阅读说明：本技术 一种水稻内含子及其活性鉴定方法 (Rice intron and activity identification method thereof ) 是由 刘鹏 张韬 于 2021-07-27 设计创作，主要内容包括：本发明公开了一种水稻内含子及其活性鉴定方法,水稻内含子的序列如SEQ ID NO.1,通过构建Pro::GFP、Pro+Intron+UTR::GFP、Pro+UTR::GFP三个载体来鉴定内含子的活性,结果显示,本发明的内含子DNA分子,104bp,对转录具有较好的增强效果。(The invention discloses a rice Intron and an activity identification method thereof, wherein the sequence of the rice Intron is shown as SEQ ID NO.1, the activity of the Intron is identified by constructing three vectors of Pro GFP, Pro + Intron + UTR GFP and Pro + UTR GFP, and the result shows that 104bp of the Intron DNA molecule has a better enhancement effect on transcription.)

1. A rice intron has a sequence shown in SEQ ID NO. 1.

2. The rice intron of claim 1, wherein: the rice is japonica rice Nipponbare (Oryza sativa L.spp.Japonica var.Nipponbare) belonging to Gramineae.

3. The rice intron of claim 2, wherein: taking the rice as the template of japonica rice Nipponbare genome DNA of Gramineae, and amplifying to obtain an amplification product of 104 bp.

4. The rice intron of claim 3, wherein: the primers for amplification have the following sequences:

and (3) forward direction F: 5'-gtcggtacgcgtctctaggc-3', respectively;

reverse R: 5'-ccacaaaacaagaaaaaaa-3' are provided.

5. The method for identifying the activity of a rice intron according to claim 1, wherein: the method comprises the following steps:

(1) constructing an expression vector of LOC _ Os04g57220 promoter driving GFP, which is named as Pro: : GFP, LOC _ Os04g57220 promoter sequence of SEQ ID NO.2,

the amplification primers are as follows:

promoter-F：5’-gagtgtcgtgctccaccatgcgctaagttaaaaatagatat-3’

promoter-R：5’-ctcttcttcttaggagccatctttcggtgctcttcgctttc-3’；

(2) constructing a promoter and a vector for regulating GFP expression by a 5' UTR region sequence (SEQ ID NO.3) comprising an Intron, wherein the vector is named as Pro + Intron + UTR: : the presence of a GFP (green fluorescent protein),

the amplification primers are as follows:

intron-F：5’-gagtgtcgtgctccaccatgcgctaagttaaaaatagatat-3’；

intron-R：5’-ctcttcttcttaggagccatcgatcaaaccctaatcaccc-3’；

(3) a promoter and a 5' UTR sequence (SEQ ID NO.4) are constructed to regulate a GFP expression vector, and the vector is named as Pro + UTR: : the presence of a GFP (green fluorescent protein),

the amplification primers are as follows:

UTR-F：5’-gagtgtcgtgctccaccatgcgctaagttaaaaatagatat-3’；

UTR-R：5’-ctcttcttcttaggagccatcggcgacggggcgctccggc-3’；

(4) preparing and transforming rice protoplast;

(5) after transformation, the activity of a 104bp intron in the 5' UTR region of the LOC _ Os04g57220 gene is verified according to the intensity of a fluorescence signal.

6. The method for identifying the activity of a rice intron according to claim 5, wherein: and (3) observing the intensity of the fluorescence signal by using a high-content cell imaging system.

Technical Field

The invention relates to an intron and an identification method, in particular to a rice intron and an activity identification method thereof.

Background

Eukaryotic genes are usually composed of several exons and introns that are linked to each other in spaced apart relation. An exon is a part of a eukaryotic gene, which is preserved after splicing, wherein a part that can be translated is called a coding region and a part that cannot be translated is called a UTR. Introns are spacer sequences located between exons in a gene, are non-coding sequences, and are removed during splicing. Upon transcription of a gene, both exons and introns are transcribed into precursor RNA molecules, which are further spliced to delete intron sequences, joining the exons together to produce a functional mature RNA molecule.

With the continuous and intensive research, scientists find that introns are no longer nonsense sequences, and the introns are used as important components of eukaryotic genomes and participate in signal pathways such as gene expression, maintenance of chromatin structures, construction and dynamic change of cytoskeletons and the like. More and more studies have confirmed that introns play an important role as regulatory elements to enhance the expression of the target gene. Therefore, introns are important research targets in genetic engineering.

Disclosure of Invention

The purpose of the invention is as follows: the present invention aims to provide an intron having an enhancing effect on transcription.

The technical scheme is as follows: the invention provides a rice intron, the sequence of which is shown as SEQ ID NO. 1.

Further, the rice is japonica rice Nipponbare (Oryza sativa L.spp.Japonica var.Nipponbare) belonging to the genus Oryza of the family Gramineae.

Further, using the rice as the template of japonica rice Nipponbare genomic DNA of Gramineae, and amplifying to obtain an amplification product of 104 bp.

Further, the primers for amplification have the following sequences:

and (3) forward direction F: 5'-gtcggtacgcgtctctaggc-3', respectively;

reverse R: 5'-ccacaaaacaagaaaaaaa-3' are provided.

The method for identifying the activity of the rice intron comprises the following steps:

(1) firstly verifying the activity of the LOC _ Os04g57220 promoter, constructing an expression vector of the LOC _ Os04g57220 promoter driving GFP, and naming the expression vector as Pro: : GFP, LOC _ Os04g57220 promoter sequence of SEQ ID NO.2,

the amplification primers are as follows:

promoter-F：5’-gagtgtcgtgctccaccatgcgctaagttaaaaatagatat-3’

promoter-R：5’-ctcttcttcttaggagccatctttcggtgctcttcgctttc-3’；

(2) analysis shows that the 5 'UTR region of LOC _ Os04g57220 gene is separated by 104bp Intron, and in order to detect whether the Intron has influence on gene expression, a promoter and a vector for regulating GFP expression by a 5' UTR region sequence (SEQ ID NO.3) comprising the Intron are constructed, and the vector is named as Pro + Intron + UTR: : the presence of a GFP (green fluorescent protein),

the amplification primers are as follows:

intron-F：5’-gagtgtcgtgctccaccatgcgctaagttaaaaatagatat-3’；

intron-R：5’-ctcttcttcttaggagccatcgatcaaaccctaatcaccc-3’；

(3) in order to detect the influence of an individual 5 'UTR sequence on gene expression, a promoter and a 5' UTR sequence (SEQ ID No.4) are constructed to regulate a GFP expression vector, which is named as Pro + UTR: : the presence of a GFP (green fluorescent protein),

the amplification primers are as follows:

UTR-F：5’-gagtgtcgtgctccaccatgcgctaagttaaaaatagatat-3’；

UTR-R：5’-ctcttcttcttaggagccatcggcgacggggcgctccggc-3’；

(4) preparing and transforming rice protoplast;

(5) after transformation, the activity of a 104bp intron in the 5' UTR region of the LOC _ Os04g57220 gene is verified according to the intensity of a fluorescence signal.

Identification principle:

the promoter of a gene plays a very important role in the regulation of expression of the gene at the transcriptional level. Promoters like "switches" determine the initiation of transcription. In the invention, the promoter of LOC _ Os04g57220 gene is found to only promote the expression of GFP gene at a background level, and the presence of intron greatly enhances the expression of GFP. The system for driving GFP expression by combining the promoter and the intron sequence verifies the function of the intron in enhancing gene expression in a transient transformation system.

Has the advantages that: the intron DNA molecule of the invention, 104bp, has better enhancement effect on transcription.

Drawings

FIG. 1 is a schematic structural diagram of the LOC _ Os04g57220 gene;

FIG. 2 is a schematic diagram of the vector for detecting the intron in the protoplast and the result of the activity detection.

Detailed Description

The method for obtaining and identifying the intron in this example is as follows:

1. intron

Japanese nitrile genome sequence and annotation information were obtained in the public database of rice genomes (RGAP, http:// rice. plant biology. msu. edu /), and open chromatin data (DNase-seq) were obtained from the plant open chromatin database, PlamDHS (http:// plant dh. org /). The LOC _ Os04g57220 gene, which encodes ubiquitin-conjugating enzyme, was analyzed to be strongly expressed in stem, leaf, inflorescence, embryo, endosperm and seed. The structure of the gene is studied, and the 5' UTR of the gene is found to have an intron sequence with the length of 104 bp.

Designing a sequence specific primer, and preparing the following primers:

and (3) forward direction F: 5'-gtcggtacgcgtctctaggc-3'

Reverse R: 5'-ccacaaaacaagaaaaaaa-3' are provided.

The above primer is used for amplifying to obtain an amplification product of 104bp by using the genome DNA of japonica rice Nipponbare as a template. The nucleotide sequence of the amplified product is shown as the first site to 104 sites from the 5' end of SEQ ID NO.1 by sequencing, as shown in FIG. 1.

SEQ ID NO.1：

gtcggtacgcgtctctaggcccccctctctctctcgatttgatcggtttgatctgtggtgccctaggtttgatctgtggatttattttttttcttgttttgtgg。

2. Detection of

To examine the effect of 104bp intron sequence on gene expression, the following three vectors were constructed:

1) to examine the activity of the LOC _ Os04g57220 promoter itself, its promoter sequence was first cloned for driving expression of the reporter GFP gene. The GFP vector used was provided by Zhang Yong topic group of the university of electronic technology. LOC _ Os04g57220 gene self promoter sequence (SEQ ID NO.2), 144bp in length, amplified using the primers:

promoter-F：5’-gagtgtcgtgctccaccatgcgctaagttaaaaatagatat-3’

promoter-R：5’-ctcttcttcttaggagccatctttcggtgctcttcgctttc-3’

SEQ ID NO.2：

cgctaagttaaaaatagatataaactctgtaaagatgcaggtgtccaggctaaacttccaagatcatccaataaaaggaacacttccttttacttttctccttaggaaaaaaaagaaaaaaaagaaagcgaagagcaccgaaag，

the constructed vector was named Pro: : GFP (as shown in FIG. 2).

2) Analysis found that LOC _ Os04g57220 gene 5 'UTR region was separated by 104bp intron, and to examine whether intron-included influence on gene expression, promoter and intron-included 5' UTR region (SEQ ID NO.3) were amplified, using the primers:

intron-F：5’-gagtgtcgtgctccaccatgcgctaagttaaaaatagatat-3’

intron-R：5’-ctcttcttcttaggagccatcgatcaaaccctaatcaccc-3’

SEQ ID NO.3：

cgctaagttaaaaatagatataaactctgtaaagatgcaggtgtccaggctaaacttccaagatcatccaataaaaggaacacttccttttacttttctccttaggaaaaaaaagaaaaaaaagaaagcgaagagcaccgaaaggcgaatctaagcgcgtccagcgtaagcatcacgcgagtcgtcggcgcgcgcggatccccgatcggacggtccacgttgccccgtcgccctataaattggtccccccgtctcccccacccaaatcctccccgactcctcgcagcttcctcttgtttttcttggccgaaccccccctcgacacgccgtcgccgccgaggggagagagagagaggccgccggccgccgctaccactgaccccccccctcgccggagcgccccgtcgccggtcggtacgcgtctctaggcccccctctctctctcgatttgatcggtttgatctgtggtgccctaggtttgatctgtggatttattttttttcttgttttgtgggggtgattagggtttgatcg

the constructed vector is named as Pro + Intron + UTR: : GFP (as shown in FIG. 2).

3) To examine the effect of the 5 'UTR sequence alone on gene expression, the promoter and 5' UTR sequence (SEQ ID NO.4) were amplified,

UTR-F：5’-gagtgtcgtgctccaccatgcgctaagttaaaaatagatat-3’

UTR-R：5’-ctcttcttcttaggagccatcggcgacggggcgctccggc-3’

SEQ ID NO.4：

cgctaagttaaaaatagatataaactctgtaaagatgcaggtgtccaggctaaacttccaagatcatccaataaaaggaacacttccttttacttttctccttaggaaaaaaaagaaaaaaaagaaagcgaagagcaccgaaaggcgaatctaagcgcgtccagcgtaagcatcacgcgagtcgtcggcgcgcgcggatccccgatcggacggtccacgttgccccgtcgccctataaattggtccccccgtctcccccacccaaatcctccccgactcctcgcagcttcctcttgtttttcttggccgaaccccccctcgacacgccgtcgccgccgaggggagagagagagaggccgccggccgccgctaccactgaccccccccctcgccggagcgccccgtcgccg，

the constructed vector is named as Pro + UTR: : GFP (as shown in FIG. 2).

3. Rice protoplast preparation and transformation

3.1 preparation of rice protoplast:

(1) the blade is prepared. Taking 40-60 fresh rice plants which grow well in about 14 days of sterile culture, and removing roots for later use.

(2) And (4) enzymolysis. Cutting the rice leaves and stem segments into strips with the width of 0.5mm by using a sharp blade, wherein the speed is high, the dehydration of the leaves is prevented, putting the cut leaves into a culture dish containing 10mL of enzymolysis liquid, putting the culture dish into a closed container, vacuumizing the closed container for 30min by using a vacuum pump, sealing the closed container by using a sealing film, and putting the closed container on a shaker with the rotating speed of 60rpm for overnight enzymolysis at 25 ℃ in the dark.

(3) And (5) filtering. The crude enzyme solution after overnight enzymolysis was filtered through a 40 μm pore size cell sieve previously rinsed with 2mL of W5 washing Buffer to remove impurities that did not undergo sufficient enzymolysis.

(4) And (5) purifying. Transferring the filtered cell enzymolysis solution into two 15mL centrifuge tubes, wherein 6mL of cell enzymolysis solution is added into each tube, 16mL of 0.55M sucrose is sucked by using a syringe with a long needle, 8mL of 0.55M sucrose is added into the bottom of each tube of cell enzymolysis solution, and attention is paid to: when adding sucrose, the needle head extends into the bottom to push the injector, and the injector is slowly moved upwards along with the rise of the liquid level, and the action is gentle to prevent the damage to the protoplast cells. Centrifuge at 1000g for 5 min.

(5) And (5) cleaning. One 50mL centrifuge tube was taken, 10mL Washing buffer was added, and placed on a tube rack. Pipette 8mL of Washing buffer with a syringe fitted with a long needle, extend the needle into the centrifuged middle cell layer from the previous step, pipette all protoplasts from this layer slowly, and then carefully transfer to a 50mL centrifuge tube containing 10mL of Washing buffer. Flicking, mixing, and centrifuging at 100g room temperature for 5 min.

(6) The washing was repeated once. The supernatant was removed, 10mL Washing buffer was slowly added along the wall, gently mixed, and centrifuged at 100g for 2 min.

(7) And (4) resuspending. The supernatant was removed and 5mL Washing buffer was added, slowly along the wall, gently flicked and mixed to keep the cells in suspension.

(8) And (6) counting. Aspirate 100. mu.L of cells for counting, place 6-7. mu.L of cells on a cell counting plate for counting, and count after diluting with MGG if the number of cells is too large.

(9) And (5) diluting the cells. Taking a proper amount of cell suspension according to counting results, centrifuging, then using MGG Buffer to resuspend cells, and finally diluting the cells to 1 × 10⁶one/mL, used for protoplast transformation.

3.2 transformation of Rice protoplasts:

(1) preparation of plasmids. The following plasmids were extracted using a plasmid miniprep kit (TIANGEN, Cat # DP-106): pro: : GFP, Pro + Intron + UTR: : GFP and Pro + UTR: : GFP. Ensuring the concentration above 500 ng/. mu.L, and the whole process is preferably carried out aseptically. The plasmid was frozen at-20 ℃ in a refrigerator. Before use, the protoplast is centrifuged for 10min at the highest rotating speed of the centrifuge, so that impurities are precipitated at the bottom of the centrifuge tube, and the influence on the protoplast conversion efficiency due to the impurities is avoided. Diluting the processed plasmid to the plasmid concentration required by the experiment by using MGG Buffer, and fully and uniformly mixing the diluted plasmid for later use.

(2) PEG-mediated transformation. 200. mu.L of protoplast cells prepared according to the preparation process of 2.2.6 was added to the prepared plasmids (Pro:: GFP, Pro + Intron + UTR:: GFP and Pro + UTR:: GFP) and gently mixed. Then, 210-230. mu.L of 40% PEG (the amount of PEG added is related to the plasmid volume to ensure equal volume addition) was added to each tube, gently mixed until the cells were uniformly suspended in PEG, and the time was counted for 20min from the time when PEG was added to the first tube. The time from the first tube to the last tube is controlled within 5min as much as possible.

(3) And (5) cleaning. After 20min of reaction by adding PEG, 1mL of W5 Washing Buffer was added to each tube to terminate the reaction, the mixture was gently inverted and mixed, and centrifuged at 250g for 5 min.

(4) The washing was repeated once. The supernatant was gently aspirated off with a gun, 800. mu.L of Washing Buffer was added again, the mixture was gently inverted and mixed, and 150g was centrifuged for 5min, and the supernatant was gently aspirated off.

(5) And (5) resuspending and culturing the cells. 2mL of W5Buffer was added to a 40mm petri dish, and then W5Buffer in the petri dish was taken to resuspend the cells in the centrifuge tube, and all were transferred to the petri dish, cultured in the dark at 25 ℃, and fluorescence was observed under a microscope after two days.

4. Observation by fluorescence microscope

Observations were made using a high content cell imaging system. GFP was used at an excitation wavelength of 488nm and an absorption wavelength of 490-540 nm. All fluorescence experiments were repeated at least three times. Pro: : GFP, Pro + Intron + UTR: : GFP and Pro + UTR: : the results of observation of transformants with the GFP plasmids after protoplast transformation are shown in FIG. 2. A sequence upstream of the transcription initiation site of the LOC _ Os04g57220 gene and located in an open chromatin region is defined as a promoter, and when the promoter regulates GFP expression, a weak fluorescent signal is observed; when the 5' UTR region including the intron and the promoter together regulated GFP expression, a significant fluorescent signal was observed in protoplasts; when only the 5' UTR region and promoter regulated GFP expression, it was still a weak fluorescent signal. Experimental results show that the LOC _ Os04g57220 gene promoter promotes the expression of GFP background level and shows weak fluorescence. The 5' UTR region had no effect on GFP expression. In the presence of introns, GFP expression was significantly enhanced and a strong fluorescent signal was observed. Indicating that the intron is capable of enhancing gene expression.

Sequence listing

<110> Yangzhou university

<120> a rice intron and method for identifying its activity

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 104

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gtcggtacgc gtctctaggc ccccctctct ctctcgattt gatcggtttg atctgtggtg 60

ccctaggttt gatctgtgga tttatttttt ttcttgtttt gtgg 104

<210> 2

<211> 144

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cgctaagtta aaaatagata taaactctgt aaagatgcag gtgtccaggc taaacttcca 60

agatcatcca ataaaaggaa cacttccttt tacttttctc cttaggaaaa aaaagaaaaa 120

aaagaaagcg aagagcaccg aaag 144

<210> 3

<211> 534

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgctaagtta aaaatagata taaactctgt aaagatgcag gtgtccaggc taaacttcca 60

agatcatcca ataaaaggaa cacttccttt tacttttctc cttaggaaaa aaaagaaaaa 120

aaagaaagcg aagagcaccg aaaggcgaat ctaagcgcgt ccagcgtaag catcacgcga 180

gtcgtcggcg cgcgcggatc cccgatcgga cggtccacgt tgccccgtcg ccctataaat 240

tggtcccccc gtctccccca cccaaatcct ccccgactcc tcgcagcttc ctcttgtttt 300

tcttggccga accccccctc gacacgccgt cgccgccgag gggagagaga gagaggccgc 360

cggccgccgc taccactgac cccccccctc gccggagcgc cccgtcgccg gtcggtacgc 420

gtctctaggc ccccctctct ctctcgattt gatcggtttg atctgtggtg ccctaggttt 480

gatctgtgga tttatttttt ttcttgtttt gtgggggtga ttagggtttg atcg 534

<210> 4

<211> 410

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cgctaagtta aaaatagata taaactctgt aaagatgcag gtgtccaggc taaacttcca 60

agatcatcca ataaaaggaa cacttccttt tacttttctc cttaggaaaa aaaagaaaaa 120

aaagaaagcg aagagcaccg aaaggcgaat ctaagcgcgt ccagcgtaag catcacgcga 180

gtcgtcggcg cgcgcggatc cccgatcgga cggtccacgt tgccccgtcg ccctataaat 240

tggtcccccc gtctccccca cccaaatcct ccccgactcc tcgcagcttc ctcttgtttt 300

tcttggccga accccccctc gacacgccgt cgccgccgag gggagagaga gagaggccgc 360

cggccgccgc taccactgac cccccccctc gccggagcgc cccgtcgccg 410