CN102125544A - Inducer for activating expression of fish lipid metabolism control gene and application thereof - Google Patents
Inducer for activating expression of fish lipid metabolism control gene and application thereof Download PDFInfo
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Abstract
The invention discloses an inducer for activating the expression of a fish lipid metabolism control gene and application thereof. The inducer is prepared from one or a mixture of more of clofibrate, 2-palmitic acid, 15-deoxidized-delta 12 and 14-prostaglandin J2. The inducer can activate the expression of the fish somatic cell lipid metabolism control gene, namely a peroxisome proliferator activated receptor gene, so that the inducer can be used for breeding fishes, maintaining the nutrition metabolization balance in the fish breeding, reducing lipidosis of the fishes and improving the quality of fish flesh.
Description
Technical field
The present invention relates to biological technology application, be specifically related to a kind of derivant and application thereof that activates Fish lipid metabolism regulate gene expression.
Background technology
Peroxisome proliferation-activated receptors (peroxisome proliferator activated receptor, PPAR) be that a class can be by endogenous fatty acid and exogenous peroxisome proliferation (peroxisome proliferator, PP), belong to II type nuclear receptor superfamily member as the activated nuclear factor of part.PPAR is also because of being called FAA (fatty acid receptor) by endogenous fatty acid and metabolite activation thereof.In multiple animal body, all there are three class PPARs receptors, i.e. PPAR α, PPAR β also claims PPAR δ and PPAR γ, these three kinds of hypotypes all have specificity, can produce different biological actions.PPAR can regulate and control the destination gene expression of the inside and outside lipid metabolism of many participation cells, especially the gene of some important enzymes in the beta-oxidation process of encoding, PPAR also participates in the differentiation of adipose cell in addition, and the growth of possible pair cell, differentiation even apoptosis have material impact.PPARs is the important regulatory factor of adipose cell differentiation and lipid metabolism, playing central action aspect the regulation and control energy metabolism, can control fatty acid absorption, lipidosis and lipogenesis, be the main regulatory factors of stablizing interior environment of lipid metabolism and insulin resistant.Therefore, PPARs function and Its Mechanisms have been caused the interest of many researcheres.
The application of artifical compound feed in pisciculture is very extensive.In the pond culture,, usually can cause fatty liver and the fertile tendency of fat of Fish, influence the growth of Fish and the delicate flavour of the flesh of fish because the prolonged application mixed feed is raised chinese carp.Along with the development of society, people are more and more higher to the requirement of the composition of animal products and local flavor, science, reasonably regulate and control the important topic that poultry body fat deposition becomes animal breeding and production field.PPARs plays the greasy feel receiver, and local change gene expression is to regulate lipid metabolism.Regulate and control the adipose cell differentiation simultaneously, adipose cell is all had transcriptional activation, wherein PPAR γ has the ability of the strongest promotion adipose cell differentiation.According to these biological functions of PPARs, seek more high-affinity and specific activator or antagonist, medically be used for the treatment of metabolic diseases such as obesity, hyperlipidemia, type; In herding is produced, improve the poultry lean meat percentage and reduce the lipidosis of birds abdomen all significant to improve meat etc.
Up to now, three kinds of hypotypes of PPAR gene are identified and are cloned in primates, Rodents, amphibian, birds and some Fish, and mammal is many but the research of relevant Fish PPAR is far from.Three kinds of hypotypes of present known PPAR have some common parts, as PP, polyunsaturated fatty acid, insulin, nonsteroidal antiinflammatory drug.The PPAR alpha ligands comprises that long-chain unsaturated fatty acid, side chain, polymerization and oxidized form fatty acid, eicosane compounds, leukotriene B4, fibrate lipid-lowering drugs and Wy-14563 etc. are multiple.Polyunsaturated fatty acids, prostaglandin and tretinoin are the natural aglucons of PPAR β.The part of PPAR γ comprises some prostaglandin, prostaglandin-like molecule etc., and the metabolite of arachidonic acid after cyclooxygenase, lipoxygenase effect is the native ligand of PPAR γ as 15-deoxy prostaglandin J2, derivative of fatty acid.Had not yet to see clofibrate, 2-bromine palmitic acid, 15-deoxidation-Δ 12,14-prostaglandin J2 is used to regulate the correlational study report of lipid metabolic balance in the Ctenopharyngodon idellus body.
Ctenopharyngodon idellus (
Ctenopharyngodon idella) be that object is mainly cultured by China, can effectively utilize cheap plant protein source that aquatic products are provided, but Ctenopharyngodon idellus body fat content in breeding process increase and the generation performance of fatty liver is comparatively outstanding.Under the intensive culture condition, owing to taking in artifical compound feeds in a large number and lacking exercise, the Ctenopharyngodon idellus body fat deposits, poor growth, and build is unusual, has reduced flesh quality and local flavor, has also increased aquaculture cost simultaneously.Through dissect checking, in the body cavity fatty major part be deposited on around the intestinal, on the mesentery or liver top layer, the severe patient liver loses color, is milk yellow or lark.Therefore in fish feed, add the blood fat reducing factor, reduce accumulating of its soma's fat and become aquatic animal threpsology's research focus in recent years.
Summary of the invention
The objective of the invention is to provides a kind of Fish Nutrition and Metabolism balance of keeping according to shortcomings such as the fish body fat deposition, poor growth, the build that exist in the existing fish culture are unusual, reduces the fatty,fiss deposition, improves the derivant of flesh quality.
Another purpose of the present invention is to provide the application of above-mentioned derivant.
Above-mentioned purpose of the present invention is achieved by the following technical programs:
A kind of derivant that activates Fish lipid metabolism regulate gene expression comprises clofibrate, 2-bromine palmitic acid, 15-deoxidation-Δ 12, the mixture of one or more among the 14-prostaglandin J2.
The present invention is with clofibrate (Clofibrate), 2-bromine palmitic acid (2-bromo palmitate), 15-deoxidation-Δ 12,14-prostaglandin J2(15-deoxy-Δ 12,14-prostaglandin J2 15d-J2) carries out the live body injection as derivant to Ctenopharyngodon idellus, injection trace (mg/kg level), activate Ctenopharyngodon idellus Regulation of Lipids Metabolism gene, particularly peroxisome proliferation body activated receptor (PPAR) gene expression is cultured fish Nutrition and Metabolism balance to keep.
At first the inventor has studied the interior PPAR of fish body influences for lipid metabolism:
Peroxisome proliferation-activated receptors is as the activated nuclear factor of part, it is the important regulatory factor of adipose cell differentiation and lipid metabolism, playing central action aspect the regulation and control energy metabolism, can control fatty acid absorption, lipidosis and lipogenesis, be the main regulatory factors of stablizing environment in the lipid metabolism.PPAR can regulate and control the destination gene expression of the inside and outside lipid metabolism of many participation cells, especially the gene of some important enzymes in the beta-oxidation process of encoding, PPAR also participates in the differentiation of adipose cell in addition, and the growth of possible pair cell, differentiation even apoptosis have material impact.3 kinds of hypotype expression in different tissues of PPAR have nothing in common with each other, though PPAR α, PPAR β and PPAR γ are co expression in most of histiocytes, expression differs greatly.PPAR α mainly is expressed in the active tissue of metabolism such as the heart, liver, kidney proximal tubule, skeletal muscle and brown fat.PPAR β is at multiple tissue, cellular expression, and its midbrain, adipose cell and skin are the highest, and PPAR γ mainly is present in fatty tissue and immune system, has than high expressed in tissues such as fat, intestinal and mammary gland.
The inventor utilizes fluorescence quantifying PCR method, is outer reference with beta-actin, research Ctenopharyngodon idellus different tissues PPAR expression of gene level.Result of study shows that secondly PPAR α great expression in liver is brain, in muscle and heart trace expression is arranged; PPAR β expresses in heart, liver and muscle at most, in brain, spleen and lipid trace expression is arranged; A little less than PPAR γ expresses relatively, only more expression is arranged in liver, and at brain, faint expression in muscle and the lipid.Different with mammal PPAR gene expression profile, Fish PPAR gene is great expression in liver organization mainly, and this may be that its main lipopexia position is abdominal cavity mesenteric adipose tissues, liver and muscle because Fish lack subcutaneous layer of fat.Because liver is the metabolic significant points of tallow fatty acid beta oxidation, thereby it to be Fish change lipopectic main modulability storage fat organ with nutriture that (nucleotide sequence of PPAR α is shown in SEQ ID NO:1, and its amino acid sequence coded is shown in SEQ ID NO:2; The nucleotide sequence of PPAR β is shown in SEQ ID NO:3, and its amino acid sequence coded is shown in SEQ ID NO:4; The nucleotide sequence of PPAR γ is shown in SEQ ID NO:5, and its amino acid sequence coded is shown in SEQ ID NO:6).
Then analyzed the part of three kinds of hypotypes of PPAR, they have some common parts, as PP, polyunsaturated fatty acid, insulin, nonsteroidal antiinflammatory drug.The PPAR alpha ligands comprises that long-chain unsaturated fatty acid, side chain, polymerization and oxidized form fatty acid, eicosane compounds, leukotriene B4, fibrate lipid-lowering drugs and Wy-14563 etc. are multiple.Regulate the downstream gene relevant by activating PPAR α,, and then quicken decomposition of fatty acids to reach the purpose of blood fat reducing as the expression of acyl CoA oxidase, Carnitine palmitoyltransferase I with the fatty acid beta-oxidation.Fatty acid also can activate PPAR α, quickens decomposition of fatty acids.Polyunsaturated fatty acids, prostaglandin and tretinoin are the natural aglucons of PPAR β.The part of PPAR γ comprises some prostaglandin, prostaglandin-like molecule etc., and the metabolite of arachidonic acid after cyclooxygenase, lipoxygenase effect is as 15-deoxy prostaglandin j
2, derivative of fatty acid is the native ligand of PPAR γ.
Therefore, the present invention selects clofibrate (Clofibrate), 2-bromine palmitic acid (2-bromo palmitate), 15-deoxidation-Δ 12,14-prostaglandin J2(15-deoxy-Δ 12,14-prostaglandin J2,15d-J2) respectively as the derivant of three kinds of hypotypes of PPAR, induce and activate Ctenopharyngodon idellus Regulation of Lipids Metabolism gene---peroxisome proliferation body activated receptor (PPAR) expression.
Grope to find through concrete experiment, injection Clofibrate 25-67 mg/kg fish body weight (preferred 50 mg/kg body weight), 2-bromo palmitate 35-58mg/kg fish body weight (preferred 42 mg/kg body weight), during 15d-J2 0.95-1.05 mg/kg fish body weight (preferred 1 mg/kg body weight), injection trace (mg/kg level), peroxide activator enzyme body paraphyte activated receptor (PPAR) gene expression, economical as the gene expression derivant of regulating lipid metabolic balance in the Ctenopharyngodon idellus body.
Compared with prior art, the present invention has following beneficial effect:
1. the invention provides a kind of derivant that activates Fish lipid metabolism regulate gene expression, injection trace (mg/kg level), can activate Ctenopharyngodon idellus Regulation of Lipids Metabolism gene---peroxisome proliferation body activated receptor (PPAR) gene expression, culture the fish fats metabolic balance to keep;
2. Ctenopharyngodon idellus is the distinctive phytophage cultured fishes of China, but man-made feeds are fed Ctenopharyngodon idellus after a period of time, and excessive the accumulating of fat usually taken place, and what influence feedstuff increases the meat effect, reduces its nutritive value.According to three kinds of PPAR activator provided by the invention to fish body lumbar injection, lipid metabolic balance in the scalable Ctenopharyngodon idellus body, in fish culture,, help to disclose herbivorous fishes to nutraceutical utilization and mechanism thereof for reducing lipidosis, improving meat matter important value is provided.
Description of drawings
Fig. 1 Ctenopharyngodon idellus PPAR α gene cDNA partial sequence and deduced amino acid, aminoacid sequence shows with one-letter symbol correspondence under nucleotide sequence;
Fig. 2 Ctenopharyngodon idellus PPAR β gene cDNA partial sequence and deduced amino acid, aminoacid sequence shows with one-letter symbol correspondence under nucleotide sequence;
Fig. 3 Ctenopharyngodon idellus PPAR γ gene cDNA partial sequence adds 3 ' terminal sequence and deduced amino acid.Aminoacid sequence shows with one-letter symbol correspondence under nucleotide sequence; Translation stop codon shows with asterisk (*); Underscore is represented the polyA tailing signal;
Fig. 4 is Ctenopharyngodon idellus liver, brain, spleen, muscle, fat and heart PPARs genome molding expression, PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is PPAR α/beta-actin mRNA, and 2 is PPAR β/beta-actin mRNA, and 3 is PPAR γ/beta-actin mRNA;
Fig. 5 is three kinds of concentration C lofibrate lumbar injection Ctenopharyngodon idellus after 24 hours, liver PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is PPAR α/beta-actin mRNA, and 2 is PPAR β/beta-actin mRNA, and 3 is PPAR γ/beta-actin mRNA;
Fig. 6 is three kinds of concentration C lofibrate lumbar injection Ctenopharyngodon idellus after 24 hours, mesentery fat PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is PPAR α/beta-actin mRNA, and 2 is PPAR β/beta-actin mRNA, and 3 is PPAR γ/beta-actin mRNA;
Fig. 7 is three kinds of concentration C lofibrate lumbar injection Ctenopharyngodon idellus after 24 hours, muscle PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is PPAR α/beta-actin mRNA, and 2 is PPAR β/beta-actin mRNA, and 3 is PPAR γ/beta-actin mRNA;
Fig. 8 is three kinds of concentration 2-bromo palmitate lumbar injection Ctenopharyngodon idellus after 24 hours, liver PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is PPAR α/beta-actin mRNA, and 2 is PPAR β/beta-actin mRNA, and 3 is PPAR γ/beta-actin mRNA;
Fig. 9 is three kinds of concentration 2-bromo palmitate lumbar injection Ctenopharyngodon idellus after 24 hours, mesentery fat PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is PPAR α/beta-actin mRNA, and 2 is PPAR β/beta-actin mRNA, and 3 is PPAR γ/beta-actin mRNA;
Figure 10 is three kinds of concentration 2-bromo palmitate lumbar injection Ctenopharyngodon idellus after 24 hours, muscle PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is PPAR α/beta-actin mRNA, and 2 is PPAR β/beta-actin mRNA, and 3 is PPAR γ/beta-actin mRNA;
Figure 11 is three kinds of concentration 15-deoxy-Δs 12, and 14-prostaglandin J2 lumbar injection Ctenopharyngodon idellus is after 24 hours, liver PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is PPAR α/beta-actin mRNA, and 2 is PPAR β/beta-actin mRNA, and 3 is PPAR γ/beta-actin mRNA;
Figure 12 is three kinds of concentration 15-deoxy-Δs 12, and 14-prostaglandin J2 lumbar injection Ctenopharyngodon idellus is after 24 hours, mesentery fat PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is PPAR α/beta-actin mRNA, and 2 is PPAR β/beta-actin mRNA, and 3 is PPAR γ/beta-actin mRNA;
Figure 13 is three kinds of concentration 15-deoxy-Δs 12, and 14-prostaglandin J2 lumbar injection Ctenopharyngodon idellus is after 24 hours, muscle PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is PPAR α/beta-actin mRNA, and 2 is PPAR β/beta-actin mRNA, and 3 is PPAR γ/beta-actin mRNA;
Figure 14 is PBS and 50mg/kg Clofibrate lumbar injection Ctenopharyngodon idellus after 24 hours, liver PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is lumbar injection PBS, and 2 is lumbar injection 50mg/kg Clofibrate;
Figure 15 is PBS and 50mg/kg Clofibrate lumbar injection Ctenopharyngodon idellus after 24 hours, mesentery fat PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is lumbar injection PBS, and 2 is lumbar injection 50mg/kg Clofibrate;
Figure 16 is PBS and 50mg/kg Clofibrate lumbar injection Ctenopharyngodon idellus after 24 hours, muscle PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is lumbar injection PBS, and 2 is lumbar injection 50mg/kg Clofibrate;
Figure 17 is PBS and 125umol/kg 2-bromo palmitate lumbar injection Ctenopharyngodon idellus after 24 hours, liver PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is lumbar injection PBS, and 2 is lumbar injection 125umol/kg 2-bromo palmitate;
Figure 18 is PBS and 125umol/kg 2-bromo palmitate lumbar injection Ctenopharyngodon idellus after 24 hours, mesentery fat PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is lumbar injection PBS, and 2 is lumbar injection 125umol/kg 2-bromo palmitate;
Figure 19 is PBS and 125umol/kg 2-bromo palmitate lumbar injection Ctenopharyngodon idellus after 24 hours, muscle PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is lumbar injection PBS, and 2 is lumbar injection 125umol/kg 2-bromo palmitate;
Figure 20 is PBS and 1mg/kg 15-deoxy-Δ 12, and 14-prostaglandin J2 lumbar injection Ctenopharyngodon idellus is after 24 hours, liver PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is lumbar injection PBS, and 2 is lumbar injection 1mg/kg 15-deoxy-Δ 12,14-prostaglandin J2;
Figure 21 is PBS and 1mg/kg 15-deoxy-Δ 12, and 14-prostaglandin J2 lumbar injection Ctenopharyngodon idellus is after 24 hours, mesentery fat PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is lumbar injection PBS, and 2 is lumbar injection 1mg/kg 15-deoxy-Δ 12,14-prostaglandin J2;
Figure 22 is PBS and 1mg/kg 15-deoxy-Δ 12, and 14-prostaglandin J2 lumbar injection Ctenopharyngodon idellus is after 24 hours, muscle PPARs/ beta-actin mRNA ratio block diagram; Wherein, 1 is lumbar injection PBS, and 2 is lumbar injection 1mg/kg 15-deoxy-Δ 12,14-prostaglandin J2.
The specific embodiment
Further explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.
The successful clone of the new sequence of embodiment 1 Ctenopharyngodon idellus peroxisome proliferation-activated receptors gene cDNA
Ctenopharyngodon idellus is provided by Guangdong Freshwater Famous Fish Breeding Center (Fanyu, Guangdong).
(1) total RNA extraction and cDNA first chain is synthetic
From Ctenopharyngodon idellus sharp separation liver organization, the extraction and purification of total RNA is undertaken by the SV Total RNA Isolation System test kit recommend method of Promega company.The synthetic ReverTra Ace-α-TM test kit that uses TOYOBO company of cDNA first chain is a template with the Ctenopharyngodon idellus liver total RNA, and oligo (dT) 20 is the reverse transcription primer, and operation is undertaken by the test kit recommend method.
(2) clone of Ctenopharyngodon idellus PPAR gene cDNA core sequence
Synthesize 3 couples of degenerate primer PPAR α 01F and PPAR α 02R, PPAR β 01F and PPAR β 02R, PPAR γ 01F and PPAR γ 02R according to the conservative region design of known vertebrates PPAR α, PPAR β and PPAR γ aminoacid sequence respectively.(Table?1)
| The primer title | Sequence (5'-3') |
| partial cDNA sequence | ? |
| PPARα01F | 5’- ACC ATTCGG CTC AAA CTG GAG TA(C/T) GA(T/C) AA(G/A) -3’ |
| PPARα02R | 5’-CACCAGAAGACCGTCTTTGTTCAT(G/A)CA(C/T/A/G)(C/G) -3’ |
| PPARβ01F | 5’- GCA ATAAGG TAC GGA CGT ATG CC(A/T/C/G) GA(A/G) GC -3’ |
| PPARβ02R | 5’-CTCCAGAGCATTGAATTTCAC(C/A/G/T)GC(G/A)AA(T/C)TC-3’ |
| PPARγ01F | 5’- CAC TGCGAT CTG CAC TGT CGC AT(A/T/C) CA(C/T) AA(G/A) -3’ |
| PPARγ02R | 5’-GCGCAGACTCTTGAGGAACTC(C/T/A/G)C(G/T)(C/T/A/G)GTCAT-3’ |
| RACEs | ? |
| Gc PPARγ3’01F | 5’-CCTACGGTCAGATCTTCAT-3’ |
| Gc PPARγ3’02F | 5’-GGCTCCGCTCATGAACAA-3’ |
| FQ | ? |
| FQGCPPARα01F | 5’-AGCAGAGAAGGACGTCAG-3’ |
| FQGCPPARα02R | 5’-TTCCTTCTCGGCATGCTG-3’ |
| FQGCPPARβ01F | 5’-TCTGAAGTCTCTCGCCAA-3’ |
| FQGCPPARβ02R | 5’-GAGCTCCGTTAATCACCT-3’ |
| FQGCPPARγ01F | 5’-GATGGTTGGCATGTCACAC-3’ |
| FQGCPPARγ02R | 5’-TTCCTGACAGTATGGCTC-3’ |
| FQGC ACT01F | 5’-CGTGACATCAAGGAGAAG-3’ |
| FQGC ACT02R | 5’-GAGTTGAAGGTGGTCTCAT-3’ |
Expection PCR product clip size is about 630 bp respectively, about 600 bp and 650 bp.With above-mentioned cDNA is template, and (TaKaRa) carries out pcr amplification with the Taq archaeal dna polymerase, and amplification condition is: 94 ℃ of pre-degeneration 3 min, and 94 ℃ of 1 min, 40 ℃ of 1 min, 72 ℃ of 1 min, totally 30 circulations, last 72 ℃ are extended 5 min.
(3) clone of PCR product and sequence analysis
The PCR product is through 2% sepharose electrophoresis purification, H.Q.﹠amp; .Q. Gel Extraction Kit II (U-gene) reclaims rear clone to pMD 19-T carrier (TaKaRa), transformed competence colibacillus E.coli DH5 α, utilize the forward and reverse primer of M13, obtain positive colony by the PCR reaction detection, PCR detects and to serve extra large Ying Jun Bioisystech Co., Ltd behind the positive colony and check order with ABI 3730 sequenators.Sequencing result is carried out analysis and arrangement obtain Ctenopharyngodon idellus PPARs gene cDNA core sequence, infer and obtain its aminoacid sequence, the various biological PPARs aminoacid sequence of delivering on the GenBank of NCBI website carries out sequence analysis with BLAST and DNA analysis software Vector NTI suite 8.0.
The Ctenopharyngodon idellus PPAR sequence that obtains is asked for an interview Figure of description 1-3.
With beta-actin is outer reference, with PPAR α, PPAR β and the PPAR γ gene mRNA relative expression level of fluorescence quantifying PCR method comparison Ctenopharyngodon idellus liver, brain, spleen, muscle, fat and heart, establishes 5 for every group.According to Ctenopharyngodon idellus PPAR α, PPAR β and PPAR γ gene cDNA core sequence design special primer FQPPAR α 01F and FQPPAR α 02R, FQPPAR β 01F and FQPPAR β 02R, FQPPAR γ 01F and FQPPAR γ 02R (table 1), PCR product clip size is respectively 202 bp, 185 bp and 206 bp. are according to the Ctenopharyngodon idellus beta-actin design special primer FQACT01F and the FQACT02R (table 1) that have delivered. and the PCR reaction condition is 94 ℃ of pre-degeneration 3 min, 94 ℃ of 1 min, 57 ℃ of 1 min, 72 ℃ of 1 min, totally 30 circulations, last 72 ℃ are extended 5 min. Ctenopharyngodon idellus livers, brain, spleen, muscle, PPAR/beta-actin mRNA (%) data of fat and heart are asked for an interview Figure of description 4. results and are shown, wherein Ctenopharyngodon idellus PPAR α is in liver, and PPAR β is in liver, muscle, heart is relative higher in the constitutive expression of liver with PPAR γ.
Ctenopharyngodon idellus is supported in the crawl that has constant flow filtering water, every day is in the coccoid feedstuff (Guangzhou Hai Wei) that floats of 10:00-11:00 rationing fish body weight 2%, collect residual feed in 11:00-12:00, tested preceding 7 days, again weigh, the fish that body weight is close is classified as one group, and every group 5 tail is used for test injection.Derivant Clofibrate(clofibrate) be dissolved in dimethyl sulfoxide, 2-bromo palmitate(2-bromine palmitic acid), 15-deoxy-Δ 12,14-prostaglandin J2(15-deoxidation-Δ 12,14-prostaglandin J2) be dissolved in dehydrated alcohol.Phosphate buffer PBS is a diluent, and derivant is diluted to corresponding concentration.Begin the anesthesia injection after once in the 10:00-11:00 feeding, matched group is only injected 200ulPBS, experiment component be 3 greatly the group, the concentration of injecting the 200ulPBS. Clofibrate group of three kinds of derivants that contain variable concentrations respectively is respectively 50mg/kg(n=1), 250mg/kg(n=1), 1.25g/kg(n=1); The concentration of 2-bromo palmitate group is respectively 25umol/kg(n=1), 125umol/kg(n=1), 250umol/kg(n=1); 15-deoxy-Δ 12, the concentration of 14-prostaglandin J2 group is respectively 100ug/kg(n=1), 500ug/kg(n=1), 1mg/kg(n=1). frost anesthesia behind 24 h, separate liver, mesentery fat, muscular tissue.Method for extracting total RNA carries out fluorescent quantitation and detects the various expression of gene of PPAR as described in example 1 above behind the reverse transcription.Sequential design special primer according to embodiment 1 acquisition, the synthetic use ToYoBo ReverTra Ace-α-TM test kit of cDNA first chain, be template with the total RNA of Ctenopharyngodon idellus respectively, oligo (dT) 20 is the reverse transcription primer, and operation is carried out .RT-PCR by the test kit recommend method and used DyNAmo
TMFlash SYBR Green qPCR Kit (Finnzymes, Finland) (MJ Research, USA), abide by description and carry out for test kit and Chromo4 Real-Time detection system by experimental design.The PCR system comprise Master Mix (2 * concentration) 10 μ l, 0.5 μ M forward primer, 0.5 μ M reverse primer, ROX 0.3 μ l, an amount of by the cDNA that the total RNA reverse transcription of 1 μ g obtains, supply ddH
2O is to totally being the solubility curve of amplified production to be analyzed after the each PCR of 20 μ l.. and the quantitative PCR of each sample repeats 3 times. and reaction is step by step: 95 ° of C 3 min; 95 ° of C 20 s, 55 ° of C 20 s, 72 ° of C 35 s, 40 circulations.(MJ research, USA) the delta delta Ct method of software is calculated the Expression of Related Genes amount to adopt the Opticon Monitor software 2.03 Version.Genes of interest mRNA transcriptional level is with genes of interest/beta-actin mRNA (%) expression.Specimen repeatability and group difference relatively adopt SPSS 13.0 to add up, and all results represent with mean ± standard deviation.PPAR/beta-actin mRNA (%) data are asked for an interview Figure of description 5-13 in Ctenopharyngodon idellus liver, mesentery fat, the muscle.The result shows that Clofibrate can activate the expression of PPAR under 50mg/kg concentration; 2-bromo palmitate can activate the expression of PPAR under 125umol/kg concentration; 15-deoxy-Δ 12,14-prostaglandin J2 can activate the expression of PPAR under 1mg/kg concentration.
According to the result of embodiment 3, three kinds of derivants have been selected more effective concentration respectively.Matched group is only injected 200ulPBS(n=5), totally 3 groups of experimental grouies, injection contains 50mg/kg Clofibrate(n=5 respectively), 125umol/kg 2-bromo palmitate(n=5), 1mg/kg 15-deoxy-Δ 12,14-prostaglandin J2(n=5) 200ulPBS.Its liver, mesentery fat, muscle extraction RNA are put to death and got to fish with all fishes after experiment is handled 24 hours, carry out fluorescent quantitation behind the reverse transcription and detect the various expression of gene of PPAR, and method as described in example 3 above.The result shows that the Clofibrate of 50mg/kg has significant inducing action to the PPAR β expression of Ctenopharyngodon idellus mesentery fat, and trend is induced in the PPAR β expression of liver and muscle, and the PPAR alpha expression of liver and mesentery fat is induced trend.The 2-bromo palmitate of 125umol/kg has significant inducing action to the PPAR β expression of Ctenopharyngodon idellus mesentery fat.The 15-deoxy-Δ 12 of 1mg/kg, 14-prostaglandin J2 has significant inducing action to the PPAR β expression of liver and mesentery fat.Therefore we find the 15-deoxy-Δ 12 of 2-bromo palmitate, 1mg/kg of Clofibrate, the 125umol/kg of 50mg/kg, 14-prostaglandin J2 improves its expression as the activator of PPAR, cultures fish Nutrition and Metabolism balance thereby regulate the Ctenopharyngodon idellus lipid metabolism to reach.
Correlated results is asked for an interview Figure of description 14-22.
SEQUENCE?LISTING
＜110〉Ji'nan University
＜120〉a kind of derivant and application thereof that activates Fish lipid metabolism regulate gene expression
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<160> 6
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Thr?Ile?Arg?Leu?Lys?Leu?Glu?Tyr?Asp?Lys?Cys?Glu?Arg?Asn?Cys?Lys
1 5 10 15
Ile?Gln?Lys?Lys?Asn?Arg?Asn?Lys?Cys?Gln?Tyr?Cys?Arg?Phe?Arg?Lys
20 25 30
Cys?Leu?Ala?Val?Gly?Met?Ser?His?Asn?Ala?Ile?Arg?Phe?Gly?Arg?Ile
35 40 45
Pro?Gln?Ser?Glu?Lys?Gln?Arg?Leu?Lys?Ala?Glu?Lys?Asp?Val?Ser?Gly
50 55 60
Lys?Glu?Glu?His?Gln?Ser?Pro?Gln?Leu?Asp?Thr?Lys?Ser?Leu?Ala?Arg
65 70 75 80
Gln?Met?His?Glu?Ala?Tyr?Leu?Lys?His?Phe?His?Met?Asn?Lys?Ala?Lys
85 90 95
Ala?Arg?Val?Phe?Leu?Thr?Gly?Lys?Thr?Ser?Thr?Pro?Pro?Phe?Val?Ile
100 105 110
His?Asp?Met?Asp?Pro?Leu?Gln?His?Ala?Glu?Lys?Glu?Leu?Leu?Thr?Gln
115 120 125
Leu?Leu?Gly?Asn?Met?Thr?Ser?Gly?Asp?Ala?Ser?Thr?Leu?Arg?Glu?Arg
130 135 140
Glu?Val?Glu?Ala?Arg?Leu?Phe?Leu?Phe?Cys?Gln?Tyr?Ala?Ser?Val?Ala
145 150 155 160
Thr?Val?Thr?Glu?Leu?Thr?Glu?Phe?Ala?Lys?Ala?Val?Pro?Gly?Phe?Ala
165 170 175
Thr?Leu?Asp?Leu?Asn?Asp?Gln?Val?Thr?Leu?Leu?Lys?Tyr?Gly?Val?Tyr
180 185 190
Glu?Ala?Leu?Phe?Ala?Leu?Leu?Ala?Ser?Cys?Met?Asn?Lys?Asp?Gly?Leu
195 200 205
Leu?Val
210
<210> 3
<211> 604
<212> DNA
＜213〉artificial sequence
<400> 3
gcaataaggt?acggacgtat?gcccgaagca?gagaagcgta?agctagtcgc?gggtctgtta 60
gcgggggaaa?agagctctca?gacctccagc?ggttcagatc?tgaagtctct?cgccaaacgg 120
gtcaacaacg?cctacctgaa?gaatctgaac?atgaccaaga?agaaagctcg?caacatcctg 180
acggggaaga?ccaacgcaag?cccgccattt?gtcattcatg?acatggactc?gctgtggcag 240
gcggaaaacg?gactggtctg?gaatcaggtg?attaacggag?ctccgcccaa?taaggagatt 300
ggcgtgcatg?tgttttaccg?ctgtcaatgt?acaaccgtgg?aaaccgtgcg?agagctcacc 360
gagtttgcca?aaaacatccc?tgggtttgtg?gatctcttcc?taaatgatca?ggtaacactg 420
ttaaaatatg?gagtccacga?ggccatattt?tcaatgctcc?catctctcat?gaataaagac 480
gggctgctgg?tggccaatgg?gaagggcttt?gtgacgagag?agttcctgcg?tagtctacgc 540
aaacctttca?gtgagatcat?ggagcccaag?tttgaatttg?ctgtgaaatt?caatgctctg 600
<210> 4
<211> 201
<212> PRT
＜213〉artificial sequence
<400> 4
Ala?Ile?Arg?Tyr?Gly?Arg?Met?Pro?Glu?Ala?Glu?Lys?Arg?Lys?Leu?Val
1 5 10 15
Ala?Gly?Leu?Leu?Ala?Gly?Glu?Lys?Ser?Ser?Gln?Thr?Ser?Ser?Gly?Ser
20 25 30
Asp?Leu?Lys?Ser?Leu?Ala?Lys?Arg?Val?Asn?Asn?Ala?Tyr?Leu?Lys?Asn
35 40 45
Leu?Asn?Met?Thr?Lys?Lys?Lys?Ala?Arg?Asn?Ile?Leu?Thr?Gly?Lys?Thr
50 55 60
Asn?Ala?Ser?Pro?Pro?Phe?Val?Ile?His?Asp?Met?Asp?Ser?Leu?Trp?Gln
65 70 75 80
Ala?Glu?Asn?Gly?Leu?Val?Trp?Asn?Gln?Val?Ile?Asn?Gly?Ala?Pro?Pro
85 90 95
Asn?Lys?Glu?Ile?Gly?Val?His?Val?Phe?Tyr?Arg?Cys?Gln?Cys?Thr?Thr
100 105 110
Val?Glu?Thr?Val?Arg?Glu?Leu?Thr?Glu?Phe?Ala?Lys?Asn?Ile?Pro?Gly
115 120 125
Phe?Val?Asp?Leu?Phe?Leu?Asn?Asp?Gln?Val?Thr?Leu?Leu?Lys?Tyr?Gly
130 135 140
Val?His?Glu?Ala?Ile?Phe?Ser?Met?Leu?Pro?Ser?Leu?Met?Asn?Lys?Asp
145 150 155 160
Gly?Leu?Leu?Val?Ala?Asn?Gly?Lys?Gly?Phe?Val?Thr?Arg?Glu?Phe?Leu
165 170 175
Arg?Ser?Leu?Arg?Lys?Pro?Phe?Ser?Glu?Ile?Met?Glu?Pro?Lys?Phe?Glu
180 185 190
Phe?Ala?Val?Lys?Phe?Asn?Ala?Leu?Glu
195 200
<210> 5
<211> 1331
<212> DNA
＜213〉artificial sequence
<400> 5
cactgcgatc?tgcactgtcg?catccataag?aagagtcgca?acaagtgcca?gtactgccgc 60
tttcagaagt?gcctgatggt?tggcatgtca?cacaacgcca?ttcgttttgg?tcgaatgccc 120
caagccgaga?aagagaagct?cttggccgag?ttctccactg?acatggacca?catgcaccca 180
gaatcggctg?atctccgagc?gctggccagg?catctgtacg?agtcctatct?gaagtatttc 240
cccctgacca?aagccaaggc?cagagccata?ctgtcaggaa?agaccagcga?caatgcacct 300
ttcgttatcc?acgacatgaa?gtctctgatg?gaaggagagc?acatgatcaa?ctgccggcag 360
atgcccatgc?aggagcaccg?gagatccgac?atgggcatca?tgcaagaagt?ggagcttcgt 420
ttcttccaca?gctgccagtc?gcgttcggcc?gaagctgtca?gcgaagtcac?tgaatttgcc 480
aagagcgtcc?cgggctttgt?caacctggac?ttgaacgacc?aagtgacgct?cctgaagtac 540
ggcgtcatcg?aggtgctcat?catcatgatg?gctccgctca?tgaacaaaga?cggcacgctc 600
atctcctacg?gtcagatctt?catgacccgc?gagttcctca?agagcctgcg?caaacccttc 660
tgtgaaatga?tggagcccaa?gttcgagttc?tccgtcaagt?tcaacatgct?ggagttggac 720
gacagcgaca?tggcgctgtt?cctggccgtc?atcatcctga?gcggagatcg?tcccggactg 780
ctgaacgtca?agcccattga?ggatctacag?gagacggttc?ttcactctct?ggagctgcag 840
ctgaagacca?accatccaga?ctcgctccag?cttttcgcca?aggtcctgca?gaagatgacg 900
gacctgcggc?agctggtgac?cgaccacgtc?cagctgatcc?agctgatgaa?ggagacggag 960
ttggactggt?gcttacaccc?gctcctgcag?gagatcatga?gggacttgta?ctagtctgtg 1020
tttgtgatcc?tgagctttgg?tttactgctc?tgaagttttt?gcactgtaac?agacagagaa 1080
accagcagag?agtgatactg?gttcgacaga?gattcgtttt?tgcgtcgtgt?ccactgctgc 1140
ggcgctggtt?aaagagagca?gtaccactgt?gaaaatgttt?tttagcaggt?gatttttgca 1200
ttcatggtcc?ttacagataa?agacaaggac?ataaaagtga?cactccatat?ttaattttga 1260
atacagcggt?gtccaaaagt?ctgagactac?atttgtaaag?ataaaaaaaa?aaaaaaaaaa 1320
aaaaaaaaaa?a 1331
<210> 6
<211> 337
<212> PRT
＜213〉artificial sequence
<400> 6
His?Cys?Asp?Leu?His?Cys?Arg?Ile?His?Lys?Lys?Ser?Arg?Asn?Lys?Cys
1 5 10 15
Gln?Tyr?Cys?Arg?Phe?Gln?Lys?Cys?Leu?Met?Val?Gly?Met?Ser?His?Asn
20 25 30
Ala?Ile?Arg?Phe?Gly?Arg?Met?Pro?Gln?Ala?Glu?Lys?Glu?Lys?Leu?Leu
35 40 45
Ala?Glu?Phe?Ser?Thr?Asp?Met?Asp?His?Met?His?Pro?Glu?Ser?Ala?Asp
50 55 60
Leu?Arg?Ala?Leu?Ala?Arg?His?Leu?Tyr?Glu?Ser?Tyr?Leu?Lys?Tyr?Phe
65 70 75 80
Pro?Leu?Thr?Lys?Ala?Lys?Ala?Arg?Ala?Ile?Leu?Ser?Gly?Lys?Thr?Ser
85 90 95
Asp?Asn?Ala?Pro?Phe?Val?Ile?His?Asp?Met?Lys?Ser?Leu?Met?Glu?Gly
100 105 110
Glu?His?Met?Ile?Asn?Cys?Arg?Gln?Met?Pro?Met?Gln?Glu?His?Arg?Arg
115 120 125
Ser?Asp?Met?Gly?Ile?Met?Gln?Glu?Val?Glu?Leu?Arg?Phe?Phe?His?Ser
130 135 140
Cys?Gln?Ser?Arg?Ser?Ala?Glu?Ala?Val?Ser?Glu?Val?Thr?Glu?Phe?Ala
145 150 155 160
Lys?Ser?Val?Pro?Gly?Phe?Val?Asn?Leu?Asp?Leu?Asn?Asp?Gln?Val?Thr
165 170 175
Leu?Leu?Lys?Tyr?Gly?Val?Ile?Glu?Val?Leu?Ile?Ile?Met?Met?Ala?Pro
180 185 190
Leu?Met?Asn?Lys?Asp?Gly?Thr?Leu?Ile?Ser?Tyr?Gly?Gln?Ile?Phe?Met
195 200 205
Thr?Arg?Glu?Phe?Leu?Lys?Ser?Leu?Arg?Lys?Pro?Phe?Cys?Glu?Met?Met
210 215 220
Glu?Pro?Lys?Phe?Glu?Phe?Ser?Val?Lys?Phe?Asn?Met?Leu?Glu?Leu?Asp
225 230 235 240
Asp?Ser?Asp?Met?Ala?Leu?Phe?Leu?Ala?Val?Ile?Ile?Leu?Ser?Gly?Asp
245 250 255
Arg?Pro?Gly?Leu?Leu?Asn?Val?Lys?Pro?Ile?Glu?Asp?Leu?Gln?Glu?Thr
260 265 270
Val?Leu?His?Ser?Leu?Glu?Leu?Gln?Leu?Lys?Thr?Asn?His?Pro?Asp?Ser
275 280 285
Leu?Gln?Leu?Phe?Ala?Lys?Val?Leu?Gln?Lys?Met?Thr?Asp?Leu?Arg?Gln
290 295 300
Leu?Val?Thr?Asp?His?Val?Gln?Leu?Ile?Gln?Leu?Met?Lys?Glu?Thr?Glu
305 310 315 320
Leu?Asp?Trp?Cys?Leu?His?Pro?Leu?Leu?Gln?Glu?Ile?Met?Arg?Asp?Leu
325 330 335
Tyr
Claims (10)
1. a derivant that activates Fish lipid metabolism regulate gene expression is characterized in that described derivant is clofibrate, 2-bromine palmitic acid, 15-deoxidation-Δ 12, the mixture of one or more among the 14-prostaglandin J2.
2. the application of the described derivant of claim 1 is characterized in that described derivant carries out the live body injection to Fish or feedstuff adds, and is used to activate Fish lipid metabolism regulate gene expression.
3. according to the application of the described derivant of claim 2, the addition that it is characterized in that described clofibrate is a 25-67 mg/kg fish body weight.
4. according to the application of the described derivant of claim 3, the addition that it is characterized in that described clofibrate is a 50mg/kg fish body weight.
5. according to the application of the described derivant of claim 2, the addition that it is characterized in that described 2-bromine palmitic acid is a 35-58mg/kg fish body weight.
6. according to the application of the described derivant of claim 5, the addition that it is characterized in that described 2-bromine palmitic acid is a 42mg/kg fish body weight.
7. according to the application of the described derivant of claim 2, it is characterized in that described 15-deoxidation-Δ 12, the addition of 14-prostaglandin J2 is a 0.95-1.05 mg/kg fish body weight.
8. according to the application of the described derivant of claim 7, the addition that it is characterized in that described 2-bromine palmitic acid is a 1mg/kg fish body weight.
9. according to the application of the described derivant of claim 2, it is characterized in that described activation Fish lipid metabolism controlling gene is a peroxisome proliferation body activated receptor gene.
10. according to the application of the described derivant of claim 2, it is characterized in that described Fish are Ctenopharyngodon idellus.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105934809A CN102125544B (en) | 2010-12-17 | 2010-12-17 | Inducer for activating expression of fish lipid metabolism control gene and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105934809A CN102125544B (en) | 2010-12-17 | 2010-12-17 | Inducer for activating expression of fish lipid metabolism control gene and application thereof |
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| Publication Number | Publication Date |
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| CN102125544A true CN102125544A (en) | 2011-07-20 |
| CN102125544B CN102125544B (en) | 2013-04-10 |
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| CN2010105934809A Expired - Fee Related CN102125544B (en) | 2010-12-17 | 2010-12-17 | Inducer for activating expression of fish lipid metabolism control gene and application thereof |
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| CN (1) | CN102125544B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102813092A (en) * | 2012-09-11 | 2012-12-12 | 北京市水产科学研究所 | Salmon and trout feed based on peroxisome proliferator activated receptor (PPAR) and method for preparing salmon and trout feed |
| CN103931897A (en) * | 2013-08-22 | 2014-07-23 | 华中农业大学 | Feed additive for reducing freshwater fish body fat content and preparation method thereof |
| CN105995032A (en) * | 2016-06-17 | 2016-10-12 | 合肥丰瑞隆生物科技有限公司 | Feed additive for reducing fat content of freshwater fish and preparation method thereof |
| CN106071384A (en) * | 2016-06-17 | 2016-11-09 | 合肥丰瑞隆生物科技有限公司 | Reduce feed additive of wall pollack fish fats content and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6474995A (en) * | 1987-09-16 | 1989-03-20 | Nippon Oils & Fats Co Ltd | Production of prostaglandin |
| RU1769826C (en) * | 1990-05-28 | 1992-10-23 | Univ Dagestanskij | Method for improving viability of fish eggs |
| RU1792597C (en) * | 1990-03-05 | 1993-02-07 | Свердловский Сельскохозяйственный Институт | Method for fish sperm stimulation |
| CN101002740A (en) * | 2005-09-26 | 2007-07-25 | 刘凤鸣 | Slow release preparation of compactin |
-
2010
- 2010-12-17 CN CN2010105934809A patent/CN102125544B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6474995A (en) * | 1987-09-16 | 1989-03-20 | Nippon Oils & Fats Co Ltd | Production of prostaglandin |
| RU1792597C (en) * | 1990-03-05 | 1993-02-07 | Свердловский Сельскохозяйственный Институт | Method for fish sperm stimulation |
| RU1769826C (en) * | 1990-05-28 | 1992-10-23 | Univ Dagestanskij | Method for improving viability of fish eggs |
| CN101002740A (en) * | 2005-09-26 | 2007-07-25 | 刘凤鸣 | Slow release preparation of compactin |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102813092A (en) * | 2012-09-11 | 2012-12-12 | 北京市水产科学研究所 | Salmon and trout feed based on peroxisome proliferator activated receptor (PPAR) and method for preparing salmon and trout feed |
| CN102813092B (en) * | 2012-09-11 | 2014-07-02 | 北京市水产科学研究所 | Salmon and trout feed based on peroxisome proliferator activated receptor (PPAR) and method for preparing salmon and trout feed |
| CN103931897A (en) * | 2013-08-22 | 2014-07-23 | 华中农业大学 | Feed additive for reducing freshwater fish body fat content and preparation method thereof |
| CN105995032A (en) * | 2016-06-17 | 2016-10-12 | 合肥丰瑞隆生物科技有限公司 | Feed additive for reducing fat content of freshwater fish and preparation method thereof |
| CN106071384A (en) * | 2016-06-17 | 2016-11-09 | 合肥丰瑞隆生物科技有限公司 | Reduce feed additive of wall pollack fish fats content and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102125544B (en) | 2013-04-10 |
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