CN105132430B - Corn nutritive organ-specific promoter and its application - Google Patents
Corn nutritive organ-specific promoter and its application Download PDFInfo
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Abstract
The invention discloses a kind of Corn nutritive organ-specific promoter and its applications.The corn nourishment organ expresses promoter, which can express in corn nourishment organ, with the nucleotide sequence shown in SEQ ID NO.1.The present invention includes the plant expression vector of the corn nourishment organ expression promoter.The plant expression vector is the plant expression vector built with the CaMV35S promoters on corn nourishment organ expression promoter substitution plant binary expression vector pCAMBIA1301.The present invention only expresses in the nutrition organs of transgenic paddy rice (in spend 11), is not expressed in seed, it will has good application prospect in the corn of transgenic pest-resistant, disease-resistant etc. is cultivated.
Description
Technical field
The present invention relates to plant genetic engineering fields, and in particular to Corn nutritive organ-specific promoter and its application.
Background technology
Promoter is important cis-acting elements, the transcript and expression of controlling gene, according to the transcriptional profile of promoter
Difference is classified as composing type, induction type and tissue-specific promoter's three classes.Which kind of promoter is selected to drive the table of gene
It reaches, is the hot spot of current genetic engineering research.It is promoter and enhancer that tissue-specific promoter, which is also known as organ specific promoters,
One of, under the driving of this kind of promoter, the expression of gene often only certain specific organs and with the efficient table of tissue site
It reaches, can effectively avoid the unnecessary waste of plant nutrient and the adverse effect caused by plant, and show growth adjustment etc.
Characteristic.
Isolated different types of organ specific promoters from plant at present, including root, stem, leaf, pollen,
Vascular bundle, seed or fruit, separate living tissue, stomata etc..Keller etc. (1989) drives GUS bases with 427bp GRPI.8 promoters
Cause, it is in vascular-specific expression in transgene tobacco to find gus gene.Azria etc. (2011) is isolated to one from rice
Kind Orys1 pollen specific expression promoters；The embryo that Vincent etc. (2002) also takes the lead in obtaining cypress PtNIP1 genes sends out
Educate specificity promoter；Cai etc. (2007) separation has cloned the chlorenchyma specificity promoter PD540 in a rice source simultaneously
It is blended with cry1AC genes, obtained transgenic paddy rice has good insect resistace, and can't detect albumen in seed
Expression；
China, which is one, the big country of 1,300,000,000 populations, wherein there is 700,000,000 peasants.Therefore, country will solve population problem of food and clothing
The first place of developing national economy is placed on the problem of increasing farmers' income.Corn is made as the highest important grain of whole world total output
Object and important feed resource have great importance to the research of its genetic engineering.Simultaneously as food plant, foreign gene
Preferably directly expression in other positions other than Leaf-feeding insects, is more in line with the safety of genetically modified plants.At present in corn
It is middle lack it is a kind of can regulate and control target gene and expressed at nutrition organs position, and the promoter that reproductive organs is not expressed.
Invention content
There is provided the corn nourishments that target gene can be controlled specific expressed for the technical problems to be solved by the invention
Organ specific promoters and its application.
To achieve these goals, the present invention is achieved through the following technical solutions：A kind of corn nourishment organ of the present invention
Specific expressing promoter PZmCCR has the nucleotide sequence shown in SEQ ID NO.1.PZmCCR promoters are beautiful by extracting
The total DNA clone of rice Zea mays L.B73 self-mating systems obtains, by it is sequenced as 2145bp.
Promoter of the present invention is used for the purposes of the specific expressed target gene in rice nutrition organ.
The present invention includes the plant expression vector of the corn nourishment Organ specific expression promoter.The promoter energy
It is specific expressed in the nutrition organs of transgenic paddy rice, it is not expressed in genitals official rank other organs.
Further, the plant expression vector is to replace plant binary with corn nourishment Organ specific expression promoter
CaMV35S promoters on expression vector pCAMBIA1301, the plant expression vector pCAM-PZmCCR built.
The application of promoter of the present invention or the plant expression vector in prepare transgenosis plant.
Further, the genetically modified plants are transgenic paddy rice.
The present invention includes the host cell of the plant expression vector, wherein the host cell is Agrobacterium tumefaciems place
Chief cell.
A kind of preparation of genetically modified plants containing the corn nourishment Organ specific expression promoter of the present invention
Method includes the following steps：
(a) by the plant expression vector Introduced into Rice tissue described in promoter described in claim 1 or claim 3
Cell；
(b) the rice tissue cell is cultivated under conditions of plant growth is promoted, obtains genetically modified plants.
By Agrobacterium-mediated transformation rice, 18 plants of transfer-gen plants are obtained altogether, and PCR detections there are 10 plants of positive strains.And
GUS histochemical stains have all been carried out to the different tissues position of positive plant.GUS histochemical stains identification confirms
PZmCCR promoters are nutrition organs specific expressing promoter, and pCAM-PZmCCR are specific expressed for a kind of nutrition organs
Carrier.
Advantageous effect：A nutrition organs specific expressing promoter PZmCCR and its expression vector provided by the invention,
The promoter is replaced to the CaMV35S promoters on plant binary expression vector pCAMBIA1301, the new plant built
Expression vector, can in the nutrition organs of rice specifically expressing.The promoter can control target gene in normal rice nutrition
Specifically expressing in organ can instruct foreign gene specific expressed in nutrition organs, have with this method screening transgenic rice
There is the advantages of strong stage, high sensitivity.And downstream gene is only driven in the various nutrition organs of rice according to the promoter
Middle expression, and the characteristics of do not expressed in reproductive organs, various beneficial target gene can be connected and be transferred in plant, from
And the anti-environment stress ability of plant is improved, enhance diseases and insect pests resistance and absorption of nutrient ingredients ability etc..Meanwhile in reproduction
It is not expressed in organ, does not influence the quality of seed, reduce worry of the public to genetically modified plants.Therefore the present invention corn or its
He, which plants, will have potential commercial value and good application value in the breeding of subclass crop.
Description of the drawings
Fig. 1 PZmCCR promoter molecules testing results；A be PCR as a result, B be double digestion as a result,
M：DL-5000 1：Stripe size is 2145bp 2：HindIII and NcoI double digestion bands；
Fig. 2 is the T-DNA areas collection of illustrative plates of the expression vector PZmC2H2 of the present invention；
Fig. 3 is the histochemical stain result figure of the gus gene of the present invention；
Wherein：1 young root；2 young stems；3 spires；4 matured roots；5 ripe stems；6 climax leaves；7 glumes；8 anther；9 endosperm.
Specific embodiment
The present invention is described further with reference to the drawings and specific embodiments.It should be understood that these embodiments are merely to illustrate
Purpose rather than the limitation scope of the invention.
A kind of corn nourishment Organ specific expression promoter PZmCCR of the present invention has shown in SEQ ID NO.1
Nucleotide sequence.PZmCCR promoters clone acquisition by extracting the total DNA of corn Zea mays L.B73 self-mating systems, by surveying
Sequence its be 2145bp.
Promoter of the present invention is used for the purposes of the specific expressed target gene in rice nutrition organ.
The present invention includes the plant expression vector of the corn nourishment Organ specific expression promoter.The promoter energy
It is specific expressed in the nutrition organs of transgenic paddy rice, it is not expressed in genitals official rank other organs.
The plant expression vector is to replace plant binary expression vector with corn nourishment Organ specific expression promoter
CaMV35S promoters on pCAMBIA1301, the plant expression vector pCAM-PZmCCR built.
The application of promoter of the present invention or the plant expression vector in prepare transgenosis plant.
The genetically modified plants are transgenic paddy rice.
The present invention includes the host cell of the plant expression vector, wherein the host cell is Agrobacterium tumefaciems place
Chief cell.
A kind of preparation of genetically modified plants containing the corn nourishment Organ specific expression promoter of the present invention
Method includes the following steps：
(a) by the plant expression vector Introduced into Rice tissue described in promoter described in claim 1 or claim 3
Cell；
(b) the rice tissue cell is cultivated under conditions of plant growth is promoted, obtains genetically modified plants.
By Agrobacterium-mediated transformation rice, 18 plants of transfer-gen plants are obtained altogether, and PCR detections there are 10 plants of positive strains.And
GUS histochemical stains have all been carried out to the different tissues position of positive plant.GUS histochemical stains identification confirms
PZmCCR promoters are nutrition organs specific expressing promoter, and pCAM-PZmCCR are specific expressed for a kind of nutrition organs
Carrier.
Fig. 1 PZmCCR promoter molecules testing results.A is PCR results；B is double digestion result.
M：DL-5000 1：Stripe size is 2145bp 2：HindIII and NcoI double digestion bands
Fig. 2 is the T-DNA areas collection of illustrative plates of the expression vector PZmCCR of the present invention；
LB and RB is expressed as the left margin and right margin of T-DNA；Hyg represents hygromycin gene；Mcs represents more
Cloning site；Nos represents the terminator of gene；Hind III and NcoI represents the enzyme of restriction enzyme Hind III and NcoI respectively
Enzyme site；
Fig. 3 is the histochemical stain result figure of the gus gene of the present invention；Wherein：1 spire；2 young stems；3 young roots；4 is ripe
Leaf；5 ripe stems；6 matured roots；7 glumes；8 anther；9 endosperm.
The primer is synthesized by Shenzhen Hua Da bioengineering Co., Ltd, Shanghai Sangon Biotech Company's sequencing；pTEAY-T1、
Taq enzyme, Trans5 α competence and relevant kit purchase Beijing Quan Shi King Companies；Restriction enzyme Hind III and NcoI,
T4 ligases are purchased from TaKaRa companies；Corresponding antibiotic is from Shanghai life work and SIGMA companies；Remaining reagent is domestic point
It analyses pure.Method therefor is conventional method unless otherwise instructed in following embodiments.
Embodiment 1
The clone of Corn nutritive organ-specific promoter PZmCCR
According to the PZmCCR promoter full length sequences announced on NCBI websites, the primer of the design PCR amplification segment, upstream
Primer adds the AAGCTT restriction enzyme sites of HindIII, and downstream primer adds the CCATGG restriction enzyme sites of NcoI.
Primer sequence is as follows：
Primer 1 (sense primer)：5’-CCCAAGCTT CAGGCTGTGGGACACCTCCATTCTA-3’
Primer 2 (downstream primer)：5’-CATGCCATGG GTGCTCCTCTGCTCTAGCTCTT-3’
Using the corn B73 genomic DNAs of Quan Shi King Companies Plant Genome kit extraction as template, with primer 1 and draw
Object 2 carries out PCR amplification, and PCR reaction systems are as shown in table 1：
Table 1
PCR reaction conditions are：Pre-degeneration：94℃ 5min；Denaturation：94℃ 30s；Annealing：62℃ 30s；Extension：72℃
2min, 27 cycles；Overall elongation：72℃ 10min.
After reaction, PCR product is detected with 2% agarose gel electrophoresis.Recycle and purify the mesh of 2145bp
Segment；The recycling segment of Quan Shijin Bioisystech Co., Ltd with pEASY T1 Cloning Vector is connect, is transformed into
In the E. coli competent Trans5 α cells of Quan Shijin Bioisystech Co., Ltd；PCR and positive gram of digestion detection screening
It is grand；To testing result, tentatively judicious junction fragment, sends to Shanghai Sangon Biotech Company and is sequenced, and sequencing result is such as:Sequence
SEQ ID NO in table:Shown in 1 DNA sequence dna, by 2145bp base composition, by itself and the sequence alignment reported on NCBI, knot
Fruit is completely the same.
Embodiment 2
The foundation of pZmCCR gene promoter expression vectors
The PZmCCR segments for the small fragment being already connected on pEASY T1 Cloning Vector and Agrobacterium binary are carried
The CaMV35S promoters of large fragment on body pCAMBIA1301 carry out double digestion with Hind III and NcoI enzymes, in 37 DEG C of water-baths
In, 3h, digestion 20uL systems are as shown in table 2：
Table 2
Above-mentioned size segment T4DNA ligases are connected, 25 DEG C of 2~12h of connection, linked system is as shown in table 3：
Table 3
Carrier pCAM-PZmCCR the plasmids built of 4~6 μ L is taken gently to be driven into the impression of 200 μ L EHA105 Agrobacteriums
In state cell, ice bath 5min after liquid nitrogen flash freezer 1min, 37 DEG C of water-bath 5min, adds in 200 μ L YEP fluid nutrient mediums, 28 DEG C,
220rpm expresses 4~5h in advance；10000rpm centrifuges 30s, abandons supernatant, adds in 100 μ L YEP fluid nutrient mediums, suspends again thin
Born of the same parents are coated on the YEP solid plates containing 100 μ g/mL Kan and 50 μ g/mL Rif, 28 DEG C of culture about 24~48h；Picking is put down
The yellowish single bacterium colony grown on plate is inoculated in the YEP liquid mediums containing 100 μ g/mL Kan and 50 μ g/mL Rif,
Shake 24~48h of bacterium；It treats bacterium solution muddiness, is shown as orange-yellow, extract plasmid；It is verified respectively with PCR and double digestion, sees Fig. 1.
Embodiment 3
Agrobacterium-Mediated Transformation in Oryza sativa
PCAM-PZmCCR expression will be contained by Agrobacterium tumefaciems (mono- mediated of Agrobacterum) agrobacterium-mediated transformation
In the Agrobacterium Introduced into Rice of carrier.The process of rice transformation is：Induction；It infects；Selection；Differentiation；It takes root and transplants.
The acquisition of 3.1 Mature Embryos of Rice callus inductions
Before experiment, its decladding is handled to testing after mature seed used shines 3~4h under strong sunlight.First with going out
Bacterium is washed, and water impregnates 5min to clear, then with 75% ethyl alcohol, and the sterilized water then prepared with aqua sterilisa, hypochlorous acid and tween is (specific
Ratio：Aqua sterilisa：Hypochlorous acid=1：1, total volume：Tween=1mL:30min 1ul) is impregnated, is during which ceaselessly rocked, thus into
Mature seed later with aseptic water washing, until water becomes clarification, then is placed on suck dry moisture in sterilizing filter paper by row surface sterilizing
Afterwards, take it in callus on inducing culture, 26 ± 1 DEG C are cultivated；After 10~15d, by the milk yellow callus induced be transferred to after
For squamous subculture on culture medium.Squamous subculture is primary every two weeks, and subculture selects 5~7d of squamous subculture afterwards twice, by pale yellow
Callus for co-culturing.
3.2 are used for the preparation of the Agrobacterium bacterium solution of rice transformation
By the Agrobacterium inoculation containing pCAM-pPZmGL6 expression vectors in YEP fluid nutrient mediums (containing 50 μ g/mL cards that
Mycin and 50 μ g/mL rifampins) in, 28 DEG C of shaken cultivations to OD600=0.6~1.0；5000rpm centrifuges 5min and receives at room temperature
Collect thalline, be then suspended in liquid and co-cultured in base, adjustment cell concentration to OD600=0.4 as co-cultures conversion water
The agrobacterium suspension of rice.
3.3 Agrobacteriums infect rice nutrition organ
It selects that color and luster is fresh and tender, callus in yellow fraction, is concentrated into the sterile triangular flasks of 100mL, add in above-mentioned prepare
Agrobacterium suspension (suspension is to flood preferably callus)；28 DEG C, 20~30min is cultivated on 220rpm shaking tables；Bacterium solution is outwelled, it will
Callus after infecting, which is placed on the culture dish containing aseptic filter paper, sucks extra bacterium solution, is transferred on solidified co-cultivation medium immediately, 22
DEG C 2~3d of light culture.
3.4 selection cultures
The callus of common training is first cleaned until water change clarification, discards cleaning solution, be transferred to containing carbenicillin with aqua sterilisa
More than oscillation cleaning 30min in the solution of (100mg/ml), is blotted with aseptic filter paper, is placed in the culture dish containing aseptic filter paper and is done
Dry processing；Callus is selected containing on carbenicillin (500mg/ml) and hygromycin (50mg/L) screening and culturing medium again, 28
DEG C illumination cultivation 30d or so, until growing new kanamycin-resistant callus tissue.
The differentiation of 3.5 kanamycin-resistant callus tissues
Growing way is selected from screening and culturing medium preferably in the new callus of milk yellow in the differentiation training containing 50mg/L hygromycin
It supports on base, first 28 DEG C of light culture 3d, full exposure culture is carried out under the conditions of being then transferred into 30 DEG C, 15~30d has green point to occur；
Seedling can be differentiated after 30~40d.
3.6 take root, strong sprout and transplanting
It when the seedling h >=3cm differentiated, is transferred on root media, cultivates 2~3 weeks；Select h >=15cm, root
It is flourishing seedling, after adding 2~3d of hydroecium temperature lower refining seedling, culture medium is washed away with warm water, is moved into the bucket of the compost containing rice
Cultivation.It moves into paddy field and grows after seedling robust growth.
Embodiment 4
The identification of transgenic paddy rice
1. the PCR Molecular Detections of transgenic paddy rice
In order to detect transgenic paddy rice, using the transgenic paddy rice total DNA of extraction as template, started with target gene PZmCCR
Contained hygromycin gene and gus gene are to detect object on sub-piece and expression vector, design primer, amplified fragments, to
Preliminary Identification transfer-gen plant.
The primer sequence detected with target gene is as follows：
Primer 1 (sense primer)：5’-CCCAAGCTT CAGGCTGTGGGACACCTCCATTCTA-3’
Primer 2 (downstream primer)：5’-CATGCCATGG GTGCTCCTCTGCTCTAGCTCTT-3’
PCR reaction systems and reaction condition are identical with clone's condition of Corn nutritive organ-specific promoter PZmCCR；
It is as follows for detecting the primer sequence of hygromycin gene：
Primer 3 (sense primer)：5’-TAGGAGGGCGTGGATATGGC-3’
Primer 4 (downstream primer)：5’-TACACAGCCATCGGTCCAGA-3’
The clone of PCR reaction systems reference PZmCCR, reaction condition are as follows：PCR reaction conditions are：Pre-degeneration：94℃
10min；Denaturation：94℃ 30s；Annealing：60℃ 30s；Extension：72 DEG C of 1min, 34 cycles；Overall elongation：72℃ 10min.
The histochemical stain of 2.GUS genes
After Molecular Detection is as a result, be initially identified as transgenic line, respectively to turn PZmCCR promoter plant different parts into
Row GUS histochemical stains.Concrete operation step is as follows：Take the tissue that transgenic paddy rice is different：Young root, young stem, spire, maturation
Root, ripe stem, climax leaves, glume, anther, endosperm, each position is moved into test tube, adds in suitable GUS buffer solutions submergence group
Block is knitted, adds GUS dyeing liquors, 4-12h is preserved at 37 DEG C after mixing, after, stained tissue is first placed in the drift of 75% ethyl alcohol
Color is eluted, then bubble more than 20min is respectively invaded with 50% and 20% ethyl alcohol, until material is white；Naked eyes or micro- Microscopic observation, group
It is GUS expressive sites to have knitted blue dot.Coloration result is as shown in figure 3, PZmCCR promoters can drive gus gene seeking
It supports and is expressed in organ, do not expressed in reproductive organs.
Basic principle, main feature and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, the present invention
Claimed range is delineated by the appended claims, the specification and equivalents thereof from the appended claims.
Claims (8)
1. a kind of corn nourishment Organ specific expression promoter PZmCCR, it is characterised in that have shown in SEQ ID NO.1
Nucleotide sequence.
2. promoter described in claim 1 is for the purposes of the specific expressed target gene in rice nutrition organ.
3. include the plant expression vector of corn nourishment Organ specific expression promoter described in claim 1.
4. plant expression vector according to claim 3, it is characterised in that：The plant expression vector is to use corn nourishment
CaMV35S promoters on Organ specific expression promoter substitution plant binary expression vector pCAMBIA1301, structure obtain
Plant expression vector pCAM-PZmCCR.
5. the plant expression vector described in promoter described in claim 1 or claim 3 is in prepare transgenosis plant
Using.
6. application according to claim 5, the genetically modified plants are transgenic paddy rice.
7. the host cell of the plant expression vector described in claim 3 or 4 is included, wherein the host cell is crown gall agriculture bar
Bacterium host cell.
8. a kind of preparation side of the genetically modified plants containing corn nourishment Organ specific expression promoter described in claim 1
Method, it is characterised in that include the following steps：
(a) by the plant expression vector Introduced into Rice histocyte described in promoter described in claim 1 or claim 3；
(b) the rice tissue cell is cultivated under conditions of plant growth is promoted, obtains genetically modified plants.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667290A (en) * | 2013-11-25 | 2014-03-26 | 安徽农业大学 | Corn nutritive organ-specific promoter and application thereof |
| CN103789312A (en) * | 2014-02-13 | 2014-05-14 | 安徽农业大学 | Corn endosperm tissue specificity promoter and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667290A (en) * | 2013-11-25 | 2014-03-26 | 安徽农业大学 | Corn nutritive organ-specific promoter and application thereof |
| CN103789312A (en) * | 2014-02-13 | 2014-05-14 | 安徽农业大学 | Corn endosperm tissue specificity promoter and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Accession NO.：NW_007617764.1，Zea mays cultivar B73 chromosome 8 genomic scaffold, B73 RefGen _v3 8;Lowe,T.M. and Eddy,S.R.;《Genbank Datebase》;20140802;全文 * |
| 木质素生物合成途径中关键酶肉桂酰辅酶A还原酶研究进展综述;杨硕知;《安徽农学通报》;20100625;第16卷(第12期);45页左栏第4段 * |
| 白桦BpCCR基因的功能研究和应力木转录组分析;张岩;《中国优秀硕士学位论文全文数据库农业科技辑》;20130115;正文0027页最后一段~0032页图3-8 * |
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