CN105296499A - Cyrinus carpio IGF3 gene as well as amplification primer group and amplification method thereof - Google Patents
Cyrinus carpio IGF3 gene as well as amplification primer group and amplification method thereof Download PDFInfo
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Abstract
The invention relates to a cyrinus carpio IGF3 gene as well as an amplification primer group and an amplification method thereof, and belongs to the technical field of molecular biology. A full-length cDNA sequence of the IGF3 gene is cloned from cyrinus carpio ovarian tissue by the primer group provided by the invention and a 3' and 5'-end rapid amplification (RACE) technology; the gene comprises two transcripts IGF3-1 and IGF3-2; the full length of cDNA of the IGF3-1 is 753bp; 205 amino acids are encoded; the full length of the cDNA of the IGF3-2 is 1199bp; and 198 amino acids are encoded. According to the cyrinus carpio IGF3 gene, an important basis is laid for research of functions of the cyrinus carpio IGF3 gene, especially an important role in gonad development; and the cyrinus carpio IGF3 gene is of great significance in understanding excellent economic characters of the cyrinus carpio.
Description
Technical field
The present invention relates to a kind of good fortune auspicious carp IGF3 gene and amplimer group thereof and amplification method, belong to technical field of molecular biology.
Background technology
Growth and reproduction are the most basic features of organism.The regulation and control of vertebrate growth is subject to " Hypothalamus-pituitary-liver " axle, reproduction activity is mainly by " brain/hypothalamic-pituitary-gonadal " axle regulation and control (woods is great, 2007).Growth is closely related distinguished again mutually with reproduction, and insulin-like growth factor system (IGFs) is the key factor that growth axis is cross-linked mutually with Reproductive Axis.IGFs comprises 3 part (type-1 insulin like growth factor IGF1, IMA-IGF2BP3-001 IGF2 and rhIGF-1 3IGF3), 2 acceptors (type-1 insulin like growth factor acceptor IGF1R and IMA-IGF2BP3-001 acceptor IGF2R) and 6 igf binding proteins (IGFBPs).Fish IGFs(IGF1, IGF2) basic function be growth promotion, but since IGF3 gene (Wangetal., 2008 after the ovary of fish is found; Lietal., 2011), they start to receive much concern in the function of sexual gland, start to turn to reproduction by growth to the visual angle of fish GH-IGFs axle.Nearest research shows, the growth of fish sexual gland and maturation are along with cytodifferentiation and tissue growth, and traditional growth factors I GF1, IGF2 and the IGF3 recently found, play an important role (Reinecke, 2010 to fish sexual gland; Yang Huirong etc., 2013).At present, the IGF3 gene only in zebra fish, bolti and perch is cloned, and the IGF3 gene of carp have not been reported.
The auspicious carp of good fortune (FFRCCyprinuscarpio) is this laboratory with jian carp and the Yellow River carp for original parent, adopt the method for total fertilization failure, PIT mark auxiliary under through 1 generation unexpected mass incident and continuous 4 generation BLUP family selective breedings and the aquaculture new variety that obtain, obtain larger genetic progress, growth traits is significantly improved, large cultured freshwater fish new variety that the Ministry of Agriculture " 12 " promotes mainly, cultivation is promoted in overwhelming majority area at home at present, become one of the important channel (Dong Jie, 2014) of China's growth of agricultural efficiency, increasing peasant income.Along with molecular biological development, inquire into the molecular mechanism that seed selection improves economic characters, thus the development trend of fishery breeding technology will be become to molecular breeding technology transition.And sexual gland is the oogenetic place of fish essence, it is the critical organ of life procreation.The research carrying out fish gonadal development acceleration mechanism is also the important research topic of life science always.
Summary of the invention
The object of the invention is to overcome above-mentioned weak point, a kind of good fortune auspicious carp IGF3 gene cDNA full length sequence, its amplimer group and amplification method are provided, important foundation is established to the research of the vital role of good fortune auspicious carp IGF3 gene function especially in gonad development, and significant to the molecular mechanism understanding the excellent economic characters of the auspicious carp of good fortune.
According to technical scheme provided by the invention: a kind of good fortune auspicious carp IGF3 gene, comprise IGF3-1, its nucleotide sequence is as shown in SEQIDNO.1; IGF3-2, its nucleotide sequence is as shown in SEQIDNO.2.
For the primer sets of the auspicious carp IGF3 of the good fortune described in pcr amplification gene, it is made up of following primer:
3 ' forward outer primer IGF3-F1: sequence is as shown in SEQIDNO.3;
3 ' forward inner primer IGF3-F2: sequence is as shown in SEQIDNO.4;
5 ' reverse outer primer IGF3-R1: sequence is as shown in SEQIDNO.5;
5 ' reverse inner primer IGF3-R2: sequence is as shown in SEQIDNO.6.
Obtained the method for described good fortune auspicious carp IGF3 gene by amplification, step is:
(1) obtain the total serum IgE of the auspicious carp ovary tissue of good fortune, reverse transcription is 3 ' cDNA template and 5 ' cDNA template respectively;
(2) respectively organize high-flux sequence transcript profile library from the auspicious carp of good fortune and screen IGF3 gene fragment, in this, as IGF3 gene intermediate sequence;
(3) with step (1) gained cDNA for template, with primer sets according to claim 2 for primer, obtained 3 ' end fragment and the 5 ' end fragment of good fortune auspicious carp IGF3 gene by RACEPCR amplification;
(4) 3 ' end fragment of step (2) described IGF3 gene intermediate sequence, step (3) described IGF3 gene and the splicing of 5 ' end fragment are obtained good fortune auspicious carp IGF3cDNA full length nucleotide sequence IGF3-1, SEQIDNO.1 and IGF3-2, SEQIDNO.2.
Step (3) described amplification is shell type two-wheeled pcr amplification.
Described amplification obtains the method for good fortune auspicious carp IGF3 gene, and concrete steps are as follows:
(1) Total RNAs extraction: the total serum IgE extracting the auspicious carp ovary tissue of good fortune, and reverse transcription is 3 ' cDNA template and 5 ' cDNA template respectively, the first chain cDNA synthesis completes according to Takara company PrimerScriptRTMasterMixPerfectRealTime test kit specification sheets operation steps;
(2) segment amplification in the middle of: respectively organize high-flux sequence transcript profile library from the auspicious carp of good fortune and screen IGF3 gene fragment, in this, as IGF3 gene intermediate sequence; After carrying out homology comparison according to transcript profile sequencing result, design pair of primers
IGF3-F：5’-GTAGAGCAGTGTTGTGTGCG-3’；
IGF3-R：5’-TGCATGCTTCTGGTATCGCT-3’；
Then intermediate sequence is increased, adopt round pcr to verify intermediate segment, confirm the exactness of IGF3 gene intermediate sequence;
(3) 3 ' and 5 ' acquisition of sequence end fragment: the good fortune auspicious carp IGF3 intermediate segment obtained according to step (2), design 3 ' forward outer primer according to claim 2,3 ' forward inner primer, 5 ' reverse outer primer and 5 ' reverse inner primer; With step (1) gained cDNA for template, obtained 3 ' end fragment and the 5 ' end fragment of good fortune auspicious carp IGF3 gene by RACEPCR amplification;
Wherein, universal primer in 3 ' forward outer primer and RACE test kit forms primer pair and carries out first round pcr amplification, with 3 ' forward inner primer and universal primer, second is carried out to amplified production and takes turns pcr amplification, take turns pcr amplification product 1.5% agarose gel electrophoresis by second and detect acquisition single band; Equally, 5 ' reverse outer primer and universal primer carry out first round pcr amplification, carry out second take turns pcr amplification to amplified production with 5 ' reverse inner primer and universal primer, and second takes turns pcr amplification product obtains 2 bands clearly through agarose gel electrophoresis;
(4) splice: after the two band of gained cuts glue purification after the single band of gained after described for step (3) 3 ' end amplification and 5 ' end amplification, be connected to pMD18-T carrier, be transformed in DH5 α competent cell, screen through blue hickie, select positive plasmid, obtain required object band through agarose gel electrophoresis qualification, obtain good fortune auspicious carp 3 ' nucleotide sequence fragment and 5 ' nucleotide sequence fragment by order-checking; Finally by 25 ' fragment sequences respectively with intermediate segment and 3 ' fragment assembly, obtain good fortune auspicious carp IGF3cDNA full length nucleotide sequence IGF3-1 and IGF3-2.
The present invention utilizes construction cDNA library, adopt reverse transcriptase polymerase chain reaction (RT-PCR), nested polymerase chain reaction (nested-PCR), RACE technology, IGF3 full length gene cDNA sequence has been cloned into first from the auspicious carp ovary tissue of good fortune, comprise two kinds of transcript IGF3-1 and IGF3-2, and IGF3 aminoacid sequence feature, homology, evolutionary degree and the express spectra coded by it is analyzed, determine that it is good fortune auspicious carp IGF3 gene.Described IGF3-1cDNA total length 753bp, open reading frame 615bp, 205 amino acid of encoding; IGF3-2cDNA total length 1199bp, open reading frame 594bp, 198 amino acid of encoding, have polyadenylic acid tail, and this gene plays a significant role in good fortune auspicious carp gonad development process.
Aminoacid sequence coded by the auspicious carp of described good fortune IGF3-1 and IGF3-2 is all the highest with the IGF3 homology of zebra fish through comparison, use MEGA5.1 software building NJ phylogenetic tree (as Fig. 1) visible, first the auspicious carp IGF-3 of good fortune gathers with zebra fish is one, then meets with other teleostei.
Beneficial effect of the present invention: the present invention has been cloned into IGF3 full length gene cDNA sequence first from the auspicious carp ovary tissue of good fortune, comprise two kinds of transcript IGF3-1 and IGF3-2, and IGF3 aminoacid sequence feature, homology, evolutionary degree and the express spectra coded by it is analyzed, important foundation is established in the research of these results to the vital role of good fortune auspicious carp IGF3 gene function especially in gonad development, and significant to the molecular mechanism understanding the excellent economic characters of the auspicious carp of good fortune.The cloning process of IGF3 gene provided by the invention also can use for reference be applied to screening other fish IGF3 gene research among.
Accompanying drawing explanation
The NJ systematic evolution tree of the IGF3 of Fig. 1 various animals;
The expression characterization figure (Semiquatitative RT-PCR assay) of Fig. 2 good fortune auspicious carp IGF3mRNA in each tissue.Band is from left to right followed successively by Marker, heart, muscle, brain, skin, spleen, kidney, intestines, liver, blood, ovary and spermary.
Embodiment
Below in conjunction with embodiment, the present invention is further described, but be not construed as limiting the invention.
Embodiment 1: the clone of good fortune auspicious carp IGF3cDNA full length sequence
(1) Total RNAs extraction: the auspicious carp of good fortune is from cultivation base, China Aquatic Science Research Academy Fresh Water Fishery Research Center Yixing.Yuanpinghao (Tianjin) Biological Technology Co., Ltd. EASYspin tissue/cell ultrapure RNA rapid extraction test kit specification sheets is adopted to operate.And reverse transcription becomes cDNA, the first chain cDNA synthesis completes according to Takara company PrimerScriptRTMasterMixPerfectRealTime test kit specification sheets operation steps.
(2) segment amplification in the middle of: after carrying out homology comparison according to transcript profile sequencing result, design pair of primers IGF3-F:5 '-GTAGAGCAGTGTTGTGTGCG-3 ';
IGF3-R:5 '-TGCATGCTTCTGGTATCGCT-3 '; Adopt the middle segment of conventional molecular biology method amplification good fortune auspicious carp IGF3 gene, adopt round pcr to verify intermediate segment, PCR system is as following table:
| Takara Premix Ex Taq TM hot Start version | 25 μL |
| CDNA template | 2 μL |
| IGF3-F（20μM） | 1 μL |
| IGF3-R（20μM） | 1 μL |
| Sterile purified water | Up to 50μL |
PCR reaction conditions is as follows: 98 DEG C of 10s, 59 DEG C of 30s, 72 DEG C of 1min, 30 circulations; 72 DEG C, 10min.PMD18-T carrier is connected to after the PCR primer purifying of amplification, be transformed in DH5 α competent cell, screen through blue hickie, select positive plasmid, required object band is obtained through agarose gel electrophoresis qualification, then deliver to the order-checking of Shanghai Sheng Gong Bioisystech Co., Ltd, confirm the exactness of the middle segment of IGF3 gene.
(3) 3 ' and 5 ' acquisition of terminal sequence fragment: utilize RACE technology to obtain 3 ' and 5 ' sequence, RACE (rapid-amplificationofcDNAends) is the technology of being carried out cDNA end quick clone by PCR, by known one section of cDNA fragment, by extending amplification toward two ends thus obtaining 3 ' complete end fragment and the method for 5 ' end fragment, amplification method refers to CLONTECHSMARTRACEkit specification sheets.Concrete steps are as follows:
A, according to the explanation in RACE test kit, by the total serum IgE of auspicious for the good fortune of extraction carp ovary tissue respectively reverse transcription be acquisition 3 ' and 5 ' cDNA template.
B, according to obtain good fortune auspicious carp IGF3 intermediate segment, design 3 ' forward outer primer, 3 ' forward inner primer, 5 ' reverse outer primer and 5 ' reverse inner primer.Wherein, universal primer in 3 ' forward outer primer and RACE test kit forms primer pair and carries out first round pcr amplification, with 3 ' forward inner primer and universal primer, second is carried out to amplified production and takes turns pcr amplification, take turns pcr amplification product 1.5% agarose gel electrophoresis by second and detect acquisition single band.Equally, 5 ' reverse outer primer and universal primer carry out first round pcr amplification, carry out second take turns pcr amplification to amplified production with 5 ' reverse inner primer and universal primer, and second takes turns pcr amplification product obtains 2 bands clearly through agarose gel electrophoresis.
3 ' forward outer primer IGF3-F1:5'-GCTGGCGAGGCTGCCAAAGCACG-3';
3 ' forward inner primer IGF3-F2:5' – CCAAAGCACGCTGTGGACGAGAACT-3';
5 ' reverse outer primer IGF3-R1:5'-CATCACGCTGCACCCTTTTGGGTTT-3';
5 ' reverse inner primer IGF3-R2:5' – TTCCCACGAGAGCGGGGGCCG-3'.
C, the single band of 3 ' and two bands of 5 ' are cut glue purification after be connected to pMD18-T carrier, be transformed in DH5 α competent cell, screen through blue hickie, select positive plasmid, required object band is obtained through agarose gel electrophoresis qualification, then deliver to the order-checking of Shanghai Sheng Gong Bioisystech Co., Ltd, obtain the auspicious carp 3 ' of good fortune and 5 ' nucleotide sequence.By 25 ' fragment sequences respectively with intermediate segment and 3 ' fragment assembly, obtain good fortune auspicious carp IGF3cDNA full length nucleotide sequence IGF3-1(SEQIDNO.1) and IGF3-2(SEQIDNO.2).
Embodiment 2: bioinformatic analysis
Carry out on-line analysis to gained sequence NCBI website blastx, confirm the exactness of good fortune auspicious carp IGF3 sequence, it has the distinctive structural domain of IGF family.IGF3-1cDNA total length 753bp, open reading frame 615bp, 205 amino acid of encoding, IGF3-2cDNA total length 1199bp, open reading frame 594bp, 198 amino acid of encoding, have polyadenylic acid tail.Aminoacid sequence coded by IGF3-1 and IGF3-2 is all the highest with the IGF3 homology of zebra fish through comparison, use MEGA5.1 software building NJ phylogenetic tree (as Fig. 1) visible, first the auspicious carp IGF-3 of good fortune gathers with zebra fish is one, then meets with other teleostei.
Embodiment 3: semi-quantitative RT-PCR analysis
Carry out IGF3 gene expression amount in different tissues with semiquantitive RT-PCR to detect, according to IGF3cDNA full length sequence design primer pair sequence be:
IGF3-F3：5′-AGCAGCGATACCAGAAGCAT-3′；
IGF3-R3：5′-GAGTTCCACCGGTAAAGCGT-3′。
Extract the RNA of the auspicious carp heart of good fortune, muscle, brain, skin, spleen, kidney, intestines, liver, blood, ovary and spermary tissue respectively, and reverse transcription is cDNA template.Reaction system is identical with the checking of IGF3 intermediate segment, and PCR reaction conditions is as follows: 98 DEG C of 10s, 58 DEG C of 30s, 72 DEG C of 1min, 30 circulations; 72 DEG C, 10min.PCR primer 1.5% agarose gel electrophoresis detects, result is (band is from left to right followed successively by Marker, heart, muscle, brain, skin, spleen, kidney, intestines, liver, blood, ovary and spermary) as shown in Figure 2, and the expression amount of IGF3 gene in good fortune auspicious carp blood, ovary and spermary is the highest.
The foregoing is only the preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., should be considered as protection scope of the present invention.
SEQIDNO.1
<210>1
<211>753
<212>DNA
<213>IGF3-1
<400>1
TCTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGTACATGGGAGTAGCT60
CTTTAGAGAGACCAAACCATTAAAAAAATAGACATGCCGTCAGACGGAATGCCAACCTGT120
CATGCCAGACGGCTTCAGATGCTTAGAGGATTCCTGCTGAAGGTGCCCAGCTGGCGAAGT180
GTGTGTGTCCTCTATTCCCTGTACTGCGTCCTGATCCTGCCAGACGCCGGCGAGGCTGCC240
AAAGCACGCTGTGGACGAGAACTAGTTGCTGACCTGGAGTTTGTGTGTGGGGACCGTGGC300
TTTTACAGAGGCAAACCTGGAGCAGCCCGTAGCGGCGGCCCCCGCTCTCGTGGGAAAGGG360
ATCGTAGAGCAGTGTTGTGTGCGTGGATGTGACCTCCAGCATTTGGAGTCGTACTGTGCA420
AAACCCAAAAGGGTGCAGCGTGACGTCCCTGCATCTCTGCAACAGAATCTGGAAGATCAG480
TTTTGGCTTGTGTTTCTGAGGCAATACCAGAAGCATGCAGAGATGAACAGAGATGAGGAG540
GCTGCTTCTGAAAGACTCAGAGAGCGAATGCTTTACCGATGGCACTCCAGAGATTCAGTA600
TTACTAACCAACCCACCCCCCTCAACACAACAGCATCCTTCCACTGACACAGTGACATCA660
GGACCAACATTTATCCACCACATCCTTTTTGGATCTGTGTGGACCCTATGAATCACCTTC720
CTTTTCAAAAAAAAAAAAAAAAAAAAAAAAAAA753
SEQIDNO.2
<210>2
<211>1199
<212>DNA
<213>IGF3-2
<400>1
TCTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGTACATGGGAGTAGCT60
CTTTAGAGAGACCAAACCATTAAAAAAATAGACATGCCGTCAGACGGAATGCCAACCTGT120
CATGCCAGACGGCTTCAGATGCTTAGAGGATTCCTGCTGAAGGTGCCCAGCTGGCGAAGT180
GTGTGTGTCCTCTATTCCCTGTACTGCGTCCTGATCCTGCCAGACGCCGGCGAGGCTGCC240
AAAGCACGCTGTGGACGAGAACTAGTTGCTGACCTGGAGTTTGTGTGTGGAGACCGTGGC300
TTTTACAGAGGCAAACCTGGAGCAGCCCGTAGCGGCGGCCCCCGCTCTCGTGGGAAAGGG360
ATCGTAGAGCAGTGTTGTGTGCGTGGATGTGACCTCCAGCATTTGGAGTCATATTGTGCC420
AAACCCAAGAGGCTGCGGCGTGACATCCCTGCATCTCTGCAGCAGACTTCAGAAGATCAG480
TTTTGGCTGGTGTTTCAGCAGCGATACCAGAAGCATGCACAGATGAACAGAGATGAGGAC540
GCTGCTTCTCAAAGACTCAGAGAGCGGACGCTTTACCGGTGGAACTCCAGAGATTCAGTT600
TTACTAACCAACCAACCCTCCTCAACACAGCAGCAACCTTCCACTGAGAGAATGACATCA660
AGACCAACATTTCTCCCCCACATCCGGTAGTCCTGGCTCATTTTGGATCTGTGTGGATCC720
TATGAATCACCTTCCTTCACTAAAAAAAGACACTTCTTTCCTGGCAGATGTATAAACACT780
ATGTAAGAGAAACTAGAGGACAACATGCCTAAGATGACATAAAATAGTGAAGAGCAGGGT840
GTACCTGAACTCTCACGGGCTGTACTAGTTTCATTAAGCATATGCTGAATACAAGATAAA900
TTGTATTTTTCCATTCAGTTTTGTTTTCAGCATCCCTCTGTCTTTGGGTAAACGAGTTCA960
AAGCAAGTTAAAGTTGAATGTGTTTCTATGATGGCTGAGGTAAGTCATTTTGAATGTAAA1020
TGTCACATTTTTAATGAAAGATTACTGATGTAAGTGCATATATTTTATGTTTAATTTATG1080
TTCTATAAGATTACAGTTTTCTGGTCCTTGTAAAATATGTATCTAAAGCAGATTTGTAAA1140
GAATTTTAAATAAAAGAAAACTGTGAGGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAA1199
SEQIDNO.3
<210>3
<211>23
<212>DNA
<213>3 ' forward outer primer IGF3-F1
<400>1
GCTGGCGAGGCTGCCAAAGCACG23
SEQIDNO.4
<210>4
<211>25
<212>DNA
<213>3 ' forward inner primer IGF3-F2
<400>1
CCAAAGCACGCTGTGGACGAGAACT25
SEQIDNO.5
<210>5
<211>25
<212>DNA
<213>5 ' is outer primer IGF3-R1 oppositely
<400>1
CATCACGCTGCACCCTTTTGGGTTT25
SEQIDNO.6
<210>5
<211>21
<212>DNA
<213>5 ' is inner primer IGF3-R2 oppositely
<400>1
TTCCCACGAGAGCGGGGGCCG21
Claims (5)
1. a good fortune auspicious carp IGF3 gene, is characterized in that: comprise IGF3-1, and its nucleotide sequence is as shown in SEQIDNO.1; IGF3-2, its nucleotide sequence is as shown in SEQIDNO.2.
2., for the primer sets of pcr amplification good fortune according to claim 1 auspicious carp IGF3 gene, it is characterized in that: it is made up of following primer:
3 ' forward outer primer IGF3-F1: sequence is as shown in SEQIDNO.3;
3 ' forward inner primer IGF3-F2: sequence is as shown in SEQIDNO.4;
5 ' reverse outer primer IGF3-R1: sequence is as shown in SEQIDNO.5;
5 ' reverse inner primer IGF3-R2: sequence is as shown in SEQIDNO.6.
3. obtained the method for the auspicious carp IGF3 of good fortune described in claim 1 gene by amplification, it is characterized in that step is:
(1) obtain the total serum IgE of the auspicious carp ovary tissue of good fortune, reverse transcription is 3 ' cDNA template and 5 ' cDNA template respectively;
(2) respectively organize high-flux sequence transcript profile library from the auspicious carp of good fortune and screen IGF3 gene fragment, in this, as IGF3 gene intermediate sequence;
(3) with step (1) gained cDNA for template, with primer sets according to claim 2 for primer, obtained 3 ' end fragment and the 5 ' end fragment of good fortune auspicious carp IGF3 gene by RACEPCR amplification;
(4) 3 ' end fragment of step (2) described IGF3 gene intermediate sequence, step (3) described IGF3 gene and the splicing of 5 ' end fragment are obtained good fortune auspicious carp IGF3cDNA full length nucleotide sequence IGF3-1, SEQIDNO.1 and IGF3-2, SEQIDNO.2.
4. amplification obtains the method for good fortune auspicious carp IGF3 gene as claimed in claim 3, it is characterized in that: step (3) described amplification is shell type two-wheeled pcr amplification.
5. as described in claim 3 or 4, amplification obtains the method for good fortune auspicious carp IGF3 gene, it is characterized in that concrete steps are as follows:
(1) Total RNAs extraction: the total serum IgE extracting the auspicious carp ovary tissue of good fortune, and reverse transcription is 3 ' cDNA template and 5 ' cDNA template respectively, the first chain cDNA synthesis completes according to Takara company PrimerScriptRTMasterMixPerfectRealTime test kit specification sheets operation steps;
(2) segment amplification in the middle of: respectively organize high-flux sequence transcript profile library from the auspicious carp of good fortune and screen IGF3 gene fragment, in this, as IGF3 gene intermediate sequence; After carrying out homology comparison according to transcript profile sequencing result, design pair of primers
IGF3-F：5’-GTAGAGCAGTGTTGTGTGCG-3’；
IGF3-R：5’-TGCATGCTTCTGGTATCGCT-3’；
Then intermediate sequence is increased, adopt round pcr to verify intermediate segment, confirm the exactness of IGF3 gene intermediate sequence;
(3) 3 ' and 5 ' acquisition of terminal sequence fragment: the good fortune auspicious carp IGF3 intermediate segment obtained according to step (2), design 3 ' forward outer primer according to claim 2,3 ' forward inner primer, 5 ' reverse outer primer and 5 ' reverse inner primer; With step (1) gained cDNA for template, obtained 3 ' end fragment and the 5 ' end fragment of good fortune auspicious carp IGF3 gene by RACEPCR amplification;
Wherein, universal primer in 3 ' forward outer primer and RACE test kit forms primer pair and carries out first round pcr amplification, with 3 ' forward inner primer and universal primer, second is carried out to amplified production and takes turns pcr amplification, take turns pcr amplification product 1.5% agarose gel electrophoresis by second and detect acquisition single band; Equally, 5 ' reverse outer primer and universal primer carry out first round pcr amplification, carry out second take turns pcr amplification to amplified production with 5 ' reverse inner primer and universal primer, and second takes turns pcr amplification product obtains 2 bands clearly through agarose gel electrophoresis;
(4) splice: after the two band of gained cuts glue purification after the single band of gained after described for step (3) 3 ' end amplification and 5 ' end amplification, be connected to pMD18-T carrier, be transformed in DH5 α competent cell, screen through blue hickie, select positive plasmid, obtain required object band through agarose gel electrophoresis qualification, obtain good fortune auspicious carp 3 ' nucleotide sequence fragment and 5 ' nucleotide sequence fragment by order-checking; Finally by 25 ' fragments respectively with intermediate segment and 3 ' fragment assembly, obtain good fortune auspicious carp IGF3cDNA full length nucleotide sequence IGF3-1 and IGF3-2.
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| CN109055356A (en) * | 2018-08-08 | 2018-12-21 | 中国水产科学研究院淡水渔业研究中心 | The preparation method of the auspicious carp igf3 gene dsRNA of good fortune a kind of and the application in living body interference |
| CN111217902A (en) * | 2020-01-20 | 2020-06-02 | 中国水产科学研究院淡水渔业研究中心 | Preparation and application of carp insulin-like growth factor IGF3 recombinant protein |
| CN112625118A (en) * | 2020-11-26 | 2021-04-09 | 广东海洋大学 | Pseudosciaena crocea growth promoting cell maturation gene IGF3 and application thereof |
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