CN106434692A - Applications of rice OsPCF7 gene in culturing high-tillering rice varieties - Google Patents
Applications of rice OsPCF7 gene in culturing high-tillering rice varieties Download PDFInfo
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Abstract
The invention discloses applications of a rice OsPCF7 gene in culturing high-tillering rice varieties, wherein the nucleotide sequence of the rice OsPCF7 gene is shown as SEQ ID No.3. The experiment proves that the tiller number of the transgenic positive plants obtained by transforming the rice by adopting the rice OsPCF7 gene is obviously increased compared with that of the non-transgenic rice plants, and therefore, the rice OsPCF7 gene can be used for culturing the high-tillering rice varieties. The invention further discloses applications of plant expression vectors and microbial conversion bodies which contain the rice OsPCF7 gene in culturing the high-tillering rice varieties, therefore, a foundation is laid for improving the tillering capacity of rice and promoting the stable and high yield of rice, and the application prospects are very good.
Description
Technical field
The invention belongs to biological technical field is and in particular to rice Os PCF7 gene is in cultivating high tiller rice varieties
Application.
Background technology
Oryza sativa L., as monocotyledonous Typical Representative, is one of most important Three major grain crops in the whole world, full generation
Boundary treaty has more than 1/2nd people to eat Oryza sativa L., has more than 1/3rd population with Oryza sativa L. as staple food.Social economy is quick
Development promote the growth to grain demand, increased our needs to grain.Nowadays, grain security produces and has become as
One serious problems that must solve, but the developmental morphology of rice plant builds up the yield that can directly affect plant.Oryza sativa L. is
The crop of tiller, tiller is the physiological inheritting characteristic of Oryza sativa L. itself.Rice tillering is exactly substantially the branch of Culm of Rice, is water
The important component part of rice plant type, is also the determiner that rice yield is formed simultaneously.Substantial amounts of research shows, tiller is to Oryza sativa L.
Group structure, photosynthesis, material produces has substantial connection with distribution and structure of output.
The morphogenesis of plant mainly passes through to regulate and control growth, the differentiation of organ, and the size of plant organ and character depend on
The quantity of the cell of its sustainable growth, size and character, that is, the process of cell division, propagation and differentiation.Produce a plant
The labyrinth of thing is requisite with the regulation and control of these processes.However, the main moderator of cell division, propagation and differentiation
It is transcription factor, transcription factor is combined, with DNA or promoter, the expression to start this gene, finally determine cell and complete
Tissue signature, thus regulate and control the growth course of plant.The specific transcription factor of TCP protein family plant the most it has already been proven that
It is the major regulatory person that phytomorph is built up, participate in the related process of many growth and development of plants, such as fetal development, leaf
The growth of son, floral symmetry, pollen development, circadian rhythm, Leaf pattern generation and aging etc..
In recent years, with engineered develop rapidly with transgenic technology increasingly perfect, genetic engineering breeding becomes
For an important branch in breeding field, in terms of orderly improvement Oryza sativa L. characteristic, also show that great potentiality.Using molecule life
Thing learns to do section, the key gene of adjusting and controlling rice growth promoter is imported in target rice varieties, orderly improvement target rice varieties
Characteristic, be improvement rice varieties a kind of effective means.This is to improving rice yield, improve rice quality, ensure people
Life, the income of raising people have great significance.
The existing document report of the nucleotide sequence of the OsPCF7 gene of Oryza sativa L. TCP protein family, but so far, yet there are no
Using this gene in the relevant report cultivating high tiller rice varieties.
Content of the invention
In view of this, an object of the present invention is to provide rice Os PCF7 gene in cultivating high tiller rice varieties
Application；The second object of the present invention is to provide the plant expression vector containing rice Os PCF7 gene cultivating high tiller water
Application in rice varieties；The third object of the present invention is to provide the microbial transformant containing rice Os PCF7 gene cultivating
Application in high tiller rice varieties.
For achieving the above object, the present invention provides following technical scheme：
1st, rice Os PCF7 gene cultivate high tiller rice varieties in application it is characterised in that：Described Oryza sativa L.
The nucleotide sequence of OsPCF7 gene is as shown in SEQ ID No.3.
In the present invention, described rice varieties are to spend No. 11 in Oryza sativa L..
2nd, application in cultivating high tiller rice varieties for the plant expression vector containing rice Os PCF7 gene, described water
The nucleotide sequence of rice OsPCF7 gene is as shown in SEQ ID No.3.
In the present invention, described rice varieties are to spend No. 11 in Oryza sativa L..
In the present invention, the described plant expression vector containing rice Os PCF7 gene contains by 35S promoter, Oryza sativa L.
OsPCF7 gene and Tnos terminate molecular expression cassette.
In the present invention, the described plant expression vector containing rice Os PCF7 gene is to be replaced by rice Os PCF7 gene
Gus gene reading frame on pCAMBIA1301 carrier and obtain.
In the present invention, the described plant expression vector containing rice Os PCF7 gene is prepared by following methods：
A. cloning rice OsPCF7 gene：First extract rice total RNA, obtain cDNA first chain through reversion, then with cDNA
First chain is template, and SEQ ID No.1 and nucleotides sequence shown in SEQ ID No.2 are classified as primer and enter performing PCR amplification, obtain Oryza sativa L.
OsPCF7 gene；
B. the structure of plant expression vector：The difference of the rice Os PCF7 gene with restriction enzyme site that the amplification of step a is obtained
Reclaim Oryza sativa L. OsPCF7 gene endonuclease bamhi with NcoI and BstEII double digestion, use NcoI and BstEII double digestion simultaneously
PCAMBIA1301 carrier recovery carrier framework, then by the rice Os PCF7 gene endonuclease bamhi after enzyme action and pCAMBIA1301
Carrier framework connects, and obtains the plant expression vector containing rice Os PCF7 gene.
3rd, application in cultivating high tiller rice varieties for the microbial transformant containing rice Os PCF7 gene, described water
The nucleotide sequence of rice OsPCF7 gene is as shown in SEQ ID No.3.
In the present invention, described rice varieties are to spend No. 11 in Oryza sativa L..
In the present invention, described microbial transformant is Agrobacterium.
The beneficial effects of the present invention is：Present invention firstly discovers that rice Os PCF7 gene has adjusting and controlling rice morphogenesis
Function, the therefore sequence characteristic according to rice Os PCF7 gene, choose rice Os PCF7 gene reading frame fragment, construct
The plant expression vector of rice Os PCF7 gene, is proceeded to by the agriculture bacillus mediated plant expression vector by rice Os PCF7 gene
In spend in No. 11, obtain transgenic paddy rice positive plant, testing result shows, OsPCF7 base in transgenic paddy rice positive plant
The expression of cause dramatically increases compared with non-transgenic rice plant, and the tillering quantity of transgenic paddy rice positive plant dramatically increases, and shows
Rice Os PCF7 gene can be used for the breeding of high tillering ability Oryza sativa L., promotes Oryza sativa L. stable and high yields Oryza sativa L. to have acquisition important
Meaning.
Brief description
In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below and attached
Table：
Fig. 1 is the agarose gel electrophoresis figure of OsPCF7 gene reading frame fragment pcr amplification product, and wherein M is DNA molecular
Amount standard, 1 is OsPCF7 gene PCR amplified production.
Fig. 2 is the PCR qualification figure of overexpression binary vector pCAMBIA1301-OsPCF7, and wherein M is DNA molecular amount mark
Standard, 1 is the pcr amplification product being obtained for template with overexpression binary vector pCAMBIA1301-OsPCF7.
Fig. 3 is the enzyme action qualification figure of overexpression binary vector pCAMBIA1301-OsPCF7, and wherein M is DNA molecular amount mark
Standard, 1 is the digestion products of overexpression binary vector pCAMBIA1301-OsPCF7.
Fig. 4 is the structure chart (LB of OsPCF7 gene plant expression vector pCAMBIA1301-OsPCF7：Left margin, RB：Right
Border；T35S：35S terminator；HPT：Hygromycin phosphotransferase gene；Pnos：Rouge alkali synthetase promoter；MCS：Many grams
Grand restriction enzyme site；P35S：35S promoter；OsPCF7：Rice Os PCF7 gene；Tnos：Terminator).
Fig. 5 is the agarose gel electricity of transgenic positive plant and nontransgenic plants genomic DNA pcr amplification product
Swimming figure, wherein M is DNA molecular amount standard, and L1, L2, L3 and L4 are respectively the transgenic positive plant of different strains, and NT is non-turn
Gene plant.
Fig. 6 is rice seedling figure, and wherein NT is nontransgenic plants, and what L1, L2, L3 and L4 were respectively different strains turns base
Because of positive plant.
Specific embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.Unreceipted concrete in embodiment
The experimental technique of condition, generally according to normal condition, such as Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brooker etc. writes)
Described in condition, or according to the condition proposed by manufacturer.
Used in preferred embodiment, rice strain spends No. 11 (Oryza sativa cv.Zhonghua 11), matter in being
Grain pCAMBIA1301 (carrying CaMV35S promoter and terminator) and bacillus coli DH 5 alpha are preserved by this laboratory.
TaKaRaRNA PCR Kit (AMV) Ver.3.0 test kit, restricted enzyme, Taq DNA synzyme etc. are Dalian TaKaRa
Products.Ultra-thin/plain agar sugar gel DNA QIAquick Gel Extraction Kit (centrifugation column type) is TIANGEN Products.Remaining reagent
It is import or domestic pure analysis pure reagent.
Embodiment 1, the clone of rice Os PCF7 gene reading frame fragment
According to the OsPCF7 gene candidate sequences (NM_001050819) finding in GenBank, online with series processing
Tool kit (SMS) biosoftware net http://www.bio-soft.net/sms searches its maximum open reading frame sequence, adopts
PrimerPremier 5.0 software Design primers OsPCF7-f：5’-catgccatgggcatgcgcaacgccaagg-3’(SEQ
ID No.1, underscore part is NcoI restriction enzyme site) and OsPCF7-r：5’-gggtaacctcacctgatcacctcacttcc-
3 ' (SEQ ID No.2, underscore part is BstEII restriction enzyme site).Take and in difference, spend No. 11 rice seedlings, extract total serum IgE,
Then the total serum IgE extracting is carried out cDNA first chain according to TaKaRa RNA PCR Kit (AMV) Ver.3.0 kit specification
Synthesis, then with this cDNA first chain as template, expand the reading code of OsPCF7 gene using primer OsPCF7-f and OsPCF7-r
Frame sequence.PCR amplification system consists of：2×Taq Mix 25μl、OsPCF7-f 2.5μl、OsPCF7-r 2.5μl、cDNA
2.5μl、ddH2O 17.5 μ l, totally 50 μ l.Sequence amplification condition is 95 DEG C of denaturations 30s；95 DEG C of degeneration 30s, 58 DEG C of annealing
30s, 72 DEG C of extension 50s, circulate 35 times；Extend 10min after last 72 DEG C.Pcr amplification product adopts agarose gel electrophoresiies to reflect
Fixed, result is as shown in Figure 1.Result shows, PCR amplifies the DNA fragmentation that a size is 833bp.
Pcr amplification product carries out recovery purifying using agarose gel DNA QIAquick Gel Extraction Kit, according to kit specification behaviour
Make.
Embodiment 2, the structure of rice Os PCF7 gene plant expression vector
The pcr amplification product purified with NcoI and BstEII double digestion, obtains the opening code-reading frame fragment of OsPCF7 gene,
It is connected with the carrier pCAMBIA1301 skeleton through same double digestion, obtain plant overexpression binary vector pCAMBIA1301- again
OsPCF7.By overexpression binary vector pCAMBIA1301-OsPCF7 primer OsPCF7-f and OsPCF7-r of structure expand into
Performing PCR is identified, result is as shown in Figure 2.Result shows, obtains the DNA fragmentation of long 833bp, the open reading with OsPCF7 gene
Code frame clip size is consistent.Meanwhile, overexpression binary vector pCAMBIA1301-OsPCF7 NcoI and BstEII that will build
Carry out double digestion identification, result is as shown in Figure 3.After enzyme action, product length is respectively 9771bp and 813bp, respectively with carrier
PCAMBIA1301 skeleton fragment is consistent with the opening code-reading frame clip size of rice Os PCF7 gene, shows rice Os PCF7 base
Because being successfully connected on pCAMBIA1301 skeleton, the structure of OsPCF7 gene plant expression vector pCAMBIA1301-OsPCF7
As shown in Figure 4.Then positive recombinant is entrusted Invitrogen (Shanghai) trade Co., Ltd to carry out sequence verification, result shows
Show, the plant overexpression binary vector pCAMBIA1301-OsPCF7 sequence of structure is correct, its opening code-reading frame sequence such as SEQ
Shown in ID No.3.
Embodiment 3, the preparation of Oryza sativa L. PCF7 gene Agrobacterium tumefaciens attachment
Overexpression binary vector pCAMBIA1301-OsPCF7 is adopted the conversion Agrobacterium tumefaciems AGL0 sense of liquid nitrogen cold shock method
By state cell, coat YEB solid medium (agar containing 1.5% (w/w), the kanamycin (Kan) of 50mg/L,
The streptomycin (Sm) of 500mg/L and the rifampicin (Rif) of 50mg/L), pH 7.0) on, cultivate 2 under 28 ± 2 DEG C, dark condition
My god, picking single bacterium colony, with YEB fluid medium (Rif of Sm and 50mg/L of Kan, 500mg/L containing 50mg/L, pH
7.0) cultivate 2 days, take bacterium solution to enter performing PCR identification, positive recombinant as rice Os PCF7 gene Agrobacterium tumefaciens attachment, -80
DEG C freezen protective, standby.
Embodiment 4, agriculture bacillus mediated rice Os PCF7 gene plant expression vector rice transformation and identification
11 seeds are spent peelling off in NBD in shell2On calli induction media, after growing wound healing, choose spherical in shape,
The bright orange calluss of color proceed to NBD2Successive transfer culture on wound healing subculture medium.Subculture Time is 20d.Subculture
Choose that consolidation, spherical in shape or lamellar, color be bright orange, a diameter of 10 μm about sizes wound healing immerse above-mentioned OsPCF7 bases twice afterwards
Because co-culturing in the AAM suspension of Agrobacterium tumefaciens attachment.The regeneration of transformed plant and culture carry out (Hu by literature method
TZ.OsLEA3,a Late embryogenesis abundant protein gene from rice,confers
tolerance to water deficit and salt stress to transgenic rice.Russ J Plant
Physiol.2008,55:530-537).
PCR detects the presence of hygromycin phosphotransferase gene (HPT) in transgenic paddy rice：Take turning of different strains respectively
Trans-genetic hybrid rice plant and non-transgenic rice plant, extract genomic DNA, then with gained genomic DNA as template, with HPT-
F (SEQ ID No.4) and HPT-r (SEQ ID No.5) enters performing PCR detection, screening transgenic positive plant for special primer.
PCR amplification system is as previously mentioned.PCR amplification cycles parameter is：94 DEG C of denaturations 2min；Then 94 DEG C of degeneration 30s, 58 DEG C of annealing
30s, 72 DEG C of extension 40s, totally 35 circulations；Last 72 DEG C extend 10min eventually.Pcr amplification product enters row agarose gel electrophoresis,
Result is as shown in Figure 5.The pcr amplification product of the different strain transgenic positive plant of result display is all in the special electrophoresis of 1026bp
Band, and electrophoretic band in relevant position in the pcr amplification product of nontransgenic plants, shows transgenic positive plant
Middle OsPCF7 gene is successfully integrated in rice plant.
Embodiment 5, transgenic positive plant are compared with nontransgenic plants tiller situation
Respectively transgenic positive plant strains L1, L2, L3, No. L4 and non-transgenic rice seedling plant are planted in field
In, observe the state of plant, result is as shown in Fig. 6 and Biao 1.
Table 1, transgenic positive plant are compared with the tiller number of nontransgenic plants and heading number
| Pepper plant | The mean tillering quantity | Average heading quantity |
| Nontransgenic plants | 25.4 | 22.6 |
| Transgenic positive plant strains L1 | 36.2 | 34.2 |
| Transgenic positive plant strains L2 | 44.7 | 44.3 |
| Transgenic positive plant strains L3 | 35.8 | 34.0 |
| Transgenic positive plant strains L4 | 36.0 | 34.5 |
In same growth period, transgenic positive plant strains L1, L2, L3, the tillering quantity of No. L4 are planted compared with non-transgenic
Strain is many.Transgenic positive plant strains L1, L2, L3, No. L4 final tiller number and heading number are also above nontransgenic plants (table
1).The above results show that OsPCF7 improves the effect of rice tillering, can be used for cultivating high tiller rice varieties.
Finally illustrate, preferred embodiment above only in order to technical scheme to be described and unrestricted, although logical
Cross above preferred embodiment the present invention to be described in detail, it is to be understood by those skilled in the art that can be
In form and various changes are made to it, without departing from claims of the present invention limited range in details.
Claims (10)
1. rice Os PCF7 gene cultivate high tiller rice varieties in application it is characterised in that：Described rice Os PCF7 base
The nucleotide sequence of cause is as shown in SEQ ID No.3.
2. according to claim 1 application it is characterised in that：Described rice varieties are to spend No. 11 in Oryza sativa L..
3. application in cultivating high tiller rice varieties for the plant expression vector containing rice Os PCF7 gene, its feature exists
In：The nucleotide sequence of described rice Os PCF7 gene is as shown in SEQ ID No.3.
4. according to claim 3 application it is characterised in that：Described rice varieties are to spend No. 11 in Oryza sativa L..
5. the application according to claim 3 or 4 it is characterised in that：The described expression of the plant containing rice Os PCF7 gene
Carrier contains to be started by 35S promoter expresses, the expression cassette of rice Os PCF7 gene and the termination expression of Tnos terminator.
6. the application according to claim 3 or 4 it is characterised in that：The described expression of the plant containing rice Os PCF7 gene
Carrier is to be obtained by gus gene reading frame on rice Os PCF7 gene replacement pCAMBIA1301 carrier.
7. the application according to claim 3 or 4 it is characterised in that：The described expression of the plant containing rice Os PCF7 gene
Carrier is prepared by following methods：
A. cloning rice OsPCF7 gene：First extract rice total RNA, obtain cDNA first chain through reversion, then with cDNA first
Chain is template, and SEQ ID No.1 and nucleotides sequence shown in SEQ ID No.2 are classified as primer and enter performing PCR amplification, obtain Oryza sativa L.
OsPCF7 gene；
B. the structure of plant expression vector：The rice Os PCF7 gene with restriction enzyme site that the amplification of step a is obtained is used respectively
NcoI and BstEII double digestion reclaims Oryza sativa L. OsPCF7 gene endonuclease bamhi, uses NcoI and BstEII double digestion simultaneously
PCAMBIA1301 carrier recovery carrier framework, then by the rice Os PCF7 gene endonuclease bamhi after enzyme action and pCAMBIA1301
Carrier framework connects, and obtains the plant expression vector containing rice Os PCF7 gene.
8. application in cultivating high tiller rice varieties for the microbial transformant containing rice Os PCF7 gene, its feature exists
In：The nucleotide sequence of described rice Os PCF7 gene is as shown in SEQ ID No.3.
9. according to claim 8 application it is characterised in that：Described rice varieties are to spend No. 11 in Oryza sativa L..
10. according to claim 8 application it is characterised in that：Described microorganism is Agrobacterium.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111172174A (en) * | 2020-03-06 | 2020-05-19 | 沈阳农业大学 | Application of OsUGE3 gene in improving rice traits |
| CN112745376A (en) * | 2019-10-31 | 2021-05-04 | 华中农业大学 | Function and application of transcription inhibitor LIP1 for regulating and controlling rice yield |
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