CN102241761A - Nile tilapia cholecystokinin and coding nucleic acid thereof and application of functional octopeptide - Google Patents

Abstract

The invention provides Nile tilapia cholecystokinin and a nucleic acid sequence, a cloning method and application thereof. In the invention, the amino acid sequence for coding the Nile tilapia cholecystokinin is shown as SEQ ID NO:1. The invention also provides application of a CCK(Cholecystokinin)-8 octopeptide sequence in digestion ingestion, and the fact that the CCK-8 polypeptide can promote pyloric caecum to secrete amylase, trypsin and pepsinogen and promote hypothalamus to secrete NPY (neuropeptide Y) and orexin is discovered after being abdominally injected into tilapia. The invention provides a theoretical basis for clarifying an effect of CCK in a fish digestion and ingestion process and provides some gene resources for developing novel fish feed additives in the future.

Description

The application of a kind of bolti cholecystokinin and coding nucleic acid thereof and function octapeptide

Technical field

The present invention relates to new albumen and the coding nucleic acid thereof of a kind of bolti, simultaneously, the invention still further relates to the application of this new proteic octapeptide residue.

Background technology

Tilapia ( Tilapia), be commonly called as African crucian, be under the jurisdiction of Perciformes, Percoidei, Callichthyidae Cichlidae, tilapia and belong to Tilapia (also claim beautiful porgy section, beautiful porgy belongs to).Tilapia is as the substitute of cod, and the demand of world market constantly increases.In global fresh-water fishes trade, tilapia accounts for the 3rd, is only second to salmon and trout, has high economic worth.Along with the continuous expansion of breed and export volume, the requirement that bait is disposed also improves constantly, because feed cost constantly increases, research trend is in reducing cost with the vegetable-protein substituted fish meal.But fish are taken in plant protein fodder, the phenomenon that food ration reduces, loses weight can occur, and this result and original intention are runed counter to.Yet, the tilapia regulation mechanism of ingesting is not studied clear as yet, though many peptide classes are proved and can influence fish and ingest, as NPY, orexin, ghrelin, melanochrome acrasin (melanin concentration hormone, the short factor of ingesting such as MCH), bombesin (bombesin, BBS), the factor but leptin etc. ingest.But, ingest also not deeply for hormone regulation in the fish.

(Cholecystokinin CCK), is a kind of by small intestinal mucosa I cell and axoneure excretory braingut petide to cholecystokinin.It is by single-gene coding in the human genome, the precursor protein that produces can cut into various ways, extensively be present in gi tract, maincenter and peripheral nervous system, and participate in regulating important physical effects such as pain sensation impression, appetite and pituitrin release with the form of neurotransmitter or neuromodulation, can promote pancreatin secretion and gall-bladder to shrink at Digestive tract, slow down stomach emptying, strengthen many-sided functions such as small intestine and colon motion.In view of the critical function of CCK in Mammals, more scholars pay close attention to the function of CCK in fish, in multiple fish, cloned CCK cDNA at present, there is other form A CK cDNA precursor in discovery in fish, confirm that fish CCK has the function similar with higher animal, as suppress food intake, and promote pancreatin secretion and gall-bladder to shrink, short-term promotes growth hormone secretion etc.

In the existing research, find that can regulate tilapia ingests and the gastro-intestinal digestion small peptide.

Summary of the invention

One object of the present invention is to provide a kind of bolti cholecystokinin.

Another object of the present invention is to provide a kind of coding above-mentioned proteic nucleotide sequence.

A further object of the present invention provide above-mentioned bolti cholecystokinin octapeptide residue regulate that tilapia ingests and gastro-intestinal digestion in application.

The technical solution used in the present invention is:

A kind of bolti cholecystokinin, its aminoacid sequence is shown in SEQ ID NO:1.

The encode nucleotide sequence of above-mentioned bolti cholecystokinin, special, its sequence is shown in SEQ ID NO:2.The cloning process of this sequence comprises the steps:

Extract total RNA of the full brain of bolti, reverse transcription obtains cDNA; Method by RT-PCR and RACE obtains the cDNA total length, the last cDNA that obtains with reverse transcription is a template, with CCK-2-QF:5 '-GATCTCTCCCTTCTCCCAGCC-3 ' (SEQ ID NO:4) and CCK-2-QR:5 '-TAATCTCTGTCCTTTATCCTGTG-3 ' (SEQ ID NO:5) is primer, and amplification obtains its opening code-reading frame.

The terminal octapeptide DYLGWMDF(SEQ of the C-of bolti cholecystokinin ID NO:3) regulate that tilapia ingests and gastro-intestinal digestion in application.

The invention has the beneficial effects as follows:

Novel bolti cholecystokinin provided by the invention and nucleic acid sequence encoding thereof are provided fundamental basis for illustrate the effect that CCK taken place in the fish digestion and the process of ingesting, and provide some genetic resourceses for developing the fish novel fodder additive in the future.

The terminal octapeptide DYLGWMDF of C-is behind abdominal injection, can stimulate ingest in tilapia pyloric caecum amylase, trypsinase and propepsin and the brain factor NPY and orexin expression, have and regulate the dual function of ingesting with gastro-intestinal digestion, be expected to as a kind of novel fodder additives, make tilapia can more effectively utilize vegetable-protein, and then reduce the production cost of tilapia.

Description of drawings

Fig. 1 is a CCK-2 intermediate segment clone electrophoresis result;

Fig. 2 is CCK-2 a 3 ' RACE clone electrophoresis result;

Fig. 3 is CCK-2 a 5 ' RACE clone electrophoresis result;

Fig. 4 is a CCK-2 ORF clone electrophoresis result;

Fig. 5 is amylase, trypsinase, propepsin cDNA part fragment cloning electrophoresis result;

Fig. 6 is amylase, trypsinase, propepsin solubility curve result;

Fig. 7 is amylase mRNA expression level result;

Fig. 8 is Trypsin mRNA Expression level result;

Fig. 9 is stomach en-original mRNA expression level result;

Figure 10 is NPY and orexin part fragment cloning electrophoresis result;

Figure 11 is NPY and orexin solubility curve result;

Figure 12 is NPY mRNA expression level result;

Figure 13 is orexin mRNA expression level result.

Embodiment

The present invention takes RT-PCR and RACE technology to clone.At first utilize the product Trizol Reagent of Invitrogen company to carry out the extraction of the total RNA of full brain, utilize the product Rever Tra Ace-a-Tm First Strand cDNA Synthesis Kit of Toyabo company to carry out RT-PCR again, carry out the segmental clone of this gene cDNA part with the degenerated primer that designs at last.After obtaining the part fragment, utilize TaKaRa company's T dT tailing test kit to add the preceding paragraph dCTP, design the terminal special primer of cDNA5 ', and carry out 5 ' RACE with joint primer AAU and AUAU at cDNA3 ' end.Utilize product SMART 3 ' the RACE ThermoScript II SMARTScribe Reverse Transcriptase of CLONTECH company to carry out reverse transcription, utilize joint primer UPM(to comprise Long-UPM and Short-UPM) carry out 3 ' RACE.At 3 ' and 5 ' non-coding region design special primer, carry out the clone of opening code-reading frame ORF at last, determine sequence.

Below in conjunction with embodiment, further specify the present invention.

The clone of bolti CCK-2 cDNA

(1) design of primers

(previous experiments obtains acquired bolti CCK-1 sequence, delivered GenBank:HM222539.1), compare with salmon, black blue spot filefish, rainbow trout sequence, but find salmon, black blue spot filefish, the rainbow trout homology the is higher Position Design degenerated primer lower with the CCK-1 homology, according to acquired intermediate segment, design CCK-2 RACE special primer.

(2) bolti CCK-2 cDNA intermediate segment clone

Utilize the Rever Tra Ace-a-Tm First Strand cDNA Synthesis Kit of Toyobo company, synthetic the 1st the chain cDNA of total RNA.The intermediate segment pcr amplification carries out according to the product polysaccharase blend tap plus of Toyobo company.First round PCR reaction conditions: 94 ℃ of 3min; 94 ℃ of 15s, 62 ℃ of (0.3 ℃ of every circulation landing) 15s, 72 ℃ of 30s, 40 circulations; 72 ℃ of 7min.Second takes turns the PCR reaction conditions: 94 ℃ of 3min; 94 ℃ of 15s, 58 ℃ of 15s, 72 ℃ of 30s, 40 circulations; 72 ℃ of 3min.

CCK-2 intermediate segment clone electrophoresis result as shown in Figure 1, through first round PCR, to having occurred the expection band between the 400bp, second takes turns at 200bp and goes out to expect band to nested between the 300bp at 300bp.The expection band of the first round is reclaimed the fragment that order-checking obtains 385bp, prove the CCK-2 sequence through the blast comparison.

(3) bolti CCK-2 3 ' RACE and 5 ' RACE

3 ' end carries out 3 ' RACE PCR reaction conditions: the first round: 94 ℃ of 3min according to the RACE of Clontech company test kit; 94 ℃ of 15s, and thermograde (56 ℃, 58 ℃, 60 ℃, 62 ℃) 15s, 72 ℃ of 40s, 40 circulations; 72 ℃ of 10min.Second takes turns: 94 ℃ of 3min; 94 ℃ of 15s, 58 ℃ of 15s, 72 ℃ of 40s, 40 circulations; 72 ℃ of 10min.Utilize the RACE primer and the joint primer AUAP that have designed, AAP amplification CCK-1 5 ' end sequence, 5 ' RACE PCR reaction conditions: first round P:94 ℃ 3min; 94 ℃ of 15s, 58 ℃ of 15s, 72 ℃ of 30s, 40 circulations; 72 ℃ of 5min.Second takes turns: 94 ℃ of 3min; 94 ℃ of 15s, 58 ℃ of 15s, 72 ℃ of 30s, 40 circulations; 72 ℃ of 5min.CCK-2 3 ' RACE and 5 ' RACE clone electrophoresis result shown in Fig. 2 and 3, obtain the fragment that length is respectively 421bp and 335bp respectively, and be identical with CCK-2 intermediate segment coincidence part.

(4) bolti opening code-reading frame (Opening Reading Frame, clone ORF)

In 3 ' and 5 ' non-coding region design upstream and downstream special primer clone CCK-2 ORF sequence, the PCR reaction conditions is: 94 ℃ of 3min; 94 ℃ of 15s, 58 ℃ of 15s, 72 ℃ of 30s, 40 circulations; 72 ℃ of 10min.The expection band of 500bp occurs in the first round, be 489bp through reclaiming order-checking.The result as shown in Figure 4.

The ORF cloned sequence of CCK-2 is 508bp through the size that checks order, and ORF length is 396bp, the precursor protein of a 132aa of coding.This precursor protein comprises 20 amino acid whose signal peptides and 112 amino acid whose mature peptides of a supposition, terminal octapeptide (CCK-8): the DYLGWMDF of C-, and gastrin structural domain.

CCK-8 is to amylase, trypsinase, propepsin and the maincenter factor NPY that ingests, the influence of orexin

(1) experimental design

CCK-8 albumen is synthetic by Invitrogen company.The experimental design scheme is bolti 200 tails, body weight 40-60g, during raising and train, throw something and feed every day 2 times, the morning 9:30 and afternoon 16:30, throw something and feed according to body weight 2%, raise and train 10 days after, hungry 5 days, bolti after hungry 5 days earlier with the fish anaesthetic treatment, is handled 3-4min with fish with the narcotic Eugenol before injection, the CCK octapeptide is dissolved in the PBS, dosage is 10ng/g respectively, 100ng/g, 1000ng/g BW fish.

Experiment is grouped as follows table:

(2) the part fragment cloning of bolti amylase, trypsinase, propepsin cDNA

The Mozambique tilapia trypsin trypsin AY510093.1 that has delivered according to the GeneBank database), amylase (amylase AJ555243.2), propepsin sequence (pepsin AY513876.1), the design Auele Specific Primer carries out the segmental amplification of part, and primer is as follows.

Tilapia amylase, propepsin, trypsinase part fragment cloning, through first round PCR, band has appearred about 200bp respectively, as shown in Figure 5, conform to expected results, will expect that band reclaims the order-checking size and is respectively 173bp, 209bp, 197bp, will obtain sequence and Mozambique's tilapia amylase, propepsin and trypsinase sequence alignment, homology reaches 99%, proves bolti amylase, propepsin, trypsinase sequence.

(3) fluorescence quantitative PCR detection bolti amylase, trypsinase, stomach en-original mRNA change

Inject the PBS(negative control respectively to the tilapia abdominal cavity), 10ng/g, 100ng/g, three kinds of dosage CCK-8 of 1000ng/g.Get all tilapia pyloric caecum tissues, extract synthetic cDNA first chain of RNA, according to the SYBR of Invitrogen company Green Real-time PCR Mix test kit explanation application of sample reacts reaction conditions with the product LightCycler of Roche company 480 real time PCR system: amylase: 94 ℃ of 3min; 94 ℃ of 15s, 60 ℃ of 15s, 72 ℃ of 30s, 40 circulations; 72 ℃ of 10min; Trypsinase: 94 ℃ of 3min; 94 ℃ of 15s, 62 ℃ of 15s, 72 ℃ of 30s, 40 circulations; 72 ℃ of 10min; Propepsin: 94 ℃ of 3min; 94 ℃ of 15s, 58 ℃ of 15s, 72 ℃ of 30s, 40 circulations; 72 ℃ of 10min.

It is PCR specificity product that solubility curve presents the unimodal amplified production that shows, as shown in Figure 6.Negative control is amplification not.The process software analysis obtains the typical curve of amylase, trypsinase, propepsin, and approaching-3.33 explanation PCR efficient of the slope of typical curve are near desirable PCR efficient.CP value according to goal gene in each reaction calculates the concentration that changes gene, draw the CP value of three kinds of digestive ferments and internal control gene according to Q-PCR, with the conversion of comparing of typical curve CP value and concentration, draw the relative concentration of amylase, trypsinase, propepsin and internal control gene, concentration and internal control gene concentration with amylase, trypsinase, propepsin compares again, draws the relative expression quantity of amylase, trypsinase, stomach en-original mRNA.Adopt SPSS software analysis gained result, compare analysis with the Duncan method, think that there were significant differences when P value＜0.05, P value＜0.01 o'clock thinks that difference is extremely remarkable.

As Fig. 7, shown in 8 and 9, in 1h, the mRNA level of three kinds of digestive ferments (amylase, trypsinase, propepsin) has no significant change.From 1h, the mRNA level of amylase and trypsinase 100ng/g treatment group significantly raises, in the 1h-3h, amylase mRNA raise 5.5 times (P＜0.01), the Trypsin mRNA level has raise 9.1 times (P＜0.01), and 10ng/g and 1000ng/g treatment group amylase and Trypsin mRNA level all do not have considerable change.Propepsin 10ng/g and 100ng/g treatment group mRNA level all significantly raise, in 1h-3h, 10ng/g treatment group stomach en-original mRNA level raise 12.9 times (P＜0.01), the 100ng/g treatment group has raise 5.6 times, and 1000ng/g treatment group stomach en-original mRNA level does not have considerable change.

(4) bolti NPY, the part fragment cloning of orexin cDNA

According to tilapia orexin sequence (GenBank:FJ871159.1) and the NPY sequence (this laboratory obtains) that the GenBank database has been delivered, the design Auele Specific Primer carries out the segmental amplification of Partial cDNA, and primer is as follows:

Tilapia NPY, orexin 2 part fragment clonings through first round PCR, band occurred respectively about bp more than 100 and 200bp, see accompanying drawing 8, conform to expected results, will expect that band reclaims the order-checking size and is respectively 130bp, 165bp.With this sequence and bolti NPY, orexin sequence alignment,, prove bolti NPY, orexin sequence with wherein a part of identical.

(5) fluorescence quantitative PCR detection bolti NPY, orexinmRNA changes

Inject the PBS(negative control respectively to the tilapia abdominal cavity), 10ng/g, 100ng/g, three kinds of dosage CCK-8 of 1000ng/g.Get all tilapia hypothalamuses, extract synthetic cDNA first chain of RNA, according to the SYBR of Invitrogen company The explanation carrying out of Green Real-time PCR Mix test kit Q-PCR, reaction conditions: 94 ℃ of 3min; 94 ℃ of 15s, 60 ℃ of 15s, 72 ℃ of 30s, 40 circulations; 72 ℃ of 10min.

It is PCR specificity product that solubility curve presents the unimodal amplified production that shows, negative control is amplification not, as shown in Figure 9.The process software analysis obtains the typical curve of NPY, orexin, and the CP value of goal gene calculates the concentration that changes gene in reacting according to each, and approaching-3.33 explanation PCR efficient of the slope of NPY, orexin typical curve are near desirable PCR efficient.Cp value according to NPY that Q-PCR draws, orexin and internal control gene 18s, with the conversion of comparing of typical curve cp value and concentration, draw the relative concentration of NPY, orexin and internal control gene, concentration and internal control gene concentration with NPY, orexin compares again, draws the relative expression quantity of two kinds of NPY, orexinmRNA.Adopt SPSS software analysis gained result, compare analysis with the Duncan method, think that there were significant differences when P value＜0.05, P value＜0.01 o'clock thinks that difference is extremely remarkable.

As Figure 10, shown in 11 and 12, causing behind the abdominal injection CCK-8 that NPY and orexin mRNA are expressed in the 1h in the tilapia hypothalamus raises, the half an hour of maximum expression amount after injection, this effect occurs over just the 1000ng/g treatment group, NPY has raise when 0.5h 3.3 times (P＜0.01), orexin raise 3.4 times (P＜0.01).And other dosage group fails all to cause that the expression of orexin, NPY mRNA changes.

According to this experimental result, in conjunction with more existing reports, we reason out the model of ingesting of tilapia: behind the food arrival stomach that tilapia is taken in, stimulating gastrointestinal road intestines CCK secretion, excretory CCK or import in the brain by blood circulation or by vagus nerve stimulates the secretion of hypothalamus appetite peptide, and this time continued in the half an hour that begins to take food, after half an hour, appetite peptide expression amount descends.Influenced by this, tilapia is constantly feed in one hour, descend along with appetite peptide expression amount after half an hour, feeding behavior weakens gradually, and CCK continues to express in the brain, produces full sense, approximately behind the 1h, tilapia stops to ingest gradually, and the CCK in the enteron aisle begins the enzyme secretion that stimulates digestion simultaneously, with digest food.

As if ingest and weight increase is controlled by endocrine system, the fish appetite stimulator by complicated hypothalamus neural network, comprises the multiple correlation factor of ingesting from maincenter and peripheral tissues.Our result shows, CCK-8 is as the neurotransmitter in the neural system, the regulation and control between the factor of ingesting of possible participation maincenter.In sum, the CCK endocrine regulation factor participates in ingesting, growth and possible energy balance are regulated, and has the dual function of full sense of adjusting maincenter and gastro-intestinal digestion.

＜110〉Zhongshan University

＜120〉application of a kind of bolti cholecystokinin and coding nucleic acid thereof and function octapeptide

<130>

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 131

<212> PRT

＜213〉tilapia (Tilapia)

<400> 1

Met Asn Val Gly Ile Cys Val Cys Val Ile Leu Ala Ala Leu Ser Ser

1 5 10 15

Gly Ser Leu Ser Leu Pro Ser Gln Ser Ile Thr Ala Glu Ser Thr Ala

20 25 30

Leu Leu Ser Gly Ser Gly Ser Leu Pro Ser Pro Ser Leu Lys Arg Gln

35 40 45

Ala Arg Ser Ala Pro Ala Pro Leu Ser Gly His Leu Ile Asn Tyr Ser

50 55 60

Gln His Gln Glu Asp Ala Asp Thr Pro Asp Ser Leu Ser Gln Leu Leu

65 70 75 80

Ala Arg Leu Ile Ser Arg Lys Gly Ser Ala Tyr His Thr Arg Ser Ser

85 90 95

Leu Ser Ser Arg Ala Ser Gly Leu Pro Pro Asn His Arg Ile Lys Asp

100 105 110

Arg Asp Tyr Leu Gly Trp Met Asp Phe Gly Arg Arg Ser Ala Glu Glu

115 120 125

Tyr Glu Tyr

130

<210> 2

<211> 908

<212> DNA

＜213〉tilapia (Tilapia)

<400> 2

ctcactatct tcaaatcagc ctctcctggt ttacttcaac agctgcagcc atgaatgtag 60

gcatctgtgt ttgtgttatc ctggctgctt tgtccagtgg ttccctgagt ctgccttcac 120

aatcaattac agctgaaagt acagctcttc tctcaggcag cggatctctc ccttctccca 180

gcctgaaacg gcaggctcgc tccgctccag cgcccctctc agggcatctt atcaactaca 240

gccagcacca agaggacgca gacactccag acagcctgag ccagcttctg gccagactca 300

tctctaggaa aggctctgcc taccacacca gatcctccct cagcagcaga gccagtggtc 360

tcccccccaa ccacaggata aaggacagag attatctcgg ctggatggac ttcggacgac 420

gtagtgcaga ggagtacgaa tactagtcct gaaggcacag ctttgtttct tcgaacgctg 480

agcccaaaac ctccaacatc cccagttaaa gtttttattt taaaaaatct gaagcctttt 540

tcacactgca ctctcagccc accagagctg acttagtgag aagtacatct cagcacatat 600

ccttcaatct acaaaccaca aacttcaatc caaaacttac ggcacattat tgtatatttg 660

tgttactgtt aaggccaaag ccagcaatgc ttgtccattc cacagtgctt tcttagtccc 720

tcattctctc tatggtttgg atggtaatca ctaaaaatga gtcacaaaaa cagttttttc 780

atggaaaaaa ggtgttttga tttcttaaaa tatggggtca ttgtaatttt ctaaccagca 840

tagagttttt aatctgtttg ggagtacttt cagagtttgg taaaaaaaaa aaaaaaaaaa 900

aaaaaaaa 908

<210> 3

<211> 8

<212> PRT

＜213〉artificial polypeptide

<400> 3

Asp Tyr Leu Gly Trp Met Asp Phe

1 5

<210> 4

<211> 21

<212> DNA

＜213〉artificial primer

<400> 4

gatctctccc ttctcccagc c 21

<210> 5

<211> 23

<212> DNA

＜213〉artificial primer

<400> 5

taatctctgt cctttatcct gtg 23

<210> 6

<211> 21

<212> DNA

＜213〉artificial primer

<400> 6

cagggtcatc cgtcaccaaa g 21

<210> 7

<211> 21

<212> DNA

＜213〉artificial primer

<400> 7

ggtcagggta gttgcttcca c 21

<210> 8

<211> 20

<212> DNA

＜213〉artificial primer

<400> 8

ttcatgtgga agctggtttg 20

<210> 9

<211> 21

<212> DNA

＜213〉artificial primer

<400> 9

ccagggcaag atccagcaga c 21

<210> 10

<211> 21

<212> DNA

＜213〉artificial primer

<400> 10

gtgggtccct tcagtgtatt g 21

<210> 11

<211> 20

<212> DNA

＜213〉artificial primer

<400> 11

cttctgtctc gctcaatcca 20

<210> 12

<211> 22

<212> DNA

＜213〉artificial primer

<400> 12

acaagacaga ggtatgggaa ga 22

<210> 13

<211> 18

<212> DNA

＜213〉artificial primer

<400> 13

ggcagcatca ccacattg 18

<210> 14

<211> 18

<212> DNA

＜213〉artificial primer

<400> 14

ccctgccgcc tttatgtg 18

<210> 15

<211> 18

<212> DNA

＜213〉artificial primer

<400> 15

ttggttcctg gagccttg 18

Claims (5)

1. bolti cholecystokinin, its aminoacid sequence is shown in SEQ ID NO:1.

2. the nucleotide sequence of coding claim 1 described bolti cholecystokinin.

3. the nucleotide sequence of the described bolti cholecystokinin of coding claim 1 according to claim 2 is characterized in that: described sequence is shown in SEQ ID NO:2.

4. the terminal octapeptide DYLGWMDF(SEQ of the C-of the described bolti cholecystokinin of claim 1 ID NO:3) regulate that tilapia ingests and gastro-intestinal digestion in application.

5. the nucleotide sequence of bolti cholecystokinin according to claim 3 is characterized in that: its cloning process comprises the steps:

Extract total RNA of the full brain of bolti, reverse transcription obtains cDNA; Method by RT-PCR and RACE obtains the cDNA total length, the last cDNA that obtains with reverse transcription is a template, with CCK-2-QF:5 '-GATCTCTCCCTTCTCCCAGCC-3 ' (SEQ ID NO:4) and CCK-2-QR:5 '-TAATCTCTGTCCTTTATCCTGTG-3 ' (SEQ ID NO:5) is primer, and amplification obtains its opening code-reading frame.

Images