CN103497995B - A kind of method of rDNA ITS2 sequence mark Rapid identification Mactraantiquata Spengler colony - Google Patents
A kind of method of rDNA ITS2 sequence mark Rapid identification Mactraantiquata Spengler colony Download PDFInfo
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Abstract
Do you the present invention relates to a kind of rDNA? the method of ITS2 sequence mark Rapid identification Mactraantiquata Spengler colony, comprising: it is individual that (1) gathers Mactraantiquata Spengler, extracts DNA; (2) the ITS2 sequence mark primer of rDNA is synthesized; (3) Mactraantiquata Spengler population sample is carried out to the pcr amplification of ITS2 sequence; (4) purifying is carried out to PCR primer; (5) PCR primer of purifying is checked order and sequence alignment, draw specific position, belonging to judgement sample.Result of the present invention is stablized, repeatable strong, and discrimination method is simple, to the plasm resource protection of this shellfish and scientific utilization significant.
Description
Technical field
The present invention relates to qualification Mactraantiquata Spengler kind matter field, particularly a kind of method of rDNAITS2 sequence mark Rapid identification Mactraantiquata Spengler colony.
Background technology
Mactraantiquata Spengler [Coelomactraantiquata(Spengler, 1802) mollusk (mollusca) lamellibranchiata (Lamellibranchia)] is under the jurisdiction of, different tooth subclass (Heteridonta), curtain clam order (Veneroida), clam section (Mactridae), chamber clam belongs to (Coelomactra) shellfish, and Chinese popular name has: " extra large freshwater mussel ", " red eggs ", " highest-ranking imperial concubine freshwater mussel " etc.; Shell appearance is slightly triangular in shape, shell thin and fragile, and the nearly top of shell table is purple powder; In natural waters, 1 age shell length be 4 ~ 6cm, heavily about 20g; 2 age shell length can reach 8cm, heavy about 110g; 3 age shell length can reach more than 10cm, heavily more than 150g, large person can reach more than 250g.Mactraantiquata Spengler is individual large, fine and tender taste, delicious flavour not only, and good-looking containing multiple nutritional components, shell appearance luster, is a kind of famous and precious shellfish of marine products eating medicine and have both.
Mactraantiquata Spengler all has distribution, with ground resource comparatively horn of plenties such as Shandong, Jiangsu, Fujian, Guangdong China north and south is coastal.But in recent ten years, Mactraantiquata Spengler natural resources amount constantly reduces, and its growth district constantly reduces and presented fragmentation trend.Because output is little, Mactraantiquata Spengler is also difficult to see on the market in these places of production, and supply falls short of demand, and price rises year after year.For the resource situation of Mactraantiquata Spengler, Fujian Province established in 1985 in permanently happy sea area " permanently happy extra large freshwater mussel stock enhancement Provincial Nature Reserve, Fujian Province ", limited amount of fishing.Large quantity research shows, due to the Geographical environment that sea area is unique separately, the north and south Different population of Mactraantiquata Spengler has existed obvious genetic variation and genetic differentiation in kind of matter information, should as different kind matter Administrative Units.Because different groups Mactraantiquata Spengler phenotype is close, therefore rely on phenotype to carry out colony and differentiate quite difficulty.Draw along with the seed strange land propagated artificially is moved, this easily causes obscuring and detrimentally affect of kind of matter to a certain extent; How to distinguish different areas source seedling and later stage close shellfish to select, become Mactraantiquata Spengler plasm resource protection and the important problem of fine-variety breeding work.
There is not the molecular marker identification method for Mactraantiquata Spengler different groups at present.ITS2 sequence is the non-coding sequence between 5.8S and 28SrRNA gene, because it is rrna rDNA one-piece construction functionally special, both there is certain conservative property, there is again higher mutability, all may larger difference be there is in its sequence in different sorts or Different Individual of the same race, therefore, utilize ITS2 sequence as the not only simple and clear but also reliable characteristic of Mactraantiquata Spengler colony identifying mark tool.
Summary of the invention
The object of the invention is to provide a kind of method of rDNAITS2 sequence mark Rapid identification Mactraantiquata Spengler colony, and the method result is stablized, and repeatable strong, discrimination method is simple.
For realizing object of the present invention, technical scheme of the present invention is:
Utilize a method for rDNAITS2 sequence mark Rapid identification Mactraantiquata Spengler colony, the method comprises the following steps:
(1) Mactraantiquata Spengler gathering different groups is individual, gets the closed shell muscular tissue of Mactraantiquata Spengler sample, and extract genome DNA, wash-out is dissolved in TE solution or distilled water surely ,-20 DEG C of preservations, in 4 DEG C of preservations time for subsequent use;
(2) with ITS2-F:5 '-GGGTCGATGAAGAACGCAG-3 ' and ITS2-R:5 '-GCTCTTCCCGCTTCACTCG-3 ' for primer, carry out the pcr amplification of ITS2 sequence; Then pcr amplification product detects through 1.2%-1.5% agarose gel electrophoresis, detect and completely carry out cutting glue and utilize PCR primer Purification Kit to reclaim, purified product carries out two-way order-checking row, wherein cannot the PCR primer of direct Sequencing, checks order after cloning and carry out bacterium colony PCR inspection again; Application software carries out different sequence alignment, draws specific position, belonging to judgement sample.
TE strength of solution in described step (1) is 100ng/ μ l.
The reaction system of the pcr amplification in described step (2) is 30 μ L, and each reaction system includes: the template DNA 1 μ L of 50ng/ μ L, 20mMMg
2+10 × buffer2.5 μ L, 5mMeachdNTP1.0 μ L, each 1.0 μ L of primer of 5 μMs, Taq enzyme polysaccharase 1U, adds ddH
2o to 30 μ L.
Pcr amplification reaction in described step (2) is specially: react front 94 DEG C of denaturation 4min, and be then the reaction of 35 circulations, condition is 94 DEG C of 40s, 52 DEG C of 30s, 72 DEG C of 60s, and last 72 DEG C extend 7min.
Carry out cutting glue for molecular weight identifies with DL500 standard DNA Marker in described step (2).
Clone in described step (2) is specially: PCR primer fragment good for purifying is connected into T-easyVECTOR in carrier, system 10 μ L: wherein T4 ligase enzyme 1U, 2 × connect buffer5 μ l fast, PCR primer 2 μ L, carrier T 1 μ L; 16 DEG C of reaction 1h, transform the JM109 competent cell all connecting product and add 100 μ l, the transformed bacterium liquid containing competent cell be spread evenly across add IPTG and X-gal ampicillin/LB plates on, cultivate overnight incubation for 37 DEG C; Select white single bacterium colony, be inoculated in the LB nutrient solution of 450 μ L, 37 DEG C concussion cultivate to exponential growth or visible cloud vaporific; Get 1 μ L nutrient solution and be bacterium colony PCR, detect the fragment inserted, select 2 positive colonies to check order.
Described IPTG consumption is 100 μ l, and concentration is 10mmol/L; X-gal consumption is 100 μ l, and concentration is 2%; Overnight incubation is specially cultivates 16h.
Bacterium colony PCR reaction conditions in described step (2) is: the reaction system of 15 μ L: Mg
2+2.5mmol/L, dNTP0.25mmol/L, primer 0.25 μm of ol/L, Taq enzyme 1U; Reaction conditions is 94 DEG C of denaturation 5min, carries out 30 circulations afterwards: 94 DEG C of sex change 45s, 52 DEG C of annealing 45s, and 72 DEG C extend 120s; Finally, 72 DEG C extend 5min.
beneficial effect
The present invention adopts ITS2 sequence as identifying mark, because the variant sites of sequence is stablized again less, rapidly and efficiently can realize the precise Identification to different groups sample; Be conducive to avoiding different sources Mactraantiquata Spengler seed or become obscuring of shellfish, especially in genetic breeding to the qualification selecting close shellfish; The kind matter of this shellfish Different population natural yielding area is managed, conservation of resources and scientific utilization significant.
Accompanying drawing explanation
Fig. 1 is that the specific base of the rDNAITS2 sequence of different groups Mactraantiquata Spengler individual sample compares; Wherein: Hap-1-Hap-5 is QD colony, Hap-6-Hap-10 is CL colony.
The comparison result of Fig. 2 A-E to be Mactraantiquata Spengler length be 38 rDNAITS2 sequences of 400bp; Wherein: ". " represents the conserved positions of Shandong, Jiangsu colony and Changle of Fujian Province colony, "
" represent the base position differentiated from Shandong, Jiangsu colony and Changle of Fujian Province colony (CL); RZ, JM, QD and GY represent from sunshine in Shandong, Jimo respectively; and the Qidong in Jiangsu, Ganyu the individual sequence of Mactraantiquata Spengler, all the other sequences are used for comparing for downloading from ncbi database.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1
1) sample collection of different sources and DNA extraction:
At China's Shandong Jimo (JM), sunshine (RZ), the Ganyu (GY) in Jiangsu Province, Qidong (QD), and to gather the Mactraantiquata Spengler of self-sow individual in the sea area such as Fujian Province permanently happy (CL).With the scalper of sterilizing and the closed shell muscular tissue of tweezers sample thief.Genome DNA is extracted can adopt DNA extraction kit or phenol chloroform extraction.Finally, OD260, OD280 value of ultraviolet spectrophotometer working sample DNA, determines its concentration and purity, and mother liquor is placed in-20 DEG C of preservations.Time for subsequent use, DNA concentration 100ng/ul is in 4 DEG C of preservations.
2) pcr amplification condition:
Primer base sequences is respectively:
ITS2-F(2A):5′-GGGTCGATGAAGAACGCAG-3′
ITS2-R(2B):5′-GCTCTTCCCGCTTCACTCG-3′
PCR reaction system is 30 μ L, and each reaction system includes: the template DNA 1 μ L of 50ng/ μ L, 20mMMg
2+10 × buffer2.5 μ L, 5mMeachdNTP1.0 μ L, each 1.0 μ L of primer of 5 μMs, Taq enzyme polysaccharase 1U, adds ddH
2o to 30 μ L.
React front 94 DEG C of denaturation 4min, be then the reaction of 35 circulations, condition is 94 DEG C of 40s, 52 DEG C of 30s, 72 DEG C of 60s, and last 72 DEG C extend 7min.
3) PCR primer purifying and clone:
The pcr amplification product of ITS2 sequence detects through 1.2%-1.5% agarose gel electrophoresis, EB dyes, with DL500 standard DNA Marker for molecular weight identifies, under gel imaging system, PCR primer carried out cutting glue and utilize PCR primer purification kit to carry out purifying recovery (concrete operation step is with reference to purification kit specification sheets).Purified product is sent and is carried out two-way order-checking row.Cannot the PCR primer of ITS2 of direct Sequencing, after cloning and carry out bacterium colony PCR inspection, send order-checking again.
Clone: PCR primer fragment good for purifying is connected into T-easyVECTOR(Promega in carrier), system 10 μ L: wherein T4 ligase enzyme 1U, 2 × connect buffer5ul fast, PCR primer 2 μ L, carrier T 1 μ L.16 DEG C of reaction 1h.The JM109 competent cell (Takara) all connecting product and add 100ul is transformed.The transformed bacterium liquid containing competent cell is spread evenly across and adds IPTG(100 μ l10mmol/L solution) and X-gal(100 μ l2% solution) ampicillin/LB plates on, 37 DEG C of cultivations overnight incubation (16h).The single bacterium colony of random choose white, the LB being inoculated in 450 μ L trains in liquid, 37 DEG C of concussions cultivate exponential growth or visible cloud vaporific.Get 1 μ L nutrient solution and be bacterium colony PCR, detect the fragment inserted, select 2 positive colonies to send order-checking company to check order.
Bacterium colony PCR reaction conditions: the reaction system of 15 μ L: Mg
2+2.5mmol/L, dNTP0.25mmol/L, primer 0.25 μm of ol/L, Taq enzyme 1U; Reaction conditions is 94 DEG C of denaturation 5min, carries out 30 circulations afterwards: 94 DEG C of sex change 45s, 52 DEG C of annealing 45s, and 72 DEG C extend 120s; Finally, 72 DEG C extend 5min.
4) order-checking and sequence data analysis
Adopt ABI3730 sequenator to carry out the two-way order-checking of purified product, surveying aligning primer is pcr amplification primer.Sequencing result Chromas231 program reads, and each base of artificial each sequence of check and correction and waveform, to ensure correctness, and remove the primer sequence etc. at sequence two ends.DNA sequence dna, with ClustalX1.81 software (Thompsonetal., 1997) comparison, obtains the conservative and variation base position between different sequence, by the Mactraantiquata Spengler colony of peculiar variant sites qualification Different producing area.(file that comparison is formed can in analytic sample sequence preservative, variant sites in DnaSP4.10.9 software (Rozasetal., 2003).Mactraantiquata Spengler different groups ITS2 sequence (GenBank sequence accession number: DQ523265CL5 can be downloaded from GenBank database, DQ523266CL7, DQ875817GX23, or FJ827075-FJ827079 etc.), carry out specific site comparison as canonical sequence and search.）
5) interpretation of result
If there is the base insertion/deletion the same with figure or special site in the sequence alignment result that sample obtains, be the sample belonging to corresponding geographical population source.Wherein, have if detect in the ITS2 sequence of sample the nucleotide variation the same with Hap6-Hap10 underscore place in figure or insertion/deletion site (see arrow "
"), be then CL colony place of production sample; If sample sequence exists the grey parts base deletion the same with Hap-1-Hap5 (site 21 and or have 1-12 to lack), be from QD colony place of production sample.
In Fig. 1, grey has underscore place to be that the sequence specific of Mactraantiquata Spengler CL colony and QD colony identifies site.Wherein: Hap-1-Hap-5 is QD colony, there is a base deletion in QD colony in the site 21 of arrow indication entirely; Hap-6-Hap-10 is CL colony, namely have arrow indication place 13,14-16,19 and 20 specific sites have base transition, have 2 base deletions, and 21 have the insertion of 1 base " A " in 17-18 site; Separately, the 1-12 site of arrow indication is the intragroup specific site of CL.
Above-mentioned pcr amplification and comparison are carried out to Different population Mactraantiquata Spengler 38 samples, the results are shown in Figure 2.In Fig. 2: ". " represents the conserved positions of Shandong, Jiangsu colony and Changle of Fujian Province colony,
represent the base position differentiated from Shandong, Jiangsu colony and Changle of Fujian Province colony (CL), RZ, JM, QD and GY represent from the Jimo in Shandong, sunshine respectively, and the Qidong in Jiangsu, Ganyu the individual sequence of Mactraantiquata Spengler (other sequence is for downloading as comparing from ncbi database).After comparison, remove two end portions primer sequence and there is no changing unit sequence, obtain the sequence length of Mactraantiquata Spengler Different population between 385 ~ 400bp, ITS2 sequence variations site is mainly at the 3 ' end near sequence, sequence differences is mainly manifested in the insertion/deletion of base, base difference has 15bp, have the specific site that 7 places are qualification colony, be respectively: site 1-12(208-219, be 12 base insertion/deletions), site 13(the 226th, TC changes), site 14-16(273-275, TCT is converted to CTC), site 17-18(279-280, 2 base insertion/deletions), site 19(the 317th, TC changes), site 20(the 381st, GA changes), site 21(the 395th, a base insertion/deletion).ITS2 sequence variations site is less, main 3 ' the end near ITS2, sequence differences is mainly manifested in conversion and the insertion/deletion of base, also proves that this regional sequence is guarded relatively, embodies the not only simple and clear but also reliable characteristic of ITS2 sequence as Mactraantiquata Spengler colony identifying mark.
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the scope of protection of present invention.The scope of protection of present invention is defined by appending claims and equivalent thereof.
Claims (4)
1. a method for rDNAITS2 sequence mark Rapid identification Mactraantiquata Spengler colony, comprising:
(1) gather the Mactraantiquata Spengler individuality of Jimo, sunshine, Ganyu, Qidong, permanently happy different groups, get the closed shell muscular tissue of Mactraantiquata Spengler sample, extract genome DNA, wash-out is dissolved in TE solution or distilled water surely ,-20 DEG C of preservations, in 4 DEG C of preservations time for subsequent use;
(2) Mactraantiquata Spengler different groups ITS2 sequence is downloaded from GenBank database, GenBank sequence accession number: DQ523265CL5, DQ523266CL7, DQ875817GX23, or FJ827075-FJ827079, carry out specific site comparison as canonical sequence and search;
(3) with ITS2-F:5 '-GGGTCGATGAAGAACGCAG-3 ' and ITS2-R:5 '-GCTCTTCCCGCTTCACTCG-3 ' for primer, carry out the pcr amplification of ITS2 sequence; Then pcr amplification product detects through 1.2%-1.5% agarose gel electrophoresis, detect and completely carry out cutting glue and utilize PCR primer Purification Kit to reclaim, purified product carries out two-way order-checking row, wherein cannot the PCR primer of direct Sequencing, check order again after cloning and carry out bacterium colony PCR inspection; Application software carries out different sequence alignment, draws specific position, belonging to judgement sample; Wherein, described specific position is respectively: site 1-12: the 208-219, is 12 base insertion/deletions; Site 13: the 226, TC changes; Site 14-16: the 273-275, TCT is converted to CTC; Site 17-18: the 279-280,2 base insertion/deletions; Site 19: the 317, TC changes; Site 20: the 381, GA changes; Site 21: the 395, a base insertion/deletion; Site 21 have a base deletion for Qidong colony, 13,14-16,19 and 20 specific sites have base transition, have 2 base deletions in 17-18 site, and 21 sites have 1 base " A " to insert is permanently happy colony; Standard sequence is:
2. the method for a kind of rDNAITS2 sequence mark Rapid identification Mactraantiquata Spengler colony according to claim 1, it is characterized in that: the reaction system of the pcr amplification in described step (3) is 30 μ L, each reaction system includes: the template DNA 1 μ L of 50ng/ μ L, 20mMMg
2+10 × buffer2.5 μ L, 5mMeachdNTP1.0 μ L, each 1.0 μ L of primer of 5 μMs, Taq enzyme polysaccharase 1U, adds ddH
2o to 30 μ L.
3. the method for a kind of rDNAITS2 sequence mark Rapid identification Mactraantiquata Spengler colony according to claim 1, it is characterized in that: the pcr amplification reaction in described step (3) is specially: react front 94 DEG C of denaturation 4min, then be the reaction of 35 circulations, condition is 94 DEG C of 40s, 52 DEG C of 30s, 72 DEG C of 60s, last 72 DEG C extend 7min.
4. the method for a kind of rDNAITS2 sequence mark Rapid identification Mactraantiquata Spengler colony according to claim 1, is characterized in that: the bacterium colony PCR reaction conditions in described step (3) is: the reaction system of 15 μ L: Mg
2+2.5mmol/L, dNTP0.25mmol/L, primer 0.25 μm of ol/L, Taq enzyme 1U; Reaction conditions is 94 DEG C of denaturation 5min, carries out 30 circulations afterwards: 94 DEG C of sex change 45s, 52 DEG C of annealing 45s, and 72 DEG C extend 120s; Finally, 72 DEG C extend 5min.
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Non-Patent Citations (2)
| Title |
|---|
| 海洋贝类基因组DNA的提取改进及ITS2扩增;张波等;《江苏农业科学》;20081231;第59-61页 * |
| 蛤蜊科3种贝类16S rRNA基因片段及ITS2核苷酸序列分析;孟学平等;《湛江海洋大学学报》;20060830;第26卷(第4期);第9页左栏倒数第10行至右栏倒数第4行 * |
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