CN114561385B - Plant inducible promoter and application thereof - Google Patents
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Abstract
The invention discloses a plant inducible promoter and application thereof, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the promoter is shown as SEQ ID NO.1, and the promoter is a dark inducible and/or green light inducible promoter. The promoter element is obtained by first identifying from rape, and further, the promoter is modified, so that the inducibility of a downstream reporter gene under dark and green light irradiation is obviously enhanced. The invention utilizes the promoter to construct the biological self-luminous system module, successfully induces plants to emit light at night, and provides a useful starting element for releasing the light at night by accumulating energy of the luminous plants in the daytime and further improving the luminous intensity of the plants. The promoter has important effects on plant genetic engineering research and application, and has very broad application prospects.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a plant inducible promoter induced by dark and green light and application thereof.
Background
Plant gene promoters are DNA sequences that recognize, bind to, and initiate transcription by RNA polymerase and contain conserved sequences required for specific binding and transcription initiation by RNA polymerase. The growth and development and the growth cycle of plants are the results of orderly expression of different genes in time and space, and one of key links of the expression control of the genes is the control of the transcription level under the control of a specific promoter. Plant tissue-specific or condition-inducible promoters are the main factors for the rational distribution of energy flow and environmental adaptation.
Promoters currently employed in genetic engineering techniques include constitutive promoters and inducible promoters. The constitutive promoter can drive the target gene to continuously express in each tissue of the plant, but can excessively consume substances and energy in receptor cells, break natural rules of the plant, and often can not well regulate and control the expression of the gene, so that certain defects exist in application. In the design of published bioluminescent plants, researchers have used constitutive promoters 35S to drive the expression of genes of the fungal light emitting system, creating tobacco emitting at a wavelength of about 520nm, khakhar A et al have integrated the NPGA (4' -phosphopantetheinyl transferase) gene in the genome of Aspergillus nidulans (Aspergillus nidulans), the H3H (hispidin-3-hydrolytic enzyme) gene and Hisps (Hispidin synthase) gene in the genome of Tricholoma matsutake (Neonothopanus nambi), and the fungal luciferase (Luz) gene into the Moclo plasmid, all of which use constitutive promoters 35S to drive the expression of these genes (Mitiouchkina et al 2020). Since the introduced light-emitting system is a constitutive promoter, the tobacco can continuously emit green light, and a large amount of energy in plants can be consumed in the daytime, so that adverse effects on the growth and development or stress resistance of the plants can be generated.
The inducible promoter can rapidly induce the on and off of gene transcription under the induction of exogenous physical and chemical factors and the like, so that the expression of the transgene in plants can be regulated and controlled according to experimental requirements. The induction expression systems which are widely used at present are as follows: tetracycline-inducible expression systems, steroid-inducible systems, dexamethasone-inducible systems, hormone-inducible expression systems, and pesticide and ethanol expression systems. The reaction conditions of the chemically induced promoters require the artificial addition of chemically inducing substances to activate the expression of the target genes, which is difficult and inefficient to control and may be toxic to plants to some extent. Therefore, the development of naturally inducible promoters has become an essential requirement for current genetic engineering (Kusunoki & Yamamoto, 2017).
In the growth and development process of higher plants, light is involved in photosynthesis to accumulate biomass, and is also used as an important signal to regulate and control the expression of related genes. Numerous studies have shown that a range of endogenous genes exist in plants that are expressed by light induction. Therefore, research on promoters of the genes and development of a photoinduction type promoter-induced gene expression system are of great significance to plant genetic engineering research and application. At present, the dark inducible promoter is not systematically excavated and utilized, the dark inducible promoter with application value is developed, and the luminescent plants are induced to emit light at night, so that the method has important significance for the development and the breeding of the self-luminous plants.
Disclosure of Invention
The invention aims to provide a promoter element which is induced by darkness and can induce the expression of a downstream target gene under the darkness condition, but can not induce the expression of the downstream target gene under natural light. The element is used for controlling the plant lighting system module to emit light only in darkness, so that the energy accumulated by the lighted plant in daytime releases light at night, and the lighting intensity of the plant is further improved.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention screens a dark induction promoter by analyzing rape dark treatment transcriptome data, and the nucleotide sequence of the dark induction promoter is shown as SEQ ID NO. 1. The research shows that compared with the normal illumination condition, the promoter can induce the expression quantity of the downstream gene to be improved by more than 10 times under the dark or green light condition.
The invention provides a method for obtaining a DNA molecule shown in SEQ ID NO.1, which comprises the following steps: firstly extracting rape genome DNA, and then cloning the DNA molecule fragments by utilizing a PCR technology; the primers used for PCR amplification are: f primer: 5'-TTATTGCCTAAGATTGATG-3', R primer: 5'-TTTTCTTATTTAATGCAAAGT-3'.
The invention makes core sequence analysis to the promoter sequence, presumes that the CACGTG at 547-551 and 726-731 is the core region of dark induction. Furthermore, the promoter sequence is modified, and a plurality of core elements are repeatedly inserted in the nucleotide sequence shown in SEQ ID NO.1 within 200bp range near a photoinduction core element G-BOX (CACGTG).
Specifically, a plurality of fragments containing core elements are inserted between 552 th and 725 th positions of a DNA molecular sequence with a nucleotide sequence shown as SEQ ID NO.1, and the sequence of the core elements is 5'-CACGTG-3'.
Preferably, the insert is selected to comprise 5 to 6 bases upstream and downstream of the core element in addition to the core element. In particular, the sequence comprising the core element fragment is 5'-GATACCACGTGTATGAC-3' and/or 5'-ATACAACACGTGGCAACA-3'.
The two fragments were inserted between the 558 th to 719 th sites of the nucleotide sequence shown in SEQ ID NO.1, and each fragment was repeated 1 to 2 times.
Preferably, the nucleotide sequence of the modified promoter is shown as SEQ ID NO. 2. Studies show that the modified promoter obviously enhances the induction of downstream genes to more than 40 times under dark or green light conditions.
The invention provides a related biological material containing the promoter, which is any one of the following A1) -A3):
a1 A gene expression cassette comprising said promoter;
a2 A recombinant vector containing said promoter.
A3 A recombinant cell comprising said promoter.
The invention provides application of the promoter in serving as a plant inducible promoter, wherein the inducible promoter is a dark inducible and/or green light inducible promoter.
Further, the plant is a herbal plant such as tobacco, butterfly orchid, rape, rice, chrysanthemum and the like.
The invention also provides application of the promoter in promoting expression of a target gene in plants. By integrating the promoter upstream of the target gene, the target gene can be induced to be expressed under dark or green light conditions.
The invention provides a cultivation method of self-luminous plants, which comprises the following steps: and introducing the promoter and the self-luminous gene element into a receptor plant to obtain a plant with the self-luminous gene expressed by dark induction and/or green light induction.
Further, the cultivation method of the self-luminous plant comprises the following steps:
(1) Integrating the Hisps gene, the CPH gene, the H3H gene, the NPGA gene and the Luz gene into a receptor vector by utilizing a polygene assembly technology, and constructing a polygene vector, wherein the promoter is integrated at the upstream of the Luz gene in the polygene vector;
(2) The target gene segment in the polygene carrier is led into the receptor plant by using the transgenic technology, and the transgenic plant of which the biological self-luminescence is induced by darkness and/or green light is obtained by cultivating.
Wherein the sequence information of Hisps is shown in gene accession number: QJQ48095.1; the sequence information of CPH is shown in gene accession number: QJQ48093.1; sequence information of H3H is shown in gene accession number: QJQ48094.1; sequence information of NPGA is shown in gene accession number: QJQ48097.1; sequence information for Luz is found in gene accession number: QJQ48096.1; sequence information of BnC H is shown in gene accession number: XP_009133464.2.
Further, in step (1), the polygene assembly was performed using a TransGene Stacking II system. pYL322 and pYL D1 and pYL D2 are used as donor carrier I and II, respectively, and pYLTAC380GW is used as acceptor carrier.
Specifically, the construction method of the polygene vector comprises the following steps:
1) Inserting the Hisps gene fragment and the H3H gene fragment into multiple cloning sites of a donor vector pYL and 322d1 respectively to obtain donor vectors pYL d1-35S-Hisps and pYL and 322d1-35S-H3H, and replacing the 35S promoter of the donor vector pYL and 322d1 with the Luz gene fragment with the promoter to obtain a donor vector pYL d1-eDIE-Luz;
inserting the CPH gene fragment and the NPGA gene fragment into the multiple cloning site of the donor vector pYL d2 to obtain a donor vector pYL d2-35S-CPH, pYL322d2-35S-NPGA;
2) Mixing donor vector pYL322d1-35S-Hisps and acceptor vector pYLTAC380GW according to a ratio of 1:1 to 2:1, co-transferring into competent cells of escherichia coli NS3529, coating the competent cells in a double-antibody culture medium containing kanamycin and chloramphenicol, culturing, and taking positive strain to extract plasmids;
3) Performing enzyme digestion on the plasmid extracted in the step 2) by using homing enzyme I-Sce I, then converting the escherichia coli strain NEB 10-beta, culturing, screening, extracting the plasmid, and obtaining a positive clone pYLTAC380GW-Hisps containing a target gene Hisps;
4) Mixing donor vector pYL d2-35S-CPH and acceptor vector pYLTAC380GW-Hisps prepared in the step 3) according to the ratio of 1:1 to 2:1, co-transferring into competent cells of escherichia coli NS3529, coating the competent cells into a double-antibody culture medium containing kanamycin and ampicillin, culturing, and taking positive strains to extract plasmids;
5) Enzyme cutting is carried out on the plasmid extracted in the step 4) by using homing enzyme PI-Sce I, then the escherichia coli strain NEB 10-beta is transformed, culturing, screening and extracting are carried out on the plasmid, and positive clone pYLTAC380GW-Hisps-CPH containing target genes Hisps and CPH is obtained;
6) Repeating the steps 2) -5), taking the new plasmid containing the target genes obtained in the previous step as a receptor carrier, carrying out recombination by using d1 and d2 donor carriers containing different genes in a crossing way until all the target genes are assembled on the receptor carrier, and finally connecting a BP recombination reaction to remove a screening marker gene expression cassette element to construct a polygenic carrier pYLTAC380GW-Hisps-CPH-H3H-NPGA-eDIE-Luz.
In the step (2), the constructed polygene fragments are introduced into a receptor plant to be expressed in the plant body, and each expressed protease participates in caffeic acid cycle, wherein the Luz gene induces expression only under dark or green light conditions, so that the plant only emits light in the dark.
Further, agrobacterium-mediated techniques are used to introduce the multiple gene segments into recipient plants. The agrobacterium employs EHA105.
Further, the recipient plant is a tobacco, butterfly orchid, rape, rice or chrysanthemum plant.
Furthermore, the coding sequence of the Hisps gene is shown as SEQ ID NO.3, the coding sequence of the CPH gene is shown as SEQ ID NO.4, the coding sequence of the H3H gene is shown as SEQ ID NO.5, the coding sequence of the NPGA gene is shown as SEQ ID NO.6, and the coding sequence of the Luz gene is shown as SEQ ID NO. 7.
The invention has the beneficial effects that:
(1) The invention firstly identifies and obtains the promoter sequence shown in SEQ ID NO.1 from rape, and the promoter is a dark inducible and/or green light inducible promoter. The promoter has important effects on plant genetic engineering research and application, and has very broad application prospects.
(2) The promoter obtained by modification of the invention has obviously enhanced inducibility to downstream reporter genes under dark and green light irradiation.
(3) The invention utilizes the promoter to construct the biological self-luminous system module, successfully induces plants to emit light at night, and provides a useful starting element for releasing the light at night by accumulating energy of the luminous plants in the daytime and further improving the luminous intensity of the plants.
Drawings
FIG. 1 is a schematic diagram of a transient transformation vector for the dark inducible promoter DIE.
FIG. 2 is a graph showing the intensity measurement of the DIE-induced reporter gene Luz of the dark-induced promoter.
FIG. 3 is a schematic diagram of a transient transformation vector for the dark inducible promoter eDIE.
FIG. 4 is a Luz intensity assay for the dark inducible promoter eDIE inducible reporter gene.
FIG. 5 shows the detection of each round of cleavage during construction of the dark and green inducible transgenic vector 380 GW-eDIE-G6.
FIG. 6 shows qPCR detection of dark and green-induced Luz gene expression.
FIG. 7 shows the seedling stage detection of dark and green light induced transgenic tobacco.
FIG. 8 is a fluorescent intensity analysis of dark and green light induced transgenic tobacco seedling stage.
Detailed Description
The invention will be further illustrated with reference to specific examples. The following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
EXAMPLE 1 identification of dark inducible promoters
1. A dark inducible promoter (dark induce element, DIE) with the nucleotide sequence shown in SEQ ID NO.1 was selected by analysis of rape dark transcript data.
The promoter sequence was amplified by PCR technique using the F primer: 5'-TTATTGCCTAAGATTGATG-3', R primer: 5'-TTTTCTTATTTAATGCAAAGT-3' the amplified promoter element is connected into a promoter analysis carrier (figure 1), after the sequencing is verified to be correct, the amplified promoter element is transferred into agrobacterium EHA105, EHA105 bacterial liquid containing the verified correct vector plasmid is respectively streaked and activated on LA+Kana+Rif plates, cultured for 36h at 28 ℃, colonies are picked from the plates, transferred into LB+Kana+Rif+15 mu M As culture medium, and cultured at 28 ℃ at 200rpm until OD 600 Bacterial cells were collected at 4000rpm for 10min, and agrobacterium cells were suspended in an infection solution (containing 10mM MgCl,10Mm MES,150 μm As) and allowed to stand at room temperature for 2-3 h.
2. And verifying transient expression in tobacco leaves, lightly clicking a small opening on the surface of the tobacco leaves by using a 1mL needle head, sucking bacterial liquid by using a needle tube with the needle head removed, and injecting the bacterial liquid into the leaves from a wound of the tobacco leaves. Culturing in a normal white light incubator (25 ℃ C., 12 h/12 h darkness, 6000 lux) for 48h, continuing to illuminate for 3h after illumination for 12h, beginning to illuminate for 3h after illumination for 12h in darkness, changing to green fluorescent lamp for 3h after illumination for 12h in green light treatment, taking bacterial liquid to infect leaf samples, grinding samples with liquid nitrogen, taking 0.02g, adding 100 mu L of lysis buffer (Promega, luciferase Assay System), mixing for 30s, and placing on ice. mu.L of the cell lysate was added to the ELISA plate, 45. Mu.L of the substrate (Promega, luciferase Assay System) was added, and after mixing for 2s,10s, the signal value of fluorescence emission was measured by detection with an ELISA reader.
As shown in FIG. 2, the dark or green light 3h treatment can significantly induce the expression of the reporter gene to more than 10 times, and the promoter is determined to be dark-inducible.
Example 2 engineering of dark inducible promoters
We performed a core sequence analysis of the Dark Induce Element (DIE) promoter selected in example 1, and showed that the promoter portion had a core element Gbox (CACGTG), possibly a dark-induced core region, and we performed multiple repeats of the dark-induced core elements GATACCACGTGTATGAC and ATACAACACGTGGCAACA, designated Engineered dark induce element (eDIE), with the nucleotide sequence shown in SEQ ID NO. 2.
According to the sequence entrusting company, the corresponding promoter fragment is synthesized, and is connected into a promoter analysis carrier (figure 3), after the sequencing verification is correct, the promoter fragment is transferred into agrobacterium, and the promoter fragment is verified through a tobacco leaf transient transformation experiment (the operation method is the same as that of example 1).
The results are shown in FIG. 4, and the inducibility of the modified eDIE promoter to the downstream reporter gene is obviously enhanced to more than 40 times under dark and green light irradiation, which indicates that the modified eDIE element can be applied to the expression of the target gene which needs dark or green light induction.
EXAMPLE 3 construction of dark inducible luminous plant transgenic vector
Based on the above results, expression of the Luz gene in the plant self-luminous system was driven using the modified dark enhanced promoter element eDIE, and in this operation, the TransGene Stacking II system developed by the university of agricultural in south China, liu Yaoguang institute of subject, was used for polygenic assembly, see chinese patent No. 2017103841977, pYL322d1, pYL322d2 and pYLTAC380GW for professor laboratory donation at university of agricultural in south China Liu Yaoguang.
The NPGA (4' -phosphopantetheinyl transferase) gene in the genome of Aspergillus nidulans (Aspergillus nidulans), the H3H (hispidin-3-hydrolytic enzyme) gene and the milk tree alkali synthase gene Hisps (Hispidin synthase) in the genome of Tricholoma matsutake (Neonothopanus nambi), the caffeoyl pyruvate hydrolase gene CPH (caffeoyl pyruvate hydrolase) and the fungal luciferase (Luz) gene are integrated into the pYLTAC380GW plasmid, wherein the NPGA and the H3H, hisps, CPH are both driven by a 35S promoter, the Luz is driven by a dark inducible promoter eDIE promoter, and finally, a screening marker gene expression cassette element is removed by a one-step BP recombination reaction, so that a dark inducible vector pYLTAC380 GW-Hisps-CPH-3H-NPGA-eDIELuz (pYLTAC 380 GWeide-G6) is formed.
The coding sequence of the Hisps gene is shown as SEQ ID NO.3, the coding sequence of the CPH gene is shown as SEQ ID NO.4, the coding sequence of the H3H gene is shown as SEQ ID NO.5, the coding sequence of the NPGA gene is shown as SEQ ID NO.6, and the coding sequence of the Luz gene is shown as SEQ ID NO. 7.
The specific process is as follows.
(1) Construction of donor vectors pYL322d1-35S-Hisps, pYL d2-35S-CPH, pYL322d1-35S-H3H, pYL322d2-35S-NPGA, pYL322d1-eDIE-Luz.
The target gene fragments are synthesized by biological company, and Hisps and H3H gene fragments are respectively inserted into the multiple cloning sites of pYL322d1 to obtain pYL322d1-35S-Hisps and pYL322d1-35S-H3H; the CPH and NPGA gene fragments are respectively inserted into the multiple cloning sites of pYL and 322d2 to obtain pYL and 322d2-35S-CPH and pYL322d2-35S-NPGA; the eDIE promoter was synthesized with Luz gene fragments and the pYL322d1-eDIE-Luz was constructed by replacing the 35S promoter in the pYL d1 base vector using homologous recombination techniques.
(2) Mixing donor vector pYL d1-35S-Hisps and acceptor vector pYLTAC380GW (1:1 to 2:1) in NS3529 competent, performing cotransformation by heat shock method, ice bath for 30min, heat shock for 90S, ice bath for 2-3min, in LB without antibiotic, at 37deg.C, at 200rpm for 2h for reactivation, applying on LA plate containing kanamycin (Km, 25 mg/L) and chloramphenicol (Chl, 15 mg/L), growing monoclonal after about 18h, and using ddH 2 O all the monoclonal were washed into the tube and the mixed plasmid was extracted.
(3) 100-200ng of the mixed plasmid was digested with 0.5uL of I-Sce I (NEB) in a10 uL system for 4-5h, transformed into E.coli strain NEB 10-beta (Bomeide), smeared on LA plates containing kanamycin (Km, 25 mg/L), after 15h, monoclonal was picked up, cultured in LB (containing 25mg/L Km and 0.5mM IPTG) and subjected to bacterial liquid PCR identification, further extracted plasmids capable of amplifying the bright band were obtained by using Green Taq Mix, 200ng of each plasmid was digested with 0.2uL of Not L in a 20uL reaction system, five bands were generated, and four framework bands containing the genes of interest of 6.2 kBp and 1290, 1447bp, 1532bp and 13816bp were obtained as the required positive clone pYLTAC380GW-Hisps (FIG. 5, GW 380-G1).
(4) The donor vector pYL322d2-35S-CPH and (3) the acceptor vector pYLTAC380GW-Hisps (1:1 to 2:1) were mixed in NS3529 competence and co-transformed according to (2) method, spread on LA plates containing kanamycin (Km, 25 mg/L) and ampicillin (Amp, 70 mg/L) and after about 18h a single clone was grown, using ddH 2 O all the monoclonal were washed into the tube and the mixed plasmid was extracted.
(5) 100-200ng of the mixed plasmid is taken, 0.5uL of PI-Sce I (NEB) is added with 0.5uL of BSA and digested for 4-5 hours in a10 uL system, then the transformation and verification are carried out according to the method in (3), six bands appear, and the band with the sizes of 1.7k bp CPH and 6.2k bp Hisps of the target gene is positive clone pYLTAC380GW-Hisps-CPH (FIG. 5, 380 GW-G2).
(6) More rounds of recombination were performed by cross-transferring d1, d2 donor vectors containing different genes with the acceptor vector from the previous round of construction to complete pYLTAC380GW-eDIE-G6, and finally pYLTAC380GW-eDIE-G6 (200 ng) was mixed with PYLTAC380GW-eDIE-G6 (100 ng) in 5. Mu.l reaction with 1. Mu.L of 5 XBP enzyme mixture for 5 hours by BP reaction. Mu.l of protease K solution was then added to terminate the reaction at 37℃for 10 minutes. Transferring into NEB 10-beta (Bomaide) competence, and selecting monoclonal identification. Plasmid restriction assays were extracted from the monoclonal strains as shown in FIG. 5, confirming that the correct strain was used in the subsequent transgenic experiments.
EXAMPLE 4 phenotype identification of dark inducible transgenic luminous plants
1. EHA105 bacterial liquid containing the verified correct vector plasmid pYLTAC380GW-eDIE-G6 is streaked on LA+Rif+Kana plates, and then is subjected to monoclone culture in 3-5ml LB medium at 200rpm for 36h at 28 ℃ for 36h, 50ml is subjected to expansion culture for 3-5h to OD=0.6 according to the proportion of 1:100-1:50, and then the bacterial liquid is centrifuged, and bacterial cells are suspended in MS0 liquid medium (MS+3%subculture+PH5.8, 50 ml) to OD=0.6 for infection.
2. Wild tobacco ZY100 seeds were selected, grown for 4-5 weeks on sterile MS medium to fully expanded tobacco healthy leaves, cut to 0.5cm square size (cut out margin to avoid main vein) with a scalpel, and dark-cultured on MS1 solid medium (ms+0.5 mg/L iaa+2.0mg/L ba+3% sucrose+0.6-0.8% phytagel, ph=5.8) with the upper surface of the leaf down for 2-3 days at 25 ℃.
3. Adding the pre-cultured tobacco leaves into the bacterial liquid, carrying out vortex oscillation to ensure that leaf cuts are immersed by the bacterial liquid, standing for 5-30min, and sucking the attached bacterial liquid by using sterile filter paper; placing the upper surface of the infected leaf on an MS1 solid culture medium downwards, and culturing for 2d at 28 ℃ in a dark way; placing the upper surface of leaf upward on MS1 screening culture medium containing Tintin and glyphosate, and culturing at 25deg.C; when She Yuanchang buds and can be separated (more than 1 cm), cutting off the buds, transferring the buds to MS2 (MS+0.5 mg/L IAA+3%s of cross+0.6-0.8%s of Phytagel, PH=5.8) solid medium containing antibiotics (TM+basta), growing roots after two weeks, opening the cover of a seedling raising box, training seedlings for one week, transferring the seedlings into planting soil, culturing the seedlings into T0 generation, and collecting the seedlings of the T0 generation transgenic plants.
Using T0 generation transgenic plant seeds as T1 generation, taking T1 generation seeds for germination, growing in a normal white light incubator (25 ℃,12h illumination/12 h darkness, 6000 lux) for 15 days, continuing illumination for 3h after illumination treatment is 12h illumination, beginning 3h darkness after illumination for 12h, changing green light treatment into green fluorescent lamp illumination for 3h after illumination for 12h, taking leaf samples, extracting total RNA by liquid nitrogen grinding samples for reverse transcription, and qPCR analysis, and determining that dark-induced Luz expression is subjected to dark and green light-induced expression (figure 6). And (3) photographing and analyzing part of T1 generation plants under a full-automatic luminescence detection system, wherein the luminescence brightness of the transgenic luminescence tobacco is obviously enhanced after being induced by dark or green light (figure 7), and the luminescence brightness of the bioluminescence tobacco is obviously enhanced to more than 40 times after being induced by dark or green light (figure 8) through luminescence intensity analysis.
The result shows that the eDIE promoter has good application prospect in dark induction and green light induction.
Sequence listing
<110> Hangzhou International science center of Zhejiang university
<120> a plant inducible promoter and use thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 906
<212> DNA
<213> rape (Brassica rapa)
<400> 1
ttattgccta agattgatga ccagaaaaag tttgtcttcc atattccatc gcatgagttg 60
aatttaagga agtattttgt gattttggat ctagtgtaaa tctcgtttta aaaataatta 120
tagacaaact gggcactata gatgttgagc atcttcagct gactctagct tttgccaact 180
cttctacagt tctctatggc atcattcgta accttcttgt ccaagccgga gactacattt 240
gcatactgaa aaacaaacac gatataactt tactaatata tatttctttt cttattaata 300
tagatttctc aggcatattt ttcactattt tttgcgtgtg ctattatttt ccaaaatttc 360
aaatattttg gaattagagt agtaaaagaa agtcccttcg tgtcatccaa taataaaaaa 420
atgataggcc gaagaaaatg atagaaatta gatttgtgaa atctatttta aaacatttca 480
atctgaattt atatactgtt ttaagatagc aaaatgctta tctacgatca accattctcc 540
agataccacg tgtatgactc taaatcaatc gtaagagtca tttaaaatat tttccaacgt 600
cagatcaagt atctatccag tgctgactca gccggataaa cctaaaccgg agctgcctca 660
ccattaaaat tagaataaac cggcgaaagt aaagaaatat ccaatcagta gttggggaaa 720
tacaacacgt ggcaacagct acccggttct tatccggttc gggggtaaaa aataaaattg 780
gcctcttcac acgaaacgcg acatcacgtc gccgcttgga ctttataaat agagccatcg 840
gtcgcagaga acaagacatc aaacaaagca ataccaagaa tattactttg cgattaaata 900
agaaaa 906
<210> 2
<211> 959
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
ttattgccta agattgatga ccagaaaaag tttgtcttcc atattccatc gcatgagttg 60
aatttaagga agtattttgt gattttggat ctagtgtaaa tctcgtttta aaaataatta 120
tagacaaact gggcactata gatgttgagc atcttcagct gactctagct tttgccaact 180
cttctacagt tctctatggc atcattcgta accttcttgt ccaagccgga gactacattt 240
gcatactgaa aaacaaacac gatataactt tactaatata tatttctttt cttattaata 300
tagatttctc aggcatattt ttcactattt tttgcgtgtg ctattatttt ccaaaatttc 360
aaatattttg gaattagagt agtaaaagaa agtcccttcg tgtcatccaa taataaaaaa 420
atgataggcc gaagaaaatg atagaaatta gatttgtgaa atctatttta aaacatttca 480
atctgaattt atatactgtt ttaagatagc aaaatgctta tctacgatca accattctcc 540
agataccacg tgtatgactc taaatcaatc gtaagagtca tttaaaatat tttccaacgt 600
cagatcaaga taccacgtgt atgacgtatc tatccagtgc tgactcagcc ggataaacct 660
atacaacacg tggcaacaaa accggagctg cctcaccatt aaaattagaa taaaccggcg 720
aaagtaatac aacacgtggc aacaaagaaa tatccaatca gtagttgggg aaatacaaca 780
cgtggcaaca gctacccggt tcttatccgg ttcgggggta aaaaataaaa ttggcctctt 840
cacacgaaac gcgacatcac gtcgccgctt ggactttata aatagagcca tcggtcgcag 900
agaacaagac atcaaacaaa gcaataccaa gaatattact ttgcgattaa ataagaaaa 959
<210> 3
<211> 5097
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atgaactcat ctaagaaccc accatctact ctcctcgatg ttttcctcga tactgctaga 60
aacctcgata ctgcttcaag aaacgttttg gagtgtggtg agcatagatg gtcatacagg 120
gagctggata ctgtgtcatc agctctcgct caacatctta ggtacactgt gggattgtca 180
cctactgtgg ctgttatctc agagaaccat ccatacatcc tcgctcttat gctcgctgtt 240
tggaagttgg gtggaacttt cgctccaatc gatgtgcatt caccagctga gctggtggct 300
ggaatgctta acatcgtgtc accatcatgt ctcgttatcc catcttctga tgtgactaac 360
caaactttgg cttgtgattt gaacatccca gtggtggctt tccatccaca tcaatcaact 420
atccctgagt tgaacaagaa gtaccttact gattcacaaa tctcaccaga tttgccattc 480
cctgatccaa acaggccagc tttgtacctt ttcacttctt cagctacttc taggtcaaac 540
cttaagtgtg tgccattgac tcatactttc atcctcagaa actctttgtc taagagggct 600
tggtgtaaga gaatgagacc agagactgat ttcgatggaa tcagggtttt gggatgggct 660
ccctggtcac atgttttggc tcacatgcaa gatattggtc cacttactct tttgaacgct 720
ggatgttacg ttttcgctac tactccatct acttacccta ctgagttgaa ggatgatagg 780
gatgttatct catgtgctgc taacgctgtt atgtacaagg gagtgaagtc attcgcttgt 840
ttgccattcg ttttgggagg acttaaggct ctctgtgagt ctgagccatc agtgaaggct 900
caattgcaag ttgaggagag ggctcaactc cttaagtcat tgcaacacat gaaaatatta 960
gaatgtggtg gagctatgtt ggaggtgtca gtggcttcat gggctatcga gaacagaatc 1020
ccaatctcaa tcggaatcgg tatgactgag actggtggtg cgcttttcgc tggtccagtg 1080
caagctatcc aaactggttt ctcttctgag gataagttca tcgaggatgc tacttacctc 1140
ctcgtgaagg atgattacga gtcacatgct gaggaggata ttaacgaggg tgaattagtt 1200
gttaagtcca gaatgttgcc taggggttac ctcggataca acgatccatc attctcagtg 1260
gatgatgctg gttgggtgac tttcaagact ggtgataggt actcagtgac tccagatgga 1320
aagttctcat ggctcggtag aaacactgat ttcatccaaa tgacttctgg tgagactctt 1380
gatcctagac caatcgagtc actcctctgt gagtcttcac ttatctctag ggcttgtgtt 1440
atcggtgata agttccttaa cggaccagct actgctgtgt gtgctatcat cgagttggag 1500
ccaactactg ttgagaaggg acaagctcat tctagggaca tagctagaat cttcgctcca 1560
atcaacaggg atttgccacc accacttaga atcgcttggt cacatgtttt ggttttgcaa 1620
ccttctgaga agatccctat gactaagaag ggaactatct tcagaaagaa gatcgagcaa 1680
gttttcggtt cagctctcgg tggatcttct ggtgataact cacaagctac tactgatgct 1740
tcagtggtta gaagggatga gttgtctaac actgtgaagc atatcatcag cagagttctc 1800
ggagtgtctg atgatgagtt gctctggact ttgtcattcg ctgagttggg tatgacttct 1860
gctctcgcta ctagaatcgc taacgagttg aacgaggttt tggtgggagt gaacctccct 1920
atcaacgctt gttacatcca tgttgatttg ccttcattgt caaacgctgt gtacgctaag 1980
ttggctcatt tgaagttgcc agatagaact ccagagccta gaaaggctcc agtggagaac 2040
cctggtggaa aggagatcgt tatcgtggga caggcattca gattgccagg ttctatcaac 2100
gatgtggctt cacttaggga tgctttcctt gctaggcagg cctcttctat catcactgag 2160
atcccaccag atagatggga tcatgcttct ttctacccta aggacatcag gttcaacaag 2220
gctggacttg ttgatatagc taactacgat cattctttct tcggtttgac tgctactgag 2280
gctctttacc tttcaccaac tatgagactc gctcttgagg tgtctttcga ggctctggaa 2340
aacgctaaca tcccagtgtc acaattgaag ggttcacaaa ctgctgttta cgtggctact 2400
actgatgatg gtttcgagac tctccttaac gctgaggctg gttacgatgc ttacactagg 2460
ttctacggaa ctggtagggc tgcttcaact gcttctggta gaatctcata ccttttggat 2520
gtgcatggtc catcaatcac tgtggatact gcttgttcag gaggagctgt gtgtatcgat 2580
caagctatcg attacctcca atcttcatca gctgctgata ctgctatcat ctgtgcttct 2640
aacactcatt gttggcctgg ttcattcatg ttcctctcag ctcaaggtat ggtgtcttct 2700
ggaggtaggt gtgctacttt cactactgat gctgatggat acgtgccatc tgagggtgct 2760
gtggctttca tccttaagac tagagaggct gctatgaggg ataaggatac tatcctcgct 2820
actatcaagg ctactcaaat ctcacataac ggtaggtcac aaggtcttgt ggctccaaac 2880
gttaactcac aagctgattt gcataggtca ctcctccaaa aggctggttt gtcaccagct 2940
gatatacatt tcatcgaggc tcatggtact ggaacttcac tcggtgattt gtcagagatc 3000
caagctatca acgatgctta cacttcatca caacctagga ctgctggacc acttatcgtg 3060
tcagcttcta agactgttat cggacatact gagcctgctg gaccactcgt gggtatgttg 3120
tcagttttga actcattcaa ggagggagct gtgccaggtc ttgctcattt gactgctgat 3180
aaccttaacc cagctctcga ttgttcttca gtgccattgc ttatcccata ccaaccagtg 3240
catttggctg ctccaaagcc acatagggct gctgttatgt catacggatt ctcaggaact 3300
ctcggtggaa tcgttttgga ggctccagat gaggagagac ttgaggagga gccaccaaac 3360
gataagccta tgttgttcgt ggtgtcagct aagactcata ctgctcttat cgagtacctt 3420
ggtcggtatc ttgagttcct tttgcaagct aacccacaag atttctgtga tatatgttac 3480
acttcttgtg tgggtagaga gcattacagg tacaggttcg cttgtgtggc taacgatatg 3540
gaggatttga tcggacaatt gcaaaagaga ttgggttcta aggtgccacc aaagccatct 3600
tacaagaggg gcgcactcgc tttcgctttc tctggacaag gaactcaatt caggggaatg 3660
gctactgagc tggctaaggc ttactcaggt ttcagaaaga tcgtgtcaga tttggctaag 3720
agagcttctg agttgtctgg acatgctatc gataggttcc tcctcgctta cgatattggt 3780
gctgagaacg tggctccaga ttcagaggct gatcaaatct gtatcttcgt ttaccaatgt 3840
tctgttctta gatggttgca aactatggga atcaggccat cagctgttat cggacattca 3900
ctcggagaaa tatctgcttc agttgctgct ggagctttgt cacttgattc agctctcgat 3960
ctcgttatct ctagggctag actccttagg tcttctacta acgctcctgc tggaatggct 4020
gctatgtctg cttcacaaga tgaggtggtt gagttgatcg gaaagttgga tctcgataag 4080
gctaactcat tgtctgtgtc agttatcaac ggaccacaaa acactgttgt gtctggatct 4140
tcagctgcta tcgagtcaat cgtggctctt gctaagggta gaaagatcaa ggcttctgct 4200
cttaacatca accaggcctt ccattcacca tacgttgatt cagctgtgcc tggacttagg 4260
gcttggtcag agaagcatat ctcttcagct aggccactcc aaatcccatt gtactctact 4320
cttttgggtg ctcaagtgtc tgagggacaa atgttgaacc cagatcattg ggttgatcat 4380
gctagaaagc ctgtgcaatt cgctcaagct gctactatca tgaaggagtc attcactgga 4440
gttatcatcg acataggacc acaagtggtg gcttggtcac ttttgttgtc taacggattg 4500
acttcagtga ctgctttggc tgctaagagg ggtaggtcac aacaagtggc tttcctctca 4560
gctctcgctg atttgtacca agattacgga gtggtgccag atttcgttgg attgtacgct 4620
caacaagagg atgcatcacg gcttaagaaa actgacattc ttacttaccc attccaaagg 4680
gttagacggt atccatcatt catcccatca agaagggctc caactcatgc tcatgtgcaa 4740
gatgaggaga ctttgtcttc aggttcttca actccaactc ttgagaacac tgatttggat 4800
tcaggtaagg agtcacttat gggacctact aggggtctgc ttagagttga tgatcttaga 4860
gattcaatcg tgtcttcagt taaggatgtt ttggagttga agtctaacga ggatcttgat 4920
ttgtcagagt cacttaacgc tttgggtatg gattctatca tgttcgctca attgagaaag 4980
agaatcggtg agggtcttgg attgtcagtg cctatggttt tcctttctga tgctttctca 5040
atcggagaga tggtgtctaa cctcgttgag caagctgagg cttcagagga taactaa 5097
<210> 4
<211> 921
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
atggctccta tctcatctac ttggtctagg cttatcagat tcgtggctgt tgagacttca 60
ctcgtgcata tcggtgagcc aatcgatgct actatggatg tgggactcgc tagaagagag 120
ggtaagacta tccaggccta cgagatcatc ggttcaggtt cagctcttga tttgtctgct 180
caagtgtcta agaacgtttt gactgttagg gaacttctta tgccattgtc tagggaggag 240
atcaagactg ttaggtgttt gggtcttaac tacccagtac acgctactga ggctaacgtt 300
gctgtgccta agttcccaaa ccttttctac aagccagtga cttcactcat cggaccaggt 360
ggattgatca ctatcccttc agttgtgcaa ccaccaaagg agcatcaatc tgattacgag 420
gctgaattgg tgatcgttat cggaaaggct gctaagaacg tgtcagagga tgaggctctc 480
gattacgttt tgggatacac tgctgctaac gacatatctt tcaggaagca tcaactcgct 540
gtgtcacaat ggtcattctc aaagggtttc gatggtacta acccattggg tccatgtctt 600
gtgtcagctt cttctatccc tgatccacaa gatattccta tccaatgtaa gttgaacggt 660
ggagttgtgc aaaacggaaa cactagagat caaatcttca acgtgaagaa aactatctca 720
ttcctctcac aaggtactac gctcgaacct ggttcaatca tcctcactgg tactccagat 780
ggagtgggat tcgttagaaa cccacctctt taccttaagg atggtgatga ggttatgact 840
tggatcggtt ctggaatcgg aactttggct aacactgtta gagaggagca aacttgtttc 900
gcttctggtg gacatgagta a 921
<210> 5
<211> 1269
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
atggcttcat tcgagaactc attgtcagtt cttatcgttg gtgctggact aggtggactc 60
gctgctgcta tcgctcttag aaggcaagga catgttgtta agatttacga ttcatcttca 120
ttcaaggctg agttgggagc tggacttgct gtgccaccaa acactcttag gtcactccaa 180
caattgggat gtaacactga gaaccttaac ggtgttgata acctttgttt cactgctatg 240
ggttacgatg gttctgtggg tatgatgaac aacatgactg attacaggga ggcttacggt 300
acttcttgga tcatggtgca tagagtggat ttgcataacg agcttatgag agtggctctc 360
gatcctggag gcttgggacc accagctact ttgcatttga accatagggt gactttctgt 420
gatgttgatg cttgtactgt gactttcact aacggtacta ctcaatctgc tgatcttatc 480
gtgggagctg atggaatcag gtctactatc agaagattcg tactggagga ggatgtgact 540
gttccagctt ctggaatcgt gggtttcagg tggctcgtgc aagctgatgc tctcgatcca 600
tacccagagt tggattggat cgttaagaag cctccactcg gagctagact tatctcaact 660
ccacaaaacc cacaatctgg tgttggactt gctgatagaa gaactatcat catctacgct 720
tgtaggggag gtactatggt taacgttttg gctgtgcatg atgatgagag ggatcaaaac 780
actgctgatt ggtcagtgcc agcttctaag gatgatttgt tcagagtttt ccatgattac 840
catcctagat tccgcaggct ccttgaatta gctcaagaca ttaacctctg gcaaatgaga 900
gttgtgccag tgttgaagaa gtgggtgaac aagagggtgt gtcttttggg agatgctgct 960
catgcttcat tgcctacttt gggacaaggt ttcggaatgg gtcttgaaga tgctgtggct 1020
ctcggaactc tcctcccaaa gggaactact gcttcacaaa tcgagactag gctcgctgtt 1080
tacgagcaac ttagaaagga tagggctgag ttcgtggctg ctgagtcata cgaggagcaa 1140
tacgtgccag agatgagggg tttgtacctc aggtctaagg agcttagaga tagagttatg 1200
ggttacgaca taaaggttga gtcagagaag gttcttgaga ctctccttag gtcatctaac 1260
tcagcttga 1269
<210> 6
<211> 1041
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
atggtgcaag atacttcatc tgcttcaact tcaccaatct tgactagatg gtacatcgat 60
actaggccat tgactgcttc tactgctgct ttgcctctcc ttgagacttt gcaaccagct 120
gatcaaatct ctgtgcaaaa gtactaccat ttgaaggata agcacatgtc actcgcttct 180
aacctcctta agtacctttt cgtgcataga aactgtagaa tcccttggtc atctatcgtt 240
atctctagga ctcctgatcc acatagaagg ccatgttaca tcccaccttc tggatcacaa 300
gaggattcat tcaaggatgg ttacactgga atcaacgttg agttcaacgt gtcacatcaa 360
gcctctatgg ttgctatcgc tggaactgct ttcactccaa actcaggagg tgattctaag 420
ttgaagcctg aggtgggtat cgatataact tgtgtgaacg agagacaagg tagaaacgga 480
gaggagaggt cattggagtc tcttaggcaa tacatcgaca ttttctcaga ggttttctca 540
actgctgaga tggctaacat ccgtagactc gatggagtgt cttcttcatc attgtcagct 600
gatagactcg ttgattacgg ttacagattg ttctacactt actgggctct taaggaggct 660
tacatcaaga tgactggaga ggctctcctc gctccgtggc tcagggagct ggagttctca 720
aacgttgtgg ctccagctgc tgtggctgag tctggtgatt cagctggtga tttcggtgag 780
ccatacactg gagttaggac tactttgtac aagaacctcg ttgaggatgt tagaatcgag 840
gttgctgctt tgggtggtga ttaccttttc gctactgctg ctaggggtgg tggaatcgga 900
gcttcatcta ggccaggtgg tggaccagat ggatcaggta tcaggtcaca agatccttgg 960
agaccattca agaagctcga tattgagagg gatatacaac catgtgctac tggtgtttgt 1020
aactgtttgt ctaggggatg a 1041
<210> 7
<211> 804
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
atgagaatca acatctcact ctcttctctt ttcgagagat tgtcaaagtt gtcatctagg 60
tcaatcgcta tcacttgtgg tgtggttttg gcttctgcta tcgctttccc aatcatcaga 120
agggattacc agactttctt agaagttgga ccttcttacg ctccacagaa cttcaggggt 180
tacatcatcg tgtgtgtttt gtcattgttc agacaggagc agaagggttt agctatctac 240
gatagattgc ctgagaagag aagatggctt gctgatttgc cattcaggga gggtactagg 300
ccttcaatca cttcacatat catccagaga cagagaactc agctcgtgga tcaggagttc 360
gctactaggg agcttatcga taaggttatc cctagagtgc aggctaggca tactgataag 420
actttcttgt ctacttcaaa gttcgagttc catgctaagg ctatcttcct cctcccttca 480
atcccaatca acgatcctct taacatccca tctcatgata ctgttagaag gactaagagg 540
gagatcgctc acatgcatga ttaccatgat tgtactctcc atttggctct tgctgctcag 600
gatggtaagg aggtgcttaa gaagggatgg ggacagaggc atccactcgc tggacctggt 660
gtgcctggac caccaactga gtggactttc ctttacgctc ctaggaacga ggaggaggct 720
agagtggttg agatgatcgt tgaggcttct atcggataca tgactaacga tccagctgga 780
aagatcgttg agaacgctaa gtaa 804
Claims (7)
1. A promoter is characterized in that the nucleotide sequence of the promoter is shown as SEQ ID NO. 2.
2. The biological material related to the promoter of claim 1, which is any one of the following A1) to A2):
a1 A gene expression cassette comprising the promoter of claim 1;
a2 A recombinant vector comprising the promoter according to claim 1.
3. Use of a promoter according to claim 1 as a plant inducible promoter, wherein the plant is tobacco or canola and the inducible promoter is a dark-inducible and/or green-inducible promoter.
4. A method of growing self-luminous plants, comprising: introducing the promoter and the self-luminous gene element according to claim 1 into a recipient plant, so as to obtain a plant in which the self-luminous gene is expressed under the induction of darkness and/or green light, wherein the recipient plant is tobacco or rape.
5. The cultivation method of self-luminous plants as claimed in claim 4, comprising the steps of:
(1) Using multiple basesWill be due to the assembly techniqueHispsGenes (gene),CPHGenes (gene),H3HGenes (gene),NPGAGene and geneLuzIntegration of the gene into the recipient vector, construction of the resulting multigenic vector, in whichLuzThe promoter according to claim 1 incorporated upstream of the gene;
(2) The target gene segment in the polygene carrier is led into the receptor plant by using the transgenic technology, and the transgenic plant of which the biological self-luminescence is induced by darkness and/or green light is obtained by cultivating.
6. The method for cultivating self-luminous plants as claimed in claim 5, wherein in the step (1), the construction method of the polygenic vector comprises the steps of:
1) Inserting the Hisps gene fragment and the H3H gene fragment into multiple cloning sites of the donor vector pYL and 322d1 respectively to obtain donor vectors pYL d1-35S-Hisps and pYL and 322d1-35S-H3H, and replacing the 35S promoter of the donor vector pYL and 322d1 with the Luz gene fragment containing the promoter as set forth in claim 1 to obtain a donor vector pYL d1-eDIE-Luz;
inserting the CPH gene fragment and the NPGA gene fragment into the multiple cloning site of the donor vector pYL d2 to obtain a donor vector pYL d2-35S-CPH, pYL322d2-35S-NPGA;
2) Mixing donor vector pYL322d1-35S-Hisps and acceptor vector pYLTAC380GW according to a ratio of 1:1 to 2:1, co-transferring into competent cells of escherichia coli NS3529, coating the competent cells in a double-antibody culture medium containing kanamycin and chloramphenicol, culturing, and taking positive strain to extract plasmids;
3) Using homing enzyme I-SceI, carrying out enzyme digestion on the plasmid extracted in the step 2), then converting the escherichia coli strain NEB 10-beta, culturing, screening and extracting the plasmid to obtain a positive clone pYLTAC380GW-Hisps containing the target gene Hisps;
4) Mixing donor vector pYL d2-35S-CPH and acceptor vector pYLTAC380GW-Hisps prepared in the step 3) according to the ratio of 1:1 to 2:1, co-transferring into competent cells of escherichia coli NS3529, coating the competent cells into a double-antibody culture medium containing kanamycin and ampicillin, culturing, and taking positive strains to extract plasmids;
5) Using GuiNest enzyme PI-SceI, carrying out enzyme digestion on the plasmid extracted in the step 4), then converting the escherichia coli strain NEB 10-beta, culturing, screening and extracting the plasmid to obtain a positive clone pYLTAC380GW-Hisps-CPH containing target genes Hisps and CPH;
6) Repeating the steps 2) -5), taking the new plasmid containing the target genes obtained in the previous step as a receptor carrier, carrying out recombination by using d1 and d2 donor carriers containing different genes in a crossing way until all the target genes are assembled on the receptor carrier, and finally connecting a BP recombination reaction to remove a screening marker gene expression cassette element to construct a polygenic carrier pYLTAC380GW-Hisps-CPH-H3H-NPGA-eDIE-Luz.
7. The method for cultivating self-luminous plants as claimed in claim 5, wherein the coding sequence of the Hisps gene is shown in SEQ ID NO.3, the coding sequence of the CPH gene is shown in SEQ ID NO.4, the coding sequence of the H3H gene is shown in SEQ ID NO.5, the coding sequence of the NPGA gene is shown in SEQ ID NO.6, and the coding sequence of the Luz gene is shown in SEQ ID NO. 7.
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| CN101952435A (en) * | 2008-02-01 | 2011-01-19 | 塞瑞斯公司 | Promoter, promoter control elements, and combinations, and use thereof |
| CN107177616A (en) * | 2017-05-26 | 2017-09-19 | 华南农业大学 | A kind of multiple gene assembly carrier system and its multiple gene assembly method |
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| CN101952435A (en) * | 2008-02-01 | 2011-01-19 | 塞瑞斯公司 | Promoter, promoter control elements, and combinations, and use thereof |
| CN107177616A (en) * | 2017-05-26 | 2017-09-19 | 华南农业大学 | A kind of multiple gene assembly carrier system and its multiple gene assembly method |
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