CN112375772A - Construction method and application of plant expression vector suitable for sugarcane - Google Patents
Construction method and application of plant expression vector suitable for sugarcane Download PDFInfo
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Abstract
The invention relates to the field of molecular biology and biotechnology, and particularly discloses a construction method and application of a plant expression vector suitable for sugarcane. A method for constructing a plant expression vector suitable for sugarcane comprises the following steps: (1) amplifying the ubi promoter target sequence; (2) carrying out enzyme digestion on the target fragment and the vector respectively to obtain an EcoRI-BamHI enzyme digested target fragment and an EcoRI-BamHI enzyme digested vector; (3) marking the transformed vector, picking a single bacterial colony, carrying out PCR identification on the bacterial liquid by using a primer after shaking the bacterial, and screening out a positive bacterial liquid; sequencing the positive bacteria liquid, and carrying out plasmid enzyme digestion verification to obtain the plant expression vector pHU. The plant expression vector is suitable for genetic transformation of sugarcane crops, can stably and efficiently introduce genes into sugarcane, can carry out genetic improvement on sugarcane varieties by a molecular means, and can effectively shorten the breeding period of the sugarcane fine varieties.
Description
Technical Field
The invention belongs to the field of molecular biology and biotechnology (plant genetic engineering technology), and particularly relates to a construction method and application of a plant expression vector suitable for sugarcane.
Background
Sugarcane is a main sugar crop in the world, cane sugar accounts for more than 70% of the total sugar consumption in the world, and the sugarcane is also one of the most important biomass energy crops and is a C4 plant with the largest biomass per unit area and fuel ethanol yield in field crops. Sugarcane belongs to a medium-day plant, is difficult to bloom, and is relatively difficult to cross-breed; sugarcane is also polyploid, the genetic background is complex, the breeding period is long, conventional sexual cross breeding is adopted, and 1 variety meeting the identification requirement can be cultivated from 10-30 ten thousand segregating populations after 10-13 years from the seedling. Because the sugarcane can be propagated asexually in production, compared with other transgenic sugarcane, the sugarcane has higher biological safety, belongs to transgenic safety I type plants, and has good commercial planting prospect. Therefore, the method for improving the characteristics of the sugarcane through genetic transformation is an important means for cultivating excellent varieties and has unique advantages. The construction of an efficient expression vector suitable for sugarcane is the basis of genetic transformation, the genetic transformation of the sugarcane can be stably and efficiently carried out through the vector, and the variety of the sugarcane is improved by utilizing a molecular means.
Disclosure of Invention
The invention aims to provide a construction method and application of a plant expression vector suitable for sugarcane, which are suitable for sugarcane genetic transformation.
In order to achieve the above object, the present invention provides a method for constructing a plant expression vector suitable for sugarcane, comprising the steps of:
(1) amplification of ubi promoter sequence of interest: and (3) taking EcoRI-BamHI as an insertion site, synthesizing a primer, and carrying out PCR amplification to obtain a target fragment, wherein the primer sequence is as follows:
M1300R:5’-GCACCCCAGGCTTTACAC-3’
PL515R:5’-CGCGGATCCTCCCCGCGGTAGACTAGTccgggtaccgagctcgta-3’
(2) taking pCAMBIA1300-MCS-E9 ter as a vector framework, and performing enzyme digestion on the target fragment and the vector respectively to obtain an EcoRI-BamHI enzyme-digested target fragment and an EcoRI-BamHI enzyme-digested vector; carrying out in-vitro connection on the EcoRI-BamHI enzyme-cut target fragment and an EcoRI-BamHI enzyme-cut vector, and transforming the connection product into a DH5 alpha competent cell to obtain a transformed vector;
(3) marking the transformed vector, picking a single bacterial colony, carrying out PCR identification on the bacterial liquid by using a primer after shaking the bacterial, and screening out a positive bacterial liquid; sequencing the positive bacteria liquid, and carrying out plasmid enzyme digestion verification to obtain the plant expression vector pHU.
The use of a plant expression vector pHU in the genetic transformation of sugarcane.
Preferably, in the above application, the plant expression vector pHU plasmid DNA is extracted, and sugarcane is genetically transformed by a particle gun method.
Preferably, in the application, hygromycin is used for screening out positive plants in the process of genetic transformation of sugarcane by a gene gun method.
Compared with the prior art, the invention has the following beneficial effects:
the plant expression vector obtained by the invention has a plurality of enzyme cutting sites and rare sites, and can be connected with almost all target genes to be introduced to carry out sugarcane genetic transformation. The plant expression vector is suitable for genetic transformation of sugarcane crops, can stably and efficiently introduce genes into sugarcane, can carry out genetic improvement on sugarcane varieties by a molecular means, and can effectively shorten the breeding period of the sugarcane fine varieties. According to the invention, hygromycin is selected as a screening marker, so that the transgenic sugarcane can be efficiently screened out.
Drawings
FIG. 1 is a diagram of the electrophoresis of PCR amplification agarose detection in example 1 of the present invention, wherein M is Marker, which is 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom, and 1 and 2 are target fragments.
FIG. 2 is a PCR detection diagram of the transformed vector bacterial liquid in example 1, wherein M is Marker, which is 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom, 1-24 are test bands of different single colonies, and the 14 th is screened positive clone.
FIG. 3 is a restriction enzyme digestion verification diagram of the plant expression vector pHU in example 1 of the present invention, wherein M is Marker, 1 is before restriction enzyme digestion, and 2 is after restriction enzyme digestion.
FIG. 4 is a photograph of resistant callus selection.
FIG. 5 is a photograph of the differentiation of resistant callus.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments.
Example 1
A construction method of a plant expression vector suitable for sugarcane specifically comprises the following steps:
(1) amplification of ubi promoter sequences of interest
EcoRI-BamHI is used as an insertion site and a primer is synthesized, and the sequence of the primer is as follows:
M1300R:5’-GCACCCCAGGCTTTACAC-3’
PL515R:5’-CGCGGATCCTCCCCGCGGTAGACTAGTccgggtaccgagctcgta-3’
the PCR amplification reaction is carried out by using high fidelity enzyme, the amplification system is prepared in the following table 1, and the PCR reaction procedure is as follows: pre-denaturation at 98 ℃ for 5 min; the cycle is denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 30s, and extension at 68 ℃ for 1min for 10s, and the cycle is 30 cycles; extension at 68 ℃ for 5 min. The PCR products were then separated by l.8% agarose gel electrophoresis, with the specific procedure: adjusting voltage to 150V, performing electrophoresis for 15min, photographing, recording electrophoresis result as shown in figure 1, observing under ultraviolet lamp, rapidly cutting off target strip, and recovering target segment with gel recovery kit (OMEGA), according to kit instruction.
TABLE 1 amplification System preparation Table
The 5'primer is primer M1300R, and the 3' primer is primer PL 515R.
(2) And (2) taking pCAMBIA1300-MCS-E9 ter as a vector framework, and performing enzyme digestion treatment on the target fragment obtained in the step (1) and the vector respectively to obtain an EcoRI-BamHI enzyme-digested target fragment and an EcoRI-BamHI enzyme-digested vector, wherein an enzyme digestion reaction system is shown in a table 2.
Enzyme digestion system of target fragment and vector in Table 2
The target EcoRI-BamHI enzyme-cut fragment and the EcoRI-BamHI enzyme-cut vector are subjected to in vitro ligation by adopting T4-DNA ligase to obtain a ligation product, wherein a ligation system is shown in a table 3, and the specific operation is as follows: sucking and beating the reaction liquid of the connection system, mixing uniformly, slightly centrifuging, adding a drop of mineral oil, placing at 16 ℃ for 2 hours, and then placing in a refrigerator at 4 ℃ for storage.
TABLE 3 connection System
Transforming the ligation product into DH5 alpha competent cells to obtain a transformed vector, which comprises the following specific operations:
taking out 100 mu L of DH5 alpha competent cells, placing on ice, and precooling for 10 min;
placing an EP tube on ice, adding 80 mu L of precooled DH5 alpha competent cells, then adding 10 mu L of connecting products, sucking and uniformly mixing by using a liquid transfer gun, and carrying out ice bath for 30 min;
thirdly, after the ice bath is finished, putting the ice-bath into a constant-temperature water bath kettle at 42 ℃ for 90s by heat shock, and then quickly putting the ice-bath into ice blocks for 2 min;
adding 500 mu L of LB liquid culture solution without Kan into an EP tube, mixing uniformly, and placing in a shaking table for culturing for 1h at the temperature of 37 ℃ and at the speed of 160 r/m;
fifthly, taking out the EP tube, centrifuging for 5min at 2000-3000 r/m, discarding 300 mu L of supernatant, gently sucking and uniformly mixing the residual bacterial liquid, adding the mixture into an LB solid culture dish containing Kan, uniformly coating the mixture by using a glass coating rod, and drying the mixture;
sixthly, culturing for 16-20 hours in a constant temperature incubator at 37 ℃.
(3) Marking the transformed vector, picking single colony, shaking the bacteria, and performing PCR identification on the bacteria liquid by using a primer, wherein the PCR detection system of the single colony is shown in table 4, the PCR identification result of the bacteria liquid is shown in figure 2, and screening positive bacteria liquid (positive clone) of which the 14 th is a positive clone; sequencing the positive bacterial liquid, and carrying out plasmid enzyme digestion verification, wherein an enzyme digestion verification diagram is shown in figure 3, so as to obtain the plant expression vector pHU.
TABLE 4 Single colony detection PCR System
Example 2
The application of the plant expression vector pHU in sugarcane genetic transformation,
1. plasmid DNA extraction: the constructed pHU vector plasmid DNA was extracted.
2. Transforming sugarcane by gene gun method:
(1) preparing callus tissues: sugarcane embryogenic callus is selected and pretreated on MS culture medium added with 0.2 mol.L-1 mannitol and 0.2 mol.L-1 sorbitol.
(2) Wrapping of DNA: 50 μ L of gold powder suspension was placed in an Eppendorf tube, and the extracted plasmid DNA and 50 μ L of 2.5MCaCl were added in order2And 20. mu.L of 0.1M methyleneSpermine.
(3) Vortex the mixed sample for 1min, stand on ice for 1min, and centrifuge to remove supernatant.
(4) Add 75% ethanol, centrifugate and remove the supernatant, repeat this step 3 times, resuspend the pellet with absolute ethanol finally.
(3) Transformation of callus: bombarding the pretreated callus according to the gene gun transformation step, wherein the bombardment pressure is 1100psi, the bombardment distance is 6cm, and the bombardment time is 1.
(4) Screening and culturing: after the bombarded callus is subjected to recovery culture, the callus is transferred to an MS subculture medium containing hygromycin, and the transformation efficiency is counted by screening as shown in figure 4. After 3 weeks of culture, differentiation was carried out by transferring the cells into a hygromycin-containing MS differentiation medium, and the results are shown in FIG. 5.
(5) And (3) counting the conversion efficiency: and carrying out PCR detection on the screened positive plants, and counting the transformation efficiency. Through detection, the constructed vector is used for transforming sugarcane, and the transformation efficiency reaches 2.9%.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (4)
1. A method for constructing a plant expression vector suitable for sugarcane is characterized by comprising the following steps:
(1) amplification of ubi promoter sequence of interest: and (3) taking EcoRI-BamHI as an insertion site, synthesizing a primer, and carrying out PCR amplification to obtain a target fragment, wherein the sequence of the primer is as follows:
M1300R:5’- GCACCCCAGGCTTTACAC-3’
PL515R:5’-CGCGGATCCTCCCCGCGGTAGACTAGTCCGGGTACCGAGCTCGTA-3’
(2) taking pCAMBIA1300-MCS-E9 ter as a vector skeleton, and performing enzyme digestion on the target fragment and the vector skeleton respectively to obtain an EcoRI-BamHI enzyme digested target fragment and an EcoRI-BamHI enzyme digested vector; carrying out in-vitro connection on the EcoRI-BamHI enzyme-cut target fragment and an EcoRI-BamHI enzyme-cut vector to obtain a ligation product, and transforming the ligation product into a DH5 alpha competent cell to obtain a transformed vector;
(3) marking the transformed vector, picking a single bacterial colony, carrying out PCR identification on the bacterial liquid by using a primer after shaking the bacterial, and screening out a positive bacterial liquid; sequencing the positive bacteria liquid, and carrying out plasmid enzyme digestion verification to obtain the plant expression vector pHU.
2. The use of a plant expression vector pHU in the genetic transformation of sugarcane.
3. Use according to claim 3, characterized in that the plant expression vector pHU plasmid DNA is extracted and sugarcane is genetically transformed by the biolistic method.
4. The use according to claim 4, wherein hygromycin is used to screen positive plants during the biolistic genetic transformation of sugarcane.
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| CN116200523A (en) * | 2022-12-21 | 2023-06-02 | 南通大学 | Identification method and application of plant tissue specific promoter |
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| 郭志鸿等: "ubi 启动子驱动的花生芪合酶基因表达载体的构建及玉米遗传转化初报", 《甘肃农业大学学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114164230A (en) * | 2022-01-11 | 2022-03-11 | 广西壮族自治区农业科学院 | Expression vector suitable for sugarcane genetic transformation and construction method and application thereof |
| CN116200523A (en) * | 2022-12-21 | 2023-06-02 | 南通大学 | Identification method and application of plant tissue specific promoter |
| CN116200523B (en) * | 2022-12-21 | 2024-02-06 | 南通大学 | Identification method and application of plant tissue specific promoter |
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