CN114517199A - Promoter of Pteria penguin Mitf gene and application thereof - Google Patents
Promoter of Pteria penguin Mitf gene and application thereof Download PDFInfo
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Abstract
The invention discloses a promoter of a Pteria penguin Mitf gene and application thereof. The invention provides a promoter of a Mitf gene of Pteria penguin, and provides a method for screening and identifying the Mitf transcription factor by using the promoter on the basis. The invention overcomes the defect that a promoter and a method for screening and identifying the Mitf transcription factor of the Pteria penguin are not available at present, and has important significance for color regulation and selective breeding of Pteria penguin.
Description
Technical Field
The invention belongs to the technical field of aquatic science and genetic engineering, and particularly relates to a promoter of a Pteria penguin Mitf gene and application thereof.
Background
Pteria penguin is a high-quality mother shell used for cultivating large seawater pearls, and the expression level of melanin of the Pteria penguin can influence the color of nacre and further influence the quality of the pearls. In order to cultivate pearls with specific colors, the synthesis of melanin can be regulated and controlled by regulating and controlling the expression level of a melanin synthesis signal pathway gene of the pteria penguin, so that the color and the shell color of the nacre of the pteria penguin are influenced, and a foundation is laid for breaking through a key technology for optimizing the color of the pearl and cultivating pearls with rare colors.
The synthesis of melanin in pteria penguin has a complex signal path. Among them, tyrosinase (Tyr) is a key rate-limiting enzyme, and can catalyze three key steps of melanin synthesis. The microphthalmia-associated transcription factor (Mitf) is a core transcription factor for synthesizing the melanin of mammals, and the Mitf can be combined to an M-box or an E-box of a Tyr gene promoter to activate the expression of Tyr and promote the formation of the melanin. If Tyr is said to be the driver of the command, Mitf is the decider of the operational tent. Mitf is used as a core transcription factor for melanin synthesis, is also regulated by other upstream genes, is an important factor of a melanin synthesis signal pathway, and can influence the expression of Tyr and finally influence the melanin synthesis level by regulating the expression of Mitf. Therefore, the mining of the transcription factor of Mitf has important theoretical significance for the construction of a melanin synthesis pathway and has important production significance for breaking through the key technology of color optimization of pearl oyster. In order to facilitate screening and identifying related transcription factors, a method for screening and identifying the transcription factors of the Pteria penguin Mitf is needed.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provide a promoter of Mitf gene of Pteria penguin and application thereof.
The first purpose of the invention is to provide a promoter of a Mitf gene of Pteria penguin.
The second purpose of the invention is to provide a recombinant vector containing the promoter of the Mitf gene of the Pteria penguin.
The third purpose of the invention is to provide a recombinant cell of a recombinant vector containing a promoter of a Mitf gene of Pteria penguin.
The fourth purpose of the invention is to provide the promoter, the recombinant vector and/or the application of the recombinant cell in screening/identifying the transcription factor of the Mitf gene of the Pteria penguin.
The fifth purpose of the invention is to provide a method for screening/identifying the transcription factor of the Mitf gene of the Pteria penguin.
The above purpose of the invention is realized by the following technical scheme:
the invention provides a promoter of Mitf gene of Pteria penguin, and the nucleotide sequence is shown in SEQ ID NO. 1.
The invention verifies that the promoter is a strong promoter of a Mitf gene by constructing a luciferase reporter gene recombinant vector driven by the promoter shown in SEQ ID NO. 1. On the basis, the invention constructs a screening and identifying method of the transcription factor of the Mitf gene by utilizing the constructed luciferase reporter gene recombinant vector driven by the promoter, and overcomes the defects that no promoter and method for screening and identifying the transcription factor of the Pteria penguin Mitf exist at present.
Therefore, the application of the invention protects the promoter of the Mitf gene of the Pteria penguin, and the nucleotide sequence of the promoter is shown in SEQ ID NO. 1.
The invention also applies for and protects a recombinant vector, and the vector contains a nucleotide sequence of a promoter shown in SEQ ID NO. 1.
In order to conveniently determine the activity of the promoter shown in SEQ ID NO.1 or screen and identify the transcription factor of the Mitf gene by using the recombinant vector, the recombinant vector is a vector with a fluorescein reporter gene.
Specifically, the vector is a luciferase reporter gene recombinant vector driven by a promoter shown in SEQ ID NO. 1.
In the examples of the present invention, the vector used was pGL3-Basic fluorescein reporter gene vector.
The invention also applies for a recombinant cell, which contains any one of the recombinant vectors.
Specifically, the cells used were 293T cells. Traditionally, the shellfish promoter was thought to be most efficient to express in shellfish cells, and the present inventors found that the Mitf promoter can be expressed in 293T cells and can be used for screening.
The luciferase reporter gene recombinant vector driven by the promoter shown in SEQ ID NO.1 constructed by the invention and an expression vector containing a target gene (transcription factor) sequence to be identified are co-transfected into a 293T cell line, or the expression vector containing the target gene sequence to be identified is transfected into a recombinant cell containing the luciferase reporter gene recombinant vector driven by the promoter shown in SEQ ID NO.1 constructed by the invention, and the luciferase activity in the cell is determined, so that whether the identified target gene is the transcription factor of the Mitf gene or not can be judged by comparing the luciferase activity.
Therefore, the invention also applies to protect the application of the promoter, the recombinant vector and the recombinant cell in screening/identifying the transcription factor of the Mitf gene of the Pteria penguin.
The invention also provides a method for screening/identifying the transcription factor of the Mitf gene of the Pteria penguin, which comprises the following steps:
s1, constructing a recombinant vector containing a target gene to be identified;
s2, transfecting the recombinant vector obtained in the step S1 and the luciferase reporter gene recombinant vector driven by the promoter shown in SEQ ID NO.1 constructed by the invention together, and collecting cells for dual-luciferase reporter analysis;
and S3, judging results, if the luciferase activity of the cells over-expressing the target gene is higher than that of the control, indicating that the identified target gene is a transcription factor of the Mitf gene of the Pteria penguin.
Specifically, the expression vector used in step S1 is pcdna3.1.
Specifically, in the determination of the result in step S3, the cell co-transfected by the empty vector pcDNA3.1 and the luciferase reporter gene recombinant vector driven by the promoter shown in SEQ ID NO.1 constructed by the invention is used as a negative control.
Specifically, the cells used for co-transfection were 293T cells.
Besides co-transfecting cells with the constructed expression vector containing the target gene and the luciferase reporter gene recombinant vector driven by the promoter shown in SEQ ID NO.1 to screen and identify the transcription factor of the Mitf gene, the recombinant cells containing the luciferase reporter gene recombinant vector driven by the promoter shown in SEQ ID NO.1 constructed by the invention can be transfected with the expression vector containing the target gene, and the cells are subjected to dual-luciferase reporter analysis to judge whether the identified target gene is the transcription factor of the Mitf gene.
The invention has the following beneficial effects:
the invention provides a promoter of Mitf gene of Pteria penguin, which can determine whether the identified target gene is Mitf transcription factor by cotransfecting a luciferase reporter gene recombinant vector driven by the promoter and an expression vector containing the target gene (transcription factor) sequence to be identified into cells and measuring the luciferase activity in the cells. The invention overcomes the defects that a promoter and a method for screening and identifying the transcription factor of the Mitf of the Pteria penguin are not available at present, and has important significance for screening and identifying the transcription factor of the Mitf, breaking through the key technology of optimizing the color of the Pteria penguin and culturing rare-colored pearls.
Drawings
FIG. 1 is an electrophoretogram of a single DNA fragment of the promoter of the Mitf gene amplified by genome walking.
FIG. 2 is an electrophoretogram of the promoter of the present invention.
FIG. 3 shows the results of sequence analysis of the promoter of the present invention.
FIG. 4 shows the results of activity analysis of the promoter of the present invention.
FIG. 5 shows the result of the Creb2 gene regulating the expression of Mitf gene.
Note: in fig. 4 and 5, represents P < 0.01.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
EXAMPLE 1 cloning and analysis of promoter sequences
1. Promoter sequence of Mitf gene amplified by genome walking
The invention utilizes an E.Z.N.A. tissue DNA kit (Omega) to extract genome DNA from the Pteria penguin, constructs a genome Walker library, and the specific experimental process is carried out according to the operation steps of the kit specification. On the basis, the invention designs three primers (Mitf-pro-SP1, Mitf-pro-SP2 and Mitf-pro-SP3) and amplifies a single DNA segment of the promoter of the Mitf gene by genome walking. After splicing the 3 DNA fragments, the correct splicing sequence was verified using the primer pairs Mitf-pro-F and Mitf-pro-R. The nucleotide sequences of the above primers are shown in Table 1.
TABLE 1 cloning and validation of primers used for the promoter region of Mitf
The chromosome walking is carried out in three steps, and the PCR reaction system and the program are as follows:
(1) first round PCR reaction
A first PCR reaction was performed using genomic DNA as a template, AP Primer as an upstream Primer, and Mitf-pro-SP1 as a downstream Primer.
First round PCR reaction system
First round PCR reaction procedure
(2) Second round PCR reaction
After the reaction solution of the first round of PCR reaction was diluted 100 times, 1. mu.L of the diluted solution was used as a template for the second round of PCR reaction, and the second round of PCR reaction was performed using PAD1 Primer as the upstream Primer and Mitf-pro-SP2 as the downstream Primer.
Second round PCR reaction System
Second round PCR reaction procedure
(3) Third round of PCR reaction
After the reaction solution of the second round of PCR reaction was diluted 100 times, 1. mu.L of the diluted reaction solution was used as a template for the third round of PCR reaction, and the third round of PCR reaction was performed using PAD2 Primer as the upstream Primer and Mitf-pro-SP3 as the downstream Primer, under the same reaction conditions and procedures as the second round of PCR reaction.
5 mu L of each PCR reaction solution is subjected to electrophoresis detection by using 1% agarose gel; then cutting gel-clear electrophoresis strip under ultraviolet lamp, and recovering the target fragment with gel recovery kit (OMEGA), wherein the electrophoresis detection result of the target fragment is shown in FIG. 1. After splicing the 3 DNA fragments, the primers are used to verify Mitf-pro-F and Mitf-pro-R, and the result shows that the splicing sequence is correct.
2. Sequencing analysis of Mitf Gene promoter sequence
The genome of Pteria penguin is taken as a template, the promoter sequence of the Mitf gene is amplified, and the primers are Mitf-pro-F and Mitf-pro-R. The PCR amplification product was separated by electrophoresis on 1% agarose gel using DL 2000 as Marker, and the electrophoresis results are shown in FIG. 2, which shows that a single band of about 2000bp was obtained by amplification. The amplified products were sent to the company for sequencing and then subjected to alignment analysis.
The nucleotide sequence of the Mitf gene promoter obtained by amplification of the invention is shown as follows (SEQ ID NO. 1): GAGAGGGGGTGAAGGTTGATTGACTGATTATTGTGGAGATGGGGCGTTTTCTAGATTGAACTTACCACAGGGTTTTCATTTTGCACCTTTTCCTATTGAAATACCAATAATAGATCGCTTGGTATTATAATAGAGAGGGGGGTGAAGGTTGATTGACTAATTATTGTTTTACGTCAAGTGCTGCACGATTTTTAACGTCTGTGGTTATAGCATCTCTCTGATGCATTGATAGAGGTTCATGCCTTTGCAACAGACCCACAGGCCGAAGCAGCCAGGGATCGTTTACGTGCCACACTTATTTTGACACTATACATTGGTTTTCATCGTCTCATACGAAAGATATGAAACGGGTATATCCACGACAGGAATCAAACCCGCACTTCTCAGTCACGAATCGAGCGCTTCATCCACTGAGCAACACAGGCTCCAGAGGAGGCGAAAGCCATGTTATCTTGAGTACTGTAAACGTTGCCAGTAGCCTCTCTGAAGTCTGGGTTCAAATCTTAGTTGAAGCGCGTAGAAACTCGAACAGAGTATCTTCTGAAAGTTCACAACTTATCGAAGGACGTGTGGTAGGGTTTTCCCTCTACTAAGGGAACAACCGTTTGAATTTAAAGATGAAAAGAGGAAGGGGTTAACATTGGTAGGTTTACAATACATGCATGTATTTCCCAACAAACTGGGACAGAATATTAATTTTCCTGATGTTGGAGTTTAGATTATTAATTTTCTTCAACGCACAGACCTATATCGATCGATCTATCTATCCATCGGTCCATCCATCCATGTATGAAGATATATAACTTAAAAGAAACCGGTGTGGCCTAGTGGTATCGCGTCGGACTCCTGATCGGGAGGCCGGGGTTCGAGTCTCGCTCTAGTCATGCCGGTGTCATGCCTCTCGGATGAGACAGTAAAAACCGAGGCCCCGTGTCATGATATGCATGGCACGTTAAAGATCCCTCACTGCTTTGGGTGGGTCCGTTGCAAAGGTCAAAATTTGTATACCCCGCATCAGGCAGATGCCGTCACCATATACGTGAAACAAAAATCAATCAATCAATCACATATGTCTCCCTCTTCGGAAATCCTGGATCTGCTATATTTAAAAATATAAATGGATGATATATCACAGCTCAAGCTTACTCCCCACGTATCGAATGGACAGTTTACATAAGTCGGTGTGTATAGTAATTTCATAACTAAATGTGTAGCTTGTATAGAGATTTAAAAAAAGATACATATTGAGATTATTCGTACGATTGCGTCATATCATAATGAATTATTTTGAAGTGAAGACCAGTGGCGGATCCAGGATTTCTGAAAAGGGGGCCCAAATGGGTTTTAACCACACTTCTCAGCCATTTTTGGTGCAAAATCTGGAATTTTACTAGTAAATTTGTGACCAAAGGGGGGCCCTCTAAATCCGCCACTGAAGACCTATGGTTATGTTTGATTTGAGCAACAGTGATCAATGTAAATATCACTGCGTAGAAGGAACACAGTTGAGTATGTCAGTATTGTATTAATAGACAATCAAATCAAATGCCGACACAAGTTATTTCTATATCTAGCAGTCAGTAATGGATTTATAAAAGTGAGTTTGACATCAGGGACGTGTGTTATATACGTCCCTGCTTGACATATACAAAGAGAATGTGTGATTCCCTTGCTCGACTCCTAAATCTGTCTATGATAAATAAATTATTTATAAGTAGATTGAATAAATAAATTAATTAATAAGTGCCCTGTTTTGACCACTTATATATGCTGTATGAAAAGCATAAAAACCACCCTCCCCCTCTTCTCCTTCGCTATTAAAGATGCCAAGGGCAAACTTCACGATTCATTGATCAGCAGGTTGTTGTTTGTTGGTTTGTTTGTTTTTCCTCACAAACACGTTACTCTTTCTCGAGTTGTTCCCCTTTATATGTGTATCTGACGTCTAAAAGGGACTTTCCACTATTGATTCTATATAAAGAATATTCATCATTGTCTCGAAATTTGATTGGTTAGAATGTATAGTATCATAAAGTGGTCGCGACCATCTTGTAAACGATTGTACGTTCTAAATTTTGACAAATAAAGAACGAAATGATG
The analysis result of the promoter sequence obtained by amplification is shown in FIG. 3, and as can be seen from FIG. 3, the genomic sequence of 2000bp upstream of the initiation codon (ATG) is obtained by amplification according to the present invention; the transcription initiation site of the Mitf gene is located at 96bp upstream of the ATG and is designated as the +1 position; the 2000bp sequence upstream of the transcription start site is considered a putative promoter. 5 typical CAMP Response Elements (CRE) exist in a promoter sequence obtained by amplification, and are respectively located at 1832-1824 th, 1075-1066 th, 976-967 th, 740-728 th and 61-53 th of the sequence; in addition, the polypeptide also contains 4 conserved PAX3 binding sites.
Example 2 determination of promoter Activity
1. Construction of promoter-driven luciferase reporter Gene recombinant vector
In order to verify the function of the promoter of the Mitf gene obtained by amplification in example 1, the invention constructs a luciferase reporter gene recombinant vector (Mitf-promoter-Luc) driven by the promoter, and the specific process is as follows:
The promoter sequence described in example 1 was amplified using the primers Mitf-pro-luc-F (CTTACGCGTGAGAGGGGGTGAAGGTTTG) and Mitf-pro-luc-R (CGCAGATCTCATTTCGTTCTTATTTGTC), and the cleavage site Mlu I was introduced at the 5 'end of the promoter sequence, the cleavage site Bgl I was introduced at the 3' end of the promoter sequence, after the promoter sequence and pGL3-Basic vector were cleaved simultaneously using pGL3-Basic vector, the cleaved fragments were recovered and mixed according to the ligation system in Table 2, and ligated in a water bath at 50 ℃ for 15min, and the ligation product was transformed with E.coli DH 5. alpha. competent cells of Shanghai Producer.
TABLE 2 ligation of promoter to pGL3-Basic vector
Taking out the competent Escherichia coli stored at-80 deg.C, immediately standing in ice; gently mixing appropriate volume of ligation product (50 ng DNA per 100. mu.L of competent cells, volume not more than 5% of competent cell volume) with competent cells, and standing on ice for 20 min; thermally shocking for 90s at 42 ℃, and then placing on ice for 2-3 min; adding 900 mu L of LB liquid culture medium (without antibiotics), and performing shaking culture at 37 ℃ and 150rpm for 1-1.5 h; after completion of the culture, the cells were centrifuged at 5000rpm for 3min, and the supernatant was discarded, followed by resuspending the cells in 100. mu.L of LB liquid medium. And (3) coating the heavy suspension on an LB plate containing ampicillin resistance, and carrying out inverted culture at 37 ℃ for 12-20 h. Selecting a single colony, carrying out colony PCR identification by using primers Tyr-pro-Luc-F and Tyr-pro-Luc-R, extracting a recombinant vector in a qualified bacterial liquid, and sequencing the obtained Tyr-promoter-Luc recombinant vector, wherein a sequencing result shows that a promoter sequence in the obtained recombinant expression vector is correct and is the same as a sequence shown in SEQ ID NO.1, so that the luciferase reporter gene recombinant vector (Mitf-promoter-Luc) driven by the promoter is successfully constructed, and the activity of the Mitf promoter can be determined by using a dual-luciferase reporter method.
2. Determination of promoter Activity
293T cells were cultured at 37 ℃ in DMEM medium supplemented with 10% Fetal Calf Serum (FCS), and then 0.4. mu.g of Mitf-promoter-Luc recombinant vector and 0.04. mu.g of pRL-cmv vector were mixed with 1. mu.L of Lipofectamine 2000(Invitrogen), and cells were transfected in a 24-well plate while pGL3-Basic empty vector and pRL-cmv vector were transfected as negative controls. After 48h, the cells were harvested and lysed using the dual luciferase reporter assay system (Promega) according to the protocol, fluorescence intensity measured using a Junior-LB9509 luminometer, and luciferase activity expressed as relative light units of firefly fluorescence to Renilla fluorescence (RLU).
The luciferase activity detection result is shown in FIG. 4, the cells transfected with pGL3-Basic empty vector showed low level luciferase activity, while the cells transfected with Mitf-promoter-Luc recombinant vector showed high luciferase activity, and the difference was very significant (P <0.01), indicating that the Mitf-promoter obtained by the invention is a strong promoter.
Example 3 identification of transcription factors of Mitf Gene
The following description will discuss the application of the promoter of the Mitf gene obtained by the present invention to screening and identifying a transcription factor of the Mitf gene, by taking an example of identifying whether Creb2 is a transcription factor of the Mitf gene.
The cDNA obtained by reverse transcription of the total RNA of Pteria penguin is taken as a template, and primers Creb2-F (GCGGCTAGCATGATGGATTTTTCAGTAGA) and Creb2-R (GCGCGCTCGAGTTTAATAATTTTTGTTCTTAAACGTA) are used for amplifying a Creb2 gene sequence. Then the amplified target fragment is connected with plasmid pcDNA3.1 to construct a Creb2-pcDNA3.1 recombinant expression vector. The upstream enzyme cutting site of the Creb gene sequence is Nhe I, and the downstream enzyme cutting site is Xho I. Subsequently, the Creb2-pcDNA3.1 recombinant vector and the Mitf-promoter-Luc recombinant vector were co-transfected into 293T cell line, and in addition, pcDNA3.1 and the Mitf-promoter-Luc recombinant vector were co-transfected into 293T cell line as a negative control. After 48h, the cells were harvested and lysed using a dual-luciferase reporter assay system, and the reagents and methods for detecting dual-luciferase were the same as in example 2.
The result of the Creb2 gene regulating the expression of the Mitf gene is shown in FIG. 5, and it can be seen from FIG. 5 that the cell over-expressing the Creb2 gene shows high luciferase activity, which is significantly higher than that of pcDNA3.1 cell (P <0.01), and the result shows that Creb2 has an obvious regulating effect on Mitf, and it is confirmed that Creb2 is an upstream transcription factor of Mitf and can activate the expression of Mitf.
Using the same method, screening can be performed to identify whether other melanin-related genes in Pteria penguin are transcription factors of Mitf.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Guangdong ocean university
<120> promoter of Ptf gene of Pteria penguin and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2100
<212> DNA
<213> Pteria penguin pearl shell (Pteria penguin)
<400> 1
gagagggggt gaaggttgat tgactgatta ttgtggagat ggggcgtttt ctagattgaa 60
cttaccacag ggttttcatt ttgcaccttt tcctattgaa ataccaataa tagatcgctt 120
ggtattataa tagagagggg ggtgaaggtt gattgactaa ttattgtttt acgtcaagtg 180
ctgcacgatt tttaacgtct gtggttatag catctctctg atgcattgat agaggttcat 240
gcctttgcaa cagacccaca ggccgaagca gccagggatc gtttacgtgc cacacttatt 300
ttgacactat acattggttt tcatcgtctc atacgaaaga tatgaaacgg gtatatccac 360
gacaggaatc aaacccgcac ttctcagtca cgaatcgagc gcttcatcca ctgagcaaca 420
caggctccag aggaggcgaa agccatgtta tcttgagtac tgtaaacgtt gccagtagcc 480
tctctgaagt ctgggttcaa atcttagttg aagcgcgtag aaactcgaac agagtatctt 540
ctgaaagttc acaacttatc gaaggacgtg tggtagggtt ttccctctac taagggaaca 600
accgtttgaa tttaaagatg aaaagaggaa ggggttaaca ttggtaggtt tacaatacat 660
gcatgtattt cccaacaaac tgggacagaa tattaatttt cctgatgttg gagtttagat 720
tattaatttt cttcaacgca cagacctata tcgatcgatc tatctatcca tcggtccatc 780
catccatgta tgaagatata taacttaaaa gaaaccggtg tggcctagtg gtatcgcgtc 840
ggactcctga tcgggaggcc ggggttcgag tctcgctcta gtcatgccgg tgtcatgcct 900
ctcggatgag acagtaaaaa ccgaggcccc gtgtcatgat atgcatggca cgttaaagat 960
ccctcactgc tttgggtggg tccgttgcaa aggtcaaaat ttgtataccc cgcatcaggc 1020
agatgccgtc accatatacg tgaaacaaaa atcaatcaat caatcacata tgtctccctc 1080
ttcggaaatc ctggatctgc tatatttaaa aatataaatg gatgatatat cacagctcaa 1140
gcttactccc cacgtatcga atggacagtt tacataagtc ggtgtgtata gtaatttcat 1200
aactaaatgt gtagcttgta tagagattta aaaaaagata catattgaga ttattcgtac 1260
gattgcgtca tatcataatg aattattttg aagtgaagac cagtggcgga tccaggattt 1320
ctgaaaaggg ggcccaaatg ggttttaacc acacttctca gccatttttg gtgcaaaatc 1380
tggaatttta ctagtaaatt tgtgaccaaa ggggggccct ctaaatccgc cactgaagac 1440
ctatggttat gtttgatttg agcaacagtg atcaatgtaa atatcactgc gtagaaggaa 1500
cacagttgag tatgtcagta ttgtattaat agacaatcaa atcaaatgcc gacacaagtt 1560
atttctatat ctagcagtca gtaatggatt tataaaagtg agtttgacat cagggacgtg 1620
tgttatatac gtccctgctt gacatataca aagagaatgt gtgattccct tgctcgactc 1680
ctaaatctgt ctatgataaa taaattattt ataagtagat tgaataaata aattaattaa 1740
taagtgccct gttttgacca cttatatatg ctgtatgaaa agcataaaaa ccaccctccc 1800
cctcttctcc ttcgctatta aagatgccaa gggcaaactt cacgattcat tgatcagcag 1860
gttgttgttt gttggtttgt ttgtttttcc tcacaaacac gttactcttt ctcgagttgt 1920
tcccctttat atgtgtatct gacgtctaaa agggactttc cactattgat tctatataaa 1980
gaatattcat cattgtctcg aaatttgatt ggttagaatg tatagtatca taaagtggtc 2040
gcgaccatct tgtaaacgat tgtacgttct aaattttgac aaataaagaa cgaaatgatg 2100
Claims (10)
1. The promoter of the Mitf gene of the Pteria penguin is characterized in that the nucleotide sequence of the promoter is shown as SEQ ID NO. 1.
2. A recombinant vector comprising the nucleotide sequence of the promoter of claim 1.
3. The recombinant vector according to claim 2, wherein the vector is the recombinant vector for the luciferase reporter gene driven by the promoter according to claim 1.
4. The recombinant vector according to claim 3, wherein the vector is pGL3-Basic fluorescein reporter gene vector.
5. A recombinant cell comprising the recombinant vector according to any one of claims 2 to 4.
6. The recombinant cell of claim 5, wherein the cell is a 293T cell.
7. The promoter of claim 1, the recombinant vector of any one of claims 2 to 4, and the recombinant cell of claim 5 or 6, for screening/identifying the transcription factor of Mitf gene of Pteria penguin.
8. A method for screening/identifying a transcription factor of a Mitf gene of Pteria penguin is characterized by comprising the following steps:
s1, constructing a recombinant vector containing a target gene to be identified;
s2, co-transfecting the recombinant vector obtained in the step S1 and the luciferase reporter gene recombinant vector as claimed in claim 3 or 4 to obtain cells, and collecting the cells to perform dual-luciferase reporter analysis;
and S3, judging results, if the luciferase activity of the cells over-expressing the target gene is higher than that of the control, indicating that the identified target gene is a transcription factor of the Mitf gene of the Pteria penguin.
9. The method of claim 8, wherein the expression vector used in step S1 is pcDNA3.1.
10. The method according to claim 8, wherein the negative control is a cell cotransfected with the empty vector pcDNA3.1 and the recombinant vector of claim 3 or 4 as determined in the determination of the result of step S3.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820453A (en) * | 2014-03-03 | 2014-05-28 | 中国科学院南海海洋研究所 | Pif promoter capable of being activated by pinctada martensii MSX (muscle segment homeobox) gene and application thereof |
| KR20160026180A (en) * | 2014-08-29 | 2016-03-09 | 건국대학교 산학협력단 | Use of STUB1 as MITF regulator and therapeutic agent for melanin pigmented disease |
| CN113201543A (en) * | 2021-05-06 | 2021-08-03 | 广东海洋大学 | Promoter of Pteria penguin Tyr gene and application thereof |
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- 2022-03-21 CN CN202210279043.2A patent/CN114517199B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820453A (en) * | 2014-03-03 | 2014-05-28 | 中国科学院南海海洋研究所 | Pif promoter capable of being activated by pinctada martensii MSX (muscle segment homeobox) gene and application thereof |
| KR20160026180A (en) * | 2014-08-29 | 2016-03-09 | 건국대학교 산학협력단 | Use of STUB1 as MITF regulator and therapeutic agent for melanin pigmented disease |
| CN113201543A (en) * | 2021-05-06 | 2021-08-03 | 广东海洋大学 | Promoter of Pteria penguin Tyr gene and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| YU F, 等: "Mitf Involved in Innate Immunity by Activating Tyrosinase-Mediated Melanin Synthesis in Pteria penguin", FRONT. IMMUNOL., vol. 12, pages 1 - 15 * |
| YU, F.F.等: "Pax3 Gene Regulated Melanin Synthesis by Tyrosinase Pathway in Pteria penguin", INT. J. MOL. SCI., vol. 19, pages 1 - 14 * |
| YU，F.F. 等: "Creb2 involved in innate immunity by activating PpMitf-mediated melanogenesis in Pteria penguin", FISH AND SHELLFISH IMMUNOLOGY, vol. 138, pages 1 - 10 * |
| 于非非 等: "丙戊酸通过Mitf通路促进企鹅珍珠贝黑色素合成", 水生生物学报, vol. 44, no. 5, pages 1105 - 1110 * |
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