CN100436599C

CN100436599C - Female-specific AFLP fragment of Cynoglossus semilaevis Gunther and PCR method for identification of genetic sex

Info

Publication number: CN100436599C
Application number: CNB2006100441777A
Authority: CN
Inventors: 陈松林; 李静; 田永胜; 沙珍霞; 王清印; 庄志猛; 徐建勇
Original assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Current assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Priority date: 2006-05-12
Filing date: 2006-05-12
Publication date: 2008-11-26
Anticipated expiration: 2026-05-12
Also published as: CN1880478A

Abstract

The present invention relates to a PCR method for identifying the specific AFLP fragment and the genetic sex of a female cynoglossus semilaevis, which comprises a PCR method for recovering a female specific AFLP fragment, cloning an AFLP fragment, analyzing sequences and identifying the genetic sex of the cynoglossus semilaevis. The present invention establishes a technology for separating and cloning the specific AFLP fragment of the female cynoglossus semilaevis, obtains the female specific DNA fragment, designs a specific primer, converts an AFLP label into an SCAR label and establishes a PCR technology for identifying the genetic sex of the cynoglossus semilaevis. The present invention separates the specific DNA fragment of the female cynoglossus semilaevis for the first time, ascertains the DNA sequence of the DNA fragment and establishes the PCR analysis technology for identifying the genetic sex of the cynoglossus semilaevis. The present invention has the characteristics of advancement, high efficiency, accuracy and reliability and has important application value in the sex control of the cynoglossus semilaevis and in the production of unisexual young fishes. Simultaneously, the present invention has application prospect in the research of the genetic sex identification and the genetic sex control of other fishes.

Description

The PCR method that female specific AFLP fragment of Cynoglossus semilaevis and genetic sex are identified

Affiliated technical field:

The invention belongs to fish genetic sex identification and sex control technology in the aquatic living things technical field, is a kind of molecular biology method of the fish genetic sex identification that can use in the breeding of fish unisexuality.

Background technology:

There is abundant fish stock in China, wherein many fish exist gender difference on growth velocity, for example, tilapia is male fast more about more than 40% than female growth, and seawater fishs such as lefteye flounder and Cynoglossus semilaevis, and female individuals is than the male fast 40%-100% that grows, therefore, carry out these fish genetic sexes and detect and sex-controlled research, existing important scientific meaning has great application potential and promotion prospect again.

Cynoglossus semilaevis (Cynoglossus semiliaevis) is the distinctive a kind of famous and precious economic seawater fish of China, belongs to coastal waters warm water demersal fish, and China is coastal all distribution, is many with the Huanghai Sea, the Bohai Sea.Cynoglossus semilaevis is because its delicious flavour, and fine and tender taste is nutritious, welcome by consumers in general, and its marketable value is high, and the breed prospect is boundless.Over nearly 2 years, the Cynoglossus semilaevis artificial breeding technique obtains to break through, produce nearly 1,000,000 tails of semi-smooth tongue sole offspring breed per year, but studies show that, there are huge difference in Cynoglossus semilaevis female individuals and male on growth velocity, its female individuals is than the big 2-3 of male times of (Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science, Meng Tianxiang etc., 1988).Because male was grown slow, reduced the cultured output of Cynoglossus semilaevis, increased aquaculture cost, thereby the popularization of semi-smooth tongue sole offspring breed and the formation of aquaculture industry have been had a strong impact on, owing to be difficult to identify the genetic sex of Cynoglossus semilaevis, had a strong impact on the carrying out of cynoglossus semilaevis gynogenesis and sex control research.

Relevant fish sex study of identification method was just carried out on a few fish at present.The Canada breadboard Devlin of West Vancouver (1994) has found the special dna fragmentation of chinook Y chromosome; (2000) such as the Griffiths of Britain have found two male special AFLP marks in three vertebra sticklebacks (Gasterosteusaculeatus L.), still, when being applied to other two kinds of sticklebacks, but its sex of evaluation that can not be correct.(2004) such as the Ezaz of Britain use AFLP technology to analyze bolti (Oreochromis niloticus L.) genome, find the AFLP mark of 3 Y linkages and an X-linkage, yet these marks but can not be differentiated the sex of the individuality that does not have sibship, show between these marks and the sex determination site reorganization has taken place.(2005) such as Watanabe of Japan utilize the AFLP technology, use the genomic dna of EcoRI and MseI digestion with restriction enzyme peacock fish (Poecilia reticulata), screened 32 groups of combination of primers, found 5 marks linked at last with the sex determination site.They had adopted 48 samples again these several marks had been verified afterwards, find that between sex phenotype and the genotype be not in full accord, wherein the consistence that is marked between sex phenotype and the genotype of A2-218 reaches 90%, is to get in touch 1 mark more closely with sex.But the PCR method about female specific DNA mark of Soleidae fish and genetic sex evaluation there is no report at present both at home and abroad.

Summary of the invention:

The objective of the invention is in order to obtain the female specific AFLP of Cynoglossus semilaevis (amplified fragment length polymorphism) fragments sequence, design special PCR primer, set up the simple and effective PCR detection method that Cynoglossus semilaevis genetic sex is identified, for Cynoglossus semilaevis sex control and complete female breeding research provide technique means.

Its technology contents of the present invention is as follows:

Comprise two aspects: one, the sequence of the female specific AFLP fragment of Cynoglossus semilaevis; Two, the PCR method of Cynoglossus semilaevis genetic sex evaluation.

One, the sequence of the female specific AFLP fragment of Cynoglossus semilaevis: its technology contents comprises: the 1) recovery of the female specific AFLP fragment of Cynoglossus semilaevis; 2) clone of female specific AFLP fragment; 3) sequence of female specific AFLP fragment:

1), the recovery of female specific AFLP fragment:

Choose the combination of primers that can amplify the female specific AFLP band of 382bp, carry out the AFLP amplification, amplified production carries out polyacrylamide gel electrophoresis to be separated, and downcuts the polyacrylamide gel that contains the female specific DNA band of 382bp with blade, and it is standby that the purpose fragment is reclaimed in the dissolving back.

2), the segmental clone of AFLP:

With the female specific AFLP fragment that reclaims, be cloned in the pMD 18-T carrier, the employing standard method connects and transforms, and adopts conventional PCR method screening to contain the segmental clone of purpose, length with agarose gel electrophoresis detection product meets and expects that the sample of length can be considered as positive colony.

3), sequence:

Choose positive colony, carry out sequential analysis with the ABI3730 sequenator.To carry out the multisequencing comparison with DNAMAN Version 4.0 from the nucleotide sequence of the female specific AFLP fragment of 8 positive colonies, its homology is 99.37%, shows that the band that each individuality obtains is same sequence.Comparison of consensus sequence homology and similarity searching carry out with BLAST software, do not find any homologous sequence in GenBank.

Two, the PCR method of Cynoglossus semilaevis genetic sex evaluation:

Extract the blood of Cynoglossus semilaevis fish to be measured, extract genomic dna, the sequence according to the female specific fragment of Cynoglossus semilaevis that obtains designs 1 pair of special primer, CseF382N1 and CseF382C1.Extract the genomic dna of 24 female individuals and 22 males, carry out pcr amplification with the synthetic special primer.If certain individuality has produced specific fragment, can think that these fishes are the female individuals in the heredity, there are not these segmental individualities then to be considered to male in the heredity.

The present invention and prior art contrast are characterized in: the present invention has cloned the female special AFLP fragment of Cynoglossus semilaevis, carried out sequential analysis, thereby the AFLP molecule marker is converted to the SCAR mark, design the primer of female specific fragment, set up the pcr analysis technology that China's cultured fishes genetic sex is identified first.This technology has fast, accurately, advantage such as sensitivity, opened up new technological approaches for fish sex control and unisexuality breeding, suit in other cultured fishes, to apply, to the significant and using value of cultured fishes unisexuality breeding.

Description of drawings:

The nucleotide sequence of Fig. 1, the female specific DNA fragment of Cynoglossus semilaevis;

Fig. 2, Cynoglossus semilaevis female individuals and male genetic sex qualification result.

Embodiment:

Adopt molecule clone technology, clone the special AFLP fragment of sex, set up the conventional pcr analysis technology that Cynoglossus semilaevis genetic sex is identified, this is for cultivating the complete female seed of Cynoglossus semilaevis, carrying out complete female seed cultures, improve the cultured output of Cynoglossus semilaevis, improve the economic benefit of culturing, really Cynoglossus semilaevis is developed as a good breed variety of being accepted by numerous raisers, promote the development of cynoglossus semilaevis cultivation industry and the update of marine fish culture kind, have important practical significance and huge economic benefit.

Embodiment below by ' foundation of the PCR method that the clone of the female specific AFLP fragment of Cynoglossus semilaevis and genetic sex are identified ' is elaborated to technology contents of the present invention in conjunction with the accompanying drawings:

One, the clone of the female specific AFLP fragment of Cynoglossus semilaevis and sequential analysis comprise: 1, the recovery of female specific AFLP fragment; 2, the segmental clone of AFLP; 3, sequential analysis.

1, the recovery of female specific AFLP fragment:

The selection result according to female specific AFLP mark, choose the combination of primers that can amplify the female specific AFLP band of 382bp, carry out the AFLP amplification, carry out polyacrylamide gel electrophoresis then, obtain the female specific AFLP band of 382bp, downcut the polyacrylamide gel that contains target DNA fragment with free of contamination blade, add 20 μ l ultrapure waters, 4 ℃ of dissolvings of spending the night have obtained micro-DNA, and it is standby to reclaim the purpose fragment.

2, the segmental clone of AFLP:

Adopt commercial pMD 18-T to connect the female specific AFLP fragment that test kit will reclaim, be cloned in the pMD 18-T carrier, adopt the CaCl of standard ₂Method for transformation will connect product transformed competence colibacillus bacterium, and converted product is coated with the LB culture dish, 37 ℃ of incubated overnight.Next day,, drop in 3ml LB (the containing acillin) liquid nutrient medium 37 ℃, 250rpm incubated overnight with the toothpick picking list bacterium colony of sterilization.Adopt PCR method screening purpose clone then, from each sample, get 1 μ l bacterium liquid as template, primer and response procedures with pairing selective amplification increase, and the length with agarose gel electrophoresis detection product meets and expects that the sample of length can be considered as positive colony.

3, sequential analysis:

Choose positive colony, carry out sequential analysis with the ABI3730 sequenator.To carry out the multisequencing comparison with DNAMAN Version 4.0 from the nucleotide sequence of the female specific AFLP mark of 8 positive colonies, its homology is 99.37%, shows that the band that each individuality obtains is same sequence.Comparison of consensus sequence homology and similarity searching carry out with BLAST software, do not find any homologous sequence in GenBank.The nucleotide sequence of the female specific AFLP fragment of Cynoglossus semilaevis (being shown in Fig. 1).

Two, the PCR method of genetic sex evaluation:

Extract the blood of Cynoglossus semilaevis fish to be measured, extract genomic dna, the sequence according to the female specific fragment of Cynoglossus semilaevis that obtains designs 1 pair of special primer, Cse-382N1 and Cse382C1.Extract the genomic dna of 24 female individuals and 22 males, carry out pcr amplification with the synthetic special primer.The 25.0 μ lPCR reaction systems of optimizing are: 2.5 μ l, 10 * PCR damping fluid, 1.0 μ l2.5mM dNTP, 2.0 μ l 10pM primer mixed solutions, genomic dna 0.8-1.5 μ l, distilled water 16.3-17 μ l, Taq enzyme (5Us/ μ l) 0.2 μ l.The PCR reactions steps is that the first step is at 95 ℃ of sex change 5mins; Second step, 95 ℃ of sex change 30s, 53.5-54.5 ℃ of annealing 30s, 72 ℃ are extended 60s, so carry out 30 circulations; The 3rd step was extended 5mins at 72 ℃.If certain individuality has produced specific fragment, can think that these fishes are the female individuals in the heredity, there are not these segmental individualities then to be considered to male in the heredity.PCR result shows that the female individuals of 24 supposition all amplifies the special fragment of sex, and male does not all amplify this fragment (Fig. 2).

Sequence table

＜110〉Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science

＜120〉PCR method of female specific AFLP fragment of Cynoglossus semilaevis and genetic sex evaluation

<160>1

<170>Patentlu?version?3.1

<210>1

<211>358

<212>DNA

＜213〉Cynoglossus semilaevis (Cynoglossus semilaevis)

<400>1

aattcactga?cccctgagag?cggcgaagtt?aggcagttcg?ctgagtccag?tgcaaacatc 60

aacgttctta?tatgccaaca?gactttagaa?gccaagaatt?atgtgtcata?tttgttttga 120

ctccaggttc?agtttactta?ggacaaactc?agggatttga?aatgacgagc?aacatcttat 180

cgcctcaagt?gcacatgggt?aaaaagatcc?ttccgagcca?gggctggaga?aagcaggctt 240

ctcaggtgac?acagtgggtt?tggccgagac?tagttttaca?atgatggtgc?cacaaaaaaa 300

gaaagggaaa?ctctatgagt?ggacacagac?tgagacattt?gtcgtgtgtg?agttgtta 358

Claims

1, female specific AFLP fragment of Cynoglossus semilaevis, the technology contents that it is characterized in that it comprises the nucleotide sequence of this female specific fragment, its sequence is:

Primer?CseF382N1

ATTCACTGACCCCTGAGAGCGGCGAAGTTAGGCAGTTCGCTGAGTCCAGTGCAAACATCAACGTTCTTATATGCCAACAGACTTTAGAAGCCAAGAATTATGTGTCATATTTGTTTTGACTCCAGGTTCAGTTTACTTAGGACAAACTCAGGGATTTGAAATGACGAGCAACATCTTATCGCCTCAAGTGCACATGGGTAAAAAGATCCTTCCGAGCCAGGGCTGGAGAAAGCAGGCTTCTCAGGTGACACAGTGGGTTTGGCCGAGACTAGTTTTACAATGATGGTGCCACAAAAAAAGAAAGGGAAACTCTATGAGTGGACACAGACTGAGACATTTGTCGTGTGTGA G

Primer?CseF382C1。

2, the PCR method that a kind of Cynoglossus semilaevis genetic sex is identified, the method that it is characterized in that it is: the blood that extracts Cynoglossus semilaevis fish to be measured, extract genomic dna, sequence according to the female specific fragment of Cynoglossus semilaevis that obtains, design 1 pair of special primer, CseF382N1 and CseF382C1, extract the genomic dna of female individuals and male, carry out pcr amplification with the synthetic special primer, if certain individuality has produced specific fragment, can think that these fishes are the female individuals in the heredity, not have these segmental individualities then to be considered to male in the heredity.