CN102181451A

CN102181451A - Goat TNNC1 gene and method for obtaining complete coding sequence of goat TNNC1 gene through cloning

Info

Publication number: CN102181451A
Application number: CN2011100747552A
Authority: CN
Inventors: 陈浩林; 徐刚毅; 郑程莉; 汪代华
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2011-03-28
Filing date: 2011-03-28
Publication date: 2011-09-14

Abstract

The invention discloses a goat TNNC1 gene. The nucleotide sequence of the goat TNNC1 gene is SEQ ID NO:1. The coding sequence of the gene is SEQ ID NO:2. The amino acid sequence coded by the coding sequence of the gene is SEQ ID NO:3. The invention also provides a method for cloning the goat TNNC1 gene. The method comprises the following steps: 1. extracting the total RNA in the myocardium of the goat and then reversely transcribing the total RNA to cDNA; 2. designing primers and carrying out PCR (polymerase chain reaction); 3. recovering and purifying the PCR product; and 4. cloning the product. The method has the beneficial effects of simply and quickly obtaining the complete coding sequence of the goat TNNC1 gene, filling in the gap of the gene in the goats and laying the foundation for further studying the gene in the goats.

Description

The method of goat TNNC1 gene and clone goat TNNC1 gene coding region complete sequence

Technical field

The present invention relates to biological technical field, especially the method for goat TNNC1 gene and clone goat TNNC1 gene coding region complete sequence.

Background technology

Slow muscle troponin (TNNC1) is to be present in one of calcium ion-binding protein mixture member in cardiac muscle and the slow shrinkability skeletal muscle.TnC (TNC) forms albumen composition with TnT and troponin, plays keying action in the contraction of muscle and diastole.TnC 1 is the calcium ion acceptor that shrinks skeletal muscle and cardiac muscle at a slow speed, and is the key that calcium ion activated sarcomere shrinks.The combination of calcium ion has started the variation of the structure as the thin filament fold by the thin filament constitutive protein.Cause the formation of the cross-bridges between myosin and Actin muscle, impel thin filament to produce the effect of the effect Muscle contraction of Muscle contraction to the slip of thick filament.

Fast muscle TnC 1 contains 4 calcium ion binding sites, but if calcium ion binding site II, III and IV have accepts the site that calcium ion produces effect.All by a calcium ion coupling collar of being made up of 12 amino-acid residues, the calcium ion coupling collar is present among a pair of a-helices each calcium ion binding site.Each calcium ion coupling collar all is rich in acidic amino acid (Asp and Glu) to be responsible in conjunction with single calcium ion.The TNNC1 one-piece construction comprises the spheric region of a N-end and a C-end, and these two spheric regions are formed dumb-bell shape by a spiral link flexibly.

Summary of the invention

Technical problem to be solved by this invention provides the method for a kind of goat TNNC1 gene and clone goat TNNC1 gene coding region complete sequence thereof.

Goat TNNC1 gene, nucleotides sequence are classified SEQ ID NO:1 as.

Described goat TNNC1 gene, the coding region sequence of described gene are SEQ ID NO:2.

Described goat TNNC1 gene, the coding region sequence coded amino acid of described gene are SEQ ID NO:3.

The cloning process of described goat TNNC1 gene comprises the steps:

Step 1 is extracted the total RNA in the goat cardiac muscular tissue, then total RNA reverse transcription is become cDNA;

Step 2 primer design and PCR reaction; The sequences Design pair of degenerate primers of the TNNC1 gene of the ox that foundation has been delivered, upstream primer are in the upstream region design of initiator codon, and downstream primer is in the downstream area design of terminator codon; Described degenerated primer is:

Upstream primer P1:5 '-CCTGTGAGTCGCCAGTATG-3 '

Downstream primer P2:5 '-TGGGTGAAGGTTGGTGTC-3 '

With described cDNA is that template is carried out the grads PCR reaction;

The recovery of step 3 PCR product and purifying;

The step 4 clone.

Described method, in the described step 2, described PCR reaction system is: the PCR reaction system is 20 μ l systems: 2 * Master Mix Tap of 10 μ L, each 1 μ L of upstream and downstream primer, the cDNA of 4 μ L, the ddH of 4 μ L ₂O; Reaction conditions is: 95 ℃ of pre-sex change 5min; 35 circulations (94 ℃, 40s; 50 ℃～60 ℃, 40s; 72 ℃, 50s), last 72 ℃ of 10min.

The relevant report that does not also have at present goat TNNC1 gene, for obtaining the complete coding region sequence of goat TNNC1 gene, research RT-PCR cloning is obtained the method for goat TNNC1 (slow muscle troponin) gene coding region complete sequence, to set up a kind of novel method of gene clone.

Description of drawings

Fig. 1 is the grads PCR product electrophorogram of 8 temperature spots of TNNC1 gene.

Fig. 2 is the nucleotide sequence and the amino acid sequence coded of goat TNNC1 gene coding region.

Embodiment

Below in conjunction with specific embodiment, the present invention is described in detail.

Step 1 is extracted the total RNA (Yeast Nucleic Acid) in the goat cardiac muscular tissue, then total RNA reverse transcription is become cDNA;

1. Zu Zhi collection

Choose and get about 1g cardiac muscle after the self-sufficient and strategically located region mutton sheep carotid artery bloodletting of one full year of life immediately and put into liquid nitrogen container and be used to extract total RNA.

2.RNA preparation and cDNA synthetic

In the laboratory with liquid nitrogen with the cardiac muscle EP pipe of 1.5ml of packing into of claying into power, it is standby to put into subzero 80 Ultralow Temperature Freezer.

Get the EP pipe of new 1.5ml, add the Trizol liquid of 1.1ml, add cardiac muscular tissue's powder of about 80mg then.After using hand rolling mixing immediately, shake after about 30 seconds room temperature with the concussion instrument and placed 4 minutes.

4 ℃ of following 13000rpm were transported to liquid the EP pipe of another new 1.5ml in centrifugal 3 minutes with liquid-transfering gun, and add the chloroform of 0.2ml in this new EP pipe, cover tight centrifuge tube, acutely swayed centrifuge tube about 15 seconds with hand, 4 ℃ of following 13000rpm centrifugal 8 minutes then.With liquid-transfering gun the 0.4ml that makes an appointment of the upper water in this EP pipe is moved into a new centrifuge tube after the centrifugal end, add the Virahol of 0.4ml, room temperature was placed 10 minutes, and then 4 ℃ of following 12000rpm centrifugal 10 minutes.Abandoning supernatant adds 75% ethanol (preparation of the DEPC water) mixing of 1ml, centrifugal 5 minutes of 4 ℃ of following 7500g.

Careful abandoning supernatant blots ethanol unnecessary on the tube wall with liquid-transfering gun then, notes RNA not being sopped up, and then according to the RNA amount what, adds the DEPC water of 50 to 150 μ L.Quality with 1.5% agarose electrophoresis detection RNA becomes cDNA with effective RNA reverse transcription immediately.

Step 2 primer design and PCR reaction;

Sequence (accession number is BC102995.1) the design pair of degenerate primers of the TNNC1 gene of the ox that foundation has been delivered, upstream primer are in the upstream region design of initiator codon, and downstream primer is in the downstream area design of terminator codon.The primer of design is:

Upstream primer P1:5 '-CCTGTGAGTCGCCAGTATG-3 '

Downstream primer P2:5 '-TGGGTGAAGGTTGGTGTC-3 '

With cDNA is that template is carried out the grads PCR reaction, amplification TNNC1 gene, and temperature is 8 temperature spots of scope between 50 ℃ to 65 ℃, the PCR reaction system is 20 μ l systems (2 * Master Mix Tap of 10 μ L, each 1 μ L of upstream and downstream primer, the cDNA of 4 μ L, the ddH of 4 μ L ₂O.), reaction conditions is 95 ℃ of pre-sex change 5min, and 35 circulations (94 ℃, 40s; 50 ℃～60 ℃, 40s; 72 ℃, 50s), last 72 ℃ of 10min.To have in PCR product collection to the EP pipe of 500bp approximately and cut glue and reclaim (Fig. 1) after reaction finishes, in plasmid vector, the picking positive bacteria carries out the PCR reaction then with the gene fragment clone that reclaims, and product send the order-checking of order-checking company.

The hairpin structure of PCR reaction can not appear influencing in the degenerated primer of design, primer dimer, and the upstream and downstream primer is crosslinked.Suitable PCR range of reaction temperature span is big.

The recovery of step 3 PCR product and purifying;

The PCR product reclaims the test kit purifying with precious xanthan gum and reclaims, and the concrete operations step is:

The a.PCR product downcuts and the consistent DNA band of expection clip size with the sterilization blade under UV-light after 2.0% agarose 110V electrophoretic separation, in the EP pipe of the 1.5mL that packs into, glue is softly shredded.

B. add the sol solutions A of 600 μ l in centrifuge tube, be positioned over water-bath 10min colloidal sol in 55 ℃ of water-baths, the middle centrifuge tube that takes out slowly shakes one to twice.

C. put to room temperature after peptization is separated, add 20 μ L solution B, mixing changes centrifugal post over to, the centrifugal 40s of 8000rpm.

D. abandon centrifugate, pillar is put into new 1.5Ml EP pipe, add 500 μ L elutriant C, the centrifugal 1min of 8000rpm repeats this step 3 time.

E. centrifugal post is put into new centrifuge tube, add 20 μ l sterile purified waters, soak into 10min, the centrifugal 1.5min of 10000rpm.

F. the solution in the centrifuge tube is the aqueous solution of the dna fragmentation of recovery.

G. get 2 μ L and reclaim product, detect organic efficiency and purity in 1.0% agarose gel electrophoresis detection.

The step 4 clone;

(1) the purpose fragment is connected with carrier

Purpose fragment after reclaiming, detecting is connected with the pMD-18T carrier, and according to the pMD-18T support agent box working instructions of TaKaRa, the system of being operatively connected is:

PMD-18T?Vector 1.0μL

PCR fragment 4.0 μ L after purifying returns

Connect liquid Ligase buffer 5.0 μ L

Total 10.0μL

More than operate on ice and carry out, centrifugal slightly mixing, 16 ℃ connect in the PCR instrument and spend the night, and-20 ℃ of preservations are standby.

(2) preparation (CaCl of competent escherichia coli cell ₂Method)

A. picking escherichia coli DH5a bacterial classification, setting-out on the LB agar plate, 37 ℃ of overnight incubation (12-16h).

B. picking list bacterium colony is inoculated in the 100mL LB liquid nutrient medium.37 ℃ of 190rpm shaking culture 12h work as ODD ₆₀₀When value reaches 0.4-0.5 (jog substratum naked eyes visible cloud is vaporific), stop to cultivate.

C. bacterium liquid is filled in the 100mL sterilization centrifuge tube of precooling ice bath 10-15min, 4 ℃ of centrifugal 10min of 4000rpm with 50mL/ part branch.

D. abandon supernatant, the CaCl of the 0.1Mol of the precooling of every effective 50mL ₂(have in the liquid ice pellets effect best) resuspended bacterial sediment, behind the ice bath 30min, 4 ℃ of centrifugal 3min of 4000rpm.

E. abandon supernatant, the CaCl of the 0.1M of every effective 10mL precooling ₂Resuspended bacterial sediment, ice bath 10-15min is competent cell.

F. now-making-now-using.Maybe the competent cell for preparing being added 30% glycerine to final concentration is 15-20%, and mixing is distributed into 200uL/ part, after the input liquid nitrogen quick freezing, and-80 ℃ of preservations

(3) conversion of connection product

A. take out the competent cell of preserving from-80 ℃ of refrigerators, place on ice and thaw.

B. 2.5 μ l are connected product and slowly squeeze in the competent cell of 200 μ l, it is soft that action is wanted, ice bath 20min.

C.42 ℃ water-bath heat shock 90s (centrifuge tube can not be rocked in the centre) takes out rapidly and places on ice ice bath 5-10min.

D. add 800 μ l LB liquid nutrient mediums (not adding Amp), in 37 ℃, less than 150rpm shaking culture 50min with the recovery cell.

E.3000rpm centrifugal 5min inhales and removes the part supernatant, and the bacterium that softly suspends is coated on the selectivity flat board.Treat liquid

After infiltrating agar fully, the back-off flat board is cultivated 9-16h in 37 ℃ of constant incubators.

(4) screening of positive bacterium colony

The random choose colony inoculation is in LB ammonia joint nutrient solution, and being cultured to after the intermediate concentration with bacterium liquid is template, is used for identical primer of corresponding gene pcr amplification and reaction conditions and increases.Amplified production detects with 1.5% agarose gel electrophoresis, and the bacterium liquid 0.5mL that amplified production is consistent with the purpose clip size packs in the 1.5ml EP centrifuge tube, adds 30% glycerine.Hand precious biotechnology company limited over to and finish order-checking.

The exactness of step 5 check test;

On NCBI, use the online comparison software of blast with the exactness of checking sequence and the reliability of this method sequencing result.The result shows that nucleotide sequence and other known mammiferous TNNC1 gene order homologys of the goat TNNC1 gene complete coding region sequence that this method obtains are 92.18%～99.38%.Show that this method can reach the purpose of the complete coding region sequence that obtains goat TNNC1 gene.The goat TNNC1 gene nucleotide series that obtains is SEQ ID NO:1, and coding region sequence is SEQ ID NO:2, encoding amino acid sequence SEQ ID NO:3.

The present invention has obtained the complete coding region sequence of goat TNNC1 gene simply, efficiently, has filled up the blank of this gene on goat, for this gene of further studying goat is laid a good foundation.

This technology compare segmentation clone and RACE technology, the segmental length of purpose PCR is roughly clear and definite, workload little (only needing to clone once), it is low to have a cost, the advantage that efficient is high.

Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.

Claims

1. goat TNNC1 gene is characterized in that, nucleotides sequence is classified SEQ ID NO:1 as.

2. goat TNNC1 gene according to claim 1 is characterized in that, the coding region sequence of described gene is SEQ ID NO:2.

3. goat TNNC1 gene according to claim 1 is characterized in that, the coding region sequence coded amino acid of described gene is SEQ ID NO:3.

4. the cloning process of the described goat TNNC1 of claim 1 gene is characterized in that, comprises the steps:

Step 1 is extracted the total RNA in the goat cardiac muscular tissue, then total RNA reverse transcription is become eDNA;

Upstream primer P1:5 '-CCTGTGAGTCGCCAGTATG-3 '

Downstream primer P2:5 '-TGGGTGAAGGTTGGTGTC-3 '

With described cDNA is that template is carried out the grads PCR reaction;

The recovery of step 3 PCR product and purifying;

The step 4 clone.

5. method according to claim 4 is characterized in that, in the described step 2, described PCR reaction system is: the PCR reaction system is 20 μ l systems: 2 * Master Mix Tap of 10 μ L, each 1 μ L of upstream and downstream primer, the cDNA of 4 μ L, the ddH of 4 μ L ₂O; Reaction conditions is: 95 ℃ of pre-sex change 5min; 35 circulations (94 ℃, 40s; 50 ℃～60 ℃, 40s; 72 ℃, 50s), last 72 ℃ of 10min.