CN106701755A - Application of micro RNA529a in adjusting and controlling rice plant types - Google Patents

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to application of micro RNA529a in adjusting and controlling rice plant types, namely non-coding RNA miR 529a for regulating and controlling rice plant heights, tiller numbers and spike types and isolation clone, functional verification and application of a precursor gene pri-micro RNA529a (pri-miR529a) of the non-coding RNA miR529a. A nucleotide sequence of the miR529a is shown as SEQ ID NO. 1, and a nucleotide sequence of the prim-miR529a is shown as SEQ ID NO. 2. As the pri-miR529a is expressed through transgenosis excessively, the rice plant height is shortened, tiller is increased, the spike length is shortened, and spike branches and spike kernels are reduced. The invention not only provides an active fragment of the miR529a and a genetic transformation carrier thereof , but also provides a novel rice plant type improving method.

Description

Applications of the microRNA529a in controlling plant type of rice

Technical field

The invention belongs to field of plant genetic.Specifically related to a kind of microRNA529a (hereinafter abbreviated as miR529a) and its precursor-gene Applications of the pri-miR529a in controlling plant type of rice.Change the expression of miR529a and its precursor-gene pri-miR529a by technique for gene engineering Abundance, thus it is possible to vary the economical character such as plant height, tiller number, fringe portion branch stalk number and grain number per spike of plant.

Background technology

Paddy rice is one of topmost cereal crops in the world, has more than the population of half with it as staple food, therefore the yield height of paddy rice is sent out society Exhibition has important influence.With the sustainable growth of world population, the increasingly exacerbation of continuous reduction and the environmental pollution of cultivated area, cultivating has Per unit area yield higher and environment-friendly new rice variety have important strategic importance for ensureing the grain security of China.

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)。

Rice tillering is grown under appropriate conditions by the axillary bud of basal part of stem, and the formation and tiller bud for generally comprising tiller bud grow two developments Process.The gene control tiller bud such as multiple genes relevant with tiller development, such as MOC1, LAX1, LAX2, TAB1 has been cloned at present Initial development (Komatsu et al., LAX and SPA:major regulators of shoot branching in rice.Proc Natl Acad Sci U S A, 2003,100:11765-11770；Li et al.,Control of tillering in rice.Nature,2003,422:618-621；Tabuchi et al；LAX PANICLE2of rice encodes a novel nuclear protein and regulates the formation of axillary meristems.Plant Cell, 2011,23:3276-3287；Tanaka et al.,Axillary meristem formation in rice requires the WUSCHEL ortholog TILLERS ABSENT1.Plant Cell,2015,27:1173-1184).And a kind of new plant hormone witchweed lactone for identifying in recent years is then in control paddy rice Tiller bud plays the regulating and controlling effect of core during growing.The serial Dwarf Mutant that long-term genetics research is collected in paddy rice is in parsing witchweed Be played an important role in the synthesis of ester and signal transduction.Witchweed lactone is a type sesquiterpene ene compound, is synthesized by carrotene.Water D17/HTD1 in rice, D10 and D27 control witchweed lactone synthesis (Arite et al., DWARF10, an RMS1/MAX4/DAD1ortholog, controls lateral bud outgrowth in rice.Plant J,2007,51:1019-1029；Lin et al.,DWARF27,an iron-containing protein required for the biosynthesis of strigolactones,regulates rice tillers bud outgrowth.Plant Cell,2009, 21:1512-1525；Zou et al.,The rice HIGH-TILLERING DWARF1encoding an ortholog of Arabidopsis MAX3 is required for negative regulation of the outgrowth of axillary buds.Plant J,2006,48:687-698), D3, D14 and D53 The protein of coding then controls signal transduction (Arite et al., d14, a strigolactone-insensitive mutant of rice, shows of witchweed lactone an accelerated outgrowth of tillers.Plant Cell Physiol,2009,50:1416-1424；Ishikawa et al.,Suppression of tiller bud activity in tillering dwarf of rice.Plant Cell Physiol,2005,46:79-86；Jiang et al.,DWARF 53acts a repressor of strigolactone signaling in rice.Nature,2013,504:401-405；Zhou et al.,D14-SCF(D3)-dependent degradation of D53regulates strigolactone signaling.Nature,2013,504:406-410)。

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)。

MicroRNA (miRNA) is a class length about in the 20-24 RNA molecule of the endogenous non-coding of nucleotides.Since 1993 Since first coding miRNA gene has been cloned in nematode, substantial amounts of coding miRNA is had been found that in nearly all higher organism studied Gene exist.MiRNA controls the activity of target gene mRNA often through the stability or regulation protein translation process of regulating mRNA, So as to adjust including each vital movement process including growth, development, metabolism, response environment etc..Because its powerful biological function and complexity Regulatory mechanism, miRNA related research has become one of popular domain of current life science, and it is possible to change in the heredity of crops Played an important role in good.Such as miR156 is the highly conserved tiny RNA in each higher plant species of a class, in paddy rice, there is 11 The transcription factor of individual SPL families is by its regulation and control (Xie et al., Genomic organization, differential expression, and interaction of SQUAMOSA promoter-binding-like transcription factors and microRNA156in rice.Plant Physiol,2006, 142:280-293).Overexpression miR156 causes tiller Overgrowth (Xie et al., Genomic organization, differential in paddy rice expression,and interaction of SQUAMOSA promoter-binding-like transcription factors and microRNA156in rice. Plant Physiol,2006,142:280-293)；And two target gene SPL14/IPA1 and SPL16 are also proved to have weight to the genetic improvement of paddy rice Want value (Jiao et al., Regulation of OsSPL14by OsmiR156defines ideal plant architecture in rice.Nat Genet, 2010,42:541-544；Miura et al.,OsSPL14promotes panicle branching and higher grain productivity in rice.Nat Genet,2010,42:545-549；Wang et al.,Control of grain size,shape and quality by OsSPL16in rice.Nat Genet, 2012,44:950-954)。

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.The means that the present invention passes through plant genetic engineering, study miRNAs in paddy rice Function, excavation be possibly used in agricultural production change plant type of rice new miRNAs, to provide new genetic resources for rice breeding.

The content of the invention

It is the application for providing a kind of miR529a and its precursor-gene pri-miR529a in controlling plant type of rice that the purpose of the present invention is.The present invention Another purpose be to provide a kind of carrier of overexpression pri-miR529a.Simultaneously the present invention also provide it is a kind of by adjust miR529a and its The expression quantity of precursor-gene pri-miR529a is come the method that changes plant type of rice.MiR529a has such as SEQ ID NO:Nucleotide sequence shown in 1, Also include and SEQ ID NO:Nucleotides sequence shown in 1 shows the gene order of more than 90% homology, also including because of insertion, replacement or missing one Or multiple bases and the mutant allele or derivative that produce.Present invention additionally comprises the precursor-gene pri-miR529a of coding miR529a, Pri-miR529a has such as SEQ ID NO:Nucleotide sequence shown in 2, and with SEQ ID NO:It is same that nucleotides sequence shown in 2 shows more than 90% The gene order of source property, also mutant allele or derivative including being produced because inserting, substitute or lacking one or more bases.

The genetic fragment of the precursor-gene pri-miR529a that the present invention includes miR529a by overexpression can effectively reduce rice plant Highly, increase branch stalk number and the grain number per spike of the tiller, reduction spike length and fringe portion of paddy rice, show that miR529a and its precursor-gene pri-miR529a can Effectively to control the plant type of paddy rice.MiR529a and its precursor-gene pri-miR529a can be used for and other controlling elements, such as constitutive promoter Or organ specific promoters construct expression vector；Also can be 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 technology such as CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)/Cas9 to it Manipulate to regulate and control 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 from paddy rice and includes coding miR529a's and its precursor-gene pri-miR529a DNA fragmentation；Specific separation method has a detailed description in embodiment 1.

2nd, the above-mentioned DNA fragmentation for including pri-miR529a is connected on pCAMBIA1301S carriers, builds the overexpression of miR529a The expression vector is named as 35S by carrier, applicant:miR529aOE；Specific method has a detailed description in example 2.

3rd, 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:MiR529aOE leads To enter spend in paddy rice acceptor 11 that (Institute of Crop Science, Chinese Academy of Agricultural Science obtain transformed plant；Specific method has a detailed description in embodiment 3.

4th, positive transgenic plant is analyzed and identified by the method for PCR；In the genotype and phenotype of T1 generation identification transfer-gen plants, it is divided into From detection, and statistical analysis is carried out to Relevant phenotype；Specific method has a detailed description in example 4.

5th, using the change of the expression quantity of the target gene SPL14 and SPL17 of miR529a in the method analysis transfer-gen plant of real-time fluorescence quantitative PCR Change；Specific method has a detailed description in embodiment 5.

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

The present invention has cloned the encoding gene of paddy rice miR529a, and has carried out genetic transformation research to it.Present invention discover that miR529a can be controlled The plant type of paddy rice, hereditary control is carried out by its expression quantity, can provide a kind of method of novelty for the genetic improvement of paddy rice.

Brief description of the drawings

Sequence table SEQ ID NO:1 is the nucleotide sequence of miR529a.

Sequence table SEQ ID NO:2 is the nucleotide sequence of the precursor pri-miR529a of miR529a, its 3-23 nucleotide coding miR529a.

Fig. 1：The RNA secondary structures of the precursor-gene pri-miR529a of miR529a.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 miR529a shown in red letters.

Fig. 2：Expression vector 35S:MiR529aOE 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-miR529a of miR529aOE.

B figures in Fig. 2：For construction of expression vector 35S:The structural representation of the empty carrier pCAMBIA1301S of miR529aOE.

Fragment shown in A figures in Fig. 3 is inserted the B in Fig. 3 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:miR529aOE.

Fig. 3：The expression vector 35S that the present invention builds:The structural representation of miR529aOE.

Fig. 4：11 plant (WT) and 35S are spent in wild type:The phenotype of miR529aOE (miR529aOE) transfer-gen plant compares.Accompanying drawing mark Note explanation：

A figures in Fig. 4:11 (WT) and 35S are spent in the wild type at heading stage:The plant height of miR529aOE (miR529aOE) transfer-gen plant Form with tiller compares.

B figures in Fig. 4：11 (WT) and 35S are spent in wild type:The fringe type of miR529aOE (miR529aOE) transfer-gen plant compares.

Fig. 5：11 (WT) and 35S are spent in wild type:The phenotype statistics of miR529aOE (miR529aOE) transfer-gen plant.Description of reference numerals：

A figures in Fig. 5：11 (WT) are spent in wild type with 3 35S of family:The strain of miR529aOE (miR529aOE) transfer-gen plant High statistics.Data display is from 15 average value ± standard errors of individual plant.

B figures in Fig. 5：11 (WT) are spent in wild type with 3 35S of family:MiR529aOE (miR529aOE) transfer-gen plant point Tiller number is counted.Data display is from 15 average value ± standard errors of individual plant.

C figures in Fig. 5：11 (WT) are spent in wild type with 3 35S of family:The fringe of miR529aOE (miR529aOE) transfer-gen plant Statistics long.Data display is from 15 average value ± standard errors of individual plant.

D figures in Fig. 5：11 (WT) are spent in wild type with 3 35S of family:MiR529aOE (miR529aOE) transfer-gen plant it is every Fringe Primary branch is counted.Data display is from 15 average value ± standard errors of individual plant.

E figures in Fig. 5：11 (WT) are spent in wild type with 3 35S of family:MiR529aOE (miR529aOE) transfer-gen plant it is every Fringe Secondary branch is counted.Data display is from 15 average value ± standard errors of individual plant.

F figures in Fig. 5：11 (WT) are spent in wild type with 3 35S of family:MiR529aOE (miR529aOE) transfer-gen plant it is every Fringe floret bears are counted.Data display is from 15 average value ± standard errors of individual plant.

Fig. 6：The oligonucleotide ligand of miR529a and its target gene SPL14 and SPL17 is to relation schematic diagram.

Fig. 7：11 (WT) and 35S are spent in detecting wild type using real-time quantitative PCR:In miR529aOE (miR529aOE) transfer-gen plant The expression quantity of the target gene SPL14 and SPL17 of miR529a.Description of reference numerals：

A figures in Fig. 7：SPL14 spends the 35S of 11 (WT) and two familys in wild type:MiR529aOE (miR529aOE) transgenosis Expression quantity in the blade of plant.Data display is 3 average value ± standard errors of repetition.

B figures in Fig. 7：SPL17 spends the 35S of 11 (WT) and two familys in wild type:MiR529aOE (miR529aOE) transgenosis Expression quantity in the blade of plant.Data display is 3 average value ± standard errors of repetition.

Specific embodiment

Embodiment 1：Separation includes the DNA fragmentation of the precursor-gene pri-miR529a of miR529a

Using Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/) and miRBase (http://www.mirbase.org/) two websites are retrieved, and obtain miR529a (miRBase database logins number：MIMAT0002885) and Its precursor-gene pri-miR529a (miRBase database logins number：MI0003202 nucleotide sequence).MiR529a maturation body sequences such as SEQ ID NO:Shown in 1, the sequence such as SEQ ID NO of its precursor-gene pri-miR529a:Shown in 2；The secondary structure for being formed is as shown in Figure 1.

According to the analysis result of database sequence, to expand the genetic fragment of pri-miR529a, following primer is designed：

MiR529aOEF (forward primer):5'-GGTACCTGAAAGAGATGAGTGTGCT-3'(underlined sequences are KpnI recognition sites)；

MiR529aOER (reverse primer):5'-GGATCCTGGGGTAACTAGCGTTTTGCA-3'(underlined sequences are that BamHI recognizes position Point).

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).With blade STb gene as template, enter performing PCR with primer MiR529aOEF and MiR529aOER and expand Increase.PCR reaction cumulative volumes are 20 μ l, and comprising DNA profiling 100ng, 2 μ l, 10mM dNTP of 10xPCR buffer 2 μ l, 10mM draws Each μ l of 0.3 μ l, rTaq enzyme 0.2 of thing MiR529aOEF and MiR529aOER, plus deionized water to 20 μ l (used PCR buffer, dNTP, RTaq enzymes etc. are purchased from precious bioengineering Dalian Co., Ltd).PCR reaction conditions are as follows：1. 94 DEG C of 4min, 2. 94 DEG C of 40s, 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.TBE of the PCR primer in 1% (mass/volume) Electrophoresis detection on Ago-Gel, recovery length is that (the target dna section of 359bp is plus the two restricted digestions added on primer for 371bp Site 12 bp) DNA fragmentation.The PCR primer of recovery is connected to T-A cloning vectors pGEMT-vector using T4DNA ligases It is upper that (T4DNA ligases and carrier pGEMT-vector are purchased from Pu Luomaige (Beijing) Bioisystech Co., Ltd, i.e. U.S. Promega Company).(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, specific behaviour Make the operation instructions with reference to the instrument) import in Escherichia coli DH10B (being purchased from Promega companies), plus 400 μ l LB culture mediums recovery 45 Min, is applied to the LA culture medium flat plates containing ampicillin, the chloro- 3- indoles-β-D- galactolipins of the bromo- 4- of 5- and isopropyl-β-D-thiogalactoside On, (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 is presented blue monoclonal, and Amplification Culture simultaneously extracts plasmid, by KpnI and BamHI double digestion screening positive clones, and And carry out sequence verification using the PCR primer that T7 and SP6 primer pairs on carrier are inserted.Recombinant plasmid comprising the PCR primer is named as TA-miR529a。

Embodiment 2：The structure of the overexpression vector of miR529a

With KpnI and BamHI double digestion plasmids TA-miR529a (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-miR529a comes from embodiment 1, as shown in the A figures in Fig. 2), The DNA fragmentation of the 371bp comprising pri-miR529a 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:MiR529aOE, its structure is as shown in Figure 3.

Embodiment 3：The acquisition of transgenic paddy rice

By expression vector 35S:The genetic transforming method Introduced into Rice kind that miR529aOE (coming from embodiment 2) is mediated by Agrobacterium EHA105 It is middle spend 11 callus, by preculture, infect, co-culture, screen with hygromycin (for screen the positive transgenosis callus one kind Antibiotic, purchase is from the Co., Ltd of Roche Group of Denmark) callus of resistance, break up, take root and acclimatization and transplantses crop field, obtain transgenosis plant Strain.The method of Agrobacterium genetic transformation and reagent used and formula are to optimize (Hiei et al., Efficient according to the report of Hiei et al. 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).Will be using expression vector 35S:MiR529aOE is (from implementation Example 2) T0 that is transformed is named as miR529aOE for plant.

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 4：The identification of transgenic paddy rice and Phenotypic Observation

T0 is for 35S for extracting:The STb gene of miR529aOE transformed plants (coming from embodiment 3) blade, is then converted with PCR method to T0 generations Plant carries out positive detection.DNA extracts the method system with PCR with embodiment 1.

To 35S:The transformed plant of miR529aOE carries out positive detection using beta-glucosiduronatase gene (GUS) primer, and the sequence of GUS primers is such as Under：

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

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

PCR testing results show, GUS positive transfer-gen plant shown as negative plant, all plant height become short, tiller increase, Fringe is short, fringe portion branch stalk is reduced, grain number per spike is reduced, and its phenotype is shown in Fig. 4.To T0 for positive plant point individual plant sowing, and continue to plant T1 generations Transgenic line, the PCR amplifications that gus gene is also carried out to the plant in T1 generations separate the yin and yang attribute of individual plant to detect, carry out transgenic event With the detection that isolates of character mutation, and statistics phenotypic data.

In order to carry out phenotypic analysis, the 35S that applicant carrys out 3 independent transformations:The seed of the T0 plant of miR529aOE is by seed soaking, vernalization After be seeded in rice seedling bed, transplanted after 20d to crop field obtain T1 for transfer-gen plant.Planting density is 24 centimetres of 15 cm x, during plantation place is The experimental plot of state Wuhan City, Hubei Province Hongshan District Hua Zhong Agriculture University, the paddy rice planting method under the conditions of having safety protection facility at routinely is carried out Field management.Yin and yang attribute identification is carried out using the method detection gus gene of PCR, and analyzes the corresponding relation of PCR testing results and phenotype. Result show T1 generation separate all GUS the positive individual plant shown as negative individual plant, all plant height become short, tiller increase, fringe Short, fringe portion branch stalk is reduced, grain number per spike is reduced, show the transgenic event of pri-miR529a with the signified plant type change of the present invention (plant height become it is short, Tiller increases, fringe is short, fringe portion branch stalk is reduced, grain number per spike is reduced) isolate.Investigate the character mutation sum to record T1 generation related plant According to, including plant height, every fringe Primary branch number, every fringe Secondary branch number, grains per panicle and spike length, and the GUS separated with each family Negative individual plant is control, and statistical analysis are carried out respectively, and its result is shown in Fig. 5.

Embodiment 5：The expression analysis of the target gene of miR529a

According to Bioinformatics Prediction, the binding site of miR529a is included in gene SPL14 and SPL17, therefore miR529a can regulate and control The expression of SPL14 and SPL17, the nucleotides pair relationhip between them is as shown in Figure 6.To transfer-gen plant, applicant is except utilizing PCR Carry out outside the positive detection of GUS, also have detected SPL14 and SPL17 in transfer-gen plant further with the method for real-time fluorescence quantitative PCR Expression quantity.Its specific operating method is as described below：

(1) extracting comes from 35S:The RNA of the transformed plant tillering stage blade of miR529aOE, the reagent of RNA extractings is purchase public from Invitrogen Trizol extraction agents box (concrete operation step is shown in kit specification) of department；

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

1) the μ g of total serum IgE 3 of extracting are 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 Enter 1 μ l oligo (dT)₁₅, and 10min is incubated in 65 DEG C of water-baths to destroy the secondary structure of RNA, 5min is then placed on ice；4) add 4 μ l, 0.1M DTT (mercaptoethanol) of 5x first strand buffer, 2 μ l, 10mM dNTP mixture 1 μ l, the μ l of reverse transcriptase 1, after mixing It is placed in warm bath 1.5h in 42 DEG C of water-baths；5) reverse transcription product is placed in 85 DEG C of water-bath 10min to inactivate reverse transcriptase by reaction after terminating；6) to 120 μ l water are added in reverse transcription product, -20 DEG C preserve reaction final product after mixing.The reagent used in reaction is all purchased from Invitrogen companies.

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

The method of the reverse transcription product real-time fluorescence quantitative PCR to obtaining detects that (LOC_Os08g39890, the primer is combined as SPL14 SPL14QRTF and SPL14QRTR, sequence is as follows) and SPL17 (LOC_Os09g31438, primer be combined as SPL17QRTF and SPL17QRTR, sequence is as follows) expression quantity, and using Ubiquitin expression quantity (primer is combined as UbiQRTF and UbiQRTR, Sequence is as follows) it is being balanced of internal reference.Real-time fluorescence quantitative PCR related reagent is purchased from precious bioengineering Dalian Co., Ltd, reaction system Referring to specification.PCR instrument for American AB I companies 7500, PCR parameters be 95 DEG C of predegeneration 10s, into circulation after 95 DEG C denaturation 5s, 60 DEG C of annealing extend 40s, 45 circulations.Primer sequence used by real-time fluorescence quantitative PCR is：

SPL14QRTF (forward primer):5'-TAGCCATCATGCCCACTTC-3'

SPL14QRTR (reverse primer):5'-AGACCAATCCATCGTGTTG-3'

SPL17QRTF (forward primer):5'-TGTCAACTCAGCCATGGGATAC-3'

SPL14QRTR (reverse primer):5'-GGCCGTTGACGACATTGG-3'

UbiF (forward primer):5'-AACCAGCTGAGGCCCAAGA-3'

UbiR (reverse primer):5'-ACGATTGATTTAACCAGTCCATGA-3'

35S:The testing result of SPL14 and SPL17 expression quantity is as shown in fig. 7, the two is all significantly lowered in miR529aOE transfer-gen plants.

Claims (3)

1. a kind of applications of microRNA529a in controlling plant type of rice, it is characterised in that the nucleotide sequence of the gene such as SEQ ID NO:1 institute Show.

2. application of a kind of genetic fragment of the precursor-gene pri-microRNA529a of coding microRNA529a in controlling plant type of rice, it is special Levy and be, the fragment is SEQ ID NO:Sequence described in 2.

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