CN1955312A - Albino tea tree breed 'sajin' specific DNA molecular mark and its identification method - Google Patents
Albino tea tree breed 'sajin' specific DNA molecular mark and its identification method Download PDFInfo
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- CN1955312A CN1955312A CNA2006100533463A CN200610053346A CN1955312A CN 1955312 A CN1955312 A CN 1955312A CN A2006100533463 A CNA2006100533463 A CN A2006100533463A CN 200610053346 A CN200610053346 A CN 200610053346A CN 1955312 A CN1955312 A CN 1955312A
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Abstract
The invention belongs to the field of biotechnology, involving biotechnology identification of tea varieties. It relates to the specific DNA molecular markers and identification of a light-induced albino tea varieties 'throwing', the molecular Weight of this nucleotide sequence marked by this specific DNA molecule is 285bp.. Its identification methods : from tea leaves to extract DNA, from the DNA by PCR amplification; PCR amplification products of electrophoresis, bands, 450-500bp emerging molecular weight of the bands detected tea is judged to 'throwing' varieties. Recycling molecular weight of this 450-500bp electrophoresis band plastic, amplification and sequencing; use the 285bp size public nucleotide sequence to check sequencing determine and found 'throwing' variety.
Description
Technical field
The invention belongs to biological technical field, the biotechnology that relates to tea tree breed is identified, be specially the authentication method of a kind of light induction type albino tea tree breed ' sajin ' specific dna molecular marker and breed ' sajin ', also belong to the utilisation technology in tea tree genetic breeding field.
Technical background
Famous-brand and high-quality tea production has become the main economic benefit source of China's tea industry at present.According to statistics, the famous-brand and high-quality tea output of China accounts for 30% of ultimate production, but the output value has reached about 70% of the gross output value; Zhejiang Province, famous-brand and high-quality tea major production areas even reach 90%.Popularization of tea tree improved seeds and processing technology are depended in the production of famous-brand and high-quality tea, and wherein kind plays a decisive role.The development and use of tea tree breed new resources can be injected new vitality for famous-brand and high-quality tea industry.
White tea kind young sprout has albinism, aminoacids content in its blade (especially theanine) is higher more than 2 times than common variety, make the tealeaves flavour bright especially refreshing, and, at present theanine be proved to be have strengthening immunity, hypotensive, promote effects such as brain function and prevention Parkinson's disease, add present resource scarcity, price height very.The development and utilization of white tea kind all has great importance to the development of famous-brand and high-quality tea and the development and use of tealeaves functional ingredient.
White tea has two types, and a kind of is the white tea that occurs albefaction at a lower temperature, as " No. 1, Bai Yecha " tea tree breed.Another kind is the white tea kind that produces albefaction under the intense light irradiation condition, as " spilling gold "." spill gold " when summer high temperature, high light, its young sprout blade presents white or yellow, and aminoacids content reaches as high as 7-10% during its albefaction.Produce bitter taste in summer under the effect owing to high temperature and high light because this kind can overcome common tea tree breed, so can produce the fine famous green tea equally in summer and autumn, promoting this kind is the important channel that increases green tea producing region economic benefit.
Because light induction type albino tea tree breed ' sajin ' belongs to famous and precious kind, the market seedling is in short supply.And this kind only just shows albinism at the high temperature intense light irradiation season, in the season of tea tree suitable planting---and then externally be difficult to distinguish on the morphological specificity with general tea tree breed winter.Chemical constituents determination also can be used for albino tea tree breed to be identified, but the chemical composition content of tea tree changes with the season of growth and envrionment conditions, tea tree planting phase in the winter time, the characteristic compound of albino tea tree, neither the highest season as theanine content, suitable with general tea tree breed, so on concrete the application, be subjected to season limit.Therefore at the nursery stock process of exchange, it is a kind of more accurate and effective authenticate technology is led the tea grower into a trap to prevent fake and forged seedling to press for.
It is the most accurate and stable method of identification of species that dna molecular detects, if can seek out the specific DNA molecule marker of certain kind, the verity of identification of species quickly and accurately.The invention provides the specific DNA molecular mark and the authentication method of light induction type tea tree breed ' sajin '.This method is reliable and stable, is not subjected to the influence of the season of growth and envrionment conditions difference.
Summary of the invention
Purpose of the present invention: the specific DNA sequence of light induction type albino tea tree breed ' sajin ' is provided,, carries out pcr analysis,, just can be defined as breed ' sajin ' if having the dna sequence dna of this specific molecular marker by specific primer as its molecule marker.Otherwise certified kind then is " spilling gold " other kind in addition.Reliable results, easy and simple to handle.
Technical solution of the present invention is as follows:
The specific DNA molecular mark of albino tea tree breed ' sajin ' has the described nucleotide sequence as SEQIDNO.1, and its molecular weight is 285bp.
SEQIDNO.1：
attttctgag?ctagtatcac?accattcatg?tgagctctgc?actctcctcc?gtggatagct?60
tccataaggt?gccttaattc?accttcctca?acacacaact?tatgcattcc?tagatggcct?120
attttgtaca?acttgtttct?gcagatnacg?aattgaatgg?ccagacgccg?tattcccttt?180
ttgtccttgg?cacttgcccc?tagtgggtat?gcctctttgt?ccaggaaatt?caagatgtcg?240
taatactagg?ggtaagtgtc?ttgaacctca?tctatcacgg?cgatt 285
The authentication method of above-described albino tea tree breed ' sajin ' is following steps:
1. from fresh leaves of tea plant, extract DNA;
2. the DNA that extracts is carried out the RAPD-PCR amplification by random primer CAATCGCCGT;
3. the pcr amplification product electrophoresis is identified, the banding pattern analysis, the tested tea tree that molecular weight occurs and be the 450-500bp band is judged to be " spilling gold " kind.
The present invention program can identify step 3 electrophoresis that also the molecular weight of appearance is that 450-500bp band glue reclaims, and glue is reclaimed product amplification and order-checking, the nucleotide sequence that to obtain a size be 285bp; Judge " spilling gold " kind with the described nucleotide sequence contrast of SEQIDNO.1 sequencing result.
Description of drawings
Fig. 1 is the nucleotide sequence (SEQIDNO.1) of the specific DNA molecular mark of albino tea tree breed ' sajin '.
Embodiment
1 genome NDA extracts: get and identified the about 100mg of fresh leaves of tea plant, put into mortar and add liquid nitrogen 100ml, after grinding fast; Powder after grinding is transferred to the 1.5ml centrifuge tube fast.2 * CTAB, the 500 μ l mixings that add 65 ℃ of preheatings, 65 ℃ of water-bath 5min.Chloroform-primary isoamyl alcohol (24: the 1) mixed solution that adds equivalent, fully mixing in the centrifugal 10min of 12000rpm, shifts supernatant liquor to new centrifuge tube.Add 2 times of volume dehydrated alcohols, treat that white flocks occurs, tick white precipitate and be transferred in the new centrifuge tube of another root, add 1ml 70% ethanol and clean, pour out ethanol with needle; Resuspended and washing is once dried in air at room temperature through chloroform-primary isoamyl alcohol mixed solution and ethanol again, is 100 μ g/ml with being diluted to DNA concentration in TE buffer or the sterilized water at last, and it is standby to be stored in-20 ℃ of refrigerators.
2PCR is put and is answered: add following solution in the 0.5ml centrifuge tube: 17.8 μ l ddH
2O, 10 * buffer of 2.5 μ l, the 25mmol Mg of 2.5 μ l
2+, the 10mmol dNTP of 0.5 μ l, the primer of 0.33 μ l (CAATCGCCGT) solution, the above-mentioned dna solution of 1.2 μ l, the TAG enzyme solution of 0.17 μ l.Centrifuge tube is covered tightly and places DYAD
TMOn the PTC-200PCR instrument, at 94 ℃ of pre-sex change 5min; Then through 94 ℃ of sex change 30s, 36 ℃ of annealing 1min, 72 ℃ are extended 2min, totally 40 circular treatment; Last 72 ℃ of prolonged treatment 10min; Be stored in 4 ℃, be the PCR product.
3 electrophoresis: get 1.5 gram agaroses (Agrose), add 100ml water, be heated to 80 ℃ of dissolvings, pour the gel that electrophoresis chamber is made 6-7mm thickness into, the cooling back adds above-mentioned PCR reaction product at well, adds DNA analysis amount standard substance for molecular weight identification at another well simultaneously; The about 30min of electrophoresis under the 100V constant-pressure conditions.
4 banding patterns are differentiated: place JD-801 gel electrophoresis image analysis system to carry out the banding pattern analysis above-mentioned running gel, with dna molecular amount standard substance is reference, find that under uviolizing molecular weight is that the band of 450-500bp only occurs in " spilling gold " kind, other kind does not possess.In cultivar identification, be " spilling gold " kind if possess the identification of species that supplies of this band, otherwise can be accredited as non-" spilling gold " kind.
5 specific DNA molecular mark nucleotide sequence analysises: the present invention has carried out Sequence Identification in order further to understand the nucleotide sequence of above-mentioned specific band to this sequence.Method is with aseptic disinfectant blade this specific band to be downcut from agar gel under UV-light, is recycled in the clean 1.5ml centrifuge tube, and reclaims test kit with UNIQ-10 pillar DNA glue and carry out purifying.Make template with the product behind the purifying, carry out the secondary PCR amplification with identical primer (CAATCGCCGT), amplified production detects with 1.5%Agrose 1-H gel electrophoresis (constant voltage 100V).Use full-automatic dna sequencing instrument (ABI 3700) and BigDyeTerminators Cycle Sequencing Kit then, according to single primer Sanger dideoxy chain termination order-checking.Sequencing result is seen Fig. 1.
Embodiment
1 experimental cultivar: in order to verify specific DNA molecular mark of the present invention is that light induction type albino tea tree breed ' sajin ' is peculiar, present embodiment is with common tea tree breed " Fuding white tea " (numbering No. a kind), low temperature induction type albino tea tree breed " No. 1, Bai Yecha " (numbering No. 2 kind), " snow in thousand " (numbering No. 3 kind), 2 of light induction type albino tea tree breeds---" gold bud " (numbering No. 4 kind) and " spilling gold " 5 kinds such as (numbering No. 5 kind) are test materials.Each kind is got 1 of 1 bud, 1 leaf young sprout and is identified.
2 genome NDA extract: weigh the bright leaf 100mg of above-mentioned tea tree breed respectively, put into mortar and tame liquid nitrogen 100ml, after grinding fast; Powder after grinding is transferred to the 1.5ml centrifuge tube fast.2 * CTAB, the 500 μ l mixings that add 65 ℃ of preheatings, 65 ℃ of water-bath 5min.Chloroform-primary isoamyl alcohol (24: the 1) mixed solution that adds equivalent, fully mixing in the centrifugal 10min of 12000rpm, shifts supernatant liquor to new centrifuge tube.Add 2 times of volume dehydrated alcohols, treat that white flocks occurs, tick white precipitate and be transferred in the new centrifuge tube of another root, add 1ml 70% ethanol and clean, pour out ethanol with needle; Resuspended and washing is once dried in air at room temperature through chloroform-primary isoamyl alcohol mixed solution and ethanol again, is 100 μ g/ml with being diluted to DNA concentration in TE buffer or the sterilized water at last, and it is standby to be stored in-20 ℃ of refrigerators.
2PCR is put and is answered: add following solution in the 0.5ml centrifuge tube: 17.8 μ l ddH
2O, 10 * buffer of 2.5 μ l, the 25mmol Mg of 2.5 μ l
2+, the 10mmol dNTP of 0.5 μ l, the primer of 0.33 μ l (CAATCGCCGT) solution, the above-mentioned dna solution of 1.2 μ l, the TAG enzyme solution of 0.17 μ l.Centrifuge tube is covered tightly and places DYAD
TMOn the PTC-200PCR instrument, at 94 ℃ of pre-sex change 5min; Then through 94 ℃ of sex change 30s, 36 ℃ of annealing 1min, 72 ℃ are extended 2min, totally 40 circular treatment; Last 72 ℃ of prolonged treatment 10min; Be stored in 4 ℃, be the PCR product.
3 electrophoresis: get 1.5 gram agaroses (Agrose), add 100ml water, be heated to 80 ℃ of dissolvings, pour the gel that electrophoresis chamber is made 6-7mm thickness into, the cooling back adds above-mentioned PCR reaction product at well, adds DNA analysis amount standard substance for molecular weight identification at another well simultaneously; The about 30min of electrophoresis under the 100V constant-pressure conditions.
4 banding patterns are differentiated: place JD-801 gel electrophoresis image analysis system to carry out the banding pattern analysis above-mentioned running gel, with dna molecular amount standard substance is reference, under uviolizing, find, molecular weight is that the band of 450-500bp only occurs in No. 5 breed ' sajin 's, and 4 other kinds such as " Fuding white tea ", " No. 1, Bai Yecha ", " snow in thousand ", " gold bud " that are numbered 1-4 number can not detect this band.Proof is " spilling gold " for No. 5 kinds in the identification of species, and 4 kinds that are numbered 1-4 number are not " spilling gold ".This band glue is reclaimed, and glue is reclaimed product amplification and order-checking, the nucleotide sequence that to obtain a size be 285bp; Judge " spilling gold " kind with the described nucleotide sequence contrast of SEQIDNO.1 sequencing result.
At tea tree seedling transaction or cultivar identification research process, according to the specific molecular marker of dna sequence dna provided by the invention, use aforesaid method, the tea tree breed that the is provided kind of whether " spilling gold " can accurately be provided.Because the dna molecular structure has varietY specificity, and is not subjected to the influence of envrionment conditions and seasonal variation, can carry out any time.
Claims (3)
1. the specific DNA molecular mark of albino tea tree breed ' sajin ', it is characterized in that: specific DNA molecular mark has the described nucleotide sequence as SEQIDNO.1, and its molecular weight is 285bp.
2. by the described albino tea tree breed ' sajin ' of claim 1, the authentication method that it is characterized in that it is following steps:
(1) from fresh leaves of tea plant, extracts DNA;
(2) DNA that extracts is carried out the RAPD-PCR amplification by random primer CAATCGCCGT;
(3) the pcr amplification product electrophoresis is identified, the banding pattern analysis, the tested tea tree that molecular weight occurs and be the 450-500bp band is judged to be " spilling gold " kind.
3. press the authentication method of the described albino tea tree breed ' sajin ' of claim 2, it is characterized in that: step (3) electrophoresis is identified that the molecular weight of appearance is that 450-500bp band glue reclaims, and glue reclaimed product amplification and order-checking, the nucleotide sequence that to obtain a size be 285bp; Judge " spilling gold " kind with the described nucleotide sequence contrast of SEQIDNO.1 sequencing result.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559655A (en) * | 2010-12-24 | 2012-07-11 | 云南天士力帝泊洱生物茶集团有限公司 | Method for extracting total DNA (Deoxyribonucleic Acid) of microorganisms from pile-fermentation Pu-erh tea |
| CN102776271A (en) * | 2011-05-13 | 2012-11-14 | 刘本英 | Molecular marking method for identifying tea clonal variety of tea trees |
| CN103160500A (en) * | 2011-12-14 | 2013-06-19 | 比勒陀利亚大学 | Plant-screening method based on drought resistance |
| CN106480224A (en) * | 2016-12-28 | 2017-03-08 | 中国农业科学院茶叶研究所 | The molecular marker combination of Rapid identification difference albino tea tree breed, method and application |
| CN107190082A (en) * | 2017-07-14 | 2017-09-22 | 中国农业科学院茶叶研究所 | Molecular specificity labeled primers and its discrimination method for differentiating national improved tea variety ' meeting frost ' |
| CN108220471A (en) * | 2018-01-25 | 2018-06-29 | 中国农业科学院茶叶研究所 | Differentiate the molecular specificity labeled primers and its discrimination method of national improved tea variety Fuyun No.6 |
| CN110885893A (en) * | 2019-09-04 | 2020-03-17 | 广东省农业科学院茶叶研究所 | Molecular marker locus located on WD-repetitive protein gene and linked with tea tree epicatechin content and application thereof |
-
2006
- 2006-09-12 CN CNA2006100533463A patent/CN1955312A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559655A (en) * | 2010-12-24 | 2012-07-11 | 云南天士力帝泊洱生物茶集团有限公司 | Method for extracting total DNA (Deoxyribonucleic Acid) of microorganisms from pile-fermentation Pu-erh tea |
| CN102559655B (en) * | 2010-12-24 | 2014-11-26 | 云南天士力帝泊洱生物茶集团有限公司 | Method for extracting total DNA (Deoxyribonucleic Acid) of microorganisms from pile-fermentation Pu-erh tea |
| CN102776271A (en) * | 2011-05-13 | 2012-11-14 | 刘本英 | Molecular marking method for identifying tea clonal variety of tea trees |
| CN103160500A (en) * | 2011-12-14 | 2013-06-19 | 比勒陀利亚大学 | Plant-screening method based on drought resistance |
| CN103160500B (en) * | 2011-12-14 | 2019-07-26 | 比勒陀利亚大学 | With regard to the method for drought tolerance screening plant |
| CN106480224A (en) * | 2016-12-28 | 2017-03-08 | 中国农业科学院茶叶研究所 | The molecular marker combination of Rapid identification difference albino tea tree breed, method and application |
| CN106480224B (en) * | 2016-12-28 | 2019-09-24 | 中国农业科学院茶叶研究所 | Molecular labeling combination, method and the application of Rapid identification difference albino tea tree breed |
| CN107190082A (en) * | 2017-07-14 | 2017-09-22 | 中国农业科学院茶叶研究所 | Molecular specificity labeled primers and its discrimination method for differentiating national improved tea variety ' meeting frost ' |
| CN108220471A (en) * | 2018-01-25 | 2018-06-29 | 中国农业科学院茶叶研究所 | Differentiate the molecular specificity labeled primers and its discrimination method of national improved tea variety Fuyun No.6 |
| CN108220471B (en) * | 2018-01-25 | 2020-02-18 | 中国农业科学院茶叶研究所 | Molecular specific marker primer for identifying national-grade tea tree improved variety Fuyun No. 6 and identification method thereof |
| CN110885893A (en) * | 2019-09-04 | 2020-03-17 | 广东省农业科学院茶叶研究所 | Molecular marker locus located on WD-repetitive protein gene and linked with tea tree epicatechin content and application thereof |
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