CN116162722B - InDel marker for identifying bupleurum chinense in different producing areas and application thereof - Google Patents
InDel marker for identifying bupleurum chinense in different producing areas and application thereof Download PDFInfo
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Abstract
The invention discloses an InDel marker for identifying red bupleurum roots in different producing areas and application thereof, wherein the InDel marker is located after 181bp of a trnE-UUC gene in a chloroplast genome LSC region and has or lacks a AAAATTAATTAA sequence. The InDel marker developed by the invention can be used for distinguishing and identifying the bupleurum medicinal materials from the region of the Shanxi elm road and the northeast Daqing region. The marker has important value for accurately identifying the sources of bupleurum medicinal materials and identifying the plant species of bupleurum, and has important application value in future bupleurum variety breeding and genetic improvement.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to an InDel marker for identifying bupleurum chinense schneid in different producing areas and application thereof.
Background
Bupleuri radix is the key herb for dispelling heat, soothing liver and relieving depression, and lifting yang qi. The first one is recorded in Shen nong Ben Cao Jing in Dong Han as the top grade. The bupleurum chinense (r) root source plant specified in the pharmacopoeia of the people's republic of 2020 edition is bupleurum chinense dc (commonly known as "north bupleurum") or bupleurum angustifolium Bupleurum scorzonerifoliumWilld (commonly known as "south bupleurum"). Radix bupleuri is taken as a national key medicinal material, plays an important role in relieving exterior syndrome and allaying fever, soothing liver and relieving depression, and various Chinese patent medicines developed by radix bupleuri on the market have dozens of varieties, have long administration history, have wide market and have vigorous demands (Yao Ruyu and the like, 2013). According to 2019 statistics, the cultivation area of bupleurum chinense is over 50 ten thousand mu (Keshao Ying et al 2020).
Radix bupleuri (Bupleurum scorzonerifoliumWilld), the original name of Bupleurum scorzonerifolium (still called Bupleurum scorzonerifolium in the 2020 edition of Chinese pharmacopoeia), is one of two basic source plants of Bupleurum scorzonerifolium, a medicinal material specified in pharmacopoeia. According to the examination, the bupleurum chinense is the main stream variety of the traditional Chinese medicine bupleurum chinense, the ancient medical classics is the best mode of the bupleurum chinense (Zhan Zhilai, etc., 2020), and the bupleurum chinense has long history and good quality. After the open generation, because of the drastic change of the ecological environment and the long-term and massive excavation, the wild resources are rapidly exhausted and can not meet the medication requirement, the main stream status is replaced by other bupleurum, especially north bupleurum, but is still accepted by all the old traditional Chinese medicine and expert, and the traditional Chinese people's republic pharmacopoeia revised in the past after the country is built is reserved as the main basic source plant of bupleurum medicinal materials, so that the importance status in the traditional Chinese medicine is seen (Zhao Baolin, 2013).
According to the record of Chinese plant, the natural distribution area of the red bupleurum is mainly in the vast area of north of Yangtze river and east of Qinghai-Tibet plateau, the ecological environment in most areas of China is obviously improved in recent years, and the main habitat of the red bupleurum is changed into bushes and forests in south of China and the loess plateau, so that the distribution of the red bupleurum is changed greatly. From the investigation of the team, the main distribution area is in loess plateau and northeast grassland area of inner Mongolia at present, and the other areas are scattered and distributed at intervals, so that the areas tend to be extinct. The main distribution areas of the method are the northern and the northeast grasslands in the loess plateau, the distance between the two areas is about 1500 km, the ecological environment difference is large, the loess plateau is loose in soil and is dry and less in rain, the northeast grasslands are wet and rainy and are cold in weather, and some difference characters are differentiated from the characteristics and biological characteristics of the red bupleurum root distributed in the two areas and still belong to intra-seed variation.
The bupleurum plants have 36 varieties, 17 varieties and 7 varieties in China, and the shape and the character of the bupleurum plants have small difference, so that the bupleurum plants are extremely difficult to identify and identify. Radix bupleuri and its near-source seed line leaf radix bupleuri are used as radix bupleuri drug in northeast and Shaanxi, and radix bupleuri is often mixed as pseudo product into radix bupleuri, which causes abnormal medication disorder of radix bupleuri and brings great risk to medication safety. In order to ensure medication safety and improve medicinal effects, accurate identification of bupleurum varieties and different origin sources is very necessary.
In 2013, university of Shanxi Zhang Fusheng et al developed a specific molecular marker based on ITS sequence of bupleurum chinense and bupleurum angustifolium (namely current bupleurum chinense), which can rapidly detect and identify bupleurum angustifolium and bupleurum angustifolium, set up a PCR detection system, respectively amplify PCR fragments of 336bp and 407bp in bupleurum chinense and bupleurum angustifolium, provide molecular identification basis for identifying medicinal materials of bupleurum chinense and bupleurum angustifolium, stems, leaves or other parts of plants of bupleurum angustifolium and detection (CN 201310533200.9) of medicinal materials and Chinese patent medicines (honeyed pills and water honeyed pills), and the invention is mainly used for detecting and identifying north bupleurum chinense and bupleurum angustifolium.
The current bupleurum medicinal material yield is mainly concentrated in the regions of the northern Shaanxi and the northeast China, and each region of the northern Shaanxi is a historic region, and has excellent quality; the wild domesticated varieties with a certain cultivation area in northeast Daqing are difficult to distinguish accurately after entering the market, and bring great inconvenience to drug administration.
The research lays a certain foundation for developing bupleurum molecular marker identification, but the molecular markers for bupleurum in different producing areas still lack, and the medicinal and product development of bupleurum are restricted.
Disclosure of Invention
The invention is based on classification and identification of red bupleurum distributed in two areas of Shaanxi elm and Heilongjiang Daqing and sequencing study of chloroplast genome, and finds that 1 insertion-deletion mutation (Indel) molecular marker located in the LSC region of chloroplast genome can be used for identifying red bupleurum from elm and Daqing. Based on the above, the invention protects the following technical scheme:
an InDel marker for identifying red bupleurum of different origin, said InDel marker being located downstream of the trnE-UUC gene of the chloroplast genome LSC region, having the characteristic sequence shown in SEQ ID NO.1 for Shaanxi elm red bupleurum, i.e. AAAATTAATTAA at 155 bp; for red bupleurum root of Daqing of Heilongjiang, the sequence has the characteristic sequence shown in SEQ ID NO.2, and AAAATTAATTAA is deleted at 155bp relative to the sequence shown in SEQ ID NO. 1.
A primer for identifying bupleurum chinense in different production areas, wherein the target of an upstream primer is positioned in a 1-120 bp interval of a sequence shown in SEQ ID NO. 2; the target of the downstream primer is located in the region of 180-247 bp of the sequence shown in SEQ ID NO. 2.
Preferably, the upstream primer is BSIDM-YD1-F:5'-TGCCCATTGTATGAACCGCT-3' the number of the individual pieces of the plastic,
the upstream primer is BSIDM-YD1-R:5'-AATTTCCGTAGTGGGAGCCC-3';
the characteristic band obtained by amplifying the bupleurum root of Shaanxi elm by the primer is 259bp, and the nucleotide sequence is shown as SEQ ID NO. 1;
the characteristic band obtained by amplifying the bupleurum chinense of Daqing of Heilongjiang is 247bp, and the nucleotide sequence is shown as SEQ ID NO. 2.
A kit for identifying red bupleurum of different origin, comprising a primer as described in any one of the above, and a DNA extraction reagent, a PCR amplification reaction reagent, further preferably a PCR amplification product sequencing reagent or a gel electrophoresis reagent.
The invention also protects the use of the Indel tag described above or the primer described in any of the above in any of the following:
(1) Identifying and distinguishing bupleurum root plants and medicinal materials from two main producing areas of elm forest and Daqing;
(2) Identifying and distinguishing bupleurum root specimens from two main producing areas of elm forest and Daqing;
preferably, the application is carried out using fresh or dried chloroplast-containing tissue, preferably using leaf identification.
Preferably, the application comprises the following steps: extracting genome DNA of a sample to be detected as a template, carrying out PCR amplification by using the primer, analyzing a PCR amplification product, and judging the production place of the sample to be detected.
Preferably, in the technical scheme of the application, sequencing analysis is carried out on the PCR amplified product, the sequence to be analyzed is subjected to homologous comparison with the sequence shown in SEQ ID NO.1, and the bupleurum material with the sequence of AAAATTAATTAA at the position corresponding to 155bp of the sequence shown in SEQ ID NO.1 can judge that the production place is the Shanxi elm; on the contrary, the red bupleurum material with the AAAATTAATTAA sequence deleted at the corresponding position is produced in the place of Daqing or Ulmine of Shanxi province.
Preferably, in the technical scheme of the application, the upstream primer is BSIDM-YD1-F:5'-TGCCCATTGTATGAACCGCT-3' the upstream primer is BSIDM-YD1-R:5'-AATTTCCGTAGTGGGAGCCC-3';
analyzing PCR amplified products, wherein the production area of the red bupleurum with the amplified products of 259bp is the Shanxi elm, and the production area of the red bupleurum with the amplified products of 247bp is the Heilongjiang Daqing; preferably, the PCR amplification product is subjected to sequencing analysis or polyacrylamide gel electrophoresis detection analysis.
Preferably, in the technical scheme of the application, a reaction system for PCR amplification is as follows: the total reaction system is 20 mu L, the concentration is 3-7ng mu L -1 2. Mu.L of genomic DNA at a concentration of 30-70 ng. Mu.L -1 1. Mu.L each of the primers BSIDM-YD1-F/BSIDM-YD1-R, 10. Mu.L of 2X PhantaMax Master Mix, 6. Mu.L of double distilled water;
the PCR amplification procedure was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95℃for 15 sec, annealing at 55℃for 15 sec, extension at 72℃for 30 sec, 35 cycles; incubate at 72℃for 5 min.
The beneficial effects of the invention are as follows: a molecular marker locus for distinguishing and identifying bupleurum medicinal materials from the elm plot area of shanxi and the northeast Daqing area was developed. The sequence of the locus is AAAATTAATTAA, the locus is 12bp long, and can be detected by PCR (polymerase chain reaction) by using any fresh or dried green tissues and organs of the aerial parts of the bupleurum chinense, can be detected by using a conventional DNA molecular detection method such as polyacrylamide or agarose gel electrophoresis, can also be directly sequenced, and can identify the production area by judging whether the locus contains the sequence. The marker has important value for accurately identifying the sources of bupleurum medicinal materials and identifying the plant species of bupleurum, and has important application value in future bupleurum variety breeding and genetic improvement.
Drawings
FIG. 1 shows the result of comparison of the downstream characteristic sequences of the trnE-UUC gene in the LSC region of the chloroplast genome of radix bupleuri in two places of production of elm in Shaanxi and Daqing in Heilongjiang, with Indel sites at the markers.
FIG. 2 is a schematic representation of the location of Indel sites in the genome of the invention.
FIG. 3 is the result of performing the InDel marker detection of the present invention on Shanxi elm and Heilongjiang Daqing Red Bupleurum sample in example 2.
Detailed Description
The invention is further illustrated, but is not limited, by the following examples.
The reagents used in the examples described below, unless otherwise specified, are all conventional in the art and are commercially available. The experimental conditions not specifically described are all routine experimental conditions in the art, and reference may be made to the molecular cloning laboratory Manual (Sambrook J & Russell DW, molecular cloning: a laboratory manual, 2001), or conditions recommended by the manufacturer's instructions.
Test materials
The test material used in the invention is prepared from radix bupleuri specimens collected from two areas of Shanxi elm forest and Heilongjiang Daqing, and the radix bupleuri specimens are stored in a Chinese medicine resource center of a Chinese medical college. The laboratory has the advantages of saving the test materials and ensuring that the test materials are distributed to the public for verification experiments within twenty years from the application date.
Characteristics of red bupleurum from Shaanxi elm: perennial herbs are 40-80 cm high. Developed principal root, conical shape, rare branch root, dark reddish brown color, transverse ring at the upper end, longitudinal lines at the lower part, loose and crisp texture. The stem is single or 2-3, the basal part is tightly covered with residual fibers of the petiole, and the middle upper part of the stem is branched with multiple branches. The lower part of basal leaves is slightly contracted into petioles, the other basal leaves are not provided with petioles, the length of each basal leaf is 6-16 cm, the width of each basal leaf is 2-5 mm, the mass is thick, the basal leaves are slightly stiff, 3-5 veins are protruded towards the backs of the basal leaves, the edges of the basal leaves are white, bones are made, and the upper basal leaves are small and the basal leaves are identical. Extracting umbrella-shaped inflorescences from axillary leaves, and forming loose conical inflorescences with more inflorescences and diameters of 1.2-4 cm; 3-8 cm long, 1-2 cm long, thin and arc-shaped bent; the total bract is 1-3, extremely tiny, needle-shaped, 1-5 mm long, 0.5-1 mm wide, 1-3 pulses, sometimes cling to the spoke of the umbrella, and often fall early; the diameter of the small umbrella-shaped inflorescence is 4-6 mm, the small total bract is 5, the small umbrella is tightly attached, the linear needle-like shape is 2.5-4 mm long, the width is 0.5-1 mm, the small umbrella-shaped inflorescence is thin and sharp, and the small umbrella-shaped inflorescence is equal to or slightly exceeds the time of flowers; flowers are arranged in the small umbrella-shaped inflorescences, and the flower stalks are 1-1.5 mm long; the petals are yellow, the tongue pieces are equal in length to the halves of the petals, and the top ends 2 are shallow cracked; the flower column base is thick and pad-shaped, is wider than an ovary, is deep yellow, and is bent to two sides; the oil pipe has wide oval shape, 2.5 mm long, 2 mm wide, dark brown color, 5-6 in each edge groove of the oil pipe and 4-6 in the synbiotic surface. The flowering period is 7-8 months, the fruit period is 8-9 months, and the rice wine is drought-resistant, cold-resistant and waterlogging-intolerant.
Characteristics of red bupleurum from Daqing of Heilongjiang: perennial herbs are 30-70 cm high. Developed principal root, conical shape, rare branch root, dark reddish brown color, transverse ring at the upper end, longitudinal lines at the lower part, loose and crisp texture. The stem is single or 2-3, the basal part is tightly covered with residual fibers of the petiole, and the middle upper part of the stem is branched with multiple branches. The lower part of basal leaves is slightly contracted into petioles, the other basal leaves are not provided with petioles, the length of each basal leaf is 5-14 cm, the width of each basal leaf is 4-7 mm, the basal leaves are thick, the basal leaves are slightly stiff, 3-5 veins are protruded towards the backs of the basal leaves, the edges of the basal leaves are white, and the upper basal leaves are small and have the same shape. Extracting umbrella-shaped inflorescences from axillary leaves, and forming loose conical inflorescences with more inflorescences and diameters of 1.2-4 cm; 3-8 cm long and 1-2.5 cm long, and arc-shaped; the total bract is 1-3, extremely tiny, needle-shaped, 1-5 mm long, 0.5-1 mm wide, 1-3 pulses, sometimes cling to the spoke of the umbrella, and often fall early; the diameter of the small umbrella-shaped inflorescence is 4-6 mm, the small total bract is 5, the small umbrella is tightly attached, the linear needle-like shape is 2.5-4 mm long, the width is 0.5-1 mm, the small umbrella-shaped inflorescence is thin and sharp, and the small umbrella-shaped inflorescence is equal to or slightly exceeds the time of flowers; flowers are 6-15 in the small umbrella-shaped inflorescence, and the flower stalks are 1-1.5 mm long; the petals are yellow, the tongue pieces are equal in length to the halves of the petals, and the top ends 2 are shallow cracked; the flower column base is thick and pad-shaped, is wider than an ovary, is deep yellow, and is bent to two sides; the oil pipe has wide oval shape, 2.5 mm long, 2 mm wide, dark brown color, 5-6 in each edge groove of the oil pipe and 4-6 in the synbiotic surface. The flowering period is 7-8 months, the fruit period is 8-9 months, the plant is cold-resistant, waterlogging-resistant and drought-intolerant.
Main reagent
PCR experiments used 2X Phanta Max Master Mix from Nanjinouzan Biotechnology Co., ltd; sequencing was done by the company of Biotechnology, inc.
Example 1 Indel marker development to identify two different producing areas of Red Bupleurum
The BSIDM-YD1 marker primer is prepared by sequencing and splicing chloroplast genome of 15 samples of red bupleurum varieties distributed in two areas of Shanxi elm and Heilongjiang Daqing in the laboratory to obtain chloroplast genome information, comparing chloroplast genomes of the red bupleurum varieties from two areas, and as shown in a result of FIG. 1, finding 1 insertion/deletion mutation (Indel) molecular marker located in a LSC region of the chloroplast genome, and locating at a position of 181bp-200bp behind trnE-UWC genes (the sequence is shown in FIG. 2) in the LSC region of the chloroplast genome, wherein the sequence of the red bupleurum from the elm area is 'AAAATTAATTAATAATAAA', and the sequence of the red bupleurum from the Daqing area is deleted at 'AAAATTAATTAA' after 181 bp.
A primer 5.0 software is used for designing a molecular marker primer around the molecular marker at the downstream of the trnE-UUC gene of the LSC region of the chloroplast genome:
BSIDM-YD1-F：5’-TGCCCATTGTATGAACCGCT-3’(Seq ID No.4)，
BSIDM-YD1-R:5'-AATTTCCGTAGTGGGAGCCC-3' (Seq ID No. 5) and synthesized in Beijing Productivity.
As shown in a sequence table, a characteristic sequence amplified by the molecular marker primer at the downstream of a trnE-UUC gene in a LSC region of a red bupleurum chloroplast genome in a elm region is shown as a Seq ID No. 1; the "AAAATTAATTAA" signature sequence exists at 155bp of the sequence shown in Seq ID No. 1; the characteristic sequence of the molecular marker primer pair amplified downstream of the LSC region trnE-UUC gene of the red bupleurum chloroplast genome in Daqing area of Heilongjiang is shown as a Seq ID No.2, and the primer pair lacks 'AAAATTAATTAA' relative to the sequence shown as the Seq ID No.1 at 155bp of the sequence shown as the Seq ID No. 1.
Example 2 verification of the marking
The inventor takes 3 parts of bupleurum materials respectively collected from different villages and towns in Shaanxi elm god woody city and Heilongjiang Daqing Ming water county, and takes leaf sheath residues at the head of the reed; DNA was extracted (DNA of sample leaf sheath residues was extracted according to modified CTAB method (Clark, 1998)).
The molecular marker primer BSIDM-YD1-F/BSIDM-YD1-R developed according to the invention is subjected to PCR detection.
The PCR amplified products were submitted to sequencing by Beijing division, biotechnology Co., ltd, and the sequences were multiplex aligned using Geneius prime software.
The alignment results and Indel site sequences are shown in figure 3, where the annotation tag is the Indel site.
As can be seen from the comparison result, the sequence of the red bupleurum from elm god is AAAATTAATTAATAATAAA at the downstream of the trnE-UUC gene in the chloroplast genome LSC region, and the sequence of the red bupleurum from Daqingming water is deleted AAAATTAATTAA after 181bp behind the trnE-UUC gene in the chloroplast genome LSC region, which indicates that the Indel site can accurately distinguish the red bupleurum from elm god and Daqingming water.
Example 3 identification of the origin of the Red Bupleurum root medicinal material
The inventor takes 20 total medicinal material samples collected from different areas of the Shaanxi elm representative area Shenmu city and the Mingshui county representative area of Heilongjiang Daqing (different from the sampling points in the embodiment 2), wherein 10 parts are obtained from different places of the elm Shenmu city, 10 parts are obtained from different collecting places of the Heilongjiang Daqing Mingshui county, and each sampling point is spaced more than 5 kilometers apart; because the overground parts of the plant are withered, the leaves are disabled only from the root parts and the top parts of the plant, and the production place of the plant is difficult to accurately judge.
One for each herb, the leaf sheath residue of the head of the reed rhizome was taken and DNA was extracted (DNA of the leaf sheath residue of the sample was extracted according to the modified CTAB method (Clark, 1998)).
The molecular marker primer BSIDM-YD1-F/BSIDM-YD1-R developed according to the invention is subjected to PCR detection.
PCR amplification and detection were then performed.
The PCR reaction system is as follows: the total reaction system was 20. Mu.L, 2. Mu.L of genomic DNA (5.0 ng. Mu.L -1 ) Primer BSIDM-YD1-F/BSIDM-YD1-R (50 ng. Mu.L) -1 ) 1. Mu.L each, 10. Mu.L of 2X Phanta Max Master Mix, 6. Mu.L of double distilled water.
The PCR amplification procedure was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95℃for 15 sec, annealing at 55℃for 15 sec, extension at 72℃for 30 sec, 35 cycles; preserving the temperature at 72 ℃ for 5 minutes and preserving the temperature at 4 ℃.
Through sequencing identification analysis (polyacrylamide gel electrophoresis detection can also be adopted), in the PCR amplification product, 10 materials have 259bp fragments (shown from Shanxi elm) and 10 materials have 247bp fragments (shown from Heilongjiang Daqing); is consistent with the composition of the origin of the test material.
Claims (7)
1. Use of InDel markers for identifying red bupleurum at different producing sites in any of the following:
(1) Identifying and distinguishing bupleurum root plants and medicinal materials from two main producing areas of elm forest and Daqing;
(2) Identifying and distinguishing bupleurum root specimens from two main producing areas of elm forest and Daqing;
the InDel marker is positioned at the downstream of the trnE-UUC gene in the LSC region of the chloroplast genome, and has a characteristic sequence shown as SEQ ID NO.1 for the Shanxi elm bupleurum, namely AAAATTAATTAA at 155 bp; for red bupleurum root of Daqing of Heilongjiang, the sequence has the characteristic sequence shown in SEQ ID NO.2, and AAAATTAATTAA is deleted at 155bp relative to the sequence shown in SEQ ID NO. 1.
2. The use according to claim 1, characterized in that: is identified using fresh or dried chloroplast-containing tissue.
3. The use according to claim 2, characterized in that: and adopting blade identification.
4. The use according to claim 1, characterized by the steps of: extracting genome DNA of a sample to be detected as a template, carrying out PCR amplification by adopting a primer, analyzing a PCR amplification product, and judging the production place of the sample to be detected; the target of the primer upstream of the primer is positioned in a 1-120 bp interval of a sequence shown in SEQ ID NO. 2; the target of the downstream primer is located in the region of 180-247 bp of the sequence shown in SEQ ID NO. 2.
5. The use according to claim 4, characterized in that: the upstream primer is BSIDM-YD1-F:5'-TGCCCATTGTATGAACCGCT-3' the downstream primer is BSIDM-YD1-R:5'-AATTTCCGTAGTGGGAGCCC-3';
analyzing PCR amplified product, wherein the amplified product is 259bp red bupleurum root producing area is Shaanxi elm, and the amplified product is 247bp red bupleurum root producing area is Heilongjiang Daqing.
6. The use according to claim 5, characterized in that: and (3) sequencing analysis or polyacrylamide gel electrophoresis detection analysis is carried out on the PCR amplified products.
7. The use according to claim 5, characterized in that:
the PCR amplification reaction system is as follows: the total reaction system is 20 mu L, the concentration is 3-7ng mu L -1 2. Mu.L of genomic DNA at a concentration of 30-70 ng. Mu.L -1 1. Mu.L each of primer BSIDM-YD1-F/BSIDM-YD1-R, 10. Mu.L of 2 XPhantaMaxMastermix, 6. Mu.L of double distilled water;
the PCR amplification procedure was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95℃for 15 sec, annealing at 55℃for 15 sec, extension at 72℃for 30 sec, 35 cycles; incubate at 72℃for 5 min.
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