CN103805695A - Primer pair for detecting growth rate of takifugu flavidus, kit and method - Google Patents

Abstract

The invention relates to a primer pair for detecting growth rate of takifugu flavidus, a kit and a method. The primer pair consists of an upstream primer with the base sequence of SEQ ID NO:3 and a downstream primer with the base sequence of SEQ ID NO:4. The primer pair is further used for preparing the kit. Besides the primer pair, the kit further comprises a takifugu flavidus MARKER in fast growth rate. The detection method comprises the following steps: extracting the DNA (Deoxyribonucleic Acid) of a sample takifugu flavidus, and carrying out PCR (Polymerase Chain Reaction) amplification on the DNA of the sample takifugu flavidus by the primers in the kit; and carrying out a PAGE treatment on an amplification product, observing the size of a stripe, wherein the individual which is consistent to the MARKER stripe is the one fast in growth rate while the individual which is inconsistent to the MARKER stripe is the one slow in micro growth rate. The growth rate of the takifugu flavidus is identified by means of the primer pair, the kit and the method provided by the invention, so that the invention has the advantages of high repeatability, strong accuracy, simplicity, convenience and fast speed.

Description

For detection of primer pair, test kit and the method for the yellow Fugu speed of growth of chrysanthemum

[technical field]

The present invention relates to detection primer and the method for the yellow Fugu of chrysanthemum, be specifically related to primer pair, test kit and method for detection of the yellow Fugu speed of growth of chrysanthemum.

[background technology]

The yellow Fugu of chrysanthemum (Fugu flavidus) is commonly called as ship bar, well-behaved fish, chrysanthemum Huang, magnitude all over the sky, in classification, be subordinate to Tetraodontiformes (Tetraodontif-ormes) Molidae (Tetraodontidae) Fugu and belong to (Fugu), be mainly distributed in China's Huanghai Sea, the East Sea and the Bohai Sea.Due to its delicious flavour, nutritious, rather well received, be a kind of famous and precious cultivation kind.In breeding process, breeding environment standardization degree is more and more higher, and the individuality of fast growth can shorten culturing time, improves the utilization ratio of feed, saves aquaculture cost, and therefore cultivating the yellow Fugu of screening fast growth chrysanthemum is the target that aquaculturist pursues.

Myostatin (Myostatin, MSTN) is cloned into from mice skeletal cDNA library at first, and it is great expression in muscle tissue mainly, belongs to the super family of TGF-β.To MSTN knock out mice study and show, weight ratio heterozygote and the wild-type mice body weight of mutant mice increase approximately 30%, the muscle hypertrophy that wherein myocyte's hyperplasia (hyperplasia) and myofiber hypertrophy (hypertrophy) dual function cause is the major cause that causes body weight to increase, and the sex of this characteristic and animal and age are irrelevant, and do not affect the fecundity of animal.Therefore, MSTN is an important negative regulation major gene of muscle growth.The MSTN and surface of cell membrane acceptor (activin acceptor protein II B(ACVR2B) and activin acceptor protein IB(ACVRB of activation)) combine, can activate mother's S in downstream 2/3 and p38MAPK Cellular Signaling Transduction Mediated approach, thereby regulate growing of each tissue.

At " Yellow catfish MSTN Gene polymorphism and with the correlation analysis of growth traits " (Zhu Yuanyuan etc., heredity, in January, 2012 34(1): 72-78 page) in a literary composition, disclose the impact of MSTN on yellow catfish growing speed, the muscle of testing fish by the MSTN gene discovery of transgenic technology and DNAi technology inhibition zebra fish is obviously than normal fish prosperity, therefore think that this gene plays important restraining effect in animal muscle grows, also may participate in fatty generation and adjusting.In fish, MSTN mDNA can be at muscle, eye, in brain, intestines, the cheek, kidney, the heart, spleen, find, and MSTN is also divided into two classes in different fingerlings MSTN1, the gene clone of MSTN2(Paralichthys olivaceus amicine, Xu Jianyong, Chen Songlin, the 32nd the 4th phase of volume of aquatic product journal, in July, 2008,497-498).

[summary of the invention]

In the yellow Fugu of chrysanthemum, exist gene to double phenomenon, these double gene and exist multiple copies, MSTN2(is myostatin) in the yellow Fugu of chrysanthemum, there are multiple copies, PCR can increase, and some copy unintentionally, as shown in base sequence SEQ ID NO:1, SEQ ID NO:2, this fragment is positioned at the yellow Fugu MSTN2 gene 3 untranslated region of chrysanthemum territory, has wherein just occurred repeated segments GT, the existence of these copies, has affected the analysis to result.

On the basis of above-mentioned discovery, bring the problem of interference in order to solve PC amplification copy in prior art for result, and make up the blank for the research of the yellow Fugu speed of growth of chrysanthemum in prior art.First the present invention provides a kind of primer pair for detection of the yellow Fugu of chrysanthemum, primer pair by: base sequence is that the upstream primer of SEQ ID NO:3 and the downstream primer of SEQ ID NO:4 form.

This primer pair can also be used for making test kit, except above-mentioned primer pair, also comprises the yellow Fugu mark of the chrysanthemum MARKER of fast growth in test kit.

The method that detects the yellow Fugu speed of growth of chrysanthemum with above-mentioned test kit is as follows:

Extract the DNA sequence dna of the myostatin of the yellow Fugu of sample chrysanthemum,

Utilize the primer pair in test kit that the DNA of the yellow Fugu of sample chrysanthemum is carried out to pcr amplification;

Amplified production is carried out to PAGE processing,

Observe stripe size, consistent with MARKER stripe size is the fireballing individuality of growth.

Further, aforesaid method also has following prioritization scheme:

Described pcr amplification reaction system 20 μ L, comprise 2uL10 × PCR damping fluid, the dNTP mix of 0.8ul10mmol/L, the upstream primer of 0.4 μ mol/L, the downstream primer of 0.4 μ mol/L, the yellow Fugu DNA of sample chrysanthemum of 30ng.

Described pcr amplification reaction condition optimization is, sex change 3 minutes at 94 ℃, and sex change 30 seconds at 94 ℃, renaturation 30 seconds at 64 ℃, and 72 ℃ of downward-extensions 30 seconds, totally 42 circulations, last 72 ℃ are extended 10min.

The step of described PAGE is preferably:

The sex change sample-loading buffer of getting equivalent adds respectively in PCR product and test kit in MARKER, and 99 ℃ of sex change 10 minutes, at urea-denatured PAGE(6%) in carry out electrophoresis to tetrabromophenol sulfonphthalein and run out of PAGE, carry out silver and dye, fixing, dyeing and colour developing.

Differentiate the speed of growth of the yellow Fugu of chrysanthemum by primer pair of the present invention, test kit and method, have repeatability high, accuracy is strong, simple and rapid advantage.This primer pair occupies very at high proportion in fast growth colony, and with minimum artificially breeding parent's compatible degree up to more than 96%.

[accompanying drawing explanation]

Fig. 1 is the SNPs site schematic diagram in the yellow Fugu MSTN2 of chrysanthemum of the present invention gene;

Fig. 2 is the yellow Fugu growth of chrysanthemum speed PAGE band comparison diagram.

[embodiment]

Below by specific embodiment, the present invention will be further described by reference to the accompanying drawings, and following embodiment is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.

Embodiment 1:

The selection of step 1, the yellow Fugu of fast growth chrysanthemum

Select the yellow Fugu of chrysanthemum of taking from for half age, be divided into two groups according to weight and body length, adopt according to a conventional method phenol chloroform extraction method (to translate " molecular cloning experiment guide " (third edition) referring to Huang Peitang etc., Science Press, in September, 2002) extract the yellow Fugu DNA of chrysanthemum, a pair of selective amplification primer is synthesized in design.

Upstream primer 5 '-CAAGCTTGTCTGTTGAGGCCTGTT-3 ',

Downstream primer 5 '-CAGCACCTTCGACCTCATTTGATG-3 ';

Its DNA is carried out to pcr amplification, and reaction system 20 μ L, comprise 2ul10 × PCR damping fluid, the dNTP mix of 0.8ul10mmol/L, the upstream primer of 0.4 μ mol/L, the downstream primer of 0.4 μ mol/L, the yellow Fugu DNA of sample chrysanthemum of 30ng, reaction conditions is as follows, 94 ℃ of sex change 3min, then 94 ℃ of sex change 30sec, 55 ℃ of renaturation 30 seconds, and 72 ℃ extend 30 seconds, 42 circulations, last 72 ℃ extend 10min.

Product is through 6% sex change PAGE electrophoresis, through dyeing colour developing, observations, operates as follows: get equivalent sex change sample-loading buffer and add PCR product (formula is shown in molecular cloning handbook), 99 ℃ of sex change 10 minutes, at urea-denatured PAGE(6%) in carry out electrophoresis to tetrabromophenol sulfonphthalein and run out of PAGE.Carry out silver and dye, fixing, dyeing and colour developing.Observe stripe size, the individuality that contains 187-189bp size strip, is the individuality of fast growth.

Then the individual MARKER using this individuality DNA sequence dna as fast growth.

Step 2, the yellow Fugu speed of growth of detection chrysanthemum

Above-mentioned primer pair, MARKER are made as to test kit, in the time that needs detect, extract the DNA sequence dna of the myostatin of the yellow Fugu of sample chrysanthemum, and carry out pcr amplification with the primer pair in above-mentioned test kit.

Reaction system 20 μ L, comprise 2uL10 × PCR damping fluid, the dNTP mix of 0.8ul10mmol/L, the upstream primer of 0.4 μ mol/L, the downstream primer of 0.4 μ mol/L, the yellow Fugu DNA. of the sample chrysanthemum of 30ng reaction conditions is, sex change 3 minutes at 94 ℃, sex change 30 seconds at 94 ℃, renaturation 30 seconds at 64 ℃, and 72 ℃ of downward-extensions 30 seconds, totally 42 circulations, last 72 ℃ are extended 10min.

PCR product with contrast the sex change PAGE electrophoresis of MARKER through 6%, through dyeing colour developing, observations, operate as follows: get equivalent sex change sample-loading buffer and add PCR product (formula is shown in molecular cloning handbook), 99 ℃ of sex change 10 minutes, at urea-denatured PAGE(6%) in carry out electrophoresis to tetrabromophenol sulfonphthalein and run out of PAGE.Carry out silver and dye, fixing, dyeing and colour developing.Observe stripe size, the individuality consistent with MARKER stripe size, is the individuality of fast growth.Its comparison diagram is referring to Fig. 2, and wherein L representative growth is fast individual, and S representative growth is slow individual, and M is test kit MARKER.

Claims (6)

1. for detection of a primer pair for the yellow Fugu of chrysanthemum, it is characterized in that primer pair by: base sequence is that the upstream primer of SEQ ID NO:3 and the downstream primer of SEQ ID NO:4 form.

2. for detection of a test kit for the yellow Fugu speed of growth of chrysanthemum, it is characterized in that wherein containing primer pair claimed in claim 2, and the yellow Fugu mark of the chrysanthemum of fast growth MARKER.

3. a method that detects the yellow Fugu speed of growth of chrysanthemum, is characterized in that being made up of following steps:

Extract the DNA sequence dna of the myostatin of the yellow Fugu of sample chrysanthemum,

Utilize the primer pair in claim 2 test kit that the DNA of the yellow Fugu of sample chrysanthemum is carried out to pcr amplification;

Amplified production is carried out to PAGE, dyeing,

Observe stripe size, consistent with MARKER stripe size is the fireballing individuality of growth.

4. the method for the yellow Fugu speed of growth of detection chrysanthemum as claimed in claim 3, it is characterized in that described pcr amplification reaction system 20 μ L, comprise 2uL10 × PCR damping fluid, the dNTP mix of 0.8ul10mmol/L, the upstream primer of 0.4 μ mol/L, the downstream primer of 0.4 μ mol/L, the sample globefish DNA of 30ng.

5. the method for the yellow Fugu speed of growth of detection claimed in claim 3 chrysanthemum, is characterized in that described pcr amplification reaction condition is, sex change 3 minutes at 94 ℃, sex change 30 seconds at 94 ℃, renaturation 30 seconds at 64 ℃, and 72 ℃ of downward-extensions 30 seconds, totally 42 circulations, last 72 ℃ are extended 10min.

6. the method for the yellow Fugu speed of growth of detection chrysanthemum as claimed in claim 3, is characterized in that the step of described PAGE is:

The sex change sample-loading buffer of getting equivalent adds respectively in PCR product and test kit in MARKER, and 99 ℃ of sex change 10 minutes, at urea-denatured PAGE(6%) in carry out electrophoresis to tetrabromophenol sulfonphthalein and run out of PAGE, carry out silver and dye, fixing, dyeing and colour developing.

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