JPH07107978A - Transriptional control factor gene of fish protamine gene - Google Patents

Abstract

PURPOSE:To provide a new gene coding for a transcriptional control factor of a fish protamine gene, promoting the maturation of spermatid of fish and useful for the efficient production of soft-roe of cultured fish for protamine raw material and the production of a hypoglycemic agent, antibacterial agent, etc. CONSTITUTION:A fresh soft-roe of rainbow trout is disintegrated with a homogenizer in a solution containing 4M guanidium thiocyanate, the total RNA is recovered from the homogenate and passed through an oligo-dT cellulose column to separate mRNA, a cDNA is synthesized by using the mRNA as a template and inserted into a phage vector lambdagt10 to prepare a cDNA library, the library is screened by using a probe based on the sequence of mammal SRY gene and a DNA is recovered from a positive clone and subjected to restriction enzyme treatment to obtain the objective new gene coding for a transcriptional control factor of a fish protamine gene, promoting the maturation of spermatid of fish and useful for the production of an agent for promoting the maturation of the soft-roe of cultured fish, a hypoglycemic agent, an antibacterial agent, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a gene encoding a transcriptional regulatory factor that promotes the expression of fish protamine gene.

[0002]

2. Description of the Related Art Protamine is a kind of protein and is classified into the most basic protein mainly obtained from the cell nucleus of animals. Protamine is mainly D in the nucleus of mature sperm cells of fish (salmon, trout, herring, shark, etc.).
It exists as nucleoprotamine linked to NA.

Regarding physiological functions, those related to blood pressure and respiration, effects on blood coagulation, effects on blood glucose, antibacterial effects,
Many studies have been conducted on the role of fertilization. Utilizing these effects, protamine has already been therapeutically applied as a hypoglycemic agent by mixing with insulin. The research as an antibacterial agent is also old, and there was a report in 1937 that the growth of staphylococci and lactic acid bacteria was inhibited by a dilute solution of protamine (200 to 12.5 ppm). Has been issued. A food preservative using salmon Shiroko has been developed.

[0004]

The availability of mature sperm cells of fish as a source of protamine is limited depending on the season and the amount of fish caught, and development of means for promoting spermatid maturation of fish has been desired.

[0005]

In order to investigate sex determination or differentiation in fish, the present inventor isolated SRY-related cDNA from a rainbow trout testis cDNA library. This cDNA encodes a protein containing HMGbox and leucine zipper motif, and is considered to have a transcriptional regulatory function. The present inventor succeeded in isolating a DNA encoding a transcriptional regulatory factor of the fish protamine gene, which is involved in promoting maturation of fish sperm cells, and completed the present invention. That is, the present invention provides a gene encoding a transcriptional regulatory factor of the fish protamine gene having the amino acid sequence represented by SEQ ID NO: 1.

In order to isolate the SRY-related cDNA of rainbow trout, the rainbow trout testis cDNA was templated to
In addition, the polymerase chain reaction method was performed using a primer based on the amino acid sequence of the HMGbox of the mammalian SRY gene. A fragment of approximately 200 bp in length was generated. When it was subcloned and the sequence was determined, H
It represented an amino acid sequence with 55% homology to MGbox and human SRY. Using this fragment as a probe, a cDNA clone containing a cDNA of about 3 kb in length was isolated from a rainbow trout testis cDNA library. The nucleic acid sequence is as SEQ ID NO: 2 and the deduced amino acid sequence is as SEQ ID NO: 1.

This cDNA clone (pRTSOX-L
Z1) is a 2301 bp long open reading frame (nucleic acid sequence 31
5-2616), which encodes a polypeptide of 767 amino acid residues and exhibits the following properties of a transcriptional regulator. (I) 4 leucine repeats every 7 amino acids (leucine zipper) (residues 132-15)
3): (ii) glutamine-rich 40 amino acid sequence (48%) (residues 170-209): (iii) HM
Gb0x (residues 503-581). The HMGbox-comprising peptide expressed in E. coli also showed a DNA binding specificity similar to that of human SRY.

Northern blot analysis of rainbow trout RNA revealed a testis-specific 3 kb long SOX-LZ transcript. RNA from fish of different ages was analyzed to determine at which stage in testis development a 3 kb long transcript appeared. The 3-kb long SOX-LZ transcript was not detected in 15-month-old fish. However, it was present in 18 month old fish and was even higher at 21 months. When the blot was secondary probed with protamine mRNA, which was known to initiate synthesis in the primary spermatocyte stage in trout, the expression patterns of the two mRNAs were identical.

The expression of the transcriptional regulatory factor of the fish protamine gene using the thus obtained gene of the present invention is
It may be incorporated into a known expression vector for higher animals and introduced into fish. In the fish into which the gene of the present invention has been introduced, the expression of the protamine gene is enhanced as a result of the expression of the transcriptional regulatory factor, and the maturation of the testis is promoted.

[0010]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited thereto.

Example 1 (Extraction of mRNA from rainbow trout testis): From 0.5 g of fresh rainbow trout testis, 4 M guanidinium thiocyanate, 0.5% sodium N-lauroylsarcosine, 2
5 mM sodium citrate (pH 7.0), 0.1M2
Trituration with a Teflon homogenizer in 10 ml of a solution containing mercaptoethanol. 4. The homogenate was passed through an 18 G syringe several times to disrupt the DNA, and then 5.
7M cesium chloride, 0.01M EDTA solution was overlaid in a Beckman SW40Ti ultracentrifuge tube, and ultracentrifuged at 35,000 rpm for 18 hours in a Beckman SW40Ti rotor to obtain total RNA as a precipitate. All R
NA is 20 mM Tris-HCl (pH 7.5), 0.5MN
It was dissolved in an aCl solution, treated at 65 ° C. for 10 minutes, passed through an oligo dT cellulose column, and the adsorbed mRNA having poly A was eluted with a buffer solution containing 10 mM Tris to obtain about 2 μg of mRNA.

Example 2 (Synthesis of cDNA and insertion into phage vector): cD
NA synthesis is performed by cDNA synthesis kit (Amersham)
Was used according to the protocol attached to the kit. That is, in 6.8 μl of an eluate containing 1 μg of mRNA prepared in Example 1, 4 μl of 5 × first-strand synthesis buffer contained in the above kit, 1 μl of sodium pyrophosphate solution, 1 μl of 25 units / ml human placental nuclease inhibitor, deoxyribonucleoside 3 Phosphoric acid mixture (dATP, dGTP, dCTP, dTT
P each 10, 10, 5, 10 mM) 4 μl, 2 mg / m
1 oligo (dT _12-18 ) primer 1 μl and 1
2.2 μl of 8 units / ml reverse transcriptase was added to make the total volume 20 μl, and the mixture was reacted at 42 ° C. for 40 minutes to synthesize DNA complementary to RNA.

Next, 20 μl of the obtained reaction solution was added to the second strand synthesis buffer 37. contained in the above kit.
5 μl, 0.8 unit of E. coli ribonuclease H, 23 units of E. coli DNA polymerase I and distilled water were added to make a total volume of 99 μl, and this was kept at 12 ° C. for 1 hour and then at 22 ° C.
After incubating for 1 hour at 70 ℃ for 10 minutes each,
0.25 MEDTA (pH 8.0) 10 μl and 10
The reaction was stopped by adding 10 μl of 10% SDS, followed by phenol-chloroform extraction and ethanol precipitation to obtain 2
50 μg of single-stranded cDNA was obtained.

The cDNA synthesized above was inserted into the phage vector λgt10 using a cDNA cloning system (manufactured by Amersham). That is, 13 μl of the cDNA aqueous solution obtained above was added to 4 μl of the 5 × EcoRI methylase reaction solution contained in the above system,
0.8 mM adenosylmethionine solution 2 μl, EcoRI
Add 1 μl of methylase (20 units) and mix the mixture at 37 ° C.
After the methylation reaction for 1 hour at 70 ° C., the enzyme was inactivated by heating at 70 ° C. for 10 minutes. To 20 μl of the reaction solution obtained, 3 μl of 10 × T4 DNA ligase buffer contained in the above system, and EcoRI linker 2 with phosphorylated ends
μl (1 μg), T4 DNA ligase 2 μl, and distilled water were added to make a total volume of 30 μl, and the mixture was kept warm at 15 ° C. for 16 hours to carry out an addition reaction of the EcoRI linker to the cDNA end. After the reaction, the reaction solution was heated at 70 ° C. for 10 minutes to deactivate the enzyme.

The reaction mixture thus obtained was mixed with 10 μl of 10 × EcoRI buffer contained in the above system and the restriction enzyme Eco.
Distilled water was added to RI 2 μl to make the total volume 100 μl.
After incubation at 7 ° C. for 3 hours, a cDNA having an EcoRI linker at the end was obtained. The obtained reaction solution was applied to the column included in the system, and 10 mM Tris-HCl (pH 7.
5) After elution with a buffer containing 1 mM EDTA and 100 mM NaCl to remove low molecular weight DNA,
The cDNA was recovered by ethanol precipitation.

To 6 μl of an aqueous solution containing 30 ng of the cDNA recovered above, 1 μl of 10 × ligase buffer attached to the system and 2 μl of λgt10 EcoRI arm (1 μl
g) and 1 μl of T4 DNA ligase (2.5 units) to make a total volume of 10 μl, which was incubated at 12 ° C. for 16 hours, followed by ethanol precipitation to obtain a cDNA-incorporated λg.
t10 DNA was obtained.

This DNA was dissolved in 2.5 μl and the E. coli strain BHB2688 [N205 rec attached to the system was dissolved.
A (λimm434 Clts b2 red3 Ea
m4Sam7) / λ] extract 10 μl and E. coli B
HB2690 [N205recA (λimm434 C
lts b2 red3 Dam15 Sam7) /
λ] extract solution (15 μl) was added, and inhitoro packaging was performed at 20 ° C. for 2 hours.
NaCl, 10 mM Tris-HCl (pH 7.5, 8 mM
A solution containing MgSO ₄ and 0.01% gelatin.
5 ml and 20 μl of chloroform were added to complete the encapsulation of the DNA in the phage.

Example 3 (Selection of Phage Containing Testis-Specific Transcription Regulator Sequence): Probe Preparation for Cloning Testis-Specific Transcription Factors Human, Mouse, Rabbit described in 1990 Nature. A probe was prepared by the following method for the purpose of cloning a transcription factor having an HMG-box and having an important function in sex determination from fish. Human,
Oligonucleotides 5'-CC (ACGT) ATGAA (CT) GC (AC for regions conserved in mouse and rat 3 species
GT) TT (CT) AT (ACGT) GT 5'-TA (CT) TT (AG) TA (AG) TT (A
Two types of DNA, CGT) GG (AG) AA (CT) TT, were synthesized using a DNA synthesizer type 381A (manufactured by Applied Biosystems), and the synthesized DNA was synthesized in the same manner as described above. CDNA prepared from the testis of rainbow trout was gene-amplified by the polymerase chain reaction method. The gene amplification method is the method of PNAS Yamashita et al. (Proc. Natl. Ac
ad. Sci. USA Vo189 2839-284
3, 1992). Result of gene amplification 20
A 0 bp long DNA fragment was obtained.

100 ng of this fragment digested with SmaI
Was mixed with a Bluescript IISK ⁺ digested product of Example ¹ and ligated using a ligation kit (Takara Shuzo) according to the protocol attached to the kit. Then, the ligated reaction solution was used to transform Escherichia coli JM109 strain (competent cell JM109, manufactured by Takara Shuzo) according to the attached protocol.

Transformed E. coli contained 2 ml of L-broth (1% bactotrimpton, 0.5% yeast extract,
0.5% NaCl aqueous solution), the mixture was cultured at 37 ° C. for 1 hour, and ampicillin 50 μg / ml, isopropyl-β-D-thiogalactoside 12.5 μg / ml, 5-bromo-4-.
L-broth containing 40 μg / ml of chloro-3-indolyl-β-D-galactopyranoside was added to 1.5% agar and solidified, and 100 μl was applied to each plate medium. The coated plate medium is incubated at 37 ° C for 16 hours,
White colonies formed on the plate were separated, and plasmid DNA was separated from the colonies.

Among the obtained transformants, the nucleotide sequence of PTS having an insertion fragment of 200 bp was analyzed by the Sanger method (J.S.
molecular Clonin such as ambrook
g, 2nd edition, pp13.6, Cold
Spring Harbor Laboratory
Press, 1989). DN obtained
The 66 amino acid sequence predicted from the A sequence and the amino acid sequence of human, mouse, and rabbit Sry genes are about 55.
Had a% homology. This increased the possibility that a part of the cDNA obtained from rainbow trout testis encoded a transcription factor. Therefore, the 200 bp D obtained here was obtained.
The previously prepared cDNA library was screened using NA.

Host cells NM514 were infected with the recombinant phage obtained in Example 2, and about 1 × 10 ⁵ plaques obtained were subjected to the method of Sambrook et al. (J. Sambr).
books, etc. Molecular Cloning, 2n
d edition, pp2.108, Cold Sp
ring Harbor Laboratory Pr
ess, 1989), and fixed on a nylon membrane (Hibond N, manufactured by Amersham).

Approximately 200 bp DNA obtained in Example 3
The fragment was labeled with ³² P using a random priming kit (manufactured by Amersham) and used as a probe. Sambr
methods such as J. Samook (J. Sambrook, Molec
ural Cloning, 2nd edition,
pp2.114, Cold Spring Harbo
r Laboratory Press, 1989), hybridization at 65 ° C. showed that one plaque had a strong binding ability to the probe. A large amount of phage DNA was prepared based on this plaque.

Example 4 (Determination of nucleotide sequence): 5 μg of the phage DNA obtained in Example 3 was dissolved in distilled water, and 20 μl of 10 × H-
Buffer (Toyobo) and 50 units of restriction enzyme Eco
Add RI and bring the total volume to 200 μl with distilled water.
The reaction was carried out for 3 hours, the DNA was recovered by ethanol precipitation, and 20 μl of TE buffer (10 mM Tris base p
H7.5, 1 mM EDTA). On the other hand, an aqueous solution of the plasmid vector BluscriptIISK ⁺ was similarly digested with EcoRI, recovered by ethanol precipitation, and dissolved in 20 μl of TE buffer. EcoRI fragment solution and Blues of the above phage DNA
The digestIISK ⁺ EcoRI digested product solution was bound using a ligation kit (Takara Shuzo) according to the protocol attached to the kit.

Then, using the combined reaction solution, E. coli J
The M109 strain (competent cell JM109, manufactured by Takara Shuzo) was transformed according to the attached protocol. The transformed Escherichia coli was cultured in 2 ml of L-broth (1% bactotrimpton, 0.5% yeast extract, 0.5% NaCl aqueous solution) at 37 ° C. for 1 hour to obtain 50 μg of ampicillin.
/ Ml, isopropyl-β-D-thiogalactoside 1
To L-broth containing 2.5 μg / ml and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside 40 μg / ml, 1.5% agar was added to a plate medium which was solidified, 100 μl was applied per sheet. The coated plate medium was cultured at 37 ° C. for 16 hours to separate white colonies formed on the plate, and plasmid DNA was separated from the colonies. The plasmid obtained by the above
It was named RTSOX-LZ1.

Next, the entire nucleotide sequence of the pRTSOX-LZ1 cDNA was analyzed by the Sanger method (J. Sambrook et al., Mo.
regular Cloning, 2nd editi
on, pp13.6, Cold Spring Har
bor Laboratory Press, 198
9). The results are as shown in SEQ ID NO: 1. The eukaryotic m
There is a poly A chain that is generally present in RNA, and there is an ATAAA sequence that seems to be a poly A addition code upstream of it. In addition, the base sequence contained a translation region (SEQ ID NO: 1) encoding a 767 amino acid sequence.

The SOX-LZ gene cloned this time has sry-box as a DNA binding region.
A consensus DNA binding sequence that is known to be recognized by the DNA binding regions of several transcription factors having this Sry-box has been AACAAT. When such a regulatory region of DNA is present on the promoter, it is highly possible that such (SOX-related gene) gene regulates the expression of that gene.

In fact, there are two sequences of AACAAT in the upstream region of the protamine (rainbow trout) gene.
It was confirmed that this region and the SOX-LZ protein produced in E. coli formed a DNA-protein complex in vitro. From this result, it is considered that this SOX-LZ protein may control the gene expression of protamine.

Also, Northern blotting
From the analysis, it was found that the expression timing of SOX-LZ and protamine gene is strongly expressed from the middle stage to the latter stage of the testis maturation process, and it is well co-related. From this, the mechanism of SOX-LZ and protamine expression is inferred.

[0030]

EFFECTS OF THE INVENTION When the gene of the present invention is used, the transcriptional regulatory factor of the fish protamine gene is expressed, resulting in promotion of maturation of sperm cells of fish and facilitation of availability of aquacultured salmon such as rainbow trout as a raw material for protamine. .

[0031]

[Sequence list]

[0032]

Claims (4)

[Claims] 1. A gene encoding a transcriptional regulatory factor of a fish protamine gene having the amino acid sequence represented by SEQ ID NO: 1. 2. The gene according to claim 1, which has the nucleotide sequence represented by SEQ ID NO: 2. 3. The gene according to claim 1, wherein the fish is salmon. 4. The gene according to claim 1, wherein the fish is rainbow trout.