CN102899330B - Transforming growth factor-beta (TGF-beta) type I receptor gene of chlamys farreri and single nucleotide polymorphism (SNP) locus of TGF-beta type I receptor gene - Google Patents
Transforming growth factor-beta (TGF-beta) type I receptor gene of chlamys farreri and single nucleotide polymorphism (SNP) locus of TGF-beta type I receptor gene Download PDFInfo
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- CN102899330B CN102899330B CN201210439861.0A CN201210439861A CN102899330B CN 102899330 B CN102899330 B CN 102899330B CN 201210439861 A CN201210439861 A CN 201210439861A CN 102899330 B CN102899330 B CN 102899330B
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Abstract
The invention relates to the cloning of a transforming growth factor-beta (TGF-beta) superfamily type I receptor gene Tgfbrl1 of chlamys farreri in a molecular genetic marker technology, a screening and typing technology of a single nucleotide polymorphism (SNP) locus which is relevant with the weight of adductor muscles in the gene and a method for the application of the gene to the breeding of the high-yield chlamys farreri. A total-length complementary deoxyribonucleic acid (cDNA) sequence of the Tgfbr1 gene of the chlamys farreri is obtained by utilizing a homology-based cloning strategy; an SNP lotus is discovered by blastn comparison, and primers and a probe are designed for the locus; and SNP typing is performed in a natural population of the chlamys farreri by using a high-resolution melting curve technology, and the weight of the adductor muscle of an individual is measured. Statistic analysis displays that the loci c.1815C>T are obviously relevant with the weight of the adductor muscles of the chlamys farreri, and the weight of the adductor muscles of individuals with TT genetypes is obviously higher than that of the adductor muscles of individuals with CC and CT genetypes. In the selective breeding process of the high-yield chlamys farreri, the breeding candidate colonies of the chlamys farreri can be subjected to c.1815C>T typing, and the individuals of which the loci c.1815C>T are TT genetypes are used as a breeding parent preferably by combining typing information of other loci which are relevant with growth properties.
Description
Technical field
The invention belongs to molecular genetic marker assistant breeding technical field, relate to the clone of chlamys farreri TGF-β I receptor gene Tgfbr1, in gene with the examination typing method in closed shell flesh weight related SNP site, and the method for applying in the seed selection of high yield scallop.
Background technology
Transforming growth factor-beta (transforming growth factor β, TGF-β) signal path plays a significant role in the biological procedureses such as cell proliferation, differentiation and growth.TGF-beta ligands passes through in conjunction with TGF-β I type and II receptor, and the intracellular signal molecule in downstream, passes the signal along in core the expression of goal of regulation and control gene.Wherein the activation of I receptor is considered to the important step that TGF-signal β transmits.Research in Mammals shows, the growth of Tgfbr1 gene and cell and rise in value relevantly, and single nucleotide polymorphism (SNP) site in this gene is relevant to many growth traitss.
In marine products eight delicacies, scallop is famous with its large and delicious closed shell flesh (cedductor).Cultivating high yield scallop new variety is needs of scallop culture industry fast development.Obtain and scallop growth traits, gene and gene locus that particularly closed shell flesh weight is relevant will contribute to its molecular breeding.At present, gene or the gene locus relevant to growth traits that in scallop, obtain are less, and there is no the report of TGF-signal β path acceptor gene.
Summary of the invention:
The object of the invention is to clone chlamys farreri TGF-β I receptor gene, in examination gene with the SNP site of closed shell flesh re-correlation, for the seed selection of high yield chlamys farreri provides the technological method of SNP mark and application thereof.
One aspect of the invention provides a kind of chlamys farreri TGF-β I receptor gene, and its nucleotides sequence is classified SEQ ID NO:1 as.
Above-mentioned TGF-β I receptor gene, the aminoacid sequence of its coding is SEQ ID NO:2.
Another aspect of the present invention provides the SNP site relevant to closed shell flesh weight of chlamys farreri TGF-β I receptor gene, is that sequence is that the base of 1815 of SEQ ID NO:1 gene is C or T.
The SNP of above-mentioned chlamys farreri TGF-β I receptor gene is for the breeding seed selection of chlamys farreri.
Probe and primer sequence information for detection of above-mentioned SNP are as follows:
Probe: 5 '-TTGTAAGGAAGTCGGATACCGAACT-3 ' SEQ ID NO:2;
Upstream primer: 5 '-GAGGTGTCAGACTGATTTTCAGGAG-3 ' SEQ ID NO:3;
Downstream primer: 5 '-ATGTCACAAAGGAAATTCATAAAGC-3 ' SEQ ID NO:4.
The SNP site of the present invention's screening is the chlamys farreri of TT type, closed shell flesh weight is significantly higher than CC type and CT type chlamys farreri, in the Breeding Process of high yield chlamys farreri, and can be for this site, the preferential individuality of TT type of selecting is as breeding parent, to improve the efficiency of seed selection.
Brief description of the drawings
Fig. 1: the chlamys farreri TGF-β I receptor full length gene cDNA sequence of the present invention's screening and the aminoacid sequence of coding thereof;
Wherein signal peptide sequence cleavage site marks with arrow, 10 conservative cysteine residues and cross-film district mark with shade, GS district marks with underscore, ATP land marks with shade and underscore, L45 loop marks with black surround, introne position marks with black triangle, and terminator codon represents with asterisk, and tailing signal marks with shade and black surround.SNP site c. 1815 C>T represent with bold Italic.
Embodiment
The present invention adopts homologous clone and cDNA end rapid amplifying (RACE) technology, and from chlamys farreri, clone has obtained TGF-beta superfamily I receptor gene cDNA full length sequence.This sequence total length 1908 bp, ORF length is 1575 bp, 524 amino acid of encoding, predicted protein molecular weight 59.27 KD, iso-electric point is 7.77; 5 ' UTR length is 15 bp, and 5 ' UTR length is 318 bp.At its proteins encoded N-terminal by one section of 30 signal peptide sequence that amino-acid residue forms, in 142-164 amino acids Chu Youyiduan cross-film district.There are 10 conservative cysteine residues extracellular region, and wherein 3 form typical CCX near cross-film district
5c saves structure.It in born of the same parents, is mainly serine/threonine kinase district, conservative property is higher, comprise the typical structure GS district of I receptor, the ATP at 229-250 amino acids place is in conjunction with position district, and the L45 loop (ADNKDGTW) of acceptor-Smad binding specificity is determined in the execution of 283-291 amino acids.
In chlamys farreri Tgfbr1 gene with the Screening analysis in closed shell flesh re-correlation SNP site and the method for auxiliary high yield chlamys farreri seed selection thereof:
The Tgfbr1 gene cDNA sequence obtaining by analysis, examination to a 1 SNP site, called after is c.1815C>T.Use high resolving power melting curve technology (High resolution melting, HRM) detection site polymorphism in natural population of chlamys farreri, result shows, in Suo Ce colony, this SNP site is two condition (Fig. 1).One-way ANOVA and post-hoc inspection show, c.1815C>T the heavy significant correlation of site and chlamys farreri closed shell flesh, the closed shell flesh of TT type individuality is heavily significantly higher than CC and CT type individuality (P<0.05) (table 1).Utilize site typing method c.1815C>T, can carry out gene type to chlamys farreri breeding candidate colony, in conjunction with the somatotype information of other and growth traits related locus, preferentially select site c.1815C>T for the individuality of TT type is for the parent of high yield scallop seed selection.
Below by embodiment, the present invention will be further described.
Embodiment 1.
Clone obtains chlamys farreri TGF-beta superfamily I receptor gene Tgfbr1, has the sequence shown in SEQ ID NO. 1.
Chlamys farreri Tgfbr1 gene cDNA sequence clone in the present invention comprises the following steps:
A) extraction of the total RNA of chlamys farreri closed shell flesh;
B) cDNA the first chain is synthetic;
C) acquisition of goal gene cDNA fragment;
D) chlamys farreri 5 ' RACE and 3 ' RACE library construction;
E) acquisition of goal gene full length cDNA sequence;
F) bioinformatic analysis of goal gene.
Concrete operations are as follows:
A) extraction of the total RNA of chlamys farreri: extract total RNA according to Trizol method from chlamys farreri closed shell flesh.
B) cDNA the first chain is synthetic: taking the total RNA of 2 μ g as template, add respectively 1 μ l 20 μ M Oligo d (T)
18, RNase & DNase-free H
2o complements to 13 μ l, mixes centrifugal.70 DEG C of insulation 10 min, are placed on ice, immediately to open RNA secondary structure.Add successively: 5 μ l 5 × M-MLV Buffer, 5 μ l dNTP (2.5mM), (l), (200U/ μ is l) for 1 μ l M-MLV for 40U/ μ for 1 μ l RNasin; Mix centrifugal after, 42 DEG C, 90 min.94 DEG C, 5 min, with deactivation ThermoScript II.-20 DEG C of preservations after packing.
C) homologous clone obtains the cDNA fragment of goal gene: according to TGF-β I receptor gene cDNA and the aminoacid sequence of the species such as the Pacific oyster of having reported, sea urchin, zebra fish, rainbow trout, Africa xenopus, jungle fowl, mouse and the mankind, select the high zone design degenerated primer of conservative property:
d)TGFBR1-f1:?5′-GAYAAYAARGAYAAYGGIACITGG-3′；
e)TGFBR1-r1:?5′-?GGIGCCATRTAICKYTTIGTIC-3′。
Then, taking cDNA the first chain as masterplate, carry out pcr amplification.PCR product detects with 1.2% agarose gel electrophoresis, reclaim test kit (the raw work in Shanghai) with glue and carry out PCR product purification, be connected with pMD18-T carrier (Dalian precious biotechnology company limited) again, with reference to " the molecular cloning third edition ", (Huang Peitang translates, Science Press, 2002) method transform bacillus coli DH 5 alpha.After bacterium colony PCR detects, picking positive colony checks order, and known array in sequencing result and GenBank albumen database is made to blastx homology analysis (http://blast.ncbi.nlm.nih.gov/Blast/).
F) chlamys farreri 5 ' RACE and 3 ' RACE library construction: utilize the SMART RACE cDNA Amplification Kit of Clontech company to build 5 ' RACE and 3 ' RACE library.
G) acquisition of goal gene full length cDNA sequence: the Tgfbr1 gene cDNA fragment sequence obtaining according to (c), with PrimerPremier 5.0 purpose of design gene RACE primer respectively,
3 ' ' RACE primer: 5 '-TCAACCGTACTGTGGTCAGCATCTCCG-3 '
5 ' RACE primer: 5 '-CCAATGATCTCCATGTGGAGGTGGGCG-3 '.
Utilize respectively 3 ' RACE primer and 5 ' RACE primer and joint primer NUP(5' – AAGCAGTGGTATCAACGCAGAGT – 3') carry out the amplification of 3 ' end and 5 ' end.PCR product detects with 1.2% agarose gel electrophoresis, reclaim test kit (the raw work in Shanghai) with glue and carry out PCR product purification, be connected with pMD18-T carrier (Dalian precious biotechnology company limited) again, with reference to " the molecular cloning third edition ", (Huang Peitang translates, Science Press, 2002) method transform bacillus coli DH 5 alpha.After bacterium colony PCR detects, to 5 ' RACE and 3 ' RACE, 3 positive colonies of each picking check order, and by obtaining cDNA full length sequence after the sequence two ends splicing of 5 ' RACE and 3 ' RACE order-checking acquisition, see SEQ ID NO. 1.
3 ' and 5 ' RLM-RACE, utilize 50 μ L reaction systems:
PCR response procedures used increases: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30sec, 72 DEG C of 3min, 5 circulations; 94 DEG C of sex change 30sec, 70 DEG C of annealing 30sec, 72 DEG C are extended 3min, 5 circulations; 94 DEG C of sex change 30sec, 65 DEG C of annealing 30sec, 72 DEG C are extended 3min, 28 circulations; 72 DEG C are extended 10min.
H) bioinformatic analysis of goal gene:
In ncbi database, this sequence is carried out to BLASTx (http://blast.ncbi.nlm.nih.gov/) comparison.Application ProtParam instrument is predicted SQR physics and chemistry parameter, comprises relative molecular weight, the theoretical pI value etc. (www.expasy.ch/tools/protparam.html) of protein.By internet line server http://www.cbs.dtu.dk/services/SignalP/, TGF-beta superfamily I receptor protein sequence is carried out to the prediction of signal peptide.Use the functional domain of SMART (http://smart.embl-heidelberg.de/) this albumen of software prediction.Use Phyre 2.0 (http://www.imperial.ac.uk/phyre2/) and Swiss-Pdb Viewer 4.04 (http://spdbv.vital-it.ch/) to analyze serine/threonine kinase district.Use ClustalW2 and GeneDoc (http://www.nrbsc.org/gfx/genedoc/index.html) Multiple Sequence Alignment instrument and other species TGF-beta superfamily I receptor protein sequence to compare, analyze this protein function region.Analytical results as shown in Figure 1.
Embodiment 2
In chlamys farreri Tgfbr1 gene in the present invention, the Screening analysis in closed shell flesh weight related SNP site and the method applied in the seed selection of high yield chlamys farreri thereof, comprise the following steps:
A) chlamys farreri Tgfbr1 gene SNP site examination;
B) scallop (Chlamys farreri) group DNA extraction;
C) c.1815C>T primer and probe design for somatotype of site;
D) c.1815C>T somatotype of SNP;
E) correlation analysis that c.1815C>T genotype and closed shell flesh weigh;
F) the c.1815C> method of auxiliary high yield chlamys farreri seed selection of SNP.
Concrete operations are as follows:
A) chlamys farreri Tgfbr1 gene SNP site examination: analyze 5 ' RACE clones' different from 3 ' RACE sequencing result, search candidate SNP locus.
B) extraction of chlamys farreri DNA: get closed shell flesh approximately 0.1 g, add 500 μ l STE lysis buffers, shred, add successively 50 μ l 10% SDS, 5 μ l Proteinase Ks (20 mg/ml), 56 DEG C of cracking approximately 3 h, clarify to lysate.(l), (250 μ l), rock 20 min to chloroform/primary isoamyl alcohol (24:1) to 250 μ gently, centrifugal 10 min of 12000 rpm to add the saturated phenol of same volume.Get supernatant, repeat above-mentioned steps, until between water and organic phase without protein layer.Get supernatant, add same volume chloroform/primary isoamyl alcohol, gently shake 20 min, centrifugal 10 min of 12000 rpm.Get supernatant, add 1/10 volume 3 M NaAc (pH 5.2) and 2 times of cold dehydrated alcohols of volume, after shaking up ,-20 DEG C leave standstill 20 min, centrifugal 20 min of 12500 rpm.Nucleic acid is deposited in to the pipe end.Abandon supernatant, 70% washing with alcohol precipitation.Collecting precipitation, air drying to ethanol all volatilizees.Add 20 μ l TE (containing RNase A) dissolving DNA, 37 DEG C leave standstill after approximately 30 min, 4 DEG C of preservations.1% agarose gel electrophoresis detects DNA sample, UV spectrophotometer measuring concentration and purity.
C) the c.1815C>T design of primer and probe for somatotype of site: near the gene order according to SNP site c.1815C>T, design HRM is primer and probe for somatotype.Standard is as follows: 1) probe length is 19-35 bp; Only comprise 1 candidate SNP locus, site occupy in the middle of probe, not containing deletion segment; Annealing temperature is 58 DEG C-60 DEG C; Not containing simple repeated sequence and palindromic sequence.2) primer length 19-26bp; In primer sequence, do not comprise candidate SNP locus and deletion segment; Amplified production length 70-130 bp; GC content 30%-70%.Probe TGFBR1pb1 sequence is:
5′-?TTGTAAGGAAGTCGGATACCGAACT?-3′（SEQ?ID?NO:3）；
Primer sequence is upstream primer TGFBR1sf1:
5′-?GAGGTGTCAGACTGATTTTCAGGAG-3′；
Downstream primer TGFBR1sr1:5 '-ATGTCACAAAGGAAATTCATAAAGC-3 '.
D) c.1815C>T somatotype of SNP site: taking 196 individual genomic dnas of a natural population of chlamys farreri as template, utilize primer TGFBR1sf1 and TGFBR1sr1 to carry out pcr amplification, reaction system is as follows: 10 × Buffer, 1 μ l, 2.5 mM dNTP 0.8 μ l, 2.5 mM MgCl
2, 0.6 μ l, TGFBR1sf1 (10 μ M) 0.1 μ l, TGFBR1sr1 (10 μ M) 0.5 μ l, Taq enzyme (5U/ μ is 0.1 μ l l), template 0.5 μ l, and LC Green saturated fluorescence dyestuff 0.7 μ l, adds H
2o complements to 10 μ l.Amplified reaction completes in Biometra T-Gradient PCR system, and reaction conditions is as follows: 94 DEG C of 4 min; 94 DEG C of 40 s, 63 DEG C of 40 s, 60 circulations; 72 DEG C of 5 min.In every part of PCR product, add the corresponding probe TGFBR1pb1 of 3 μ L (10 μ M), 95 DEG C of sex change 10 min.Get 10 μ l denatured products and proceed in 96 hole BLK/WHT plates (Bio-Rad), add 15 μ l mineral oil, centrifugal 1 min of 2000 rpm.In Light-Scanner, carry out somatotype detection, be warming up to 95 DEG C, continuous collecting fluorescent signal with the speed of 0.1 DEG C/s from 40 DEG C.With LightScanner Call IT v2.0 software analysis solubility curve.The genotype of recording individual: the high person of the unimodal temperature of melting curve is identical with probe sequence homozygous, and the bimodal person of melting curve is heterozygous, the low person of the unimodal temperature of melting curve is homozygous for suddenling change.
E) correlation analysis that c.1815C>T genotype and closed shell flesh weigh: by the closed shell flesh weight of this each individuality of colony of electronics balance measurement.Utilize One-way ANOVA and post-hoc inspection, site of analysis is the difference of different genotype chlamys farreri closed shell flesh weight-average value c.1815C>T, the c.1815C>T dependency heavy with chlamys farreri closed shell flesh of inspection SNP, result is as shown in the table:
Table 1:c. 1815 C>T site different genotype chlamys farreri closed shell flesh weight
In table, closed shell flesh is reused its mean+SD and is represented, represents to have significant difference (P<0.05) if numerical value upper right corner lowercase is different in every row.Result shows CC and the individual heavy significant difference of closed shell flesh of TT type (p=0.037), the individual heavy significant difference of closed shell flesh of CT and TT type (p=0.031).
SNP is the method for auxiliary high yield chlamys farreri seed selection c.1815C>T
Because c.1815C>T site is the chlamys farreri of TT type, the weight of its closed shell flesh is significantly higher than CC and CT type individuality, therefore, in the selection breeding process of high yield chlamys farreri, can carry out c.1815C>T somatotype of site to chlamys farreri breeding candidate colony, in conjunction with the somatotype information of other and growth traits related locus, preferentially select site c.1815C>T for the individuality of TT type is as breeding parent.Practical application effect shows, the parent who screens by primer and the probe of SNP of the present invention, and in its offspring who breeds, the weight of closed shell flesh is significantly higher than the not control group of screening.
Claims (1)
1. for detection of probe and the primer in the SNP site relevant to chlamys farreri closed shell flesh weight, its sequence information is as follows:
Probe sequence is SEQ ID NO:3;
Upstream primer sequence is SEQ ID NO:4;
Downstream primer sequence is SEQ ID NO:5;
Described SNP site is 1830 of the nucleotides sequence TGF-β I receptor gene of classifying SEQ ID NO:1 as, and its base is C or T.
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| CN103740729B (en) * | 2014-01-25 | 2015-07-08 | 中国海洋大学 | SNP locus related to growth characteristics of patinopecten yessoensis and detection and application thereof |
| CN110343742B (en) * | 2019-07-23 | 2023-03-21 | 中国海洋大学 | Trace shellfish DNA extraction method for high-throughput sequencing library preparation |
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| .2008,139. * |
| .2011,296. * |
| 2008年中国水产学会学术年会论文摘要集> * |
| 2011中国遗传学会大会论文摘要汇编> * |
| 郭慧慧等.栉孔扇贝myostatin信号通路相关基因的克隆及表达分析.< * |
| 郭慧慧等.栉孔扇贝myostatin信号通路相关基因的克隆及表达分析.<2008年中国水产学会学术年会论文摘要集>.2008,139. |
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