CN116970608B - Peanut seed specific gene promoter BM2Q5K and application thereof - Google Patents
Peanut seed specific gene promoter BM2Q5K and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- 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
- C12N15/823—Reproductive tissue-specific promoters
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Abstract
The invention provides a peanut seed specific gene promoter BM2Q5K and application thereof, and relates to the technical field of plant genetic engineering. The promoter BM2Q5K is selected from any one of SEQ ID NO.01, SEQ ID NO.02, a DNA or RNA sequence hybridized with the sequence of SEQ ID NO.01 or SEQ ID NO.02, a sequence with at least 95% sequence identity with the sequence of SEQ ID NO.01 or SEQ ID NO.02, and a DNA or RNA sequence complementary to any one of the sequences of SEQ ID NO.01 or SEQ ID NO. 02. The invention overcomes the defects of the prior art, and utilizes the specific gene promoter BM2Q5K to drive the target gene to express in the tissue cells of the peanut seeds, thereby realizing the purpose of expressing the target gene in the tissue cells of the specific peanut seeds and cultivating a new variety of crops with high value.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a peanut seed specific gene promoter BM2Q5K and application thereof.
Background
A promoter is a DNA sequence that recognizes, binds to, and initiates transcription by RNA polymerase and contains conserved sequences required for specific binding and transcription initiation by RNA polymerase, mostly upstream of the transcription initiation point of structural genes, and is not transcribed by itself. However, some promoters (e.g., tRNA promoters) are located downstream of the transcription initiation point, and these DNA sequences can be transcribed. The nature of a promoter is initially identified by a mutation that increases or decreases the rate of transcription of the gene, and the promoter is typically located upstream of the transcription initiation site.
The crops subjected to genetic engineering breeding can have excellent characters which are difficult to obtain or stabilize in various conventional breeding modes, or various character expressions of the crops are enhanced, so that new crops with excellent disease and insect resistance, lodging resistance, herbicide resistance, high nutritional value, high effective component content or other excellent characters are obtained, and the crops have remarkable cost-reducing and efficiency-enhancing capabilities in agricultural production and application and wide application prospects. However, there are also potential problems that some exogenous components may induce immune responses in human body or destroy original nutritional components, and the avoidance of the genetic editing is required.
The most prominent problem in the production of exogenous gene expression products by using a transgenic plant cell reactor at present is insufficient gene expression quantity, which is the biggest obstacle for producing protein, enzyme and other gene expression products by using plants. On the one hand, the problem can be solved by utilizing the characteristics of high metabolism rate and continuous division of stem cells such as callus and the like, taking the stem cells as a reactor to make up for the defect, on the other hand, the expression level of the exogenous genes needs to be improved in single cells, and the expression level of the target genes needs to be improved from the aspect of molecular biology, for example, the exogenous genes of the target have higher transcription and translation activities by means of strong promoters, specific promoters or leader sequences so as to improve the yield of target products, or the high expression quantity of the exogenous genes under the conditions of seed tissues and specific external environments is controlled, so that the target products can be collected under specific conditions in production practice to replace the whole plant extraction, and the production cost is greatly reduced.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a peanut seed specific gene promoter BM2Q5K and application thereof, and the specific gene promoter BM2Q5K is utilized to drive target genes to express in peanut seed tissue cells, so that the aim of expressing the target genes in specific peanut seed tissue cells is fulfilled, and a new crop variety with high value is cultivated.
In order to achieve the above object, the technical scheme of the present invention is realized by the following technical scheme:
A peanut seed specific gene promoter BM2Q5K, wherein the promoter BM2Q5K can drive the specificity high expression of a subsequent tandem gene or DNA molecular element in peanut seed tissue cells, and the DNA nucleotide sequence of the promoter BM2Q5K is selected from any one of the following sequences:
(1) The DNA nucleotide sequence shown in SEQ ID NO.01, namely:
GTAGTCTAGTTGGTTAGGATACTCGGCTCTCACCCGAGAGACCCGGGTTCGAGTCCCGGCAACGGAATTCAAATTTTGTAAAAATCGTTTACTTTTTGGTTGAAAAAGTGTGAGAGTTGCAACTTGCAAGTGACATGTACGCAAGAAAAGGGTGACGTCCATAGCCACAATC;
(2) SEQ ID NO.02, namely, a single-stranded sequence which is reversely complementary to the DNA nucleotide sequence shown in SEQ ID NO.01 and can form complete base complementary pairing with a primary sequence to form a double-stranded structure, wherein the sequence is as follows:
GATTGTGGCTATGGACGTCACCCTTTTCTTGCGTACATGTCACTTGCAAGTTGCAACTCTCACACTTTTTCAACCAAAAAGTAAACGATTTTTACAAAATTTGAATTCCGTTGCCGGGACTCGAACCCGGGTCTCTCGGGTGAGAGCCGAGTATCCTAACCAACTAGACTAC;
(3) A nucleic acid sequence capable of hybridizing to the sequence of SEQ ID NO.01 or SEQ ID NO. 02;
(4) A sequence having at least 95% sequence identity to the sequence set forth in (1) or (2);
(5) A DNA or RNA sequence complementary to any of the sequences of (1) or (2).
Preferably, the promoter BM2Q5K is obtained by PCR amplification techniques.
Preferably, the PCR amplification process is carried out by configuring the following two primers according to the DNA nucleotide sequence shown as SEQ ID NO. 01:
（1）SEQ ID NO.03：GTAGTCTAGTTGGTTAGGATACTCG；
（2）SEQ ID NO.04：GATTGTGGCTATGGACGTCACCCTT。
Preferably, the amplification is performed using a DNA polymerase-based DNA amplification system.
The promoter BM2Q5K is applied to crop gene editing or transgenic operation to cultivate new varieties of crops.
Preferably, the application mode is to utilize or operate a gene promoter BM2Q5K to drive target genes to express in peanut seed tissue cells, so that the seed cells are used as a reactor to synthesize related proteins in the molecular biology level.
Preferably, the specific application process is that the pCAMBIA2300 plasmid sequence can be used as a vector, DNA double strand of a peanut seed specific gene promoter BM2Q 5K-target gene-NOS terminator is constructed through enzyme digestion and connection reaction, and the plant expression vector is used for transforming target plants to complete genetic transformation.
The invention provides a peanut seed specific gene promoter BM2Q5K and application thereof, which has the advantages compared with the prior art that:
The invention can utilize or operate peanut seed specific gene promoter BM2Q5K to drive target genes to express in peanut seed tissue cells, thereby realizing the aim of expressing target genes in specific peanut seed tissue cells, and can utilize the mode to take peanut seed tissue cells as a reactor for carrying out transgene and gene transformation in molecular biology level breeding of agricultural production so as to efficiently produce heterologous proteins or strengthen expression of self proteins, fatty acid production and other various nutrient substances, and is commonly used for producing products such as medicinal proteins, antigens, vaccines and the like or cultivating varieties with excellent characters such as high nutritional value, high yield and the like.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
PCR amplification procedure on the gene promoter BM2Q 5K:
1. The DNA nucleotide sequence shown in the following SEQ ID NO.01 was selected as a peanut seed specific gene promoter BM2Q5K sequence:
GTAGTCTAGTTGGTTAGGATACTCGGCTCTCACCCGAGAGACCCGGGTTCGAGTCCCGGCAACGGAATTCAAATTTTGTAAAAATCGTTTACTTTTTGGTTGAAAAAGTGTGAGAGTTGCAACTTGCAAGTGACATGTACGCAAGAAAAGGGTGACGTCCATAGCCACAATC;
2. Two primers (25 bp in length) were configured according to the existing SEQ ID NO.01 sequence:
SEQ ID NO.03 sequence: GTAGTCTAGTTGGTTAGGATACTCG A
SEQ ID NO.04 sequence: GATTGTGGCTATGGACGTCACCCTT;
3. an amount of a DNA amplification system based on Phusion DNA polymerase (Phusion DNA Polymerase) was formulated: the preparation method is to mix the following materials, wherein the mixture ratio is taken as an example that the total amount of the system is 50 mu L:
（1）dd H2O 30.5μL
(2) 10 Xbuffer Buffer 5. Mu.L
（3）dNTP（10mmol/L）1μL
（4）SEQ ID NO.03（10μmol/L） 5μL
（5）SEQ ID NO.04（10μmol/L） 5μL
（6）MgCl2（50mmol/L） 0.5μL
(7) Peanut genomic DNA 2. Mu.L
(8) Phusion DNA polymerase 1. Mu.L;
4. the PCR amplification procedure was run:
the amplification system is put into a PCR thermal cycling instrument to carry out the following thermal cycling steps:
（1）98℃，5min
（2）98℃，30sec
（3）55℃，30sec
（4）72℃，3min
(5) Repeating the steps (2) - (4) for 36 times
(6) Preserving heat for 20min at 72 ℃;
5. Electrophoresis was run, and a DNA fragment of 0.18kb in length was recovered
6. And (3) carrying out Blunt end ligation by using pEASY-Blunt, and transforming DH5 alpha escherichia coli competence to complete sequencing.
Example 2:
Peanut seed-specific gene promoter BM2Q5K obtained using example 1 above was used for crop genome integration procedure 1:
Docosahexaenoic acid, DHA, is a polyunsaturated fatty acid essential for human body, and is contained in fish oil in a large amount. The molecular formula is C 22H32O2, and is a straight-chain fatty acid containing 22 carbon atoms and 6 double bonds. The glycerophospholipids of animals contain unequal amounts of DHA and can be produced from alpha-linolenic acid in the metabolic process in vivo, but the production amount is low and the animal glycerophospholipids are mainly supplemented by food. The discovery in the 80 s of the 20 th century shows that the marine microalgae for producing DHA has unique advantages and rapidly becomes one of the hot spots for domestic and foreign research. Crypthecodinium is a high-efficiency DHA-producing strain, and does not produce other polyunsaturated fatty acids basically, so that the DHA purification process is very simple.
In the field of plant genetic engineering, DHA fatty acid desaturase genes derived from Cryptococcus coronensis are commonly used in genetic engineering of Cryptococcus coronensis DHA fatty acid desaturase.
Selecting DHA fatty acid desaturase gene of Cryptodinoflagellate cardamomum as a target gene:
1. Construction of the transgenic vector: constructing a DNA double chain of a peanut seed specific gene promoter BM2Q 5K-crypthecodinium DHA fatty acid desaturase gene-NOS terminator through enzyme digestion and ligation reaction by using a pCAMBIA2300 plasmid sequence as a vector; and transforming the target plant by using the peanut expression vector to complete genetic transformation;
2. Genetic transformation: selecting a peanut variety of the peanut 911 as a target plant, and transforming the target plant by using an expression vector to complete genetic transformation;
3. verifying the DHA fatty acid desaturase synthesis reactor peanut cultivation level of the transgenic plant:
Tissue culture and subsequent induction differentiation are respectively carried out on peanut callus of peanut-cultivated 911 in MB culture medium to obtain peanut of original peanut-cultivated 911 variety and complete plant 20 strains obtained after tissue culture of peanut of transgenic peanut-cultivated 911 (genetic transformation positive variety) variety of the DHA fatty acid desaturase gene of the cryptococcus, which is inserted by utilizing a peanut seed specific gene promoter BM2Q 5K-cryptococcus, through the NOS terminator plasmid sequence, 120d is planted under normal greenhouse condition until the plant is mature and the result is obtained, seeds of the two plants are collected in batches of 90d and 120d, the seed parts are separated independently for RNA and protein extraction, and the condition of enhanced expression of the DHA fatty acid desaturase gene of the cryptococcus is investigated by taking the transcription and expression verification result of the DHA fatty acid desaturase gene of the cryptococcus of the two seed parts as indexes.
The RNA of the extracted sample was sequenced using transcriptome Illumina deep sequencing, and expression verification was performed on each histone sample using qRT-PCR analysis, obtaining the conditions of enhanced expression of the Crypthecodinium DHA fatty acid desaturase gene of each sample as shown in Table 1 below:
TABLE 1
From the above table, the expression of the crypthecodinium DHA fatty acid desaturase gene in peanut seeds can be achieved by transforming the target plant with the expression vector.
4. Verification that the enhanced expression effect of the peanut seed specific gene promoter BM2Q5K on the Crypthecodinium DHA fatty acid desaturase gene when used for gene editing is specific to peanut seed tissue cells only:
The verification method comprises the following steps: the method comprises the steps of carrying out DNA fluorescence quantitative comparison on the cell level of the rest parts of the whole plant of the original seed-cultivating 911 peanut and the transgenic seed-cultivating 911 (genetically transformed positive variety) seed-cultivating 911 seed-cultivating positive variety, namely roots, stems, leaves and shells, namely carrying out expression quantity verification on the cryptococcus DHA fatty acid desaturase gene, carrying out quantitative analysis on the cryptococcus DHA fatty acid desaturase gene in a sample to be tested by an internal reference method, verifying the transcription quantity of the sequence relative to original seed cells, and obtaining the fact that the expression quantity of the promoter relative to the cryptococcus DHA fatty acid desaturase gene in two plants is not obvious different except seeds after comparison.
The method comprises the following steps: in the cells of the residual plants except the seeds of the two peanut varieties of the original seed-cultivating 911 variety and the peanut variety of the transgenic seed-cultivating 911 (genetic transformation positive variety) which are inserted with the DHA fatty acid desaturase gene of the cryptococcus, namely the NOS terminator plasmid sequence of the DHA fatty acid desaturase gene of the cryptococcus by utilizing a peanut seed specific gene promoter BM2Q 5K-cryptococcus, the expression of the DHA fatty acid desaturase gene of the cryptococcus is not obviously enhanced.
According to the experiment, compared with the untreated peanut seed tissue cells of the same species, the peanut seed tissue cells of the 911 species, which are bred by inserting the peanut seed specific gene promoter BM2Q 5K-cryptococcus, DHA fatty acid desaturase gene-NOS terminator plasmid sequence, have obviously improved transcription and expression quantity of the cryptococcus, DHA fatty acid desaturase gene under the same growth condition, and the related gene transcription and expression quantity in the rest of plants are not obviously changed, so that the peanut seed specific gene promoter BM2Q5K has obvious tissue specificity and expression strengthening effect on the expression of the cryptococcus, DHA fatty acid desaturase gene in the seed tissue cells when being used for gene editing.
Example 3:
Peanut seed-specific gene promoter BM2Q5K obtained using example 1 above was used for crop genome integration procedure 2:
The peanut protein contains 8 amino acids necessary for human body, is a plant protein with higher nutritive value, and has the advantages of easy digestion, less abdominal distention factors, no beany flavor and the like compared with soybean protein; compared with the rapeseed and cottonseed proteins, the protein has fewer toxic substances and is an ideal food industry basic raw material. Wherein, the content of glutamic acid and aspartic acid is higher than that of rice, flour and corn, and the effective utilization rate is up to 98.4 percent. The content of protein in peanut is 24% -36%, and the peanut protein is classified into two main types, namely water-soluble protein and salt-soluble protein according to the dissolution characteristic of peanut protein, wherein about 10% of protein is water-soluble protein, which is called albumin, and the other 90% is salt-soluble protein. It is generally considered that the increase of water-soluble protein in food is beneficial to the hydrolysis of protein by digestive enzyme, thereby being beneficial to the digestion and absorption of protein components by animals or human bodies and remarkably increasing the digestion and utilization rate of protein. Therefore, increasing the content of water-soluble protein (albumin) in food or feed has been an important issue in the food and feed industries. For peanuts, the 2s albumin synthesis capacity of the peanuts can be considered, and the expression quantity of 2s albumin synthesis genes can be increased by using a gene editing method so as to improve the albumin output of peanut tissues.
When the vector is constructed by transgenic operation, the pCAMBIA2300 plasmid sequence can be used as the vector, DNA double chains of a peanut seed specific gene promoter BM2Q 5K-2 s albumin synthetic gene-NOS terminator can be constructed by enzyme digestion and connection reaction, and the plant expression vector is used for transforming target plants to complete genetic transformation.
To verify the 2s albumin production performance of this transgenic plant, the following experiments were designed:
Tissue culture and subsequent induction differentiation are carried out on peanut callus of peanut breeding 911 in MB culture medium to obtain peanut of original peanut breeding 911 variety and complete plant 20 strains obtained after tissue culture of peanut breeding 911 (genetic transformation positive variety) variety with 2s albumin expression gene inserted by utilizing peanut seed specific gene promoter BM2Q 5K-2 s albumin synthesis gene-NOS terminator plasmid sequence, 120d is planted under normal greenhouse condition until the plant matures and results are obtained, seed parts are respectively collected and separated, and the 2s albumin gene enhanced expression condition is examined by taking the total protein content ratio detected by the seed parts as index.
When the peanut albumin content is tested, uniformly mixing peanut seed fragments subjected to biological membrane enzymolysis, separating samples, measuring the total protein content of the sample A by using a Kjeldahl nitrogen method, preliminarily precipitating required peanut albumin by using 65% ammonium sulfate solution of the sample B, purifying by using a DEAE-52 column to obtain peanut albumin, and measuring the total albumin content to obtain the relative content of albumin in total protein as shown in the following table 2:
TABLE 2
In addition, in order to verify that the enhanced expression effect of the peanut seed-specific gene promoter BM2Q5K on the 2s albumin synthesis gene when used for gene editing was specific to peanut seed tissue cells only, the following experiments were performed on the plants obtained in the above experiments:
the method comprises the steps of carrying out the same measurement on albumin and total protein content of a mixture obtained after the smashing of root, stem, leaf, shell and seed coat parts, namely, when the albumin content of the seeds is tested, uniformly mixing peanut seed fragments subjected to biomembrane enzymolysis, separating samples, measuring the total protein by using a Kjeldahl nitrogen determination method, preliminarily precipitating required peanut albumin by using a 65% ammonium sulfate solution for sample B, purifying by using a DEAE-52 column to obtain the peanut albumin, and carrying out the total albumin measurement, wherein the residual part of a whole plant of the peanut of the 2s albumin expression gene transgenic seed flower 911 (genetic transformation positive variety) obtained after the planting, namely, the albumin and total protein content of the mixture are obtained after the smashing of root, stem, leaf, shell and seed coat parts, namely, when the albumin content of the peanut is tested, carrying out the following table 3:
TABLE 3 Table 3
It can be known that in the residual plant cells of two plants except the seeds of the original peanut variety of the peanut breeding 911 and the peanut variety of the transgenic variety of the peanut breeding 911 (genetic transformation positive variety) which are inserted with the 2s albumin expression gene by utilizing the plasmid sequence of the peanut seed specific gene promoter BM2Q 5K-2 s albumin synthesis gene-NOS terminator, the synthesis amount of the 2s albumin is not obviously improved, namely the expression of the 2s albumin synthesis gene is not enhanced.
According to the experiment, compared with the untreated peanut seed tissue cells of the same species, the peanut seed tissue cells have obviously improved albumin content, and the albumin content in the rest of the plant is not obviously changed, so that the peanut seed specific gene promoter BM2Q5K has obvious tissue specificity and expression strengthening effect on the expression of 2s albumin synthesis genes in the seed tissue cells when the peanut seed specific gene promoter BM2Q5K is used for gene editing.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the 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 scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (5)
1. The peanut seed specific gene promoter BM2Q5K is characterized in that the promoter BM2Q5K can drive target genes to be specifically and highly expressed in seed tissue cells in peanut seed tissue cells, and the nucleotide sequence of the promoter BM2Q5K is shown as SEQ ID NO. 01:
GTAGTCTAGTTGGTTAGGATACTCGGCTCTCACCCGAGAGACCCGGGTTCGAGTCCCGGCAACGGAATTCAAATTTTGTAAAAATCGTTTACTTTTTGGTTGAAAAAGTGTGAGAGTTGCAACTTGCAAGTGACATGTACGCAAGAAAAGGGTGACGTCCATAGCCACAATC.
2. a peanut seed-specific gene promoter BM2Q5K according to claim 1, characterised in that: the promoter BM2Q5K is obtained by PCR amplification technology.
3. A peanut seed-specific gene promoter BM2Q5K according to claim 2, characterised in that: in the PCR amplification process, the following two primers are configured according to the nucleotide sequence shown as SEQ ID NO.01 for amplification:
（1）SEQ ID NO.03：GTAGTCTAGTTGGTTAGGATACTCG；
（2）SEQ ID NO.04：GATTGTGGCTATGGACGTCACCCTT。
4. a peanut seed-specific gene promoter BM2Q5K according to claim 2, characterised in that: the amplification process adopts a DNA amplification system based on DNA polymerase for amplification.
5. Use of the peanut seed-specific gene promoter BM2Q5K of claim 1, wherein: the specific application process is that a pCAMBIA2300 plasmid sequence is used as a vector, DNA double chains of a peanut seed specific gene promoter BM2Q 5K-2 s albumin synthetic gene-NOS terminator are constructed through enzyme digestion and connection reaction, and the plant expression vector is used for transforming a target plant to complete genetic transformation.
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| CN109706150A (en) * | 2019-01-24 | 2019-05-03 | 山东省花生研究所（山东省农业科学院花生工程技术研究中心） | A kind of peanut seed specific expression promoter AHSSP29 and its application |
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| CN109706150A (en) * | 2019-01-24 | 2019-05-03 | 山东省花生研究所（山东省农业科学院花生工程技术研究中心） | A kind of peanut seed specific expression promoter AHSSP29 and its application |
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