CN102634514B - Bay scallop heat-resistance-related metallothionein gene marker and assisted breeding method thereof - Google Patents
Bay scallop heat-resistance-related metallothionein gene marker and assisted breeding method thereof Download PDFInfo
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- CN102634514B CN102634514B CN2012100397394A CN201210039739A CN102634514B CN 102634514 B CN102634514 B CN 102634514B CN 2012100397394 A CN2012100397394 A CN 2012100397394A CN 201210039739 A CN201210039739 A CN 201210039739A CN 102634514 B CN102634514 B CN 102634514B
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Abstract
The invention belongs to the technical field of aquatics, and particularly relates to a bay scallop heat-resistance-related metallothionein gene marker and a assisted breeding method thereof. The bay scallop heat-resistance-related metallothionein gene marker is shown in a base sequence of a sequence list SEQ ID No.1, and the upstream 337th basic group A related to an initiator is a heat-resisting gene locus. Polymorphic sites of the bay scallop metallothionein gene are screened initially, the heat resistance related metallothionein gene marker is developed, and the heat resistance gene marker assisted breeding method is established. The bay scallop heat-resistance-related metallothionein gene marker and the heat resistance gene marker assisted breeding method have the advantages of high pertinence, high breeding efficiency, simplicity, convenience and quickness in operation and the like, and are applicable to screening shellfish heat-resistance-related markers and breeding improved heat-resisting breeds.
Description
Technical field
The invention belongs to the aquatic living things technical field, specifically a kind of bay scallop heat resistanceheat resistant associated metal metallothionein gene mark and auxiliary breeding means thereof.
Background technology
The scallop culture industry of China, through after initial period flourish, has exposed many problem demanding prompt solutions gradually.Long-term close relative breeds, for many generations cultivation has caused inbreeding depression, makes bay scallop poor growth, resistance reduce.Environment in recent years worsens especially summer high temperature makes the scallop organism metabolic disorder, and extensive death incident often occurs, and causes the tremendous economic loss.Therefore, accelerate the research of breeding core cutting edge technology and the seed selection of degeneration-resistant improved seeds and become the key that ensures the sustainable development of scallop culture industry.But due to less to the research of shellfish heat resistanceheat resistant mechanism and heat resistanceheat resistant functional gene both at home and abroad at present, lack the relevant molecule marker of heat resistanceheat resistant, cause the progress of shellfish heat resistanceheat resistant breed of variety slow, restricted to a great extent that the scallop culture industry is stable, health and Sustainable development.
Metallothionein(MT) is a class lower molecular weight, be rich in sulfydryl and protein that can chelated metal ions, exercise several functions in body, for example participate in the adjusting of essential metal element in organism and the detoxification of nonessential metallic element, the biochemical reactions such as the removing of active oxygen and body growth, growth, reproduction, aging, tumour generation, immunity, stress reaction.There are some researches show, when organism is subject to heat stress, some genetic expression is raised, and wherein metallothionein(MT) is particularly violent as the component reaction of redox system.Research shows that metallothionein(MT) can extensively remove the ROS class material that comprises superoxide anion, hydrogen peroxide, hydroxyl (HO), it is a kind of endogenous antioxidant, it can further regulate and control the expression of other antioxidases simultaneously, thereby assists body antagonism oxidative stress.Research about the shellfish metallothionein(MT) at present focuses mostly at it to aspects such as heavy metals regulation and oxygen scavenging activities, but the relation research between its polymorphism and shellfish heat hardiness power has not yet to see report, therefore find the polymorphism of scallop metallothionein gene, and the distribution characteristics of research polymorphic site in heat-resisting relevant different population, will and utilize metallothionein gene to lay the foundation as the candidate gene of potential heat impedance marker assisted selection for research.
Summary of the invention
The object of the invention is to a kind of bay scallop heat resistanceheat resistant associated metal metallothionein gene mark and auxiliary breeding means thereof.
For achieving the above object, the present invention adopts technical scheme to be:
A kind of bay scallop heat resistanceheat resistant associated metal metallothionein gene mark, gulf scallop heat resistanceheat resistant associated metal metallothionein gene is labeled as shown in sequence table SEQ ID No.1 base sequence, and the 337th base A of upstream from start codon is the heat resistanceheat resistant gene locus.
The acquisition of bay scallop heat resistanceheat resistant associated metal metallothionein gene mark obtains as follows:
1) clone of bay scallop metallothionein gene promoter region partial sequence; The total DNA of bay scallop closed shell flesh of take is template, according to known metallothionein gene sequence, at promoter region design primer AiMT1pF and AiMT1pR amplification, obtains DNA sequence dna, stand-by; AiMT1pF5 '-atgtaagggcacttatgcttgatctttagt-3 ' and AiMT1pR5 '-agcagtcaggaccagcacagcca-3 ';
The screening of 2) heat resistanceheat resistant associated metal metallothionein gene mark, 12 clones to each 6 individualities from heat resistanceheat resistant colony and thermo-responsive colony are checked order, and its pleomorphism site is-488C/T ,-450A/C,-447G/C ,-440A ins-del ,-431T/C,-430G/A ,-428A/T ,-375T/C,-363T/G ,-337A/C ,-311T/A,-310A/T ,-298G/T ,-265A/G,-253A/G ,-251T/C and-118C/T, after extracting again the genomic dna of 50 thermo-responsive individualities and 50 heat resistanceheat resistant individualities, for-the 375T/C site is polymorphic, at first pcr amplification metallothionein promoter region sequence, then selecting Mse I to carry out enzyme to the PCR product cuts, after polyacrylamide gel electrophoresis,-375T/T,-375T/C and-3 kinds of genotype of 375C/C, with-the 337A/C site is polymorphic, utilize Bi-PASA PCR to be analyzed, after agarose gel electrophoresis,-337A/A,-337A/C and-3 kinds of genotype of 337C/C, through Chi-square Test, analyze, the frequency that-337A/A individuality occurs in heat resistanceheat resistant colony is significantly higher than responsive colony,-337A/A is the metallothionein gene mark that heat resistanceheat resistant is relevant.
Bay scallop heat resistanceheat resistant associated metal metallothionein gene marker-assisted breeding method, using the metallothionein gene type that occurs in heat resistanceheat resistant colony medium-high frequency as heat resistanceheat resistant associated metal metallothionein gene mark, the bay scallop that carries this heat resistanceheat resistant genes involved mark is bred, cultivate the offspring, the clone obtains the metallothionein gene promoter region sequence in the offspring, studies its polymorphism; Carry out heat stress simultaneously and process experiment, the genetic development of research heat resistanceheat resistant associated metal metallothionein gene mark and with the relation of bay scallop temperature capacity, both therefrom filter out with heat resistanceheat resistant associated metal metallothionein gene mark, the spat that temperature capacity significantly improves again carry out many generation breedings and cultivate after can set up the heat resistanceheat resistant new variety.
The present invention compared with the prior art, the present invention is by Protocols in Molecular Biology, the bay scallop of take has been excavated China's cultivated shellfish heat resistanceheat resistant associated metal metallothionein gene mark first as material, tentatively set up bay scallop heat resistanceheat resistant associated metal metallothionein gene marker-assisted breeding technology, this technology has the characteristics such as simple and efficient to handle, that Breeding Efficiency is high, the cycle is short, for new molecular breeding technological approaches has been opened up in the cultivation of shellfish heat resistanceheat resistant kind, the seed selection of cultivated shellfish heat resistanceheat resistant kind is had to most important theories meaning and using value.
The accompanying drawing explanation
The bay scallop metallothionein gene promoter region partial sequence figure that Fig. 1 provides for the embodiment of the present invention.
The bay scallop metallothionein gene promoter region pleomorphism site sequence chart that Fig. 2 provides for the embodiment of the present invention.
((A) Mse I restriction enzyme mapping wherein: M1 is DL2000Marker to the somatotype collection of illustrative plates of the bay scallop metallothionein gene promoter region different genotype that Fig. 3 provides for the embodiment of the present invention, 1 is-375T/T genotype restriction enzyme mapping, 2 are-375T/C genotype restriction enzyme mapping, 3 are-375C/C genotype restriction enzyme mapping, and M2 is 20bp Marker.(B) Bi-PASA PCR collection of illustrative plates: M1 is DL2000Marker, and 1 is-337A/C genotype PCR collection of illustrative plates, and 2 are-337C/C genotype PCR collection of illustrative plates, and 3 are-337A/A genotype PCR collection of illustrative plates.)。
The distribution frequency of metallothionein(MT) different genotype and corresponding chi square test figure in the bay scallop heat resistanceheat resistant colony that Fig. 4 provides for the embodiment of the present invention and responsive colony.
Embodiment
Below, with the example that is established as of the relevant bay scallop metallothionein gene mark of bay scallop heat resistanceheat resistant and assistant breeding technology thereof, technology contents of the present invention is elaborated.
Bay scallop heat resistanceheat resistant associated metal metallothionein gene mark comprises: 1. the clone of bay scallop metallothionein gene promoter region partial sequence; 2. the screening of heat resistanceheat resistant associated metal metallothionein gene mark; 3. carry the rapid screening of heat resistanceheat resistant genes involved tagging;
1. the clone of bay scallop metallothionein gene promoter region
Extract total DNA from bay scallop closed shell flesh with reference to the described method of molecular cloning; According to known metallothionein gene sequence, at its promoter region design primer, the clone obtains the DNA sequence dna of one section 617 base, is checked order after being connected into the T carrier, obtains its nucleotide sequence.
2. the screening of heat resistanceheat resistant associated metal metallothionein gene mark
12 clones to each 6 individualities from heat resistanceheat resistant colony and thermo-responsive colony are checked order, and have found 17 place's pleomorphism sites; Choose at random 50 thermo-responsive individualities and 50 heat resistanceheat resistant scallop individualities, extract genomic dna, for-the 375T/C site is polymorphic, at first pcr amplification metallothionein promoter region sequence, then select Mse I to carry out enzyme to the PCR product and cut, after polyacrylamide gel electrophoresis, find 3 kinds of genotype,-375T/T ,-375T/C and-375C/C.For-the 337A/C site is polymorphic, utilizes Bi-PASA PCR to be analyzed, and after agarose gel electrophoresis, finds 3 kinds of genotype ,-337A/A ,-337A/C and-337C/C.The frequency that 3 kinds of genotype individualities in-375 sites occur in heat resistanceheat resistant colony and thermo-responsive colony is without marked difference, and-frequency that the 337A/A individuality occurs in heat resistanceheat resistant colony is significantly higher than thermo-responsive colony, and-frequency that the 337C/C individuality occurs in thermo-responsive colony is significantly higher than heat resistanceheat resistant colony.Therefore, using-337C/C is as thermo-responsive relevant metallothionein gene mark, and the general-337A/A metallothionein gene mark relevant as heat resistanceheat resistant.
3. carry the rapid screening of heat resistanceheat resistant genes involved tagging
Get the individual whole blood 1 μ l of bay scallop as masterplate, utilize and analyzed with Bi-PASA PCR, after the agarose gel electrophoresis somatotype, selection-337A/A individuality is as the heat resistanceheat resistant individuality.
The acquisition of bay scallop heat resistanceheat resistant associated metal metallothionein gene mark obtains as follows:
1. the clone of bay scallop metallothionein gene promoter region
With reference to the described method of molecular cloning, extract total DNA from bay scallop closed shell flesh; In promoter region design primer AiMT1pF (5 '-atgtaagggcacttatgcttgatctttagt-3 ') and AiMT1pR (5 '-agcagtcaggaccagcacagcca-3 '), carry out pcr amplification according to following program: 94 ℃ of sex change 5min according to known metallothionein gene sequence; Carry out afterwards the constant amplification (72 ℃ are extended 1min for 94 ℃ of sex change 30s, 55 ℃ of annealing 30s) of 35 circulations; Finally at 72 ℃ of insulation 10min.The clone obtains the DNA sequence dna of one section 617 base, is checked order after being connected into the T carrier, obtains its nucleotide sequence (referring to Fig. 1).Clone's MT promoter sequence total length has 617bp, and according to the prediction of NNPP database, this fragment has four possible transcription initiation sites (TSS1-TSS4), several core promoter elements TATA box and CAAT box.Patch software and the prediction of TESS database analysis, this sequence contains many potential transcription factor binding site points, comprises metal response element (MREs), glucocorticoid response element (GREs), anti-oxidant response element (AREs), heat shock element (HSE) and SP1.
2. the screening of bay scallop heat resistanceheat resistant associated metal metallothionein gene mark
12 clones to each 6 individualities from heat resistanceheat resistant colony and thermo-responsive colony are checked order, and have found 17 place's pleomorphism site (488C/T ,-450A/C,-447G/C ,-440A ins-del ,-431T/C,-430G/A ,-428A/T ,-375T/C,-363T/G ,-337A/C ,-311T/A,-310A/T ,-298G/T ,-265A/G,-253A/G ,-251T/C and-118C/T) (referring to Fig. 2).After extracting the genomic dna of 50 thermo-responsive individualities and 50 heat resistanceheat resistant individualities, for-the 375T/C site is polymorphic, pcr amplification metallothionein promoter region sequence at first, and program is as follows: 94 ℃ of sex change 5min; The then constant amplification of 35 circulations (72 ℃ are extended 1min for 94 ℃ of sex change 30s, 55 ℃ of annealing 30s); Finally at 72 ℃ of insulation 10min.Then selecting Mse I to carry out enzyme to the PCR product cuts.For-375T allelotrope, Mse I enzyme can produce size after cutting be respectively 30,54, and 55,61,65,118,122 and 8 fragments of 407bp, can produce size and be respectively 30,54 for-375C allelotrope, 55,65,118,122 and 7 fragments of 468bp.Get enzyme and cut product 10 μ L, add tetrabromophenol sulfonphthalein sample-loading buffer 2 μ L, with 180V voltage electrophoresis 120min in 12% polyacrylamide gel.With after 0.5 μ g/mL ethidium bromide staining, under ultraviolet lamp, observe whether have 468 and two bands of 407bp carry out somatotype, find altogether 3 kinds of genotype ,-375T/T ,-375T/C and-375C/C.For-the 337A/C site is polymorphic, with A iMT1pP (5 '-ttcaggttggaaagttcatgtaagggcact-3 '), AiMT1pQ (5 '-caacacaccttcggaagacatcacagc-3 '), AiMT1pA (5 '-ggggggggggcatattttgaacagttc-3 ') and AiMT1pB (5 '-ggggggggggctcagctagacatgat-3 ') be primer, utilize Bi-PASA PCR to be analyzed.The PCR reaction adopts warm start and landing procedure to strengthen the specificity of amplification, and reaction conditions is as follows: 94 ℃ of sex change 5min; Carry out afterwards the landing procedure (94 ℃ of sex change 30s, the renaturation temperature is since 63 ℃, often carries out a thermal cycling temperature and reduces by 0.8 ℃, lands vertically to 55 ℃, the sex change time is 30s, 72 ℃ are extended 30s) of 10 circulations; Then meet constant amplification (94 ℃ of sex change 30s of 25 circulations; 55 ℃ of annealing 30s, 72 ℃ are extended 30s); Last 72 ℃ of insulation 10min.For-337A allelotrope, it is 226 and 2 fragments of 559bp that Bi-PASA PCR can produce size, can produce size for-337C allelotrope and be respectively 384 and 2 fragments of 559bp.Get PCR product 5 μ L, add tetrabromophenol sulfonphthalein sample-loading buffer 1 μ L, with 120V voltage electrophoresis 30min in 2% sepharose.With after 0.5 μ g/mL ethidium bromide staining, under ultraviolet lamp, observing banding pattern, find 3 kinds of genotype ,-337A/A ,-337A/C and-337C/C (referring to Fig. 3).
Site-375 and the individual frequency (referring to table 1 and Fig. 4) occurred of-337 different genotype in statistics heat resistanceheat resistant colony and susceptible colony, utilize SPSS11.5 software to carry out after Chi-square Test analyzes finding, the frequency that-375 different genotype individualities occur in heat resistanceheat resistant colony and thermo-responsive colony is without significant difference, and-frequency that the 337C/C individuality occurs in responsive colony is significantly higher than heat resistanceheat resistant colony, and the frequency that-337A/A individuality occurs in heat resistanceheat resistant colony is significantly higher than responsive colony (referring to table 1 and Fig. 4).Therefore ,-337C/C is thermo-responsive relevant metallothionein gene mark, and-337A/A is to be the metallothionein gene mark that heat resistanceheat resistant is relevant.
Table 1: the chi square test of bay scallop metallothionein(MT) different genotype distribution frequency in heat resistanceheat resistant colony and thermo-responsive colony
3. carry the rapid screening of heat resistanceheat resistant genes involved tagging
Get bay scallop whole blood 1 μ l as masterplate, with AiMT1pP (5 '-ttcaggttggaaagttcatgtaagggcact-3 '), AiMT1pQ (5 '-caacacaccttcggaagacatcacagc-3 '), AiMT1pA (5 '-ggggggggggcatattttgaacagttc-3 ') and AiMT1pB (5 '-ggggggggggctcagctagacatgat-3 ') carry out Bi-PASA PCR for primer.The PCR reaction adopts warm start and landing procedure to strengthen the specificity of amplification, and reaction conditions is as follows: 94 ℃ of sex change 5min; Carry out afterwards the landing procedure (94 ℃ of sex change 30s, the renaturation temperature is since 63 ℃, often carries out a thermal cycling temperature and reduces by 0.8 ℃, lands vertically to 55 ℃, the sex change time is 30s, 72 ℃ are extended 30s) of 10 circulations; Then meet constant amplification (94 ℃ of sex change 30s of 25 circulations; 55 ℃ of annealing 30s, 72 ℃ are extended 30s); Last 72 ℃ of insulation 10min.Using selection-337A/A individuality after PCR product agarose gel electrophoresis somatotype as the heat resistanceheat resistant individuality.
Bay scallop heat resistanceheat resistant associated metal metallothionein gene marker-assisted breeding method:
Using metallothionein gene type-337A/A of occurring in heat resistanceheat resistant colony medium-high frequency as heat resistanceheat resistant associated metal metallothionein gene mark.The bay scallop that carries this heat resistanceheat resistant genes involved mark is bred, cultivate the offspring, the clone obtains the metallothionein gene in the offspring, studies its polymorphism; Carry out heat stress simultaneously and process experiment, the genetic development of research heat resistanceheat resistant associated metal metallothionein gene mark and with the relation of scallop heat hardiness, therefrom filter out and both contained heat resistanceheat resistant associated metal metallothionein gene mark, the spat that heat resistance improves again, carry out can setting up the heat resistanceheat resistant improved seeds after many generation breedings and cultivation.
Claims (2)
1. a bay scallop heat resistanceheat resistant associated metal metallothionein gene mark, it is characterized in that: bay scallop heat resistanceheat resistant associated metal metallothionein gene is labeled as shown in sequence table SEQ ID No.1 base sequence.
2. a bay scallop heat resistanceheat resistant associated metal metallothionein gene marker-assisted breeding method, it is characterized in that: two locis on the site of the 281st of the sequence shown in SEQ ID No.1 of usining are the genotype of A as heat resistanceheat resistant associated metal metallothionein gene mark, the bay scallop that carries this heat resistanceheat resistant genes involved mark is bred, cultivate the offspring, the clone obtains the metallothionein gene promoter sequence in the offspring, studies its polymorphism; Carry out heat stress simultaneously and process experiment, the genetic development of research heat resistanceheat resistant associated metal metallothionein gene mark and with the relation of bay scallop temperature capacity, both therefrom filter out with heat resistanceheat resistant associated metal metallothionein gene mark, the spat that temperature capacity significantly improves again carry out many generation breedings and cultivate after can set up the heat resistanceheat resistant new variety;
Metallothionein gene promoter region sequence figure is:
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