CN102604978A - ACL (ATP (Adenosine Triphosphate) citrate lyase) gene relative to intramuscular fat deposition - Google Patents
ACL (ATP (Adenosine Triphosphate) citrate lyase) gene relative to intramuscular fat deposition Download PDFInfo
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Abstract
The invention discloses an ACL (ATP (Adenosine Triphosphate) citrate lyase) gene relative to intramuscular fat deposition. The ACL gene is separated and cloned from a muscular tissue of a Bannan miniature pig and is relative to the intramuscular fat deposition. The overall length of the CDS sequence of the gene is 3276bp, the CDS nucleotide sequence of the gene is shown in SEQ.ID.NO.1 and is coded with 1091 amino acids, and the amino acid sequence is shown in SEQ.ID.NO.2. According to detection on tissue expression profiles of the ACL gene in seven tissues, namely the heart, liver, spleen, lungs, kidney, muscle and fat of the pig, the detection result shows that the expression index of the ACL gene in the fat tissue is highest, thereby initially showing the function of the ACL gene for regulating and controlling fat deposition in fat cells.
Description
Technical field
The present invention relates to the genetic engineering technique of animal molecular breeding, particularly a kind of ATP citrate-lyase relevant (ATP citrate lyase, ACL) gene engineering with porcine intramuscular fat deposition.
Background technology
At present, China's Swine Production has been ignored the decline of meat quality when pursuing the high lean ratio and the low thickness of backfat.(intramuscular fat, IMF) content is the important factor in order of meat quality to intramuscular fat, can influence the many aspects such as local flavor, succulence, tender degree and nutritive value of meat matter.Therefore, improve intramuscular fat content and become current research focus to improve meat quality.Though the local variety pig speed of growth of China is slower, has better meat matter.Receiving miniature pig like version is one of good local pig breed in Yunnan, has characteristics such as the fast fertilizer of well-balanced, the fast length of figure, the thin bone of skin is thin, back leg is plentiful, back fat is evenly distributed, and intramuscular fat is abundant, is the raw material pig that is fit to roast suckling pig.Yet, at present version is received the molecular mechanism of growing of miniature pig and fatty deposits and knows little about it.Along with the molecular biology development of technologies; Understand version from molecular level and receive the molecular basis of miniature label of pig fat deposition description; Find, the exploitation version is received the miniature pig functional gene relevant with fatty deposits, provide fundamental basis for raising market pig intramuscular fat content improves meat quality.
In the pig muscle tissue, the increase of IMF content mainly is that (triacylglycerols, the TAG) increase of content, the increase of TAG content mainly depend on the differentiation and the propagation of intramuscular fat cell to triglyceride level.In adipocyte differentiation and the breeding, synthetic de novo synthesis and the transhipment that mainly depends on lipid acid of TAG.Therefore, the differentiation of intramuscular fat cell and propagation and metabolism thereof are the sedimentary bases of IMF, and the regulatory factor of regulation and control intramuscular fat cytodifferentiation and propagation and adjusting lipid metabolism all has material impact to the deposition of IMF.
Glucose is the main energy derive of animal body, when the intravital glucose supplies of animal is superfluous, then can change fat stores into.The generation of cytolemma and the adjusting behind the protein translation also be unable to do without and depend on glucose synthetic lipid.(ATP citrate lyase is contact carbohydrate metabolism and lipid synthetic key enzyme ACL) to ATP citrate-lyase, and it can be transformed into acetyl-CoA and oxaloacetic acid by the catalysis Hydrocerol A.Acetyl-CoA generates lipid acid at last through acetyl-CoA carboxylase and Fatty acid synthetase catalysis.Therefore, ATP citrate-lyase is one of important lipese, and is closely related with the fatty deposits of pig.Some data show that the activity of ATP citrate-lyase can receive the adjusting of diet nutrient and Regular Insulin.Hi CHO nutrition can improve the expression of mouse ATP citrate-lyase at liver, and its activity is mainly regulated at transcriptional level.Discover in addition that in addition ATP citrate-lyase can be through regulating the state that its resultant velocity changes the body fat de novo synthesis.
Summary of the invention
The purpose of this invention is to provide a kind of intramuscular fat deposition ACL gene; Adopt biochip technology and bioinformatic analysis method; In research, find ACL gene participation intramuscular fat cell lipid metabolic process, and from version is received miniature pig muscle tissue, cloned the ACL gene first pig muscle tissue and intramuscular fat cellular gene expression spectrum.
A kind of intramuscular fat deposition ACL gene; Be ATP citrate-lyase (ACL) gene relevant that from version is received miniature pig muscle tissue, separates, clones with intramuscular fat deposition; The CDS sequence total length of gene is 3276bp, its CDS nucleotide sequence such as SEQ.ID.NO.1.And, nucleotide sequence coded 1091 amino acid of CDS, aminoacid sequence such as SEQ.ID.NO.2.Intramuscular fat deposition ACL gene design primer to above-mentioned characteristic; Carry out pcr amplification; Detect the distribution expression pattern of ACL gene at Pigs Hearts, liver, spleen, lung, kidney, muscle and seven tissues of fat; The result shows that ACL gene expression amount in fatty tissue is the highest, shows that tentatively the ACL gene regulates and control the function of fatty deposits in adipocyte.
The present invention clone obtains version and receives miniature pig ACL gene C DS complete sequence, and total length is 3276bp, is submitted to Genbank, and accession number is JN205459.Compare with the pig ACL gene that Genbank provides, finding has five nucleotide mutant sites, wherein has three the justice sudden change is arranged, and causes amino acid to be become by alanine mutation that Xie Ansuan, aspartic acid become l-asparagine, Threonine becomes methionine(Met).The sudden change of inferring this amino acid sites possibly cause version to receive characteristics such as the proteinic molecular weight of miniature pig ACL, iso-electric point, secondary structure, phosphorylation site all changing to some extent, and the change of these character maybe to receive the good characteristics of miniature meat quality relevant with version.
The homology of ACL albumen between species is higher; Can find out through evolutionary tree; The sibship of pig and ox, sheep is more approaching, secondly is people, mouse, and the homology of ACL amino acid sequence coded and ox, sheep, people, mouse and rat is respectively 98%, 98%, 98%, 97% and 97%.It is thus clear that ACL has high conservative property between different plant species, this is its corresponding physilogical characteristics remain unchanged in each species molecular basis.In addition, ACL Gene RT-PCR expression pattern shows that this gene is expressed at the fatty tissue camber, has proved that also it has vital role aspect intramuscular fat deposition.
The present invention adopts biochip technology and bioinformatic analysis method; In research, find ACL gene participation intramuscular fat cell lipid metabolic process, and from the pig muscle tissue, cloned the ACL gene first pig muscle tissue and intramuscular fat cellular gene expression spectrum.The research of at present relevant pig ACL gene and expression pattern thereof is also less.To the existing research of the cloning and expression of people, mouse and Mei Shan, Large White fatty tissue ACL gene cDNA sequence, but, version is not cloned into this gene from receiving miniature pig muscle tissue as yet.
Description of drawings
Fig. 1 is a pcr amplification ACL gene diagram of the present invention
Fig. 2 is the expression level post figure of ACL of the present invention in different tissues
Fig. 3 is that ACL gene enzyme of the present invention is cut the evaluation diagram
Fig. 4 is an evolutionary tree analysis of the present invention
Fig. 5 is ACL protein secondary structure prediction figure of the present invention
Fig. 6 is ACL protein phosphorylation site estimation figure of the present invention
Fig. 7 is ACL Protein Glycosylation Overview site estimation figure of the present invention
Fig. 8 is an ACL protein hydrophobic analysis of the present invention
Embodiment
A kind of intramuscular fat deposition ACL gene is the ATP citrate-lyase relevant with intramuscular fat deposition (ATP citrate lyase, the ACL) gene that from version is received miniature pig muscle tissue, separates, clones.The CDS sequence total length of gene is 3276bp, its CDS nucleotide sequence such as specification sheets Nucleotide and aminoacid sequence table SEQ.ID.NO.1.And, nucleotide sequence coded 1091 amino acid of CDS, aminoacid sequence such as specification sheets Nucleotide and aminoacid sequence table SEQ.ID.NO.2.
Intramuscular fat deposition ACL gene design primer to above-mentioned characteristic; Carry out pcr amplification; Detect the distribution expression pattern of ACL gene at Pigs Hearts, liver, spleen, lung, kidney, muscle and seven tissues of fat; The result shows that ACL gene expression amount in fatty tissue is the highest, shows that tentatively the ACL gene regulates and control the function of fatty deposits in adipocyte.
Be specially:
1, the extraction of total RNA in the muscle tissue
Take the 50kg version of growing up to receive miniature pig muscle and organize and put into liquid nitrogen immediately and preserve subsequent use.Utilize RNA to extract reagent and extract version and receive miniature pig muscle total tissue RNA, the explanation of concrete operations reference reagent box is carried out.The RNA reverse transcription of extracting is become cDNA ,-20 ℃ of preservations, for use.
2, PCR design of primers and PCR reaction conditions
According to pig ACL sequence information among the GenBank (GenBank accession number NM_001105302), utilize Primer Premier 5.0 design primers, and synthetic.In order to obtain preferable expanding effect, adopt the method for dividing two sections amplifications, at last together with two sections sequence assemblies.
Two pairs of primer sequences of the ACL gene that is used to clone are:
Upper semisection, product length are 2063bp:
F：5’?-?CCATGTCGGC?CAAGGCGATT?TT?-?3?’
R：5’?-?ATAAACGTGG?AGCCGGGGTA?-?3’
Lower semisection, product length are 1337bp:
F：5’?-?ACAGGTACCC?CGGCTCCACG?TTTAT?-?3’
R：5’?-?CCCAGGGGAG?GAGTTCTTGT?CTTCA?-?3’
The primer sequence that is used for the ACL gene of fluorescent quantitation is:
F：5’-?CGAGCAGCAG?ACCTATGACT?AT?-3’
R：5’-?ACTTCTCCCA?TCACCCGTAA?-3’
The primer sequence of confidential reference items 18S rRNA is:
F：5’-CTGCCTCCTT?GGATGTG-3’
R：5’-GCGGCTTTGG?TGACTCTA-3’
The PCR reaction conditions of ACL gene clone is: upper semisection: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 0.5min, 53 ℃ of annealing 1min, 72 ℃ of extension 3min (35 circulations); Extending 10min after 72 ℃ increases; Lower semisection: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 0.5min, 55 ℃ of annealing 0.5min, 72 ℃ of extension 2min (35 circulations); Extend 7min after 72 ℃ and increase, the reaction back is in 4 ℃ of preservations.Pcr amplification product confirms that the length scale of the product of amplification is same as the purpose fragment after 1.5% agarose gel electrophoresis detects.Like specification sheets Nucleotide and aminoacid sequence table.
The reaction conditions of quantitative fluorescent PCR is: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 0.5min, 54 ℃ of annealing 0.5min, 72 ℃ of extension 3min (35 circulations); 72 ℃ are extended 10min.The method of data processing does, detects the relative expression quantity of ACL gene in the heart, liver, spleen, lung, kidney, muscle, seven tissues of fat, and experimental data as confidential reference items, adopts two calibration curve methods to handle with 18S rRNA.The result shows that ACL has expression in 7 tissues to be detected, and a large amount is expressed in fat, and great expression on liver, lung, muscle is that volume is expressed on the heart, kidney, is trace expression on spleen.
3, the clone of ACL gene and sequential analysis
Reclaim test kit book specification sheets according to sepharose DNA and reclaim specific band; Product is connected in the pMD-18T carrier, is converted into the Top10 competent cell, ammonia benzyl screening picking positive colony; Be inoculated in respectively in the 10mL LB liquid nutrient medium; 37 ℃ of shaken overnight are extracted plasmid and are accredited as the positive through Ecor I and Hind III double digestion, check order.Clone's qualification result such as specification sheets Nucleotide and aminoacid sequence table.
4, sequencing result
Two sections extension increasing sequences of gained are through order-checking, and splice with DNAMAN software, obtain edition to receive miniature pig ACL gene C DS coding region complete sequence.CDS total length 3276bp, nucleotide sequence and are submitted to the Genbank DB shown in specification sheets Nucleotide and aminoacid sequence table SEQ.ID.NO.1, accession number is JN205459.1091 amino acid of CDS sequence encoding, its aminoacid sequence is shown in specification sheets Nucleotide and aminoacid sequence table SEQ.ID.NO.2.
5, the sequence alignment that provides among sequencing result and the Genbank
Login http://www.ebi.ac.uk/Tools/clustalw2/ carries out version and receives the CDS nucleotide sequence of miniature pig ACL gene; And the pig ACL gene information (accession number is NM_001105302) that CDS amino acid sequence coded and Genbank provide compares, and finds that the nucleotides sequence of CDS is shown five mutational sites: the 1117th C becomes T, the 1941st T and becomes C, the 2790th T and become that C, the 2911st G become A, the 3077th C becomes T.Wherein the 1117th, the 2911st and the 3077th is that justice sudden change is arranged, cause the 373rd amino acids by alanine mutation become Xie Ansuan, the 971st amino acids by aspartic acid become l-asparagine, the 1026th amino acids becomes methionine(Met) by Threonine.Like specification sheets Nucleotide and aminoacid sequence table.
6, ACL gene nucleotide and amino acid sequence homology analysis
Login http://www.ebi.ac.uk/Tools/clustalw2/ receives miniature pig ACL gene sequencing result with version and carries out the homology comparison with ox (accession number is NM_001037457), sheep (accession number is NM_001038622), people's (accession number is NM_001096), mouse (accession number is NM_001199296) and rat (accession number is NM_016987) CDS sequence.Version is received the CDS sequence of miniature pig ACL gene and the homology of ox, sheep, people, mouse and rat and is respectively 93%, 92%, 91%, 89% and 89%.Version is received the homology of the miniature coded aminoacid sequence of pig CDS sequence and ox, sheep, people, mouse and rat and is respectively 98%, 98%, 98%, 97% and 97%.Like specification sheets Nucleotide and aminoacid sequence table.
7, the evolutionary tree of ACL gene is analyzed
Utilize vicinity (Neighbor-joining) method of Meg4 software to make up version and receive the systematic evolution tree of miniature pig, ox, people, mouse, rat and sheep ACL gene; It is nearer that the result shows that version is received the affinity of miniature pig and ox and sheep, secondly is people, mouse, rat.
8, ACL molecular weight of albumen, PREDICTION FOR THE ISOELECTRIC POINT analysis
Use the online software Compute pI/MW of ExPASy to analyze version and receive that the proteic iso-electric point of miniature pig ACL (pI) is 7.12, theoretical molecular (pW) is 119843.23 Da.
9, the ACL protein secondary structure prediction is analyzed
Land http://bioinf.cs.ucl.ac.uk/psipred/, each species ACL secondary protein structure is predicted, the result shows, has 299 spirals, 173 extensions and 628 coiled structure.
10, ACL protein phosphorylation site estimation
Land http://www.cbs.dtu.dk/services/NetPhos/; Pig ACL protein phosphorylation site is predicted; The result shows, has 39 phosphorylation sites, and wherein the serine phosphorylation site has 19, Threonine phosphorylation site to have 9, tyrosine phosphorylation site to have 11.
11, ACL Protein Glycosylation Overview site estimation
Land http://www.cbs.dtu.dk/services/NetPhos/, pig ACL protein phosphorylation site is predicted, the result shows, has three glycosylation sites, respectively at the 75th, the 346th and the 440th.
12, the ACL protein hydrophobic is analyzed
Land http://web.expasy.org/protscale/, the ACL protein hydrophobic is analyzed, the result is shown as hydrophilic protein.
Claims (2)
1. intramuscular fat deposition ACL gene; Be receive from version separate the miniature pig muscle tissue, clone's the ATP citrate-lyase relevant with intramuscular fat deposition (ATP citrate lyase, ACL) gene is characterized in that: the CDS sequence total length of gene is 3276bp; Its CDS nucleotide sequence such as SEQ.ID.NO.1; And, nucleotide sequence coded 1091 amino acid of CDS, aminoacid sequence such as SEQ.ID.NO.2.
2. according to the said a kind of intramuscular fat deposition ACL gene of claim 1; It is characterized in that: to the intramuscular fat deposition ACL gene design primer of above-mentioned characteristic; Carry out pcr amplification; Detect the distribution expression pattern of ACL gene at Pigs Hearts, liver, spleen, lung, kidney, muscle and seven tissues of fat, the result shows that ACL gene expression amount in fatty tissue is the highest, shows that tentatively the ACL gene regulates and control the function of fatty deposits in adipocyte.
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Application publication date: 20120725 |