CN113774067A - Cymbidium kanran gene expression vector for promoting seed germination, construction method and application - Google Patents
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Abstract
The invention discloses a gene expression vector for promoting germination of cymbidium kanran seeds, a construction method and application thereof, wherein the gene expression vector is composed of cymbidium kanran seed germination gene CkARF and a plant expression vector, and the plant expression vector is obtained by inserting CkARF into linearized pCAMBIA 1301-220 plasmid after double enzyme digestion of a T-vector containing CkARF fragment and an expression vector pCAMBIA 1301-220 plasmid through BamHI and XbaI. The plant expression vector pCAMBIA 1301-220-CkARF containing the cymbidium kanran seed germination gene CkARF constructed in the invention can directly introduce the constructed gene expression vector into flowers through agrobacterium-mediated genetic transformation, so that the CkARF gene is excessively expressed under the starting of a CaMV35S promoter, a large amount of ARF protein is synthesized, the expression of downstream genes is regulated, the cymbidium kanran seed is germinated in advance, and the germination quality is improved.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a cymbidium kanran gene expression vector for promoting seed germination, a construction method and application.
Background
ARF (ARF) is a class of transcription factor that regulates the expression of auxin-responsive genes found in Arabidopsis in 1997. At present, the ARF transcription factor of the model plant Arabidopsis is deeply researched and is considered to be widely involved in auxin signal transduction. In the early stages of embryogenesis, this gene not only facilitates auxin transport to adjacent suspensor cells, but also includes quiescent center precursors of the root tip meristem. After mutation of the gene, the embryo cannot form hypocotyls and radicles.
Cymbidium kanran makino belongs to the orchid family (Orchidaceae), is an important member of the Cymbidium, has the advantages of diverse lip shape variations, four seasons flowering, long flowering period and the like, and is known as the king of orchid. As with other orchids, there is a significant barrier to seed germination. Orchid seeds germinate in two ways, symbiotic germination and non-symbiotic germination (aseptic seeding). The non-symbiotic germination refers to germination of orchid seeds without any fungal infection on an artificial culture medium, a large number of young plants can be obtained in a short period, the method is one of the economic and effective rapid propagation methods at the present stage, and great commercial values are obtained in orchids with high economic values, such as butterfly orchids, dendrobium candidum, Chinese orchids, cymbidium and the like. Therefore, non-symbiotic germination of orchid seeds is of great importance in the industrial production of orchids. By cloning key genes for controlling the germination of cymbidium kanran seeds, an expression vector is constructed and is over-expressed in cymbidium kanran, and a variety easy to germinate is possibly cultured.
Reference to the literature
Coen ES,Meyerowitz EM.The war of the whorls:genetic interactions controlling flower development.Nature 1991；353:31-37.
Fan J,Li W,Dong X,Guo W,Shu H.Ectopic expression of a hyacinth AGL6 homolog caused earlier lowering and ho meotic conversion in Arabidopsis.Sci China C Life Sci 2007；50:676-689.
Hsu HF,Huang CH,Chou LT,Yang CH.Ectopic expression of an orchid(Oncidium Gower Ramsey)AGL6-like gene promotes lowering by activating lowering time genes in Arabidopsis thaliana.Plant Cell Physiol 2003；44:783-794.
Lee S,Jeon JS,An K,et al.Alteration of loral organ identity in rice through ectopic expression of OsMADS16.Planta 2003；217:904-911.
Moon YH,Kang HG,Jung JY,Jeon JS,Sung SK,An G.Determination of the motif responsible for interaction between the rice APETALA1/AGAMOUS-LIKE9 family proteins using a yeast two-hybrid system.Plant Physiol 1999；120:1193-1204.
Ohmori S,Kimizu M,Sugita M,et al.MOSAIC FLORAL ORGANS1,an AGL6-Like MADS box gene,regulates loral organ identity and meristem fate in rice.Plant Cell 2009；21:3008-3025.
Disclosure of Invention
The invention aims to provide a cymbidium kanran gene expression vector for promoting seed germination, a construction method and application thereof, so as to overcome the problem of germination obstacle of cymbidium kanran seeds.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a gene for promoting cymbidium kanran seed germination, the gene for promoting cymbidium kanran seed germination is CkARF, and the gene sequence is shown as SEQ ID NO. 1.
According to the cymbidium kanran seed germination gene CkARF, the invention provides a gene expression vector for promoting cymbidium kanran seed germination, which is composed of the cymbidium kanran seed germination gene CkARF and a plant expression vector.
The plant expression vector of the invention is: the T-vector containing the CkARF fragment and the expression vector pCAMBIA 1301-220 plasmid are subjected to double enzyme digestion by BamHI and XbaI, and the CkARF is inserted into the linearized pCAMBIA 1301-220 plasmid to obtain the CkARF fragment.
The invention also provides a construction method of the gene expression vector for promoting the germination of the cymbidium kanran seeds, and the construction process of the gene expression vector comprises the following steps:
1) cloning of cymbidium kanran seed germination gene CkARF
Taking cymbidium kanran seeds as a material, extracting total RNA, performing reverse transcription to obtain cDNA, designing a primer, and amplifying a CkARF gene by using high-fidelity enzyme; taking the reverse transcription cDNA as a template, carrying out polymerase chain reaction, connecting the product to a pMD19-T Simple vector, transforming TOP10 competent cells, and carrying out sequence determination;
2) construction of plant expression vector pCAMBIA 1301-220-CkARF
Extracting pMD19-T Simple-CkARF recombinant plasmid from TOP10 strain, recovering CkARF fragment after double enzyme digestion by BamHI and XbaI, inserting the CkARF fragment into linearized pCAMBIA 1301-and 220-CkARF to obtain recombinant plasmid pCAMBIA 1301-and 220-CkARF, transforming TOP10 competent cells, extracting positive plasmid, carrying out enzyme digestion electrophoresis detection and sequencing verification.
The invention relates to a design primer and a method for amplifying a CkARF gene by using high-fidelity enzyme, wherein the design method of the primer comprises the following steps: primers were designed by analysis using Primer Premier 5 software, and the designed Primer sequences were:
upstream primer CkARF-F: 5'-ggatccATGAGGTTTAGGATGAAGTTTGA-3'
The downstream primer CkARF-R is 5'-tctagaTCATCTGCTCTCCTGAATCTTAG-3'.
The invention also provides an application of the gene expression vector for promoting cymbidium kanran seeds to germinate: the gene expression vector can be directly used for agrobacterium-mediated genetic transformation, and the constructed gene expression vector is introduced into flowers so as to improve the germination characteristics of plant seeds.
Has the advantages that:
1. the cymbidium kanran CkARF provided by the invention is a novel cymbidium kanran auxin transport gene which can regulate and control non-symbiotic germination of plant seeds;
2. the constructed cymbidium kanran CkARF gene plant expression vector is reported for the first time, and can be directly used for agrobacterium-mediated genetic transformation to improve the germination characteristics of plant seeds.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a schematic diagram of the ligation process of a gene expression vector for promoting germination of cymbidium kanran seeds according to the present invention;
FIG. 2 is a high fidelity electrophoretic map of the enzyme amplified CkARF product;
FIG. 3 is an electrophoretogram of BamH I and Xba I double cleavage products of pMD19-T Simple-CkARF plasmid;
FIG. 4 is the electrophoresis picture of the double restriction enzyme products of BamH I and Xba I of pCAMBIA 1301-220-CkARF expression vector;
FIG. 5 is a map of plant expression vector pCAMBIA 1301-220-CkARF;
FIG. 6 is a schematic diagram comparing germination rates of wild type and transgenic Arabidopsis thaliana;
FIG. 7 is a schematic diagram comparing germination quality of wild type and transgenic Arabidopsis thaliana.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without any inventive step, are within the scope of the present invention.
The solution of some embodiments of the invention is described below with reference to fig. 1-7.
Example 1
The embodiment provides a gene for promoting cymbidium kanran seed germination, wherein the gene for promoting cymbidium kanran seed germination is CkARF, and the gene sequence of the gene is shown as SEQ ID No. 1.
Example 2
According to the cymbidium kanran seed germination gene CkARF in example 1, a gene expression vector for promoting cymbidium kanran seed germination is provided in this example, and the gene expression vector is composed of the cymbidium kanran seed germination gene CkARF and a plant expression vector.
As shown in fig. 5, the plant expression vector of the present invention is: the T-vector containing the CkARF fragment and the expression vector pCAMBIA 1301-220 plasmid are subjected to double enzyme digestion by BamHI and XbaI, and the CkARF is inserted into the linearized pCAMBIA 1301-220 plasmid to obtain the CkARF fragment.
Example 3
The embodiment provides a method for constructing a gene expression vector for promoting germination of cymbidium kanran seeds, wherein the construction process of the gene expression vector comprises the following steps:
1) cloning of cymbidium kanran seed germination gene CkARF
Selecting cymbidium kanran cultivar 'Qinghuamei' as a material, extracting total RNA of seeds, taking 1 mu g of total RNA according to an M-MLV reverse transcription kit (TaKaRa) to perform reverse transcription to form cDNA, digesting a cDNA product by using RNase, and designing Primer amplification CkARF by analyzing Primer Premier 5 software according to cymbidium kanran CkARF sequence information (NCBI accession number: AB 495345);
an upstream primer CkARF-F: 5'-ggatccATGAGGTTTAGGATGAAGTTTGA-3';
a downstream primer CkARF-R: 5'-tctagaTCATCTGCTCTCCTGAATCTTAG-3';
performing PCR reaction by using bud cDNA as a template:
50 μ L reaction: 10 XRCR Buffer 5.0. mu.L, CkARF-F, CkARF-R primers 1.0. mu.L each (20. mu. mol. L)-1)，dNTP mix 4.0μL(2.5mmol·L-1)，PrimeSTARTMHS DNA Polymerase 0.2. mu.L, cDNA template 1. mu.L, ddH2O 37.8μL；
Reaction procedure: pre-denaturation at 95 ℃ for 4min, then melting at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 1min for 30sec, reaction for 33 cycles, extension at 72 ℃ for 10 min; gel recovery reagent for PCR productsRecovering and purifying the cassette (AXYGEN) with T4DNA ligase (TaKaRa) is connected to a pMD19-T Simple vector (TaKaRa), TOP10 competent cells are transformed, sequence determination is carried out, and the detection result is shown in figure 2;
2) construction of plant expression vector pCAMBIA 1301-220-CkARF
Extracting the T-vector containing the target fragment and the expression vector pCAMBIA 1301-220 plasmid DNA and carrying out double enzyme digestion, wherein the enzyme digestion system is as follows: 10 XBuffer K2.5. mu.l, Xba I2.0. mu.l, BamH I2.0. mu.l, plasmid DNA 10. mu.l, ddH2O up to 50. mu.l, and the enzyme was cleaved thoroughly overnight at 37 ℃. The fragment containing the complete open reading frame of CkARF and the fragment of pCAMBIA 1301-220 linearized plasmid were recovered separately and the two fragments were ligated as shown in FIG. 1.
The connection reaction system is as follows: 2.0. mu.l of T4 Buffer, 1.0. mu.l of T4 ligase, 1.0. mu.l of vector recovery solution, 4.0. mu.l of target gene recovery solution, dd H2O up to 20. mu.l, overnight at 4 ℃. The well-connected recombinant plasmid pCAMBIA 1301-220-CkARF is transformed into TOP10 cells, positive plasmids are extracted, enzyme digestion electrophoresis detection and sequencing verification are carried out, and the double enzyme digestion electrophoresis detection results of the pMD19-T Simple-CkARF plasmid and the pCAMBIA 1301-220-CkARF expression vector are respectively shown in figure 3 and figure 4.
Example 4
The embodiment provides an application of a gene expression vector for promoting cymbidium kanran seeds to germinate: the gene expression vector can be directly used for agrobacterium-mediated genetic transformation, the constructed gene expression vector is introduced into cymbidium kanran so as to improve the germination characteristics of plant seeds, and the effect is shown by the following comparative experiments:
plant expression vector pCAMBIA 1301-220-CkARF for transforming arabidopsis thaliana and observing flower development characteristics
First, agrobacterium strain EHA105 competence preparation and freeze-thaw method transformation
Selecting EHA105 single colony from YEB (50ug/mL rifampicin) plate, inoculating into 50mL YEB liquid culture medium containing 50ug/mL rifampicin, culturing at 200rpm and 28 deg.C to OD value of 0.5, ice-cooling for 30min, centrifuging to collect thallus, suspending in 2mL precooled 100mM CaCl2In (20% glycerol) solution, 200uL @And (5) subpackaging the mixture by pipes for later use.
Taking 10uL pCAMBIA 1301-220-CkARF vector plasmid, adding 200uL competent cells, carrying out ice bath for 30min, freezing for 5min by liquid nitrogen, carrying out 5min at 37 ℃, adding 800uL YEB liquid culture medium, carrying out pre-culture for 4h at 28 ℃ and 200rpm, coating the bacterial liquid on a YEB (50ug/mL rifampicin +50ug/mL kanamycin) solid culture medium, carrying out dark culture for 2 days at 28 ℃, picking out single clone for detection, and selecting positive clone shake bacteria for arabidopsis thaliana inflorescence transformation.
② Arabidopsis inflorescence dip-dyeing and seed screening
Positive monoclonals were inoculated into 50mL of YEB (50ug/mL rifampicin +50ug/mL kanamycin) liquid medium, cultured for 24 hours, centrifuged at 5000rpm for 20 minutes, and then the pellet was vigorously suspended with a transformation solution (1/2MS, added with 50 g/L sucrose, adjusted to pH 5.8, and then mixed with 200uL/L Silwet L-77) until completely suspended. Directly soaking the overground part of the arabidopsis thaliana in the suspension for 1 minute, then completely wrapping the plant with a preservative film to preserve moisture, putting the plant back to a culture room for culturing for 12 hours, opening the preservative film, and harvesting when the seeds are mature.
Seed disinfection and sowing: the wild type and the transgenic arabidopsis seeds are respectively put into a centrifuge tube with the volume of 1.5mL, added with 1mL of 75 percent alcohol, added with 0.1 percent Triton X-100 and shaken for 15 minutes, then washing twice with 95% alcohol in a super clean bench, directly pouring the seeds and alcohol on sterilized filter paper, blowing dry seeds (standing for 30 minutes), lightly knocking filter paper to uniformly spread the dry seeds on a screening culture medium (1/2MS +20mg/L glufosinate-ammonium +25mg/L ampicillin) for screening culture for 10 days, then the resistant seedlings were transplanted in the soil, the seedlings were covered with a transparent cover for 1 week to preserve moisture, the flowering time of the wild type and transgenic arabidopsis thaliana were recorded, the shape of the germination of the seeds, the germination rate and the germination quality (shape of germination) of the wild type and transgenic arabidopsis thaliana seeds were compared, and the results are shown in fig. 6 and 7.
According to the comparison result, the gene expression vector constructed by the invention can be directly used for agrobacterium-mediated genetic transformation, and the germination rate and the germination quality of plant seeds can be effectively improved by introducing the constructed gene expression vector into flowers.
In conclusion, the plant expression vector pCAMBIA 1301-220-CkARF containing the cymbidium kanran seed germination gene CkARF constructed by the invention can be directly used for agrobacterium-mediated genetic transformation, can effectively solve the problem of flower seed germination obstacle and improve the germination characteristics of the seeds.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (6)
1. A gene for promoting cymbidium kanran seeds to germinate is characterized in that the gene for promoting cymbidium kanran seeds to germinate is CkARF, and the gene sequence is shown as SEQ ID NO. 1.
2. A gene expression vector for promoting the germination of cymbidium kanran seeds, which consists of the cymbidium kanran seed germination gene CkARF in claim 1 and a plant expression vector.
3. The gene expression vector for promoting germination of cymbidium kanran seeds of claim 2, wherein the plant expression vector is: the T-vector containing the CkARF fragment and the expression vector pCAMBIA 1301-220 plasmid are subjected to double enzyme digestion by BamHI and XbaI, and the CkARF is inserted into the linearized pCAMBIA 1301-220 plasmid to obtain the CkARF fragment.
4. A method for constructing a gene expression vector for promoting germination of cymbidium kanran seeds is characterized by comprising the following steps:
1) cloning of cymbidium kanran seed germination gene CkARF
Taking cymbidium kanran seeds as a material, extracting total RNA, performing reverse transcription to obtain cDNA, designing a primer, and amplifying a CkARF gene by using high-fidelity enzyme; taking the reverse transcription cDNA as a template, carrying out polymerase chain reaction, connecting the product to a pMD19-T Simple vector, transforming TOP10 competent cells, and carrying out sequence determination;
2) construction of plant expression vector pCAMBIA 1301-220-CkARF
Extracting pMD19-T Simple-CkARF recombinant plasmid from TOP10 strain, recovering CkARF fragment after double enzyme digestion by BamHI and XbaI, inserting the CkARF fragment into linearized pCAMBIA 1301-and 220-CkARF to obtain recombinant plasmid pCAMBIA 1301-and 220-CkARF, transforming TOP10 competent cells, extracting positive plasmid, carrying out enzyme digestion electrophoresis detection and sequencing verification.
5. The method for constructing a gene expression vector for promoting germination of cymbidium kanran seeds according to claim 4, wherein primers are designed and high fidelity enzyme is used for amplifying the CkARF gene, and the design method of the primers is as follows: primers were designed by analysis using Primer Premier 5 software, and the designed Primer sequences were:
upstream primer CkARF-F: 5'-ggatccATGAGGTTTAGGATGAAGTTTGA-3'
The downstream primer CkARF-R is 5'-tctagaTCATCTGCTCTCCTGAATCTTAG-3'.
6. The application of the gene expression vector for promoting the germination of the cymbidium kanran seeds is characterized in that the gene expression vector can be directly used for agrobacterium-mediated genetic transformation, and the constructed gene expression vector is introduced into flowers so as to improve the germination characteristics of plant seeds.
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Application publication date: 20211210 |