CN104293766A - Method of cloning goat IGFBP3 gene cDNA coding sequence - Google Patents

Abstract

The invention discloses a method for cloning the cDNA coding sequence of goat IGFBP3 gene, the nucleotide sequence of which is shown in the sequence table SEQ ID NO: 1, comprising the following steps: A1, extracting the total RNA in goat longissimus dorsi tissue, Then reverse-transcribe the total RNA into cDNA; A2, primer design and PCR amplification: use the sequence of the sheep gene as a template to design primers in the upstream region of the start codon and the downstream region of the stop codon, and the designed primers are: Forward primer: 5'AGCTCGCTGCCGCCCAGT3', reverse primer: 5'GCTGATCATGTCCTTGGCAG3'; A3, purification and recovery of PCR products; A4, cloning. The invention provides a simple method for obtaining the cDNA coding sequence of goat IGFBP3 gene, which lays a foundation for further research on the function of the gene in the growth and development process of goats.

Description

Cloning Method of Goat IGFBP3 Gene cDNA Coding Sequence

technical field

The invention relates to the technical field of animal genetic engineering, more specifically to a method for cloning the cDNA coding region sequence of IGFBP3 (Insulin-like growth factor binding protein 3) gene from goat muscle tissue.

Background technique

As a member of the insulin-like growth factor system, insulin-like growth factor-binding proteins (IGFBPs) and insulin-like growth factors (IGFs) are a complex growth regulation axis: IGFBPs bind to IGFs and acid-sensitive subunits (ALS) Form a ternary complex to carry IGFs, and bind to IGFs through different affinities of IGFBPs to regulate the biological activity of free IGFs. IGFBP-3 is the insulin-like growth factor binding protein with the highest concentration in blood, and it is also the main carrier of serum IGFs , IGFBP-3 is mainly combined with IGF-I, which helps to maintain the bioavailability of IGF-I and ensure the biological efficacy of IGF-I. Therefore, the IGFBP3 gene plays an important role in the growth, development and metabolism of animals.

RT-PCR cloning: It is a technology that combines RNA reverse transcription (RT) with cDNA polymerase chain reaction and molecular cloning. First, use reverse transcriptase to reverse transcribe RNA into cDNA, then use cDNA as a template to amplify the target gene fragment we need, and then connect this fragment with the vector and transform it into competent cells for cloning, and obtain the plasmid PCR product Perform sequencing. This technology utilizes PCR technology to carry out effective gene amplification in vitro without endonuclease digestion and ligase treatment, and can quickly obtain other PCR products that are difficult to obtain by restriction endonuclease digestion; at the same time, this method can overcome Common PCR product sequencing primer proximal more than a dozen bases can not be sequenced to obtain the disadvantage. Compared with RACE (rapid amplification of cDNA ends) technology, the length of the target gene fragment is clear, the cost is low, and the workload is small. The key to this technology is the design of PCR primers. A forward primer is designed upstream of the start codon, and a reverse primer is designed downstream of the stop codon, so that the PCR product can contain the entire sequence of the complete gene coding region.

Segmented PCR cloning method: the target gene is divided into several fragments to be cloned separately, and then spliced together by subcloning and sequencing. Cloning several times, followed by sequence assembly.

Cloning or RACE technology is generally used to obtain the coding region sequence of the target gene. RACE technology is based on PCR technology to obtain a complete 3' end and 5' end by extending and amplifying a known cDNA fragment to both ends. However, compared with the cloning technique, the length of the target gene fragment is not clear, the test cost is high, the workload is heavy, and the requirements for the quality of the extracted RNA are relatively high, so the application is relatively seldom.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for cloning the cDNA coding sequence of goat IGFBP3 gene in view of the deficiencies in the prior art. To fill in the gaps in the study of this gene in goats and to lay the foundation for further research on its function in goats.

The present invention adopts following technical scheme:

The method for cloning the cDNA coding sequence of goat IGFBP3 gene comprises the following steps:

A1, total RNA was extracted from goat longissimus dorsi tissue, and then the total RNA was reverse-transcribed into cDNA;

A2: Use the sequence of the sheep gene as a template to design primers in the upstream region of the start codon and the downstream region of the stop codon;

A3: PCR amplification using cDNA as template;

A4: Gel recovery and purification of amplification products;

A5: The purified product is cloned and sequenced, and the cDNA coding sequence of the goat IGFBP3 gene can be obtained.

The method, step A1 includes the following steps: take about 80 mg of tissue powder ground with liquid nitrogen, add it to an EP tube filled with 1 mL Trizol in advance, shake and mix well, and let stand at room temperature for 5 minutes; add 0.2 mL of chloroform (must According to 1/5 of the total volume), vortex for 15 seconds, and let stand at room temperature for 5 minutes; centrifuge at 12,000 rpm for 15 minutes at 4°C; transfer 450 μL of the upper aqueous phase to another new 1.5mLEP tube, add an equal volume of isopropanol, After inverting and mixing, let stand at room temperature for 10 minutes; centrifuge at 12,000 rpm at 4°C for 10 minutes; discard the supernatant, add 1 mL of 75% ethanol (prepared with DEPC-treated water) pre-cooled at 4°C, let stand for 5 minutes, and centrifuge at 7,500 g at 4°C 5min; Discard the supernatant, suck up the excess liquid with a gun, and air dry for about 3min; add 30μL of DEPC-treated water, mix well by pipetting, fully dissolve the RNA, and then immediately reverse transcribe into cDNA.

In the method, the primers in step A2 are SEQ ID NO: 3 and SEQ ID NO: 4.

Description of drawings

Fig. 1 is the electrophoresis diagram of the PCR product of goat IGFBP3 gene.

Fig. 2 is the nucleotide sequence and corresponding amino acid sequence of the goat IGFBP3 gene coding region.

Detailed ways

The present invention will be described in detail below in conjunction with specific embodiments.

Specific implementation steps:

1. Collection of tissue samples

Nanjiang yellow sheep at 120 days after birth were selected for slaughter, and longissimus dorsi tissue samples were taken and immediately stored in liquid nitrogen for later use.

2. Extraction of total RNA and synthesis of cDNA

(1) Take 80 mg of tissue powder ground with liquid nitrogen, add it to the EP tube filled with 1 mL Trizol in advance, shake and mix, and let stand at room temperature for 5 minutes;

(2) Add 0.2 mL of chloroform (must be 1/5 of the total volume), vortex for 15 seconds, and let stand at room temperature for 5 minutes; (3) Centrifuge at 12,000 rpm for 15 minutes at 4°C;

(4) Transfer 450 μL of the upper aqueous phase to another new 1.5mLEP tube, add an equal volume of isopropanol, invert and mix well, and let stand at room temperature for 10 minutes;

(5) Centrifuge at 12,000 rpm for 10 minutes at 4°C;

(6) After discarding the supernatant, add 1 mL of 75% ethanol (prepared with DEPC-treated water) pre-cooled at 4°C, and let it stand for 5 minutes.

(7) Centrifuge at 7500g for 5min at 4°C;

(8) Discard the supernatant, suck up the excess liquid with a gun, and air dry for about 3 minutes; add 30 μL of DEPC-treated water, mix well by pipetting, and fully dissolve the RNA.

(9) Use 1% agarose gel electrophoresis to detect the quality of the total RNA, and immediately reverse-transcribe it into cDNA if the effect is good.

3. Amplification of target gene fragments

(1) Primer design: According to the IGFBP3 (NM_001134302) gene sequence of sheep ( Ovis aries ) published in the GenBank database, primers were designed using Primer Premier 5.0 software. The designed primers were:

Forward primer: 5' AGCTCGCTGCCGCCCAGT 3'

Reverse primer: 5' GCTGATCATGTCCTTGGCAG 3'

(2) PCR reaction system and conditions: Reaction system (25 μL): 0.25 μL of TaKaRa Taq enzyme, 12.5 μL of 2×GC Buffer I, 4 μL of dNTP Mixture, 0.5 μL of forward and reverse primers, 6.25 μL of bacteria ddH ₂ O, template cDNA 1 μL. The reaction conditions were pre-denaturation at 95°C for 4 min, 35 cycles (94°C, 45 s; 53.4°C, 30 s; 72°C, 90 s), and finally 72°C for 7 min; PCR products were analyzed with 1.5% agarose gel Electrophoretic detection.

4. Recovery and purification of PCR products

The PCR product was purified and recovered with Shanghai Sangon UNIQ-10 Column DNA Gel Recovery Kit. The specific steps are as follows:

(1) Separate the target DNA fragment from other DNA as much as possible by 1.5% agarose gel electrophoresis, then cut off the agarose gel block containing the target DNA fragment with a clean scalpel, and put it into a 1.5 mL centrifuge tube .

(2) Weigh the weight of the gel, and add Binding Buffer II at a ratio of 400 μL per 100 mg of agarose according to the weight and concentration of the gel.

(3) Place the centrifuge tube in a metal constant temperature bath at 55°C for 5-10 minutes, and mix it occasionally until the glue block is completely melted.

(4) (Optional step) When the target fragment is <500bp, add 1/3 volume of isopropanol of the Binding Buffer II used and mix well. When the target fragment is >500bp, this step can be omitted, and step (5) can be performed directly.

(5) Transfer the melted gel solution to a UNIQ-10 column set in a 2ml collection tube, and place at room temperature for 2 minutes. Centrifuge at 8000 rpm for 1 min at room temperature.

(6) Remove the UNIQ-10 column, discard the waste liquid in the collection tube, put the UNIQ-10 column into the same collection tube, add 500 μL Wash Solution, and centrifuge at 10,000 rpm for 1 min at room temperature.

(7) Repeat step (6) once.

(8) Remove the UNIQ-10 column, discard the waste liquid in the collection tube, put the UNIQ-10 column into the same collection tube, and centrifuge at 12000 rpm for 2 min at room temperature.

(9) Put the UNIQ-10 column into a new 1.5 mL centrifuge tube, add 30 μL Elution Buffer to the center of the adsorption membrane, and place it at room temperature for 1~2 min. Centrifuge at room temperature at 12000 rpm for 1 min, and the liquid in the centrifuge tube is the recovered DNA fragment.

(10) Take 2 μL of the collected DNA, electrophoresis on 1.5% agarose gel, and check the quality of the recovered DNA.

4. Cloning of the product

(1) The target fragment is connected to the carrier

The gel recovered product was ligated with pMD19-T vector. Thaw the carrier on ice and centrifuge the tube containing the carrier briefly to avoid hanging the liquid on the tube wall. Configure the connecting liquid on the ultra-clean operating table according to the following system:

Place the reaction mixture in a water bath at 16°C for 3 hours.

(2) Conversion of ligated products

Take out the preserved DH5α competent cells from the -80°C refrigerator to help thaw in an ice bath, pipette 50 μL into 10 μL of the connection solution, and swirl gently to mix well.

Place on ice for 30min.

Heat shock transformation: heat at 42°C for 45 s, then place on ice for 1-2 min.

Add 800 μL of LB medium pre-stored at 37°C and shake at 37°C for 45 minutes.

Centrifuge at 4000 g for 10 min at room temperature, and suck off 600 μL of the supernatant.

Pipette the ligation product onto the plate and evenly spread it on the plate. After the liquid is completely absorbed, place it upside down in an incubator at 37°C for 12-16 h.

(3) Screening of positive clones

Pick a single colony with a sterilized toothpick, inoculate it in 800 μL LB medium containing Amp, incubate at 37 °C and 200 r/min for 5 h, then draw 1 μL of the bacterial solution as a PCR template, according to the PCR amplification system and conditions PCR identification was carried out, and the products were detected by 1.5% agarose gel electrophoresis. According to the results of bacterial liquid PCR detection, 800 μL of bacterial liquid containing positive clones was drawn and sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing.

5. Sequence verification

The sequencing results were spliced using DNAstar software to find the start codon and stop codon of the gene, and the complete coding region sequence of 882bp of the IGFBP3 gene was obtained, and compared with the sequences of cattle and sheep, the obtained goat IGFBP3 gene code The identities of the region sequences with cattle and sheep were 94.33% and 99.09%, respectively. It shows that this method can achieve the goal of obtaining the complete coding region sequence of goat IGFBP3 gene. The sequence was submitted to the GenBank database, and the accession number obtained was JQ341161.

It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should belong to the protection scope of the appended claims of the present invention.

sequence listing

SEQUENCE LISTING

the

<110> Sichuan Agricultural University

the

<120> Method for cloning goat IGFBP3 gene cDNA coding sequence

the

<130> 2013

the

<160> 4

the

<170> PatentIn version 3.5

the

<210> 1

<211> 882

<212> DNA

<213> goat

the

<400> 1

atgctgcggg cacgccccgc gctctgggct gcggctctga ccgcgctggc attgctccgc 60

the

ggaccgccgg cggcacgagc tggggcgggc acggcgggcg ccggcccggt ggtgcgctgc 120

the

gagccgtgcg acgcgcgtgc cgttgcccag tgcgcgccgc cgcccccctc gcccccgtgc 180

the

accgagttgg tgcgcgagcc gggctgcggt tgctgtctca cttgcgcgct gcgcgagggt 240

the

cagccttgcg gcgtctacac cgagcgctgt ggctccggcc tccgctgcca gccgccgcct 300

the

ggcgatccgc gcccgctaca cgcgttgttg gacggccgcg ggctctgcgc caacgccagc 360

the

gccgtcggcc gcctgagccc ctacctgctg cccgcgccac ccgcgccagg aaatggcagt 420

the

gagtcggaag aagaccacag catggggagc acggagaacc aggctctccc caccacacgc 480

the

cgggtgcccg actccaaatc ccacctcgcc cacaccaaga tggatgtcat caaaaaaggt 540

the

catgccaagg acagccagcg ctacaaggtt gactacgagt ctcagagcac agacacccag 600

the

aacttctcct ccgagtccaa gcatgagaca gaatacgggc cctgccgccg ggaaatggag 660

the

gacacactga acgacctcaa gttcctgaac acactcagcc ccagggccat ccacattccc 720

the

aactgtgaca agaagggctt ctacaagaaa aagcagtgcc gcccttccaa gggcaggaag 780

the

cggggtttct gctggtgtgt ggataagtac gggcagcccc tcccggcctt cagcgtgaag 840

the

gggaaagggg acgtgcactg cctcagcacg gagagcaagt ag 882

the

the

<210> 2

<211> 293

<212> PRT

<213> goat

the

<400> 2

the

Met Leu Arg Ala Arg Pro Ala Leu Trp Ala Ala Ala Leu Thr Ala Leu

1 5 10 15

the

the

Ala Leu Leu Arg Gly Pro Pro Ala Ala Arg Ala Gly Ala Gly Thr Ala

20 25 30

the

the

Gly Ala Gly Pro Val Val Arg Cys Glu Pro Cys Asp Ala Arg Ala Val

35 40 45 45

the

the

Ala Gln Cys Ala Pro Pro Pro Pro Pro Ser Pro Pro Cys Thr Glu Leu Val

50 55 60 60

the

the

Arg Glu Pro Gly Cys Gly Cys Cys Leu Thr Cys Ala Leu Arg Glu Gly

65 70 75 80

the

the

Gln Pro Cys Gly Val Tyr Thr Glu Arg Cys Gly Ser Gly Leu Arg Cys

85 90 95

the

the

Gln Pro Pro Pro Gly Asp Pro Arg Pro Leu His Ala Leu Leu Asp Gly

100 105 110

the

the

Arg Gly Leu Cys Ala Asn Ala Ser Ala Val Gly Arg Leu Ser Pro Tyr

115 120 125

the

the

Leu Leu Pro Ala Pro Pro Ala Pro Gly Asn Gly Ser Glu Ser Glu Glu

130 135 140

the

the

Asp His Ser Met Gly Ser Thr Glu Asn Gln Ala Leu Pro Thr Thr Arg

145 150 155 160

the

the

Arg Val Pro Asp Ser Lys Ser His Leu Ala His Thr Lys Met Asp Val

165 170 175

the

the

Ile Lys Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val Asp Tyr

180 185 190

the

the

Glu Ser Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys His

195 200 205

the

the

Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr Leu Asn

210 215 220

the

the

Asp Leu Lys Phe Leu Asn Thr Leu Ser Pro Arg Ala Ile His Ile Pro

225 230 235 240

the

the

Asn Cys Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser

245 250 255

the

the

Lys Gly Arg Lys Arg Gly Phe Cys Trp Cys Val Asp Lys Tyr Gly Gln

260 265 270

the

the

Pro Leu Pro Ala Phe Ser Val Lys Gly Lys Gly Asp Val His Cys Leu

275 280 285

the

the

Ser Thr Glu Ser Lys

  290  

the

the

<210> 3

<211> 18

<212> DNA

<213> goat

the

<400> 3

agctcgctgc cgcccagt 18

the

the

<210> 4

<211> 20

<212> DNA

<213> goat

the

<400> 4

gctgatcatg tccttggcag 20

the

the

Claims (3)

1. the method for goat IGFBP3 gene cDNA encoding sequence clone, is characterized in that, comprise the following steps:

Step one: extract total serum IgE from goat muscle tissue sample, then becomes cDNA by total serum IgE reverse transcription;

Step 2: the design of amplimer and PCR reaction: with the sequence of this gene of sheep for template designs primer at the downstream area of upstream from start codon region and terminator codon; The primer of design is:

Forward primer: 5'AGCTCGCTGCCGCCCAGT 3'

Reverse primer: 5'GCTGATCATGTCCTTGGCAG 3'

Be that template first carries out grads PCR reaction with cDNA, annealing region selects 50 DEG C to 60 DEG C totally 8 temperature spots, optimizes the condition of regular-PCR.

2.PCR reaction system is 25 μ L systems: the TaKaRa Taq enzyme of 0.25 μ L, the dNTP Mixture of 2 × GC Buffer II, 4 μ L of 12.5 μ L, each 0.5 μ L of forward and reverse primer, the sterilizing ddH of 6.25 μ L ₂o, template cDNA 1 μ L.

3. reaction conditions is 95 DEG C of denaturation 4 min, and 35 circulations (94 DEG C, 45 s; 53.4 DEG C, 30 s; 72 DEG C, 90 s), last 72 DEG C of 7 min;

Step 3: the recovery of amplified production and purifying;

Step 4: the product of purifying carries out choning and sequencing, obtains the nucleotide sequence of 882bp, as shown in sequence table SEQ ID NO:1, is the coding region complete sequence of goat IGFBP3 gene.