk number, every fringe Secondary branch number, grains per panicle, length of blade and width of blade all substantially increase, and its character mutation is shown in Fig. 9 and Figure 10.

To T0 for positive plant point individual plant sowing, and continue to plant the transgenic line in T1 generations, the plant to T1 generations is also carried out gus gene PCR amplifications the yin and yang attribute of individual plant is separated to detect, carry out the detection that isolates of transgenic event and character mutation, and count phenotypic data.

In order to carry out phenotypic analysis, applicant is by expression vector 35S:MiR172bOE and 35S:Each 3 independent transformations of MIM172 and come T0 plant The seed (carrying out embodiment 5) of strain is transplanted to crop field after 20d and obtains T1 for transfer-gen plant by being seeded in rice seedling bed after seed soaking, vernalization.Plantation Density is 15cm × 24cm, and plantation place is the experimental plot of Hongshan District Hua Zhong Agriculture University of Wuhan City of Hubei China province, has safety protection facility at one Under the conditions of paddy rice planting method routinely carry out field management.Yin and yang attribute identification is carried out using the method detection gus gene of PCR, and is analyzed The corresponding relation of PCR testing results and phenotype.Result shows in T1 generations, carrier 35S:It is all that miR172bOE transgenic progenies are separated GUS positive individual plants show as the reduction of fringe portion branch stalk, grain number per spike reduction, width of blade reduction, length of blade reduction for negative individual plant, all, Show that the transgenic event of miR172b is isolated with the change of these phenotypes.And to carrier 35S:MIM172 transgenic progenies are separated All GUS positives individual plants show as plant height for negative individual plant, all and become short, per fringe Primary branch number, per fringe Secondary branch number, per fringe Floret bears, length of blade and width of blade all substantially increase, and the transgenic event for showing MIM172 is also what is isolated with the change of these phenotypes.

Investigation and the character mutation and data of record T1 generation related plant, including plant height, every fringe Primary branch number, every fringe Secondary branch number, every fringe Floret bears, sword-like leave length and width, and the GUS feminine gender individual plants separated with each family are control, and statistical analysis are carried out respectively.

The 35S of table 1:MiR172bOE (miR172OE) and 35S:The phenotype system of MIM172 (MIM172) transgenosis T1 generations positive and negative individual plant Evaluation.

The explanation of table 1：(+) and (-) represents transgenic positive and negative individual plant respectively.Statistical analysis are carried out using t tests,^a,b,cP is represented respectively<0.05,0.01 with 0.001 The level of signifiance.

Embodiment 7：The expression analysis of miR172 in transgenic paddy rice

It is analysis 35S:MiR172bOE and 35S:Influence of the MIM172 transgenic events to endogenous miR172 expression quantity, applicant further with Method (Shen et al., the Global expression profiling of rice microRNAs by one-tube stem-loop of stem-loop RT-PCR reverse transcription quantitative PCR revealed important roles of microRNAs in abiotic stress responses.Mol Genet Genomics,2010,284:477-488) have detected the expression quantity of miR172 in transfer-gen plant.Its specific operating method is as described below：

(1) extracting comes from 35S:MiR172bOE and 35S:The RNA of the transformed plant tillering stage blade of MIM172, the reagent of RNA extractings is purchase Buy Trizol extraction agents box from Invitrogen companies (concrete operation step is shown in kit specification)；

(2) the reverse transcription synthesis chains of cDNA first, step is as follows：

1) the total serum IgE 200ng of extracting is taken, the μ l of 1 μ l, 10xDNaseI buffer of DNaseI 1, plus DEPC (pyrocarbonic acid diethyl ester, RNA is added The strong inhibition agent of enzyme, working concentration is 0.01%) treated water to 10 μ l, mixes and places 15min to remove the gene of residual after room temperature Group DNA；2) the μ l of 0.2M EDTA 1 are added after 15min, and 10min is incubated in 65 DEG C of water-baths to remove the activity of DNaseI；3) add (concentration is 10mM, and sequence is to enter 1 μ l primers miR172stemloopR: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACATGCAG-3'), or 1 μ l primers U6R (concentration is 10mM, and sequence is:5'-GGACCATTTCTCGATTTGTACGTG-3'), and in 65 DEG C of water-baths 10min is incubated with broken The secondary structure of bad RNA, then places 5min on ice；4) μ l, 0.1M DTT (mercaptoethanol) the 2 μ l of 5x first strand buffer 4 are added, The μ l of 10mM dNTP mixture 1, the μ l of reverse transcriptase 1, are placed in warm bath 1.5h in 42 DEG C of water-baths after mixing；5) by reverse transcription after reaction terminates Product is placed in 90 DEG C of dry bath 3min to inactivate reverse transcriptase；6) to 120 μ l water are added in reverse transcription product, -20 DEG C preserve reaction finally after mixing Product.The reagent used in reaction is all purchased from Invitrogen companies.

(3) real-time fluorescence quantitative PCR, step is as follows：

To the expression quantity of the method detection miR172 of the reverse transcription product real-time fluorescence quantitative PCR that obtains, (the primer combination for using is 172QRTF And miRUniverseR), and the expression quantity (the primer combination for using is U6F and U6R) of utilization U6 is being balanced of internal reference.It is glimmering in real time Fluorescent Quantitative PCR related reagent is purchased from precious bioengineering Dalian Co., Ltd, and reaction system is referring to specification.PCR instrument is the 7500 of American AB I companies, PCR parameters be 95 DEG C of predegeneration 10s, into circulation after 95 DEG C denaturation 5s, 60 DEG C annealing extend 40s, 45 circulation.Real time fluorescent quantitative PCR the primer sequences are：

172QRTF (forward primer):5'-GGCGCAGAATCTTGATGATG-3'

MiRUniverseR (reverse primer):5'-CCAGTGCAGGGTCCGAGGT-3'

U6F (forward primer):5'-TACAGATAAGATTAGCATGGCCCC-3'

U6R (reverse primer):5'-GGACCATTTCTCGATTTGTACGTG-3'

MiR172 in paddy rice body is encoded by 4 precursor-genes, in view of the height of the miR172 maturation body sequences that this 4 precursor-genes are formed Degree uniformity (being respectively miR172a, miR172b, miR172c and miR172d, each other at most only 2 differences of base), the above The result of real-time fluorescence quantitative PCR cannot be distinguished from these different miR172 members, detection be internal all miR172 whole result.It is real When quantitative fluorescent PCR result show in 35S:In miR172bOE transfer-gen plants, miR172 is raised, and its result is as shown in figure 11；And In 35S:In MIM172 transfer-gen plants, miR172 is then lowered, and its result is as shown in figure 12.

Embodiment 8：The detection of MIM172 antagonism miR172 effects

Whether in order to further analyze MIM172 can be with the effect of antagonism miR172, applicant be by 35S:MiR172bOE and 35S:MIM172's Transfer-gen plant phase mutual cross, and hybrid is analyzed relative to wild type, 35S by representative of fringe type:MiR172bOE transfer-gen plants and 35S: The character mutation of MIM172 transfer-gen plants, as a result shows the fringe type and 35S of hybrid:MIM172 transfer-gen plants are similar to, and fringe portion branch stalk and grain husk are spent Number rises relative to wild type, therefore 35S:Fringe portion branch stalk and grain number per spike reduce isophenous by 35S caused by miR172bOE transgenosis:MIM172 Transgenosis is recovered, i.e. MIM172 can be with the effect of antagonism miR172, and its result is shown in Figure 13.

Claims (7)

1. a kind of application of gene of microRNA172b in the plant height of adjusting and controlling rice, fringe type and blade dimensions, it is characterised in that the core of the gene Nucleotide sequence such as SEQ ID NO:Shown in 1.

2. a kind of genetic fragment of the precursor-gene pri-microRNA172b of coding microRNA172b is in the plant height of adjusting and controlling rice, fringe type and blade chi Application in very little, it is characterised in that the fragment is SEQ ID NO:Sequence described in 2.

3. a kind of genetic fragment of the antagonist MIM172 of microRNA172, it is characterised in that the nucleotide sequence of the genetic fragment such as SEQ ID NO:Shown in 3.

4. the microRNA172 antagonist MIM172 genetic fragments described in claim 3 are in the application in rice modification.

5. the application described in claim 4, including the application in the plant height of adjusting and controlling rice, fringe type and blade dimensions.

6. one kind includes SEQ ID NO:The plant expression vector of sequence shown in 2.

7. one kind includes SEQ ID NO:The plant expression vector of sequence shown in 3.