CN110982823B

CN110982823B - Paralichthys olivaceus oocyte specific gene figla and application thereof

Info

Publication number: CN110982823B
Application number: CN201911260186.3A
Authority: CN
Inventors: 梁少帅; 尤锋; 王雯祥; 王丽娟; 吴志昊; 邹玉霞; 谭训刚
Original assignee: Institute of Oceanology of CAS
Current assignee: Institute of Oceanology of CAS
Priority date: 2019-12-10
Filing date: 2019-12-10
Publication date: 2022-09-06
Anticipated expiration: 2039-12-10
Also published as: CN110982823A

Abstract

The invention discloses a paralichthys olivaceus oocyte specific gene figla and application thereof, wherein the nucleotide sequence is SEQ ID NO. 1, and the amino acid sequence is SEQ ID NO. 2. The invention clones the figla gene from the paralichthys olivaceus for the first time, designs a probe according to the cDNA sequence of the figwort gene, can specifically mark the oocyte by analyzing the probe through an in-situ hybridization method, and can be used for identifying the oocyte. The invention further discloses an application method for inhibiting the gene expression and directly influencing the oocyte development by injecting the flgla-dsRNA into the living ovary, and the effect of the method is that after the dsRNA is injected, the evaluation is carried out by observing through the histology cytology and combining the quantitative and the positioning gene expression change. The technical method can be used for the functional research of the gene in the ovarian oocyte of the paralichthys olivaceus and can also be popularized and applied to other fishes.

Description

Paralichthys olivaceus oocyte specific gene figla and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and relates to a paralichthys olivaceus oocyte specific gene figla and application thereof.

Background

The Basic helix-loop-helix (bHLH) protein is a bHLH superfamily transcription factor, and plays an important role in the development processes of sex determination, cell differentiation, nervous system development, cholesterol metabolism and the like. The factor in The germ cell line alpha (FILLA) is one of The members of this family, and studies have shown that FIGLA plays an important role in mammalian ovarian development. However, much of the figla gene reported in teleost is generally involved in qPCR expression analysis, and some reports have used in situ hybridization for histological cytological mapping studies. The functional research of the gene is few in fishes, only reports in model fish species zebra fish and freshwater economic fish tilapia are analyzed by microinjection, and the figla is found to be important for ovary development, but the influence on oocyte proliferation is not clear. Although the breeding cycle of the zebra fish as a model fish species is short, the zebra fish also needs 2-3 months, so that a second generation individual of homozygote needs a long time; the economic fish has a longer breeding cycle, generally 2-4 years, and the microinjection method has a longer cycle, so that the process of gene function research and application is directly influenced. The flounder as seawater economic fish has a long reproductive cycle, and particularly, although a microinjection method is tried in the floating eggs of the seawater fish, the technology is unstable, the efficiency is low, and therefore the flounder cannot be widely used at present. A simple and easy technical means is urgently needed for the research of gene functions.

RNA interference is a technique of inhibiting the expression of a target gene by inducing degradation of homologous mRNA by double-stranded RNA (dsrna). The technology is widely used for gene function research of species such as insects, shrimps, crabs and shellfish, and has obvious effect. In fish, RNA interference is involved in the research of zebra fish embryonic development genes, but only Odontobutis and Channa striatus on fish living bodies have reports of interference of target genes through intramuscular injection and intraperitoneal injection of Furui carp. The research on RNA interference of figla gene is only reported on the cynoglossus semilaevis cell level and is realized by transfecting siRNA to a pseudo-male fish spermary cell line, and the change of the gene transcription level shows that the cynoglossus semilaevis can interfere a target gene. However, the study of the function of a target gene by directly injecting dsRNA into a target tissue (gonad) has not been reported.

Paralichthys olivaceus is an important marine fish culture variety in China, and has the advantages of delicious meat, rich nutrition and higher economic value. The breeding of the paralichthys olivaceus is inseparable from the development of ovaries and the generation of ova, and the quantity and the quality of the ova can directly influence the artificial breeding and the culture yield, so that the research on the specific gene function of the paralichthys olivaceus oocytes has important significance on the breeding of the paralichthys olivaceus and the development and maturity promotion of the ovaries. Meanwhile, since female flounder individuals grow faster than male individuals, and higher economic benefits can be obtained by full-female culture, the sex of flounder can be researched, specific genes and functions of ovary germ cells can be explored, and the method has important significance for research and culture application of fish sex control and gonad development and maturation mechanisms.

Disclosure of Invention

The invention aims to provide a paralichthys olivaceus oocyte specific gene figla and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

a nucleotide sequence of the paralichthys olivaceus oocyte specific gene figla is shown as SEQ ID NO. 1.

The amino acid sequence of the flounder oocyte specific gene figla coding sequence is shown in SEQ ID NO. 2.

An application of a paralichthys olivaceus oocyte specific gene figla, in particular to an application of the paralichthys olivaceus oocyte specific gene figla in identifying oocytes.

A probe of the specific gene figla of the oocyte of the paralichthys olivaceus, the nucleotide sequence of the probe is shown as SEQ ID NO. 3. For specific labeling and identification of oocytes;

a gene segment for inhibiting the expression of the specific gene figla of the oocyte of the paralichthys olivaceus, a figla-dsRNA for inhibiting the expression of the specific gene figla of the oocyte of the paralichthys olivaceus, the nucleotide sequence of which is shown in SEQ ID NO. 4.

A preparation method of flgla-dsRNA for inhibiting the expression of flgla of a specific gene of oocyte of paralichthys olivaceus comprises the following steps:

(1) extracting total RNA of the ovary of the paralichthys olivaceus, and performing reverse transcription to obtain cDNA;

(2) screening the flgla cDNA siRNA sites, selecting flgla cDNA fragments according to the siRNA sites, confirming the specificity of the flgla cDNA fragments through Blast comparison, using the fragments as templates, carrying out PCR amplification by using specific upstream and downstream primers SEQ ID NO. 5 and SEQ ID NO. 6, and recovering target fragments to carry out in-vitro transcription to synthesize the flgla-dsRNA.

The primer is as follows:

forward primer SEQ ID NO: 5:

5’-TAATACGACTCACTATAGGGAGAGGGTTCATCCTCAGGGTTCATAC-3’

reverse primer SEQ ID NO: 6:

5’-TAATACGACTCACTATAGGGAGCAGTCTGACCATAAGTGATTGC-3', the T7 promoter sequence is underlined.

15 and 30 mu g of figla-dsRNA are injected into the ovary part of the juvenile flounder which is cultured conventionally in a living body, and 30 mu g of EGFP-dsRNA is injected into the ovary part of the control group in a living body for 3 times, wherein the injection time is respectively recorded as d1, d8 and d 20.

The inhibition of Figla expression was detected as follows: collecting ovarian tissues after d3, d10 and d27 after the injection of the figla-dsRNA, detecting the expression change of the figla gene and the development change of the ovary by adopting a method combining fluorescence quantitative PCR, in situ hybridization and tissue cytology observation, and determining the effect of the gene on the development of the oocyte.

The invention has the advantages that:

the invention clones and obtains the figla gene from the paralichthys olivaceus for the first time, obtains the specific probe of the figla gene according to the cDNA sequence of the figwort gene, can specifically mark the oocyte by analyzing through an in-situ hybridization method, and can be used for identifying the oocyte. The invention also further discloses an application method for inhibiting the gene expression and directly influencing the oocyte development by injecting the flgla-dsRNA into the living ovary, the effect of the method is that after the dsRNA is injected, the dsRNA is evaluated by observing tissue cytology and combining quantitative and positioning gene expression change, the dsRNA injected by the invention can reduce the target gene expression quantity, and the target gene expression efficiency of an experimental group is reduced by 50%; through ovary histology and in-situ hybridization analysis, the number of the oocytes in the experimental group is obviously less than that of the oocytes in the control group, and the hybridization signal intensity of the oocytes in the experimental group is weaker than that of the oocytes in the control group, so that the fact that the expression of the oocytes can be inhibited and the normal development of the oocytes can be influenced by injecting the figla-dsRNA into the living ovaries is shown.

The technical method can be used for the functional research of the gene in the ovarian oocyte of the paralichthys olivaceus and can also be popularized and applied to other fishes.

Drawings

FIG. 1 is a representation of the cytological localization of flounder figla in stage III ovaries provided in example 1 of the present invention; a, positive probe result; b, negative probe result. II, phase 2 oocytes; III, phase 3 oocytes. Og, oogonia; oc, oocyte. Bar, 50 μm.

FIG. 2 is a quantitative PCR detection map of the paralichthys olivaceus oocyte specific gene figla after knockdown according to embodiment 2 of the present invention; a and b, indicating that there is a significant difference (P < 0.05).

FIG. 3 is a histological observation and in-situ hybridization pattern of the paralichthys olivaceus oocyte specific gene figla after knockdown, provided in example 2 of the present invention; wherein, the A-C, d10 control group and the experimental group are observed by ovary histology; ovarian histological observations of the D-F, D27 control and experimental groups; G-I, d27 control and knockdown groups of ovaries were hybridized in situ. Oc, oocyte; bar, 50 μm.

Detailed Description

The embodiments of the present invention will be further described with reference to the accompanying drawings and examples, it being understood that the embodiments described herein are merely for purposes of illustration and explanation and are not intended to limit the invention.

The cDNA of the paralichthys olivaceus oocyte specific gene figla and the probe thereof are used for specifically marking and identifying oocytes; knocking down the specific gene figla of the paralichthys olivaceus oocyte can inhibit the expression of the specific gene and influence the development of the oocyte, so that the evaluation index after inhibiting the expression of the gene is obtained.

Example 1 cloning of Paralichthys olivaceus figla Gene and its use in oocyte specific expression

1. Cloning of genes

1.1 extracting and reverse transcribing RNA of the ovarian tissue of the paralichthys olivaceus: collecting ovarian tissue of Paralichthys olivaceus, extracting RNA by Trizol method, and detecting RNA quality, Nanod, by 1.2% agarose gel electrophoresisAfter RNA concentration is determined by rop2000, storing at-80 ℃ for later use; using SMART ^TM RACE cDNA Amplification Kit (Clontech), the ovarian RNA was inverted to cDNA template according to the instructions.

1.2 Gene cloning and sequence analysis: obtaining a partial mRNA sequence of the flounder figla gene according to a flounder gonad transcriptome sequencing result, wherein the length of the partial mRNA sequence is 693 bp; designing a pair of specific primers according to the sequence

figla-S:5’-GCACCTCCTCACGGTCCAAAC-3’

figla-AS:5’-CAAGCCATCTGCTCTACACATCA-3’

The ovarian cDNA template obtained in 1.1 is adopted, amplified by RACE, the obtained product is detected by 1.2 percent agarose gel electrophoresis, and the target band is cut into gel and recovered, and sent to a sequencing company for sequencing. The sequencing primer and the vector sequence are removed by EditSeq, and the sequencing sequence is spliced with the known sequence by SeqMan to obtain the full-length cDNA shown as SEQ ID NO. 1. The Open Reading Frame (ORF) was searched and translated by EditSeq to obtain the amino acid sequence shown in SEQ ID NO: 2.

1 (the full-length cDNA sequence of Paralichthys olivaceus figla, the underlined part is the effective length 309-914):

2 (amino acid sequence encoded by flounder figla):

2. gene mapping expression

2.1 ovarian tissue RNA extraction and reverse transcription: the RNA extraction procedure was as described in 1.1 of example 1; extracting total RNA with PrimeScript ^TM The RT reagent Kit reverse transcribes cDNA.

2.2 Probe Synthesis: selecting fragments in the figla cDNA sequence, and designing a pair of specific primers after confirming the specificity of the fragments through Blast comparison:

upstream primer

5’-ATTTAGGTGACACTATAGAAGCGGCTGAGGACTTCAACGAAACGGT-3' (underlined SP6 promoter sequence)

Downstream primer

5’-TAATACGACTCACTATAGGGAGACAAGCCATCTGCTCTACACAT-3' (underlined T7 promoter sequence)

PCR amplification is carried out on a 10-microliter reaction system, the obtained product is subjected to 1.2% agarose gel electrophoresis, a target fragment is recovered by gel, and sequencing is carried out to confirm the correctness of the sequence, wherein the sequence is SEQ ID NO. 3. After the sequencing is correct, PCR amplification and gel recovery are carried out through a 60 mu l reaction system, a DIG RNA Labeling Mix kit is used for carrying out in-vitro transcription synthesis on a gel recovery target fragment according to the steps of the instruction, a figla specific probe is placed at the temperature of minus 80 ℃ for storage, and the nucleic acid sequence of the probe is shown as SEQ ID NO. 3.3 (Figla specific probe sequence):

2.3 in situ hybridization of ovarian tissue:

2.3.1 sample embedding and section preparation: fresh ovarian tissue pieces 1cm ³ Put into Daviddions' stationary liquid (500ml stationary liquid component: 156.75ml absolute ethanol, 110ml formaldehyde, 57.5ml glacial acetic acid, 175.75ml distilled water), and after overnight at 4 ℃, the solution is changed to 70% ethanol for storage. Dehydrating the tissue block in 70% ethanol with gradient ethanol (85% → 95% → 100%), dehydrating the tissue block, putting the dehydrated tissue block into xylene for transparency, putting the tissue block into paraffin for wax immersion embedding after the tissue block is transparent, slicing the tissue block after the wax block is cooled and solidified, wherein the thickness of the tissue slice is 5 micrometers, spreading the tissue slice, and then baking the tissue slice at 60 ℃.

2.3.2 in situ hybridization: the whole experimental process ensures no RNA enzyme pollution.

The first day: the tissue sections baked overnight were taken out, cleared by xylene, rehydrated with gradient ethanol (100% → 85% → 70% → 50% → 30%) and rnase-free water, rinsed 2 times with phosphate buffer + 0.1% tween 20(PBT), digested for 30min with PBT solution containing 2 μ g/ml proteinase K, rinsed twice with PBT after digestion, fixed for 30min with 4% paraformaldehyde after rinsing, incubated for 3h in 60 ℃ prehybridization solution (1ml prehybridization solution is 500 μ l deionized formamide, 250 μ l 20 × sodium citrate buffer (SSC), 200 μ l blocking reagent, 3 μ l yeast total RNA, 1 μ l tween 20, 46 μ l rnase-free water), and incubated for 16h at 60 ℃ with hybridization solution containing 100ng/μ l figla-specific probe.

The next day: the tissue sections were washed 2 times at 60 ℃ in the following order of hybridization solution/2 XSSC (1:1), 2 XSSC, 1 XSSC, 0.2 XSSC (containing 0.1% Tween 20), each for 20 min. Then rinsing twice with maleic acid at room temperature, blocking for 2h with blocking buffer (blocking reagent/maleic acid, volume ratio 1:10), adding digoxin antibody into blocking buffer according to volume ratio 1:2000, and incubating for 16h at 4 ℃.

And on the third day: the above incubated tissue sections were taken out and subjected to the experiment at room temperature, and the tissue sections were rinsed 6 times with maleic acid. After washing, the tissue was rinsed twice with an antibody diluent, and 20. mu.l of NBT/BCIP was added to 1ml of the antibody diluent to prepare a developer, and the developer was uniformly dropped on the tissue to develop color in a closed state. And (3) rinsing twice with PBT after the color development is finished, and rinsing twice with PBT after 4% paraformaldehyde is fixed for 30 min. After counterstaining with 1% neutral red staining solution for 30min, dehydrating with gradient (70% → 95% → 100%) ethanol, sealing with neutral gum after xylene is transparent, and observing under microscopic examination after the tablet is dried (see fig. 1).

The results are shown in FIG. 1, showing that figla is specifically expressed in oocytes, so that a figla-specific probe can be used to identify oocytes.

Example 2 application of Figla-dsRNA to inhibition of Paralichthys olivaceus oocyte specific gene Figla expression

Preparation of dsRNA

1.1 ovarian tissue RNA extraction and reverse transcription: the RNA extraction and reverse transcription procedure was as described in 2.1 of example 1.

1.2 Figla-dsRNA synthesis: screening the siRNA sites in the website http:// siderect2. rnai. jp/screening the siRNA sites in the full-length Figla cDNA sequence, selecting partial fragments from the Figla cDNA according to the siRNA sites, and designing a pair of specific upstream and downstream primers after confirming the specificity through Blast comparison:

SEQ ID NO:5

5’-TAATACGACTCACTATAGGGAGAGGGTTCATCCTCAGGGTTCATAC-3’

SEQ ID NO:6

5’-TAATACGACTCACTATAGGGAGCAGTCTGACCATAAGTGATTGC-3' (underlined is the T7 promoter sequence)

Performing PCR amplification of a 10 mu l reaction system, performing electrophoresis on the obtained product, recovering a target fragment by gel, and determining the correctness of the sequence through sequencing, wherein the sequence is SEQ ID NO. 4, namely figla-dsRNA.

After the sequencing is correct, PCR amplification and gel recovery are carried out through a 60-microliter reaction system, and the target fragment is recovered by gel and is transcribed in vitro to synthesize the figla-dsRNA by using a T7 RNAi Transcription kit. SEQ ID NO 4(Figla-dsRNA sequence):

2 DsRNA injection

2.1 raising of the paralichthys olivaceus: culturing Paralichthys olivaceus (100g + -20 g) at 1m ³ Feeding commercial baits for 2 times every day in a culture barrel;

2.2 setting the experimental and control groups: the cultured 42 female paralichthys olivaceus were divided into 2 experimental groups and 1 control group. Wherein EGFP-dsRNA is injected into a control group, and figla-dsRNA is injected into 2 experimental groups;

2.3 injection concentration and time: EGFP-dsRNA was dosed at 30. mu.g, figla-dsRNA at 15 and 30. mu.g. The ovaries were injected 3 times in a month, with the injection times recorded as d1, d8 and d 20.

3 detection of injection Effect

3.1 fluorescence quantification: the gene expression change is detected by adopting a conventional fluorescence quantitative method, and beta-actin of the paralichthys olivaceus is taken as an internal reference gene. The d3 ovarian RNA extraction and reverse transcription procedure were as described in 2.1 in example 1.

The Figla specific primers are:

figla-qRT-S:5’-GTTTGAGCAATGACCTATGG-3’

figla-qRT-AS:5’-TCCTCTGTTACTGGTTCTGG-3

the beta-actin specific primer is as follows:

β-actin-qRT-S:5’-GGAATCCACGAGACCACCTACA-3’

β-actin-qRT-AS:5’-CTGCTTGCTGATCCACATCTGC-3’

the reaction conditions are as follows: the results are shown in FIG. 2 for 40 cycles of 95 ℃ 30s, then 95 ℃ 30, 60 ℃ 30 s.

3.2 HE staining: histological cytology of d10, d27 ovaries using HE staining and paraffin section preparation procedure as described in 2.3.1 of example 1. The HE staining adopts the conventional steps, paraffin sections are transparent through xylene, are rehydrated through gradient ethanol (100% → 95% → 80% → 50%) and tap water, are rehydrated through hematoxylin staining for 2min, then are washed through tap water for 5min and are rewashed through eosin for 1min, then are dehydrated through 95% ethanol and 100% ethanol, and are transparent through xylene, finally, the sheets are sealed through neutral gum, and are observed under a microscope after being dried in the air, and the results are shown in fig. 3 (A-F).

3.3 tissue in situ hybridization: in situ hybridization was performed on d27 ovarian tissue using in situ hybridization as described in example 1, 2.3. The results are shown in FIG. 3 (G-I).

From FIG. 2, it was confirmed that the expression level of Figla in the experimental group (Figla-dsRNA1 and Figla-dsRNA 2) was significantly decreased compared to the control group (EGFP-dsRNA) based on the fluorescent quantitative analysis, and that the expression of the gene could be inhibited by injection of the Figla-dsRNA. FIG. 3(A-F) HE staining shows that there is no difference between the d10 ovaries of the experimental group (Figla-dsRNA1 and Figla-dsRNA 2) and the control group (EGFP-dsRNA), and the number of oocytes in the d27 ovaries of the experimental group is significantly reduced compared with the control group; in situ hybridization (FIG. 3, G-I) showed that the hybridization signal intensity of the remaining oocytes of ovaries of d27 experimental group was weaker than that of the oocytes of control group, indicating that the expression of figla could be inhibited by RNA interference and the oocyte reduction was induced, therefore, it was confirmed that the figla had an important role in oocyte proliferation. The method can be applied to the functional research of the oocyte of the paralichthys olivaceus.

Sequence listing

<110> oceanographic institute of Chinese academy of sciences

<120> Paralichthys olivaceus oocyte specific gene figla and application thereof

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tagcatcagc tcactgcggg caggtagcgg gctcctgatg aagcacctgt ctcaggtgtg 180

ctcagcctca tgcctctgct gtttacgtgt gagaggtgag cggagacaga gactgtctgt 240

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aagctgcaac tgaatacatt cgattgcttg ttggagtttt gcaagacact gacagtcatg 660

atggcagtgg cactgatttc ctaaagaatg caatcactta tggtcagact gaaggtttga 720

gcaatgacct atggagaatg gatgatcttt tgaacatgtc agatgagtgc atggaagatg 780

gattcactct gcctccagaa ccagtaacag aggacggaga catgaccaga ctggtgttgc 840

aacattgtgc aatgcctgca taccagttca tcatccaagt agcacctgac cagacttcga 900

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cttcattttt tttctcattt ctatttagaa gtgttttatg ttttatgctt agtatgttta 1200

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tcacaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 1316

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Met Phe Leu Leu Lys Met Lys Val Pro Ala Glu Glu Leu Met Gly Asp

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Ile Leu Lys Arg Leu Thr Gly Glu Ser Ala Leu Pro Val Tyr Ser Asn

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Ile Glu Lys Phe Arg Arg Ala Lys Asp Gly Leu Tyr Phe Val Ala Glu

35 40 45

Asp Phe Asn Glu Thr Val Lys Lys Arg Glu Leu Val Asn Ala Lys Glu

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Arg Leu Arg Ile Arg Asn Leu Asn Thr Met Phe Ser Arg Leu Lys Arg

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Met Val Pro Leu Met Arg Pro Asp Arg Lys Pro Ser Lys Val Asp Thr

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Leu Lys Ala Ala Thr Glu Tyr Ile Arg Leu Leu Val Gly Val Leu Gln

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Asp Thr Asp Ser His Asp Gly Ser Gly Thr Asp Phe Leu Lys Asn Ala

115 120 125

Ile Thr Tyr Gly Gln Thr Glu Gly Leu Ser Asn Asp Leu Trp Arg Met

130 135 140

Asp Asp Leu Leu Asn Met Ser Asp Glu Cys Met Glu Asp Gly Phe Thr

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Leu Pro Pro Glu Pro Val Thr Glu Asp Gly Asp Met Thr Arg Leu Val

165 170 175

Leu Gln His Cys Ala Met Pro Ala Tyr Gln Phe Ile Ile Gln Val Ala

180 185 190

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195 200

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acagaggacg gagacatgac cagactggtg ttgcaacatt gtgcaatgcc tgcataccag 420

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agggttcatc ctcagggttc atacatgttc ctgctcaaaa tgaaggtgcc agcggaggaa 60

ctaatgggcg acatattgaa gcgtctgacg ggcgagtccg cgctgcctgt gtacagcaac 120

atagagaagt tcagacgagc taaagacggc ctgtacttcg tcgctgagga cttcaacgaa 180

acggtgaaaa agagggagct ggtcaacgcc aaggagcggc tgagaatccg gaacttgaac 240

actatgttct cccgcttgaa gcgtatggtg ccactaatgc gaccagaccg gaaacccagt 300

aaagtggaca cactcaaagc tgcaactgaa tacattcgat tgcttgttgg agttttgcaa 360

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cagactg 427

Claims

1. Paralichthys olivaceus oocyte specific genefiglaThe application of (2), which is characterized in that: paralichthys olivaceus oocyte specific genefiglaThe application in identifying oocytes;

paralichthys olivaceus oocyte specific genefiglaThe nucleotide sequence of (A) is shown as SEQ ID NO. 1.