A molecular marker for differentiating Liriope spicata and Ophiopogon japonicus and its application

Abstract

The present invention discloses a molecular marker for identifying Liriope spicata and Ophiopogon japonicus and its application. The molecular marker is the SMD-1 gene fragment derived from plants of the genus Liriope, and the sequence is shown in SEQ ID No.1. The present invention discovers a molecular marker for identifying Liriope spicata and Ophiopogon japonicus. This specific molecular marker is the SMD-1 gene of Liriope spicata. This specific molecular marker has extremely high specificity, so it can be used for rapid identification of Liriope spicata and Ophiopogon japonicus medicinal materials. The primer pair designed for the specific molecular marker of the present invention for identifying Liriope spicata and Ophiopogon japonicus can specifically amplify a 267bp band from Liriope spicata at an appropriate annealing temperature, thereby differentiating Liriope spicata from Ophiopogon japonicus.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a molecular marker for identifying Liriope spicata var. prolifera and Ophiopogon japonicus and its application. Background Art

[0002] Ophiopogon japonicus is a commonly used traditional Chinese medicine for nourishing yin, promoting the production of body fluid, moistening the lungs and clearing the heart, and is used for treating symptoms such as dry cough due to lung dryness, consumptive cough due to yin deficiency, sore throat, thirst due to body fluid injury, internal heat and polydipsia, restlessness and insomnia, and constipation due to intestinal dryness. The "Pharmacopoeia of the People's Republic of China" (hereinafter referred to as the "Chinese Pharmacopoeia") stipulates that the source of the medicinal material "Ophiopogon japonicus" is the dried tuberous roots of the plant Ophiopogon japonicus (L.f) Ker-Gawl. of the genus Ophiopogon in the family Liliaceae. It is also stipulated that the source of "Liriope spicata var. prolifera" is the dried tuberous roots of the plant Liriope spicata (Thunb.) Lour. var. prolifera Y.T.Ma or Liriope muscari (Decne.) Baily of the genus Liriope in the family Liliaceae. Liriope spicata var. prolifera and Ophiopogon japonicus are plants of different genera, and there are significant differences in their original backgrounds. However, their underground dried rhizomes are all grayish-yellow spindle-shaped and shrunken tuberous roots, which are not easily distinguishable from the appearance, and there is a phenomenon of mixing the two in the market.

[0003] Compared with microscopic detection and determination of index components, the application of molecular markers can quickly and accurately identify the origin qualitatively.

[0004] The invention application with the publication number of CN108315467A discloses a specific molecular marker LPMI001 for identifying Liriope platyphylla. The nucleotide sequence of the upstream primer LPGMI001-F of the molecular marker LPMI001 is shown as SEQ ID No1, and the nucleotide sequence of the downstream primer LPMI001-R is shown as SEQ ID No2; the nucleotide sequence of the specific band amplified by the marker in Liriope platyphylla individuals is shown as SEQ ID No3. This specific molecular marker LPMI001 can be used for the identification and detection of Liriope platyphylla, and can also be used for the detection of Ophiopogon japonicus medicinal materials, thereby standardizing the Ophiopogon japonicus medicinal material market, detecting adulteration behaviors, and ensuring the safety of Ophiopogon japonicus medicinal materials. However, the object detected in this technical solution is Liriope platyphylla and cannot be used to distinguish Liriope spicata var. prolifera and Ophiopogon japonicus.

[0005] The invention application with the publication number CN102251026A discloses a pair of detection primers for the molecule of medicinal plant Liriope muscari (Decne.) Baily and its detection method, including a pair of PCR reaction detection primers for amplifying the chloroplast trnL-F sequence and a pair of PCR reaction detection primers for amplifying the ribosomal ITS sequence. The reaction primers are all composed of forward and reverse primers. The detection method of the present invention can quickly and accurately judge whether the sample is the molecule of medicinal plant Liriope muscari (Decne.) Baily, and has the characteristics of being able to quickly and effectively identify Liriope muscari (Decne.) Baily and its confused species and intraspecific variations. However, in this prior art, one is used to identify Liriope muscari (Decne.) Baily from other confused species, and Liriope spicata var. prolifera Y. T. Ma which also belongs to the genus Liriope is also excluded, so the identification between Ophiopogon japonicus (Thunb.) Ker-Gawl. and Liriope cannot be done; the other is that two molecular markers, namely the chloroplast trnL-F sequence and the ribosomal ITS sequence, are needed, and two pairs of primers are used simultaneously, increasing the difficulty of the identification operation. Summary of the Invention

[0006] The present invention aims to invent a molecular marker for identifying Liriope and Ophiopogon japonicus (Thunb.) Ker-Gawl. medicinal materials for the origin identification of two medicinal materials with similar appearances.

[0007] The present invention first provides a molecular marker for identifying Liriope and Ophiopogon japonicus (Thunb.) Ker-Gawl., which is the SMD-1 gene fragment from plants of the genus Liriope, and the sequence is shown in SEQ ID No.1.

[0008] The present invention also provides a primer pair for identifying Liriope and Ophiopogon japonicus (Thunb.) Ker-Gawl., including an upstream primer and a downstream primer, and the sequences are as follows:

[0009] Upstream primer SMD-1F: 5’-AAGAGGGAAAAATAAACCAGCC-3’;

[0010] Downstream primer SMD-1R: 5’-GGAACCCTAACTTCATGCGAG-3’.

[0011] The present invention also provides the application of the primer pair in preparing a kit for identifying Liriope and Ophiopogon japonicus (Thunb.) Ker-Gawl.

[0012] The present invention also provides a kit for identifying Liriope and Ophiopogon japonicus (Thunb.) Ker-Gawl., including the primer pair. The kit also includes a positive control, and the positive control is a plasmid containing the sequence shown in SEQ ID No.1.

[0013] The present invention also provides a method for identifying Liriope and Ophiopogon japonicus (Thunb.) Ker-Gawl., using the kit, and the method includes the following steps:

[0014] (1) Extract genomic DNA from the sample to be identified;

[0015] (2) Using the genomic DNA obtained in step (1) as a template and the said primer pair as PCR amplification primers, perform PCR amplification;

[0016] (3) Detect the PCR amplification product obtained in step (2). If a 267bp band can be amplified, the sample to be identified is Liriope spicata var. prolifera, otherwise it is Ophiopogon japonicus.

[0017] The sample to be identified is Liriope spicata var. prolifera or Ophiopogon japonicus. Liriope spicata var. prolifera is the medicinal material or plant tissue with the origin of Liriope spicata var. prolifera or Liriope muscari; Ophiopogon japonicus is the medicinal material or plant tissue with the origin of Ophiopogon japonicus.

[0018] Preferably, the annealing temperature used for PCR amplification is 63°C.

[0019] More preferably, the reaction program for PCR amplification is: pre-denaturation at 94°C for 3 min; then each cycle: denaturation at 94°C for 30 s, annealing at 63°C for 30 s, extension at 72°C for 30 s, for 30 cycles; finally extension at 72°C for 7 min.

[0020] Preferably, the reaction system for PCR amplification is: 1 μL of genomic DNA template, 1 μL each of upstream and downstream primers, 1 μL of MgCl2 solution, 10 μL of Mix-taq, and 7 μL of sterilized ultrapure water, and the total volume of the reaction system is 20 μL.

[0021] The present invention has discovered a molecular marker for differentiating Liriope spicata var. prolifera and Ophiopogon japonicus. This specific molecular marker is the SMD-1 gene of Liriope spicata var. prolifera. This specific molecular marker has extremely high specificity, so it can be used for rapid identification of Liriope spicata var. prolifera and Ophiopogon japonicus medicinal materials. The primer pair designed for the specific molecular marker of the present invention for differentiating Liriope spicata var. prolifera and Ophiopogon japonicus can specifically amplify a 267bp band from Liriope spicata var. prolifera at a suitable annealing temperature, thereby differentiating Liriope spicata var. prolifera from Ophiopogon japonicus. Description of the Drawings

[0022] Figure 1Figure showing the detection results of PCR amplification band characteristics of different candidate sequences for Ophiopogon japonicus and Liriope spicata resources. Among them, A is the amplification band of SMD-2 with an annealing temperature of 58°C; B is the amplification band of SMD-2 with an annealing temperature of 63°C; C is the amplification band of SMD-3; D is the amplification band of SMD-1. Among them, lane 0 is the standard molecular weight indicator band, lane 1 is the SMD-1 amplification band of Liriope spicata DNA, lane 2 is the SMD-1 amplification band of Liriope muscari DNA, lane 3 is the SMD-1 amplification band of the mixed sample of Liriope spicata DNA and Ophiopogon japonicus DNA at a concentration ratio of 1:20, lane 4 is the SMD-1 amplification band of the mixed sample of Liriope muscari DNA and Ophiopogon japonicus DNA at a concentration ratio of 1:20, lane 5 is the SMD-1 amplification band of the mixed sample of Liriope spicata DNA and Ophiopogon japonicus DNA at a concentration ratio of 1:40, lane 6 is the SMD-1 amplification band of the mixed sample of Liriope muscari DNA and Ophiopogon japonicus DNA at a concentration ratio of 1:40, the annealing temperature is 63°C, and lane 7 is the SMD-1 amplification band of Ophiopogon japonicus DNA.

[0023] Figure 2 Figure showing the amplification detection results of the genomic DNA of Ophiopogon japonicus or Liriope spicata herbs with the molecular marker specific primer SMD-1F / SMD-1F. Among them, the annealing temperature in A is 63°C, and the annealing temperature in B is 55°C. In A and B, lane 0 is the standard molecular weight indicator band, lane 1 is the Liriope spicata herb with the origin of Ophiopogon japonicus sampled from Yidu City, Hubei Province, lane 2 is the Liriope spicata herb with the origin of Liriope muscari sampled from Xianyou County, Fujian Province, lane 3 is the Ophiopogon japonicus sampled from Santai County, Sichuan Province, lane 4 is the Ophiopogon japonicus sampled from Luzhou City, Sichuan Province, lane 5 is the Ophiopogon japonicus sampled from Cixi City, Zhejiang Province, lane 6 is the Ophiopogon japonicus sampled from Sanmen County, Zhejiang Province, and lane 7 is the Ophiopogon japonicus sampled from Hangzhou City, Zhejiang Province. Detailed implementation methods

[0024] Example 1

[0025] Through transcriptome sequencing technology, based on the significantly differentially expressed gene sequences of multiple groups of Ophiopogon japonicus, Liriope spicata and Liriope muscari tuberous roots, 51 sequences were initially screened as data. Among the species-differentiated sequences obtained by comparison, 3 sequences that may be developed to distinguish Ophiopogon japonicus and Liriope spicata after amplification with different annealing temperatures were screened. Three pairs of primers were designed specifically as shown in Table 1 below. The primers were synthesized by Hangzhou Qingke Biotechnology Co., Ltd.

[0026] Table 1 Candidate primers for sequence amplification with different annealing temperatures

[0027]

[0028] The reaction system for PCR amplification is as follows: 1 μL of genomic DNA template (200 ng / μL), 1 μL each of upstream and downstream primers (10 μM), 1 μL of MgCl2 solution (25 mM), 10 μL of Mix-taq, and 7 μL of sterilized ultrapure water. The total volume of the reaction system is 20 μL.

[0029] The amplification program for SMD-1 is pre-denaturation at 94 °C for 3 min; then each cycle: denaturation at 94 °C for 30 s, annealing at 63 °C for 30 s, extension at 72 °C for 30 s, for 30 cycles; finally, extension at 72 °C for 7 min. The PCR products are subjected to 2% agarose gel electrophoresis.

[0030] The amplification program for SMD-2 is pre-denaturation at 94 °C for 3 min; then each cycle: denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, for 30 cycles; finally, extension at 72 °C for 7 min. The PCR products are subjected to 2% agarose gel electrophoresis.

[0031] The amplification program for SMD-3 is pre-denaturation at 94 °C for 3 min; then each cycle: denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, for 30 cycles; finally, extension at 72 °C for 7 min. The PCR products are subjected to 2% agarose gel electrophoresis.

[0032] The amplification results are as Figure 1 shown: A is the amplification band of SMD-2 with annealing at 58 °C; B is the amplification band of SMD-2 with annealing at 63 °C; C is the amplification band of SMD-3; D is the amplification band of SMD-1. Among them, lane 0 is the standard molecular weight indicator band, lane 1 is the SMD-1 amplification band of Liriope spicata DNA, lane 2 is the SMD-1 amplification band of Liriope muscari DNA, lane 3 is the SMD-1 amplification band of the mixed sample of Liriope spicata DNA and Ophiopogon japonicus DNA at a concentration ratio of 1:20, lane 4 is the SMD-1 amplification band of the mixed sample of Liriope muscari DNA and Ophiopogon japonicus DNA at a concentration ratio of 1:20, lane 5 is the SMD-1 amplification band of the mixed sample of Liriope spicata DNA and Ophiopogon japonicus DNA at a concentration ratio of 1:40, lane 6 is the SMD-1 amplification band of the mixed sample of Liriope muscari DNA and Ophiopogon japonicus DNA at a concentration ratio of 1:40, with an annealing temperature of 63 °C, lane 7 is the SMD-1 amplification band of Ophiopogon japonicus DNA, with an annealing temperature of 63 °C. From Figure 1 the results, it can be seen that SMD-1 can identify samples of Ophiopogon japonicus mixed with Liriope spicata; SMD-2 cannot distinguish between Ophiopogon japonicus and Liriope spicata; SMD-3 also cannot distinguish between Ophiopogon japonicus and Liriope spicata, and when the annealing temperature is 60 °C, no bands can be amplified.

[0033] Example 2

[0034] 1. Extraction of medicinal material DNA

[0035] Extract the DNA of medicinal materials using the CTAB method. Cut the Ophiopogon japonicus or Liriope spicata medicinal materials into particles with a particle size of less than 0.2 cm, take 100 mg - 200 mg and grind them into powder in liquid nitrogen, add 800 μL of preheated 2% CTAB extraction solution, and pulverize them in a tissue grinder at 30 Hz - 50 s. After pulverization, water bath at 60 °C for 30 min, shake well every 10 min. After the water bath, add 800 μL of a mixed reagent of chloroform and isoamyl alcohol (24:1) with equal volume to each EP tube, shake vigorously, centrifuge at 12000 rpm / min for 8 min, aspirate 600 μL of the supernatant, place it in a new 2 mL EP tube, add a mixed reagent of chloroform and isoamyl alcohol (24:1), add 600 μL with equal volume to each EP tube, shake vigorously, centrifuge at 12000 rpm / min for 8 min, aspirate 400 μL of the supernatant, place it in a pointed 1.5 mL EP tube, add 400 μL of isopropanol stored at -40 °C with equal volume, slowly shake up and down 10 times, and refrigerate for more than 3 h. Take the refrigerated extract and centrifuge at 10000 rpm / min for 5 min, pour out the liquid to obtain a solid residue (crude DNA extract), add 800 μL of 70% ethanol, shake until the solid residue (crude DNA extract) floats up, soak for 5 min, centrifuge at 10000 rpm / min for 5 min, pour out the liquid, repeat this operation 2 times, then add 800 μL of absolute ethanol and soak for 5 min, centrifuge at 10000 rpm / min for 5 min, pour out the liquid, air dry, add 200 μL of TE to dissolve, and adjust the concentration value to 50 - 100 ng / μL for standby.

[0036] 2. Synthesize specific molecular marker primers

[0037] Design molecular marker primers based on specific sequences. The upstream primer SMD-1F: 5’-AAGAGGGAAAAATAAACCAGCC-3’ and the downstream primer SMD-1R: 5’-GGAACCCTAACTTCATGCGAG-3’ are synthesized by Hangzhou Qingke Biotechnology Co., Ltd.

[0038] 3. PCR amplification of specific molecular marker primers (SMD-1F / R)

[0039] The reaction system for PCR amplification is: 1 μL of genomic DNA template (200 ng / μL), 1 μL of each of the upstream and downstream primers (10 μM), 1 μL of MgCl2 solution (25 mM), 10 μL of Mix-taq, and 7 μL of sterilized ultrapure water. The total volume of the reaction system is 20 μL.

[0040] The PCR reaction program was set as follows: pre-denaturation at 94°C for 3 min; then for each cycle: denaturation at 94°C for 30 s, annealing at 63°C for 30 s, extension at 72°C for 30 s, for 30 cycles; finally, extension at 72°C for 7 min. The PCR products were subjected to 2% agarose gel electrophoresis.

[0041] Meanwhile, another set of experiments was carried out. The reaction system remained unchanged, and the remaining steps in the PCR reaction program were the same, except that the annealing temperature of 63°C was changed to 55°C.

[0042] According to the above method, the sampled Ophiopogon japonicus and Liriope spicata were detected respectively. Figure 2 It is a detection result diagram of the amplification of genomic DNA of Ophiopogon japonicus or Liriope spicata herbs using the molecular marker-specific primers SMD-1F / SMD-1F. Among them, the annealing temperature in A is 63°C, and the annealing temperature in B is 55°C. In lanes 0 of A and B are the standard molecular weight indicator bands. Lane 1 is the Liriope spicata herb with the origin of Ophiopogon bodinieri sampled from Yidu City, Hubei Province. Lane 2 is the Liriope spicata herb with the origin of Liriope muscari var. prolifera sampled from Xianyou County, Fujian Province. Lane 3 is the Ophiopogon japonicus sampled from Santai County, Sichuan Province. Lane 4 is the Ophiopogon japonicus sampled from Luzhou City, Sichuan Province. Lane 5 is the Ophiopogon japonicus sampled from Cixi City, Zhejiang Province. Lane 6 is the Ophiopogon japonicus sampled from Sanmen County, Zhejiang Province. Lane 7 is the Ophiopogon japonicus sampled from Hangzhou City, Zhejiang Province. When the annealing temperature of the PCR program is 63°C, only the Liriope spicata with the origin of Ophiopogon bodinieri or Liriope muscari var. prolifera has 1 obvious band at 267 bp; when the annealing temperature of the PCR program is 55°C, only the Liriope spicata with the origin of Ophiopogon bodinieri or Liriope muscari var. prolifera and the Ophiopogon japonicus with the origin of Ophiopogon japonicus have 1 obvious band at 267 bp.

[0043] This indicates that the specific molecular marker SMD-1 of the present invention has extremely high specificity, and thus can be used for rapid identification of Liriope spicata and Ophiopogon japonicus herbs.

Claims

1. A molecular marker for differentiating Liriope spicata (Thunb.) Lour. var. prolifera Y. T. Ma from Ophiopogon japonicus (Linn. f.) Ker-Gawl., characterized in that, The SMD-1 gene fragment from plants of the genus Liriope, and the sequence is as shown in SEQ ID No.

1.

2. A primer pair for differentiating Liriope spicata (Thunb.) Lour. var. prolifera Y. T. Ma from Ophiopogon japonicus (Thunb.) Ker-Gawl., characterized in that, It includes a forward primer and a reverse primer, and the sequences are as follows: Forward primer SMD-1F: 5’-AAGAGGGAAAAATAAACCAGCC-3’; Reverse primer SMD-1R: 5’-GGAACCCTAACTTCATGCGAG-3’.

3. The application of the primer pair according to claim 2 in the preparation of a kit for differentiating Liriope spicata and Ophiopogon japonicus.

4. A kit for differentiating Liriope spicata (Thunb.) Lour. var. prolifera Y. T. Ma from Liriope spicata (Thunb.) Lour., characterized in that, It includes the primer pair according to claim 2.

5. The kit according to claim 4, characterized in that, It further includes a positive control, and the positive control is a plasmid containing the sequence shown in SEQID No.

1.

6. A method for differentiating Liriope spicata (Thunb.) Lour. var. prolifera Y. T. Ma from Ophiopogon japonicus (Linn. f.) Ker-Gawl., characterized in that, Using the kit according to claim 4 or 5, the method includes the following steps: (1) Extract genomic DNA from the sample to be differentiated; (2) Using the genomic DNA obtained in step (1) as a template and the primer pair as PCR amplification primers, perform PCR amplification; (3) Detect the PCR amplification product obtained in step (2). If a 267bp band is amplified, the sample to be differentiated is Liriope spicata, otherwise it is Ophiopogon japonicus.

7. The method according to claim 6, wherein The annealing temperature used for PCR amplification is 63°C.

8. The method according to claim 7, wherein The reaction program for PCR amplification is: pre-denaturation at 94°C for 3 min; then in each cycle: denaturation at 94°C for 30 s, annealing at 63°C for 30 s, extension at 72°C for 30 s, for 30 cycles; finally extension at 72°C for 7 min.

9. The method according to claim 6, characterized in that, The reaction system for PCR amplification is: 1 μL of genomic DNA template, 1 μL of each of the forward and reverse primers, 1 μL of MgCl2 solution, 10 μL of Mix-taq, and 7 μL of sterilized ultrapure water, and the total volume of the reaction system is 20 μL.

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