CN107267647B - PCR reaction system for kudzu root SCoT molecular marker - Google Patents
PCR reaction system for kudzu root SCoT molecular marker Download PDFInfo
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Abstract
The invention discloses a PCR reaction system of kudzu root SCoT molecular marker, the total amount of the PCR reaction system is 20 mu L, and the PCR reaction system contains DNA50ng and Mg2+1.5mmol/L, dNTP0.25mmol/L, primer 0.8 mu mol/L, Taq DNA polymerase 0.5U, and ddH for the rest2And O. The molecular marking method applied to the kudzu root reported at present only comprises three types of ISSR, SRAP and RAPD, and the invention establishes the kudzu root SCoT-PCR reaction system for the first time, makes up the blank of research related to the kudzu root SCoT molecular marking and enriches means and methods of the research of the kudzu root molecular biology. The SCoT-PCR reaction band obtained by the method is clear, high in stability, simple to operate, low in cost and good in polymorphism, and can be used for realizing research on related fields such as radix puerariae germplasm resource identification, genetic diversity analysis and molecular marker assisted breeding, and the like, and has great scientific value and application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a PCR reaction system for a kudzu root SCoT molecular marker.
Background
Root of kudzu vine (Pueraria lobata (Willd.) OhwiPueraria lobata(wildOhwi) is a perennial plant of Pueraria DC of Leguminosae, is a first approved medicinal and edible dual-purpose plant of China Ministry of health, and has reputations of ' North ginseng, south Kudzuvine and ' Asian ginseng '. Radix Puerariae contains puerarin, flavone, polysaccharide, etc., and has effects of relieving muscles and skin, promoting eruption, relieving diarrhea, relieving restlessness, reducing blood lipid, lowering blood sugar, relieving hangover, caring skin and enlarging breast (Li Guohui et al 2010; xu sweet 2014). The kudzu root is rich in starch, can be used as a non-grain biomass new energy, avoids the bottleneck of 'competing for grains with people and competing for land with grains' in industrial development, and has great development potential. In addition, the kudzu root grows rapidly, has good adaptability and strong stress resistance, has deep root system and strong water and soil retention capacity, and can be used as a green plant and a soil improvement plant in barren areas due to the special nitrogen fixation capacity of the kudzu root. 2014 international radix puerariae health industry development forum disclosure, the area of kudzu and cultivated kudzu in China is nearly 100 hectares, and the method has good market prospect.
The pueraria lobata has a long planting history in China, but the genetic breeding work of the pueraria lobata is laggard for a long time, the pueraria lobata is basically used for medicine by harvesting wild resources, the research on the pueraria lobata is mainly focused on the aspects of pharmacological research, nutrient component analysis and the like, and germplasm resource identification, genetic diversity analysis, variety breeding and the like are not paid enough attention. In recent years, with the attention of people on medicine-food dual-purpose food, the pueraria industry develops, the development of pueraria products such as pueraria tablets, pueraria powder, pueraria scented tea, pueraria health-care beverage, pueraria anti-alcoholism beverage and the like, the collection and identification of pueraria germplasm resources and the research on genetic diversity are gradually carried out, but most of the research adopts morphological indexes such as field properties and the like for identification, and is easily influenced by factors such as environment, subjective judgment and the like. The method of molecular marking can effectively avoid the disadvantages. At present, the research on molecular markers on the kudzu roots is less, and the research is carried out only by three molecular marker methods of RAPD (Zhoujingbai and the like, 2013), ISSR (Guoyan and the like, 2013; Chenjunyi and the like, 2015; Yuanlin and the like, 2017) and SRAP (Chencaxia and the like, 2011), and the application of other molecular marker methods on the kudzu roots is not reported.
The target initiation codon polymorphism marker (SCoT) is a novel target gene molecular marker method based on SPAR (single primer amplification reaction) on rice, which is provided by Collard et al (2009), and the principle is to design a single primer and amplify a genome according to the conservation of a flanking sequence of an ATG translation initiation site in a plant gene. The SCoT marker combines the advantages of an ISSR marker and an RAPD marker, has simple operation, low cost and rich polymorphism, can effectively generate a marker linked with a character, is a novel molecular marker capable of tracking the character, and is beneficial to molecular marker-assisted breeding. The technology has been successfully applied to genetic diversity analysis of crops such as longan (old tiger and the like, 2010), orange (korean bright and the like, 2011), peanut (bear before hair and the like, 2010), chrysanthemum (plum and the like, 2013), orchid (kaolin and the like, 2013), peony (margarine and the like, 2011) and pineapple (old fragrant and the like, 2012). Although the kudzu root is researched by some people at home and abroad by molecular markers, the relevant research report of applying SCoT molecular markers to the kudzu root is not found yet. Therefore, a scientific and reasonable SCoT-PCR reaction system of the radix puerariae is established, an optimal theoretical combination is sought, the test process can be accelerated, scientific basis and guidance action are provided for subsequent work, the molecular marker assisted breeding efficiency can be improved, the breeding period is shortened, and the method has very important significance for further researching the genetic diversity and the geographic variation of the radix puerariae, the development of related markers, the genetic differentiation of provenance and the like.
Disclosure of Invention
The invention aims to: aiming at the defects, the invention provides a PCR reaction system of the kudzu root SCoT molecular marker. The invention establishes the radix puerariae SCoT-PCR reaction system for the first time, makes up the blank of related research of the radix puerariae SCoT molecular marker, and enriches means and methods for molecular biology research of the radix puerariae. The SCoT-PCR reaction band obtained by the method is clear, high in stability, simple to operate, low in cost and good in polymorphism, and can be used for realizing research on related fields such as radix puerariae germplasm resource identification, genetic diversity analysis and molecular marker assisted breeding, and the like, and has great scientific value and application value.
In order to achieve the purpose, the invention adopts the following technical scheme:
the radix puerariae SCoT molecular marker PCR reaction system is 20 muL in total, and DNA50ng and Mg are contained in the PCR reaction system2+1.5mmol/L, dNTP0.25mmol/L, primer 0.8 mu mol/L, Taq DNA polymerase 0.5U, and ddH for the rest2O。
Further, the PCR amplification procedure of the reaction system is as follows: pre-denaturation at 94 ℃ for 4 min; 35 cycles of 94 ℃ for 30s, 50 ℃ for 1min and 72 ℃ for 90 s; extending for 5min at 72 ℃; the amplification product was stored at 4 ℃.
Further, the primer is any one of SC1-SC 36.
The applicant finds that only 3 molecular markers including RAPD, ISSR and SRAP are reported to be applied to the genetic diversity analysis of the radix puerariae in the creative experimental process, wherein the RAPD marker has the defect of poor stability and repeatability; the SRAP marker needs to detect a PCR reaction product through polyacrylamide gel, and has the disadvantages of complicated process, large workload and high test cost; in contrast, the SCoT marker combines the advantages of the ISSR marker and the RAPD marker, can be detected by agarose gel, and has the advantages of simple operation, small workload, low cost, rich polymorphism, and good stability and repeatability. The invention establishes the radix puerariae SCoT-PCR reaction system for the first time, makes up the blank of related research of the radix puerariae SCoT molecular marker, enriches the means and the method of the radix puerariae molecular biological research, and lays a certain foundation for the application of the radix puerariae SCoT molecular marker in germplasm resource identification, genetic diversity analysis, linkage map construction and the like.
In conclusion, the invention has the following positive effects due to the adoption of the scheme:
(1) the molecular markers applied to the kudzu root reported at present are only 3 of RAPD, ISSR and SRAP, and the method establishes the kudzu root SCoT-PCR reaction system for the first time, makes up the blank of relevant researches on the kudzu root SCoT molecular markers and enriches means and methods for researching the kudzu root molecular biology.
(2) The stripe amplified by the kudzu root SCoT-PCR reaction system established by the invention has the characteristics of high stability, clear stripe, good polymorphism, simplicity in operation, high sensitivity, good repeatability and the like, can realize researches on kudzu root germplasm resource identification, genetic diversity analysis, linkage map construction, molecular marker assisted breeding and the like, and has great scientific value and application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate exemplary embodiments of the application and, together with the description, serve to explain the application and not to limit the application, and in which:
FIG. 1 shows the result of agarose electrophoresis of 16 reaction systems in the orthogonal design of the radix Puerariae SCoT-PCR reaction system in example 1 of the present invention;
FIG. 2 shows the screening results of the primers for the radix Puerariae SCoT molecular marker in example 2 of the present invention;
FIG. 3 shows the polymorphism analysis results of different kudzu vine root materials in example 3 of the present invention.
In the drawings: in FIG. 1, the serial numbers 1-16 of the electrophoresis bands correspond to 1-16 systems respectively; in FIG. 2, electrophoresis band numbers 1-36 correspond to SC1-SC36, respectively; the pueraria varieties represented by the electrophoresis band serial numbers 1-6 in fig. 3 are: 1 Guangxi Luzhai Pueraria lobata, 2 Gui radix Puerariae 1, 3 Teng county radix Puerariae, 4 Teng county local species, 5 Ping le Sand Zhen Ge 1, 6 Ping le sand Zhen Ge 2.
Detailed Description
The following description will further explain a PCR reaction system of the kudzu root SCoT molecular marker in the invention by combining the drawings of the specification and the examples.
Test materials and reagents: the kudzu root material is collected from a breeding base of the institute of biotechnology, Guangxi Zhuang national academy of autonomous region agricultural sciences, wherein 1 Guangxi Luzhai Pueraria, 2 Guifen Ge 1, 3 Teng county Pueraria, 4 Teng county local species, 5 Ping le Sand Zhen Pueraria 1 and 6 Ping le sand Zhen Pueraria 2. The 36 SCoT primer sequences used were referenced to Collard and Mackill (2009), numbered SC1-SC36, respectively, and synthesized by Biotechnology engineering (Shanghai) Inc. Taq DNA polymerase, dNTPs mixture, 10 XPCR Buffer (Mg)2+ plus), agarose, etc. were purchased from TaKaRa.
Example 1: method for establishing kudzu root SCoT-PCR reaction system
(1) Extraction and detection of genomic DNA
Collecting young and tender leaves of healthy plants of disease-free and pest-free kudzuvine roots, extracting DNA of the 1 st Guangxi Kazai Pueraria lobata by using a TaKaRa DNA extraction kit, detecting the DNA concentration by using an ultraviolet spectrophotometer, and diluting the extracted DNA into 50 ng/mu L, and storing at-40 ℃ for later use.
(2) Optimization of kudzu root SCoT-PCR reaction system
The SCoT-PCR reaction system is determined to be 20 mu L, a primer SC27 (5'-ACCATGGCTACCACCGTG-3') with better pre-amplification is used as a primary selection primer established by the radix puerariae SCoT-PCR amplification system, and the radix puerariae SCoT-PCR reaction system is optimized by taking No. 1 Guangxi Luzhai Pueraria lobata as a material. Wherein, DNA and Mg2+The 5 factors dNTP, primer, Taq DNA polymerase were set to 4 different levels (see Table 1 for details) respectively, and orthogonal experiments were performed for 16 combinations, each of which was set to 3 replicates and the volume was insufficient with ddH2And supplementing the O to 20 mu L.
TABLE 1 SCoT-PCR reaction System factors and levels
(3) PCR amplification and detection of amplification products
PCR amplification procedure: pre-denaturation at 94 ℃ for 4 min; 35 cycles of 94 ℃ for 30s, 50 ℃ for 1min and 72 ℃ for 90 s; extension at 72 ℃ for 5 min. The PCR product was electrophoresed on 1.0% agarose gel, set at 110V and run time 30 min. And after being dyed by a GelRed nucleic acid dyeing agent, the gel is imaged by an ultraviolet gel imaging system and then photographed.
(4) Orthogonal test result of kudzu root SCoT-PCR reaction system
The orthogonal test setup of the radix puerariae SCoT-PCR reaction system is detailed in Table 2, and the orthogonal test amplification result of the radix puerariae SCoT-PCR reaction system is detailed in FIG. 1.
TABLE 2 design of orthogonal experiments for PCR reaction systems [ L16（45）］（20µL）
As can be seen from FIG. 1 and Table 2, the amplification effects of the 16 combinations were different. Using visual analysis method to score the bands of 3 repeated electrophoretograms of 16 systems, averaging to obtain KiAnd R value. The larger the R is, the larger the influence of the factor on the SCoT-PCR reaction system is; kiThe larger the level, the better the level, and therefore, the order of the influence of 5 factors on the SCoT-PCR reaction system is: taq DNA polymerase & gtDNA & gtprimer & gtdNTP & gtMg2+In combination with KiDetermining an optimal SCoT-PCR reaction system (20 muL): DNA50ng, Mg2+1.5mmol/L, dNTP0.25mmol/L, 0.8 mu mol/L primer, 0.5U Taq DNA polymerase.
Example 2: primer screening of kudzu root SCoT molecular marker
(1) Extraction and detection of genomic DNA
Collecting young and tender leaves of healthy plants of disease-free and pest-free kudzuvine roots, extracting DNA of the 1 st Guangxi Kazai Pueraria lobata by using a TaKaRa DNA extraction kit, detecting the DNA concentration by using an ultraviolet spectrophotometer, and diluting the extracted DNA into 50 ng/mu L, and storing at-40 ℃ for later use.
(2) Optimization and PCR amplification of kudzu root SCoT-PCR reaction system
Taking No. 1 Guangxi Luzhai kudzu DNA as a material, respectively carrying out PCR reaction by using 36 SCoT primers, wherein the total weight of a PCR reaction system is 20 muL, wherein the DNA is 50ng, and the Mg is 502+1.5mmol/L, dNTP0.25mmol/L, primer 0.8 mu mol/L, Taq DNA polymerase 0.5U, and ddH for the rest2And O. The PCR amplification procedure was: pre-denaturation at 94 ℃ for 4 min; 35 cycles of 94 ℃ for 30s, 50 ℃ for 1min and 72 ℃ for 90 s; extending for 5min at 72 ℃; the amplification product was stored at 4 ℃.
(3) Detection of PCR amplification product
The PCR product was electrophoresed on 1.0% agarose gel, set at 110V and run time 30 min. And after being dyed by a GelRed nucleic acid dyeing agent, the gel is imaged by an ultraviolet gel imaging system and then photographed. The result (figure 2) shows that PCR reaction bands amplified by the method are clear, the number is rich, the background is clean, and 36 SCoT primers can amplify clear and identifiable bands in the radix puerariae, so that the 36 primers can be combined with the radix puerariae SCoT-PCR system of the invention to be applied to researches on radix puerariae germplasm resource identification, genetic diversity analysis, linkage map construction, molecular marker assisted breeding and the like, and meanwhile, the radix puerariae SCoT-PCR reaction system established by the method is proved to be efficient and feasible.
Example 3: polymorphism analysis of different kudzu vine root materials
(1) Extraction and detection of genomic DNA
Collecting healthy plant young leaves of disease-free and pest-free kudzuvine root and using a TaKaRa DNA extraction kit to extract the DNA of six kudzuvine root materials including 1 Guangxi Kazhai Yege, 2 Gui radix puerariae 1, 3 rattan county radix puerariae, 4 rattan county local species, 5 Ping le sand Zhe Ge 1 and 6 Ping le sand Zhe Ge 2, detecting the DNA concentration by using an ultraviolet spectrophotometer, diluting the extracted DNA into 50 ng/mu L, and storing at-40 ℃ for later use.
(2) Optimization and PCR amplification of kudzu root SCoT-PCR reaction system
Randomly selecting 8 SCoT primers of SC2, SC3, SC11, SC27, SC28, SC29, SC30 and SC32, carrying out PCR reaction by taking 6 kudzu root materials as templates, wherein the total system of the PCR reaction system is 20 mu L, DNA50ng and Mg2+1.5mmol/L, dNTP0.25mmol/L, primer 0.8 mu mol/L, Taq DNA polymerase 0.5U, and ddH for the rest2And O. The PCR amplification procedure was: pre-denaturation at 94 ℃ for 4 min; 35 cycles of 94 ℃ for 30s, 50 ℃ for 1min and 72 ℃ for 90 s; extending for 5min at 72 ℃; the amplification product was stored at 4 ℃.
(3) Detection of PCR amplification product
The PCR product was electrophoresed on 1.0% agarose gel, set at 110V and run time 30 min. And after being dyed by a GelRed nucleic acid dyeing agent, the gel is imaged by an ultraviolet gel imaging system and then photographed. The result (figure 3) shows that the PCR reaction bands amplified by the method are clear, the number is rich, the background is clean, and except SC30, the difference bands (shown by arrows in figure 3) are amplified by other 7 primers, which shows that the optimized radix puerariae SCoT-PCR reaction system has good and stable amplification effect and good polymorphism, and can be applied to the researches in the related fields of identification of radix puerariae germplasm resources, genetic diversity analysis, construction of radix puerariae genetic linkage maps and the like.
In conclusion, the invention establishes the kudzu root SCoT-PCR reaction system for the first time, makes up the blank of research related to kudzu root SCoT molecular markers and enriches means and methods for the research of kudzu root molecular biology. The stripe amplified by the kudzu root SCoT-PCR reaction system established by the invention has the characteristics of high stability, clear stripe, good polymorphism, simplicity in operation, high sensitivity, good repeatability and the like, can realize researches on kudzu root germplasm resource identification, genetic diversity analysis, linkage map construction, molecular marker assisted breeding and the like, and has great scientific value and application value.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (1)
1. A PCR reaction system of kudzu root SCoT molecular marker is characterized in that: the total amount of the PCR reaction system is 20 mu L, and the composition and the content of the PCR reaction system are as follows:
DNA50ng，Mg2+1.5mmol/L, dNTP0.25mmol/L, SC27 primer 0.8 mu mol/L, Taq DNA polymerase 0.5U, and ddH for the rest2O；
The sequence of the SC27 primer is 5'-ACCATGGCTACCACCGTG-3'.
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