CN106520953A - Gene sequence of daoli huoshan dendrobium moniliforme HT2 endophytes - Google Patents
Gene sequence of daoli huoshan dendrobium moniliforme HT2 endophytes Download PDFInfo
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- CN106520953A CN106520953A CN201611013106.0A CN201611013106A CN106520953A CN 106520953 A CN106520953 A CN 106520953A CN 201611013106 A CN201611013106 A CN 201611013106A CN 106520953 A CN106520953 A CN 106520953A
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- dendrobium
- huoshan
- endophyte
- endophytes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Abstract
The invention discloses a gene sequence of daoli huoshan dendrobium moniliforme HT2 endophytes. The gene is 26S rDNA, and has the gene sequence of SEQ ID NO.1. The gene sequence of the endophytes (HT2 endophytes) in the dertdrobium is used for daoli dertdrobium huoshanense type identification for the first time; dendrobium huoshanense, huoshandaodi dendrobium officinale and huoshandaodi dendrobium moniliforme can be effectively identified; the identification accuracy of the gene sequence of the endophytes is high; the one-sidedness of a conventional analysis measure is avoided; the result accuracy is improved; and a novel method is provided for the dendrobium variety identification. The conventional dendrobium type identification by the gene sequence of the dendrobium per se is creatively changed into the dendrobium type identification by the gene sequence of the dendrobium endophytes; the scientific basis is provided for the dendrobium variety identification and quality control; and a scientific method is also provided for variety identification of other plants or animals.
Description
Technical field
The present invention relates to identify and biology field, more particularly to a kind of genuine Huoshan Dendrobium Moniliforme HT2 endophytes
Gene order.
Background technology
It is in prior art, many for differentiating Huoshan rice dry measure used in former times, the genuine Dendrobium Moniliforme method of the genuine dendrobium candidum in Huoshan and Huoshan
Using plant genetic sequences or identification method, many english literatures or Chinese literature are differentiated suddenly using plant genetic sequences now
Mountain rice dry measure used in former times, the genuine Dendrobium Moniliforme of the genuine dendrobium candidum in Huoshan and Huoshan；Such as Chinese Patent Application No.：201610029560.9；It is public
Cloth number：105548404A, denomination of invention：A kind of side for differentiating Huoshan rice dry measure used in former times and huoshan dendrobium candidum kind based on metabolism group
Method, discloses a kind of method for differentiating Huoshan rice dry measure used in former times and huoshan dendrobium candidum kind based on metabolism group, and concrete steps include：
(1) collection of stem of noble dendrobium sample；(2) preparation of stem of noble dendrobium metabolism group sample；(3) detection of stem of noble dendrobium sample metabolite；(4) metabolism
Group learns the process and analysis of data.Stem of noble dendrobium type is judged using the detection to stem of noble dendrobium sample metabolite；But with regard to the stem of noble dendrobium
The method that endophyte gene order can be cultivated differentiates stem of noble dendrobium species, is rarely reported.
The content of the invention
Based on the technical problem that background technology is present, the present invention proposes a kind of genuine Huoshan Dendrobium Moniliforme HT2 endophytes
Gene order and its application.
A kind of gene order of genuine Huoshan Dendrobium Moniliforme HT2 endophytes proposed by the present invention, the gene sequence of HT2 endophytes
26S rDNA are classified as, with gene order SEQ ID NO.1 as described below：
Preferably, the PCR primer sequence of gene order is：5 ' GCA TAT CAA TAA GCG GAG of forward primer
GAAAAG 3 ', 5 ' GGT CCG TGT TTC AAG ACG G 3 ' of reverse primer.
Further, a kind of genuine Huoshan Dendrobium Moniliforme endophyte molecular biology method, including：
S1：Strain isolation：The carrying out of endophyte in the fresh cane of Huoshan Dendrobium Moniliforme is separated；
S2：Extract DNA：DNA is extracted to endophyte；
S3：Amplification gene：It is template with endophyte DNA, with pair of primers, 5 ' GCA TAT CAA TAA of forward primer
GCG GAG GAA AAG 3 ', 5 ' GGT CCG TGT TTC AAG ACG G 3 ' of reverse primer, by PCR
Amplify 26S rDNA genes；
S4：The 26S rDNA genes agarose electrophoresis separation that the PCR amplification of appropriate step S3 goes out is taken,
Jing fluorescent dyeings are observed under uviol lamp, according to the size result of determination of amplified production, if energy specific amplification goes out
The band of 611bp, the sequencing of Ze Song biotech firms；
S5：According to sequencing result, if with the homology of the gene order SEQ ID NO.1 more than 98%, you can
Judge described endophyte as HT2 endophytes.
Preferentially, Huoshan Dendrobium Moniliforme endophyte molecular biological variety identification method is differentiating Huoshan rice dry measure used in former times, Huoshan road subway
The application of the genuine Dendrobium Moniliforme of the skin stem of noble dendrobium or Huoshan.
Compared with the prior art, the present invention has the beneficial effect that：
1st, the present invention carries out the genuine stem of noble dendrobium in Huoshan using the gene order of the endophyte (HT2 endophytes) in stem of noble dendrobium body first
Species differentiated, can effectively differentiate Huoshan rice dry measure used in former times, the genuine Dendrobium Moniliforme of the genuine dendrobium candidum in Huoshan and Huoshan, endophyte
Gene order identification accuracy is high, it is to avoid the one-sidedness of traditional analysis means, improves the accuracy of result, is stem of noble dendrobium kind
Discriminating provide new method.
2nd, tradition is identified stem of noble dendrobium species with stem of noble dendrobium gene order itself by the present invention, and creativeness is changed to stem of noble dendrobium endophyte
Gene order identifies stem of noble dendrobium species, and the identification and quality control for stem of noble dendrobium kind provide scientific basis, also for other plants or
The cultivar identification of animal provides a scientific method.
Description of the drawings
Fig. 1 is HT2 bacterial strain 26SrDNA amplified production electrophoretograms；
Fig. 2 is HT2 bacterial strain 26S rDNA sequential system chadograms.
Specific embodiment
High-speed mixer uses twin-screw blending equipment or reciprocating mixing facilities are processed to cable material.
The present invention is further explained with reference to specific embodiment.
Embodiment 1
1st, sample treatment
Take the annual plant of Huoshan Dendrobium Moniliforme to be rinsed well with running water, then remove its leaf with aseptic water washing 5-8 after
With root, carry out surface sterilization with superclean bench being transferred to after aseptic blotting paper suck dry moisture, sterilization method is as follows：75% alcohol
Process 30 seconds, rinsed with sterile water 4 times, 0.l% mercuric chloride processes 8min, and rinsed with sterile water 4 times, 75% ethanol postincubation 30 seconds are aseptic
Water is rinsed 4 times, blots surface moisture with aseptic blotting paper standby.
2nd, the isolation and purification of endophyte
On superclean bench, standby Dendrobium Moniliforme cane sterilization scissors of sterilizing is cut into rod-short, is put into aseptic
Mortar is ground, and is weighed 1g and is put in 9ml sterilized waters, is prepared into 1 × 10-1Bacteria suspension, carries out gradient dilution to 1 × 10-5,
1 × 10 is taken respectively-3With 1 × 10-5Dilution factor coats LB, YEPD, and in PDA culture medium, each dilution factor is parallel three groups, treats table
Static gas wave refrigerator is inverted under the conditions of 28 DEG C are placed in after face moisture is slightly dry, after 4-5d, is had single bacterium colony to be formed in culture medium, is chosen with oese
Taking carries out purifying culture into corresponding slant tube, and is identified.Control group is carried out with the sterilized water of last time rinsing
Coating culture, observes media surface growth microorganism, it is ensured that the bacterial strain being separated to is endophyte under equal conditions.
3rd, the identification of bacterial strain
1) morphological observation：By morphological observation on culture medium, and carry out primarily determining that yeast using microscopic examination
Afterwards, molecular biology is recycled to be identified.
2) molecular biology identification：
A kind of genuine Huoshan Dendrobium Moniliforme endophyte molecular biology method, including：
S1：Strain isolation：The carrying out of endophyte in the fresh cane of Huoshan Dendrobium Moniliforme is separated；
S2：Extract DNA：DNA is extracted to endophyte；
S3：Amplification gene：It is template with endophyte DNA, with pair of primers, 5 ' GCA TAT CAA TAA of forward primer
GCG GAG GAA AAG 3 ', 5 ' GGT CCG TGT TTC AAG ACG G 3 ' of reverse primer, by PCR
Amplify 26S rDNA genes；
S4：The 26S rDNA genes agarose electrophoresis separation that the PCR amplification of appropriate step S3 goes out is taken,
Jing fluorescent dyeings are observed under uviol lamp, according to the size result of determination of amplified production, if energy specific amplification goes out
The band of 611bp, the sequencing of Ze Song biotech firms；
S5：According to sequencing result, if with the homology of the gene order SEQ ID NO.1 more than 98%, you can
Judge described endophyte as HT2 endophytes.
Huoshan Dendrobium Moniliforme endophyte molecular biological variety identification method is applied to differentiate Huoshan rice dry measure used in former times, the genuine iron sheet stone in Huoshan
The technical field of the genuine Dendrobium Moniliforme of dry measure used in former times and Huoshan.
3) PCR reaction systems：Template (genomic DNA 20-50ng/ μ l) 0.5 μ l, 10 × Buffer (with Mg 2
+) 2.5 μ l, 1 μ l of dNTP (each 2.5mM), 0.2 μ l of enzyme, 0.5 μ l of F (10uM), 0.5 μ l of R (10uM), plus double steaming μ l of H 2O to 25；
4) PCR reaction conditions：1. 94 DEG C of 4min of denaturation；2. 94 DEG C of 45s of denaturation；3. 55 DEG C of 45s of renaturation (annealing)；4. 72 DEG C are extended
1min, step 2.～4. circulate 30 times；5. repair and extend 72 DEG C of 10min 6. 4 DEG C of insulations of terminating reaction.
4th, it is sequenced
Sample sequencing is delivered and is carried out by Shanghai Sheng Gong bio-engineering corporations, sequenator Applied Biosystems
(3730XL)。
5th, the gene order of HT2 endophytes is 26S rDNA, with gene order SEQ ID NO.1 as described below：
Colony characteristicses and cellular morphology on 1 culture medium of table
No. HT2 can be primarily determined that from table 1 for Mycophyta；Tentatively judge that No. HT2 is yeast class by morphology, to which
Further identified.
The DNA sequence dna source of 2 HT2 bacterial strains of table and sibling species
Come from the 26S homeologouses fragment sequence of group and outer group in table 2 after comparison and editor, the length of sequence fragment
Spend for 582bp (including Gap), there are 294 variant sites, wherein there are 273 parsimony informative site (parsimony-
informative characters).The average G+C contents of all sequences are 49.6%.By parsimony principle and Bayesian Method institute
It is monosystem that the phylogenetic tree of structure can be seen that sample sequence and the fungal species cluster of Sporidiobolus category, shows this
Sample is the fungi of Sporidiobolus category.As the fungi is the endophyte that Dendrobium Moniliforme is produced in Huoshan, therefore, it is interior with this
The stem of noble dendrobium sample of raw bacterium is that Dendrobium Moniliforme is produced in Huoshan.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any those familiar with the art the invention discloses technical scope in, technology according to the present invention scheme and its
Inventive concept equivalent or change in addition, should all be included within the scope of the present invention.
Claims (4)
1. a kind of gene order of genuine Huoshan Dendrobium Moniliforme HT2 endophytes, it is characterised in that the gene of the HT2 endophytes
Sequence is 26S rDNA, with gene order SEQ ID NO.1 as described below：
2. the PCR primer of the gene order of a kind of genuine Huoshan Dendrobium Moniliforme endophyte according to claim 1, its feature
It is that primer sequence is：
5 ' GCA TAT CAA TAA GCG GAG GAAAAG 3 ' of forward primer
5 ' GGT CCG TGT TTC AAGACG G 3 ' of reverse primer.
3. a kind of genuine Huoshan Dendrobium Moniliforme endophyte molecular biological variety identification method, including：
S1：Strain isolation：The carrying out of endophyte in the fresh cane of Huoshan Dendrobium Moniliforme is separated；
S2：Extract DNA：DNA is extracted to endophyte；
S3：Amplification gene：It is template with endophyte DNA, with pair of primers, 5 ' GCA TAT CAA TAA GCG of forward primer
GAG GAAAAG 3 ', 5 ' GGT CCG TGT TTC AAGACG G 3 ' of reverse primer, are gone out by PCR amplification
26S rDNA genes；
S4：The 26S rDNA genes agarose electrophoresis separation that the PCR amplification of appropriate step S3 goes out is taken, Jing is glimmering
Photoinitiator dye is dyeed to be observed under uviol lamp, according to the size result of determination of amplified production, if energy specific amplification goes out 611bp
Band, Ze Song biotech firms sequencing；
S5：According to sequencing result, if with the homology of the gene order SEQ ID NO.1 more than 98%, you can judge
Described endophyte is HT2 endophytes.
4. a kind of genuine Huoshan Dendrobium Moniliforme endophyte molecular biological variety identification method according to claim 3, its feature
It is that wherein Huoshan Dendrobium Moniliforme endophyte molecular biological variety identification method is differentiating Huoshan rice dry measure used in former times, the genuine dendrobium candidum in Huoshan
Or the application of the genuine Dendrobium Moniliforme in Huoshan.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103210080A (en) * | 2010-03-11 | 2013-07-17 | 帝斯曼知识产权资产管理有限公司 | Yeast strains and their uses in the production of lipids |
| CN105199971A (en) * | 2015-09-12 | 2015-12-30 | 新疆农业科学院微生物应用研究所 | Low temperature-resistant Saccharomyces cerevisiae JM33 and application thereof in munake grape wine |
-
2016
- 2016-11-18 CN CN201611013106.0A patent/CN106520953A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103210080A (en) * | 2010-03-11 | 2013-07-17 | 帝斯曼知识产权资产管理有限公司 | Yeast strains and their uses in the production of lipids |
| CN105199971A (en) * | 2015-09-12 | 2015-12-30 | 新疆农业科学院微生物应用研究所 | Low temperature-resistant Saccharomyces cerevisiae JM33 and application thereof in munake grape wine |
Non-Patent Citations (2)
| Title |
|---|
| ELISABETE VALE´ RIO等: "Reappraisal of the Sporobolomyces roseus species complex and description of Sporidiobolus metaroseus sp. nov", 《INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY》 * |
| 周小风等: "铁皮石斛内生细菌分布规律的研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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Application publication date: 20170322 |