CN111518946B - Method for identifying maca - Google Patents

Method for identifying maca Download PDF

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CN111518946B
CN111518946B CN202010554672.2A CN202010554672A CN111518946B CN 111518946 B CN111518946 B CN 111518946B CN 202010554672 A CN202010554672 A CN 202010554672A CN 111518946 B CN111518946 B CN 111518946B
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rbcl
maca
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谭贵良
朱爽
胡敏
李雪雁
李梅
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Guangdong Tianbaotang Health Technology Industry Co ltd
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Abstract

The invention discloses a method for identifying maca, wherein the nucleotide sequence of a rbcL region sequence of the maca is SEQ ID NO: 1, the identification method comprises the following steps: extracting DNA of a sample to be detected as an amplification template; performing PCR amplification reaction by using upstream and downstream primers of the rbcL region sequence to obtain a rbcL region sequence; sequencing the amplified product, matching and comparing the sequences, and judging whether the sample to be detected is maca or not by checking the base information of 8 sites.

Description

Method for identifying maca
Technical Field
The invention relates to the technical field of plant species identification, in particular to a method for identifying maca.
Background
The Lepidium Meyenii Walp is Lepidium Meyenii Walp (Lepidium Meyenii Walp) of Lepidium of BrassicaceaeLepidium meyenii) The root of (1) is also called Peru ginseng, and the native Andes mountain range over 4000 meters in south America is one of the main food sources of local residents. Researches show that maca has the functions of improving fertility, enhancing immunity, resisting fatigue, relieving climacteric syndrome, resisting oxidation and the like, and is widely applied to health care products and food industries at home and abroad due to the special effects of maca.
The plant species of the Lepidium Sativum, which is described in the plant journal of China (1987), is about 150 species and is widely distributed all over the world, and 15 species and one variety exist in China, and are distributed all over the country, and the species are mainly common species such as Lepidium Sativum, and Lepidium Latifolium.
The ITS zone sequence of the ribosome genome has the advantages in plant identification due to the characteristics of high evolution rate, good primer universality, large number of sequences recorded in an NCBI database, capability of providing rich genetic information and the like, but is easily interfered by microbial pollution, so that the identification by using ITS has certain limitation. Therefore, the search for a simple, effective and reliable barcode is of great importance for the molecular biological identification of maca.
Disclosure of Invention
The invention aims to improve the limitation of the existing maca identification method only by using an ITS region sequence of a ribosomal genome, namely, a region sequence rbcL for maca identification and a method for accurately identifying maca by using the region sequence rbcL are provided.
The region sequence rbcL for maca identification is positioned in a chloroplast genome, and has the characteristics of high resolution, easiness in amplification and the like, so that the region sequence rbcL is applied to species identification in plant kingdom and molecular systematics research of different classification orders, and the region sequence rbcL is not polluted by microorganisms, so that the region sequence rbcL has certain advantages.
The above purpose of the invention is realized by the following scheme:
the invention provides a method for identifying maca, which adopts a region sequence rbcL, wherein the nucleotide sequence of the region sequence rbcL is marked as SEQ ID No: 1.
the method for identifying the sample to be detected comprises the following steps:
step 1: pretreating a sample to be detected, and extracting DNA of the sample to be detected to obtain the DNA of the sample to be detected;
step 2: the upstream primer and the downstream primer of the rbcL region sequence are respectively a primer F and a primer R, the primer F and the primer R of the rbcL region sequence are respectively used for carrying out PCR amplification reaction to obtain a rbcL region sequence of a sample to be detected, and the nucleotide sequence of the primer F is marked as SEQ ID No: 2, the nucleotide sequence of the primer R is represented as SEQ ID No: 3;
the total volume of the reaction system for PCR amplification is 20. mu.L, and the reaction system comprises 2.5 mmol/L MgCl 210 XPCR Buffer 2. mu.L, four 2.5 mmol/L mononucleotides 1.6. mu.L, 10. mu. mol/L rbcL primer F0.8. mu.L, 10. mu. mol/L rbcL primer R0.8. mu.L, 5U/. mu.L HiFi DNA polymerase 0.1. mu.L and template DNA 50 ng, and the balance is sterile ultrapure water; the four 2.5 mmol/L mononucleotides are adenine, thymine, guanine and cytosine.
The PCR amplification reaction process sequentially comprises the following steps: pre-denaturation at 95 ℃ for 4 min, denaturation at 92 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 1 min, 35 cycles, and extension at 72 ℃ for 10 min.
And step 3: and (3) comparing and matching the rbcL region sequence of the sample to be detected obtained in the step (2), and then introducing software MEGA (Mega) to analyze the region sequence: if the sequence is selected from SEQ ID No: 1, if the 221 th base from the 5' end is G, the 240 th base is A, the 375 th base is G, the 381 st base is G, the 432 th base is C, the 465 th base is A, the 471 th base is T and the 492 th base is A, then maca is identified, otherwise, maca is not identified.
In the step 3, the DNA of the sample to be detected is extracted after the sample to be detected is pretreated, so as to obtain the DNA of the sample to be detected, wherein the pretreatment is to soak the sample to be detected in 75% ethanol solution by mass for 5 min, then place the sample in a sterile environment for air drying, and then grind the sample to be detected into fine powder under the condition of liquid nitrogen cooling, and the fine powder is the sample to be detected.
The invention has the beneficial effects that:
the invention provides a rbcL region sequence which can be used for identifying maca, the sequence is compared, matched and analyzed with 55 sequences of 8 species of Lepidium published on NCBI, and by comparing 8 information sites of the sequence, the maca can be simply, quickly and effectively identified on a molecular biology level.
Detailed Description
The following description will be made by specific examples.
The particular techniques or conditions not mentioned in the examples are in accordance with the techniques or conditions described in the literature of the art or in accordance with the product specifications. The reagents or instruments used in this example are not indicated by the manufacturer, and are available commercially, and all reagents are analytically pure.
Example 1: obtaining sequence of rbcL area of maca sample
1. DNA extraction of a sample to be tested
(1) Multiple maca samples were collected from different production sites as shown in table 1.
Table 1: maca sample collection information
Serial number Sample (I) Sampling site Collection ground
1 Maca Root of a tree Yunnan province
2 Maca Root of a tree Tibet medicine
3 Maca Root of a tree Sichuan
(2) Performing DNA extraction on a sample to be detected in the table 1 by adopting a CTAB method, and specifically performing the following operations:
1) and soaking a sample to be tested in 75% ethanol for 5 minutes, taking out, and naturally air-drying in a sterile environment.
2) Taking 2 g of a sample to be detected, adding liquid nitrogen, grinding the sample to be detected into powder, placing the powder in a 10 mL centrifuge tube, adding 4.5 mL of a preheated 3 xCTAB extracting solution at 65 ℃, uniformly mixing 0.5 mL of SDS, carrying out water bath at 65 ℃ for 1h, and shaking the mixture evenly and lightly every 15-20 min; the formula of the 3 xCTAB extracting solution is as follows: 3% CTAB, 0.1 mol/L Tris-HCl, 1.4 mol/L NaCl, 2% PVPP and 25 mmol/L EDTA, and sterilizing at high temperature and high pressure; wherein, 1% beta-mercaptoethanol is added after sterilization and cooling, and the percentages in the system are volume ratio.
3) After the water bath is finished, taking the middle layer clear liquid for subpackaging into 1.5 mL centrifuge tubes under the condition of 12000 rpm for 10 min, adding equal volume of Tris saturated phenol-chloroform-isoamyl alcohol (25: 24: 1), evenly mixing, centrifuging for 10 min under the condition of 12000 rpm, taking the supernatant liquid to a new 1.5 mL centrifuge tube, adding equal volume of chloroform-isoamyl alcohol (24: 1), evenly mixing, then centrifuging for 5 min under the condition of 12000 rpm, taking the supernatant liquid, adding 0.6 time volume of isopropanol, adding 3 mol/L sodium acetate until the final concentration of the sodium acetate is 0.3 mol/L, precipitating for 1h under the condition of-20 ℃, centrifuging for 5 min under the condition of 12000 rpm, and discarding the supernatant liquid. The pellet was washed with 1mL of pre-cooled 70% ethanol and centrifuged at 12000 rpm for 5 min, the supernatant was discarded to give a pellet, and the washing was repeated 2-3 times. And after washing, air-drying the precipitate, adding 50 mu L of sterile water or 1 xTE solution for dissolving, and storing at-20 ℃ to obtain the DNA of the sample to be detected.
2. PCR amplification
(1) And performing rbcL region sequence amplification reaction on the DNA of the sample to be detected obtained from the sample in the table 1, wherein the upstream primer and the downstream primer of the rbcL region sequence are respectively a primer F and a primer R.
And carrying out amplification reaction on the rbcL region sequence of the DNA of the sample to be detected by using the primer F and the primer R to obtain the rbcL region sequence of the sample to be detected.
The nucleotide sequence of the primer F is SEQ ID No: 2, specifically to
F：5’ ATGTCACCACAAACAGAGACTAAAGC 3’。
The nucleotide sequence of the primer R is SEQ ID No: 3, specifically to
R：5’ GTAAAATCAAGTCCACCYCG 3’。
The above-mentioned primer F and primer R were synthesized by Biotechnology engineering (Shanghai) Ltd.
(2) And (3) PCR reaction system: the total volume of the reaction system for PCR amplification is 20 mu L, and the reaction system contains 2.5 mmol/L MgCl 2 2. mu.L of PCR Buffer, 1.6. mu.L of four 2.5 mmol/L mononucleotides, 0.8. mu.L of 10. mu. mol/L rbcL primer F, 0.8. mu.L of 10. mu. mol/L rbcL primer R, 0.1. mu.L of 5U/. mu.L HiFi DNA polymerase and 50 ng of template DNA, and the balance of sterile ultrapure water. The four mononucleotides of 2.5 mmol/L are adenine, thymine, guanine and cytosine.
(3) The PCR amplification reaction process comprises the following steps: pre-denaturation at 95 ℃ for 4 min, denaturation at 92 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 1 min, 35 cycles, and extension at 72 ℃ for 10 min.
In this example, the HiFi kit from Beijing Quanji Biotechnology Ltd was used for PCR amplification.
3. PCR product purification, ligation and transformation
The PCR amplification product was recovered by tapping using a DNA Gel recovery Kit (TaKaRa MiniBEST Agarose Gel DNA Extraction Kit). PCR amplification products of maca rbcL region sequences were from 1% agar gel competent cells, followed by ampicillin selection.
4. Sequencing of rbcL region sequences
The monoclonal colonies were picked up and sequenced by Bio-technology Co., Ltd of Borneo, and the sequencing primers were identical to the above PCR primers (primer F and primer R).
5. rbcL region sequence analysis
(1) Selecting Lepidium species recorded in Chinese plant journal (1987 version) including LepidiumLepidium apetalum) Green Lepidium Merrill (A. Merrill. Ex Fr.) KuntzeLepidium campestre) Lepidium meyenii, (Lepidium meyenii) LedumLepidium denhsiforum) Plectranthus Amboinicus (lour.) Merr (leaf of Plectranthus Amboinicus)Lepidium latifolium) Duxingchang (Duxingchang)Lepidium perfoliatum) Lepidium meyenii, (Lepidium meyenii Walp.)Lepidium ruderale) (iii) Jia Du xing Cao (Chinese Lasiocarpa)Lepidium sativum) Ducheng vegetable of North America (a)Lepidium virginicum) Qianye Duxingcai (Chinese Parietaria root and Szechwan Merrill.)Lepidium cuneiforme) Qianye Duxing herb (Chinese Thorowax herb)Lepidium cordatum) Alkali Lei xing Cao (Chinese Lawsonia herb)Lepidium cartilagineum) Duxinlisi (leaf of Chinese dodder)Lepidium obtusum) Lepidium meyenii, (Lepidium meyenii) LevlLepidium capitatum) All-reason Lei xing Cao (Chinese Lawsonia)Lepidium ferganense) A La shan Lei xing Cao (A La shan Li Zi Cao)Lepidium alashanicum) Fifteen kinds of sequences are searched and downloaded in NCBI database, 55 representative sequences are properly selected, and the sequence numbers are respectively as follows: MF786747, GQ436651, JF942212, JF942197, JF942198, JF942199, JF942200, JF942201, JF942202, JF942203, JF942204, JF942205, JF 946, JF942207, JF942208, JF942209, JF 9494942210, JF942211, JN891579, LT576829, JN890951, JN892842, HQ 5956, HQ 59015957, MG248625, MG247979, MG 24242457900, HQ 59018458, JX 8442, MG249005, MG247717, MG246641, MG 684246942, KX678710, JN 240955, JN 60246073, JN891, JF 898954, MG 2214, MG 7002214, MG 24678, MG 24598, MG 24549, JF 24898, JF 24589, JF 898, JF 24849, MG246641, MG 24589, JF 24668, JF 24589, JF 246641, JF 24668, JF 24589, JF 24668, fj 24589, JF 24589, sj 24668, MG 24668, JF 24589, JF 24668, JF 24589, MG 24589, JF 24668, JF 24589, JF 246641, JF 24668, JF 24589, JF 24668, JF 24589, JF 24668.
Among them, there are 7 species of striga asiatica (Lepidium cordium), Lepidium sativum (Lepidium cartilaginum), Lepidium obtusifolia (Lepidium oblitum), Lepidium capitatum (Lepidium capitatum), Lepidium cunatum (Lepidium cuneiforme), Allium amarum (Lepidium alaschum) and Lepidium integrifolium (Lepidium ferratum) which are not compared because there is no rbcL sequence in the database.
(2) According to sequencing results, 3 maca sequences are completely identical, and the nucleotide sequence of the maca sequences is shown in SEQ ID No. 1. Comparing the three sequences of the sample to be detected with the above 55 sequences, aligning the two ends by taking the maca sequence No.1 as a reference sequence, and completing the comparison between the sequences through MEGA (version 7.0) software to obtain 18 variable sites, wherein 8 variable sites are used for identifying maca.
6. Identification of maca
And (3) carrying out maca identification according to the obtained 8 variable sites, wherein the specific standard is that the 221 bit is G, the 240 bit is A, the 375 bit is G, the 381 bit is G, the 432 bit is C, the 465 bit is A, the 471 bit is T, and the 492 bit is A, and when all the 8 sites are consistent with the standard, the maca sample to be detected can be judged to be maca.
When the rbcL area sequence of the sample to be detected does not meet the standard, the sample to be detected can be judged to be not maca.
In the above-described method for aligning sequences using MEGA (version 7.0) software, one skilled in the art can further adjust the obtained alignment result to the most appropriate alignment result according to the characteristics of the sequenced peak pattern and the degeneracy of the codon.
Example 2
The difference from example 1 is that maca of example 1 was prepared as a powder, and DNA was extracted using the powder as a sample and identified, and as a result, the 8 base sites described above were also specifically detected.
SEQ ID NO：1
tatactcctgaatatgaaac caaggatact gatatcttgg cagcattccg agtaactcct
caacccggag ttccacctga agaagcaggg gctgcggtag ctgctgaatc ttctactggt
acatggacaa ctgtgtggac cgatgggctt accagccttg atcgttacaa aggacgatgc
taccacatcg agcccgttcc aggagaagaa agtcaattta ttgcgtatgt agcttaccca
ttagaccttt ttgaagaagg ttcggttact aacatgttta cctcgattgt gggtaatgta
tttgggttca aagccctggc tgctctacgt ctagaggatc tgcgaatccc tcctgcttat
actaaaactt tccagggacc gcctcatggt atccaagttg aaagagataa attgaacaag
tatggacgtc ccctattagg atgtactatt aaaccaaaat tgggattatc tgcgaagaac
tatggtagag cagttt
SEQ ID NO：2
atgtcaccacaaacagagactaaagc
SEQ ID NO：3
gtaaaatcaagtccaccycg

Claims (5)

1. A method for identifying maca is characterized in that a region sequence rbcL is adopted, and the nucleotide sequence of the region sequence rbcL is marked as SEQ ID No: 1, identifying a sample to be detected, comprising the following steps:
step 1: pretreating a sample to be detected, and extracting DNA of the sample to be detected to obtain the DNA of the sample to be detected;
and 2, step: the upstream primer and the downstream primer of the rbcL region sequence are respectively a primer F and a primer R, the primer F and the primer R of the rbcL region sequence are respectively used for carrying out PCR amplification reaction to obtain a rbcL region sequence of a sample to be detected, and the nucleotide sequence of the primer F is marked as SEQ ID No: 2, the nucleotide sequence of the primer R is represented as SEQ ID No: 3;
and 3, step 3: and (3) comparing and matching the rbcL region sequence of the sample to be detected obtained in the step (2), wherein if the sequence is SEQ ID No: 1, the 221 th base from the 5' end is G, the 240 th base is A, the 375 th base is G, the 381 th base is G, the 432 th base is C, the 465 th base is A, the 471 th base is T, the 492 th base is A, and the maca is identified, otherwise, the maca is not identified.
2. The method for identifying maca according to claim 1, wherein a sample to be detected is subjected to pretreatment before DNA extraction of the sample to be detected is performed to obtain the DNA of the sample to be detected, the pretreatment is to soak the sample to be detected in 75% ethanol solution by mass for 5 min, then place the sample to be detected in a sterile environment for air drying, and then grind the sample to be detected into fine powder under the condition of liquid nitrogen cooling, wherein the fine powder is the sample to be detected.
3. The method for identifying maca according to claim 1, wherein the total volume of the reaction system for PCR amplification is 20 μ L, and the reaction system contains 2.5 mmol/L MgCl 2 2. mu.L of 10 XPCR Buffer, 1.6. mu.L of four 2.5 mmol/L mononucleotides, 0.8. mu.L of 10. mu. mol/L rbcL primer F, 0.8. mu.L of 10. mu. mol/L rbcL primer R,0.1 muL of 5U/muL HiFi DNA polymerase, 50 ng of template DNA and the balance of sterile ultrapure water; the four 2.5 mmol/L mononucleotides are adenine, thymine, guanine and cytosine.
4. The method for identifying maca according to claim 1, wherein the PCR amplification reaction process sequentially comprises the following steps: pre-denaturation at 95 ℃ for 4 min, denaturation at 92 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 1 min, 35 cycles, and extension at 72 ℃ for 10 min.
5. The method for identifying maca according to claim 1, wherein sequences of rbcL areas of the sample to be detected in the step 3 are introduced into MEGA software and are aligned.
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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN102952878A (en) * 2012-09-28 2013-03-06 中国科学院过程工程研究所 ITS (Internal Transcribed Spacer) sequence and method for identifying certified lepidium meyenii walp and counterfeit lepidium meyenii walp as well as doped lepidium meyenii walp
CN105734153A (en) * 2016-04-20 2016-07-06 上海派森诺生物科技股份有限公司 Method of using genes for species identification of plants

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
CN102952878A (en) * 2012-09-28 2013-03-06 中国科学院过程工程研究所 ITS (Internal Transcribed Spacer) sequence and method for identifying certified lepidium meyenii walp and counterfeit lepidium meyenii walp as well as doped lepidium meyenii walp
CN105734153A (en) * 2016-04-20 2016-07-06 上海派森诺生物科技股份有限公司 Method of using genes for species identification of plants

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
A Two-Locus Global DNA Barcode for Land Plants: The Coding rbcL Gene Complements the Non-Coding trnH-psbA Spacer Region;W.JohnKress等;《PLoS ONE》;20070606(第6期);e508页 *
ACCESSION:LC385905,Lepidium mevenii T5/Yokohama/JPN/2017 chloroplast rbcl gene for ribulose-1.5-bisphosphate carboxylase/oxygenase large subumit,partical cds.;Ozawa,H.等;《Genebank database》;20200403;全文 *

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