CN117343931A - Plant leaf specificity high-expression promoter, cloning and application thereof - Google Patents
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- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
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Abstract
The invention discloses a leaf tissue specific high-expression Rubisco small subunit gene promoter and application thereof, belonging to the field of plant genetic engineering. The invention takes Populus xiaohei T.S. Hwang et Liang as plant material, successfully clones a leaf tissue specific high expression promoter rbcS for the first time, the sequence of which is shown as SEQ ID NO.1, successfully constructs a Rubisco small subunit gene promoter activity detection vector, and proves that the promoter is specifically and highly expressed in plant leaves through transient transformation tobacco and GUS staining verification. The promoter provides a new tool and a new choice for plant leaf development research and new variety transgenic breeding.
Description
Technical Field
The invention belongs to the technical field of biology, in particular to the technical field of plant transgenosis, and particularly relates to a promoter proPdrbcs-1 of a small subunit gene of populus xiaosii Rubisco, and further discloses a cloning method and application thereof.
Background
The black poplar is hybridized with black poplar (Populus x xiaohei T.S. Hwang et Liang) and black poplar (Populus nigra), and has better growth vigor in arid and cold areas than small She Yanghe small poplar (Populus pseudosimonii Kitag) in terms of growth speed, cold resistance, drought resistance, disease and insect resistance and other excellent characteristics, and can be used as a building material in 10 years, especially in the western arid and cold areas of Heilongjiang province, and can be a good fast-growing greening tree species in northern China.
The promoter is a DNA sequence which is positioned at the upstream of the 5' end of the structural gene and can guide RNA polymerase to be correctly combined with a template and start gene transcription, and the promoter comprises cis-acting elements which can be recognized and combined by transcription factors and plays a key role in the expression control of the gene. Promoters of plants can be divided into three classes: constitutive promoters, tissue-specific promoters, inducible promoters. In genetic engineering breeding of plants, constitutive promoters such as cauliflower virus CaMV35S can drive exogenous genes to express in all plant tissues and organs, but spatiotemporal properties of gene expression cannot be effectively controlled, so that certain defects exist in practical operation. However, tissue-specific high-expression promoters are only efficient at promoting gene expression in a particular growth stage or tissue site. The use of a tissue-specific high-expression promoter to control the expression of a target gene in genetic transformation of plants can more effectively avoid potential negative effects caused by the use of constitutive promoters. Therefore, research and application of tissue-specific high-expression promoters are increasingly emphasized. The leaf is a main place for photosynthesis of plants, and determines biomass of the plants, so that the promoter with specific high expression of the plant leaf is cloned and identified, and the leaf is applied to genetic operation for improving photosynthesis of the plants, so that photosynthesis of the plants can be effectively improved, biomass of the plants is increased, and the leaf has very important and profound significance for improving carbon fixation efficiency of woody plants.
Disclosure of Invention
Aiming at the current research situation, the invention aims to provide a cloning method and application of a DNA molecule of a small subunit gene promoter proPdrbcs-1 of populus xiaonivea.
One of the technical schemes adopted for solving the technical problems is as follows: provides a promoter proPdrbcs-1 of a small subunit gene of populus xiaonivea, and the nucleotide sequence of the promoter proPdrbcs-1 is shown as SEQ ID NO. 1.
The nucleotide sequence of the populus nigra Rubisco small subunit gene promoter proPdrbcs-1 comprises a DNA sequence of about 2000bp before the upstream of the Rubisco small subunit gene.
The second technical scheme adopted by the invention for solving the technical problems is as follows: there is provided a method for cloning the promoter proPdrbcs-1 of the populus nigra Rubisco small subunit gene, comprising the steps of:
(1) The following specific primers are designed by taking the aseptic seedling DNA of populus xiaoniveus as a template:
proPdrbcs-1-F：GTGGGGATATAAGTTCTATCCCT
proPdrbcs-1-R：GTGCGGTTAACTGTGGCAAC
(2) PCR cloning was performed in a 50. Mu.L system using KOD enzyme, and the reaction procedure for PCR amplification was: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 52℃for 30s, extension at 72℃for 1min15s,35 cycles, final extension at 72℃for 7min, and storage at 16 ℃.
(3) Cloning the amplified product onto a Zero TOPO-Blunt vector, transforming escherichia coli DH5 alpha, and selecting and recombining monoclonal sequencing to obtain a promoter proPdrbcs-1 of the Rubiosco small subunit gene with the length of 2240bp respectively.
The third technical scheme adopted by the invention for solving the technical problems is as follows: provides a promoter proPdrbcs-1 of a small subunit gene of populus nigra and application thereof in the technical field of plant molecular breeding.
The invention has the following beneficial effects: cloning and obtaining a promoter proPdrbcs-1 of a small subunit gene of the small black poplar in the small black poplar for the first time, carrying out fusion expression on the promoter proPdrbcs-1 of the small subunit gene of the small black poplar and a GUS gene to transiently transform tobacco embryo seedlings, verifying that the promoter proPdrbcs-1 of the small subunit gene of the small black poplar can be efficiently expressed on the tobacco leaves, and providing an efficient promoter sequence for plant leaf specific part expression research.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a cloning electrophoresis diagram of a small subunit gene promoter proPdrbcs-1 of populus nigra, and a 2240bp fragment of the small subunit gene promoter proPdrbcs-1 of populus nigra is obtained through PCR amplification;
in FIG. 2, the right panel shows GUS staining of the soil-cultured seedlings of Nicotiana benthamiana, and the left panel Control shows a Control group transiently infected with Agrobacterium GV 3101.
Detailed Description
For a better understanding of the present invention, reference will now be made in detail to the following examples and accompanying drawings, which are included to provide a further understanding of the invention, but it should be understood by those skilled in the art that the following examples are not intended to limit the scope of the invention and that any changes and modifications that would be made to the present invention are within the scope of the invention.
In the following examples, the experimental methods used are conventional methods unless otherwise specified.
The promoter activity detection vector pBI121-GUS in the examples below was a laboratory vector, and the biological material was used only for repeated experiments related to the present invention, and was not used for other purposes.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1:
the populus deltoides Rubisco small subunit gene promoter proPdrbcs-1 is obtained by the following specific operations:
the genomic DNA of the small black Yang Mojun seedling is used as a template, the upstream and downstream specific primer pair sequences of the promoter proPdrbcs-1 are designed about 2000bp before the small subunit gene of the small black poplar Rubisco, the sequences of the specific primer pair are shown as proPdrbcs-1-F and proPdrbcs-1-R in the table 1, and the PCR reaction system is shown in the table 2.
TABLE 1 nucleotide primer sequences
TABLE 2 PCR reaction System
PCR cloning was performed in a 50. Mu.L system using KOD enzyme, and the reaction procedure for PCR amplification was: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 52℃for 30s, extension at 72℃for 1min15s,35 cycles, final extension at 72℃for 7min, and storage at 16 ℃.
DNA fragments (amplified product fragments) are recovered through agarose gel electrophoresis, the gel recovery kit is purchased from Beijing qing biological Co., ltd, amplified products are cloned on a Zero TOPO-Blunt carrier, escherichia coli DH5 alpha is transformed, recombinant monoclonal sequencing is selected, and finally the populus nigra Rubisco small subunit gene promoter proPdrbcs-1 with the length of 2240bp shown in figure 1 is obtained. The DNA fragment obtained by recovery is sequenced, and the sequence is correct. The resulting DNA fragment was named rbcs-1 promoter (rbcs-1 pro).
The sequence of the rbcs-1 promoter is as follows:
EXAMPLE 2 construction of recombinant expression vectors
Construction of a recombinant expression vector of a populus deltoides small subunit gene promoter proPdrbcs-1, which comprises the following specific operations:
(1) Cleavage of pBI121 (containing GUS) vector and amplification product
The recovered amplified product obtained in example 1 and plasmid PBI121 (containing GUS) were digested simultaneously with restriction enzymes ClaI and BamH I, and the digestion system is shown in Table 3.
TABLE 3 enzyme digestion system
(2) The amplified product after cleavage and the vector backbone (about 40 kb) were ligated with T4 ligase to give recombinant plasmid pBI121Pdrbcs-pro-GUS. The connection system is shown in Table 4.
Table 4 connection system
Connection reaction conditions: overnight at 16 ℃.
(3) mu.L ligation product was used to transform 100. Mu.L DH 5. Alpha. Competent cells: mixing the product with competent cells, ice-bathing for 25min, heat-shocking at 42 deg.C for 45s, immediately placing on ice for 2min, adding 250 μL LB culture medium preheated to room temperature, shake-culturing at 37 deg.C for 1h, centrifuging at 12000rpm for 1min, discarding 200 μL culture supernatant, mixing the rest 150 μL with a pipettor, uniformly coating on LB plate containing 50 μg/mL kana resistance, inverting, and culturing overnight in a 37 deg.C constant temperature incubator.
(4) The positive single colony extracted plasmid was selected and sent to the engineering company, inc. for sequencing.
Sequencing results showed that Pdrbcs-1pro promoter sequence in pBI121-Pdrbcs-1pro-GUS cloning vector was obtained.
Agrobacterium transformation verification of the small black Yang Jiyin promoter proPdrbcs-1:
the constructed promoter activity detection vector pBI-121-pro: pdrbcs-1 was transformed into the Agrobacterium GV3101 strain to obtain recombinant Agrobacterium (GV 3101/pBI121-Pdrbcs-1 pro-GUS). Taking the leaf of Nicotiana benthamiana cultivated in soil for 4 weeks as a transient transformation explant material, carrying out transient infection of agrobacterium, carrying out dark cultivation at 22 ℃ for 2 days, and taking out for GUS staining observation.
Adding GUS dye solution into the soil-cultured seedling of Nicotiana benthamiana for detection, vacuum-filtering for 45min, placing in a 37 ℃ incubator for 8 hours, transferring the material into a bottle containing carnot fixing solution, and replacing the fixing solution until the color of the material becomes white. After the decoloring is completed, the material is transferred into a transparent agent, and transparent treatment is carried out until the material is transparent, and GUS dyeing condition is observed. The soil-cultivated seedlings of Nicotiana benthamiana infected with empty GV3101 were used as a control (FIG. 2, left panel), and the staining conditions were as shown in FIG. 2 (right panel).
Therefore, the promoter proPdrbcs-1 of the Rubisco small subunit gene of the populus xiaonivea is obtained in the populus xiaonivea, and is verified to have the expression activity. Therefore, the invention provides a high-efficiency promoter sequence for transformation research of populus deltoidea and related plants.
While the foregoing describes specific embodiments of the present invention, it will be appreciated by those skilled in the art that the specific embodiments described are illustrative only and not limiting of the scope of the invention, as modifications and variations may be made by those skilled in the art without departing from the principles of the invention, and such modifications and variations are to be regarded as being within the scope of the invention as defined in the claims.
Claims (4)
1. A populus xiaoensis Rubisco small subunit gene promoter PdrbcS-1, characterized by the DNA nucleotide sequence of promoter pro PdrbcS-1:
(1) A nucleotide sequence shown in SEQ ID NO. 1;
(2) A nucleotide sequence capable of hybridizing to the nucleotide sequence of (1) under stringent conditions;
(3) A nucleotide sequence having at least 90% identity to the nucleotide sequence set forth in (1) or (2).
2. A method for cloning a gene promoter, characterized in that the method for cloning the small subunit gene promoter PdrbcS-1 of populus nigra Rubisco according to claim 1 comprises the following steps:
(1) Using the populus xiaohei DNA as a template, and designing a specific primer:
PdrbcS-1-F：GTGGGGATATAAGTTCTATCCCT
PdrbcS-1-R：GTGCGGTTAACTGTGGCAAC
(2) PCR amplification was performed in a 50. Mu.L system using KOD enzyme;
(3) Cloning the amplified product onto a zeroTOPO-Blunt vector, transforming escherichia coli DH5 alpha, and selecting and sequencing recombinant monoclonal to obtain the gene promoter PdrbcS-1 of the small subunit of the Rubiosco of the populus nigra with the length of 2240 bp.
3. The method of cloning the populus nigra Rubisco small subunit gene promoter PdrbcS-1 according to claim 2, wherein in step (2), the PCR amplification reaction procedure is as follows: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 52℃for 30s, extension at 68℃for 1min15s,35 cycles, extension at 68℃for 7min, and storage at 16 ℃.
4. An application of a small black Yang Qi promoter is characterized in that the promoter is a pro rbcS-1 gene of a small black poplar Rubisco as claimed in claim 1 and is applied to the technical field of molecular breeding of poplars.
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