CN108754018B - Screening method and application of acanthopanax senticosus target gene SSR molecular marker - Google Patents
Screening method and application of acanthopanax senticosus target gene SSR molecular marker Download PDFInfo
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- 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
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Abstract
The invention discloses a screening method and application of an acanthopanax senticosus target gene SSR molecular marker, wherein the screening method comprises the following specific steps: 1) respectively extracting total RNA of tissues of acanthopanax senticosus; 2) performing RNA-seq sequencing after total RNA reverse transcription, and performing unigene assembly by using Trinity software; 3) screening unigene related to synthesis and accumulation of eleutheroside, SOD and the like, and screening SSR sites on the unigene by MicroSatellite software; 4) designing primers by Primer3.0 software, and synthesizing; 5) performing PCR amplification by using the genome DNA as a template; 6) and (4) carrying out electrophoresis detection on the PCR product to develop a target gene SSR primer. The invention develops the candidate SSR molecular marker related to the target character with high efficiency and more pertinence, and provides an effective marker for molecular identification of excellent acanthopanax germplasm resources. Has important significance for early identification of excellent acanthopanax germplasm resources and accelerating breeding process.
Description
Technical Field
The invention belongs to the field of acanthopanax senticosus molecular marker development and molecular biotechnology, and relates to a screening method and application of an acanthopanax senticosus target gene SSR molecular marker.
Background
Acanthopanax senticosus is deciduous shrub of Araliaceae and Araliaceae, and the fruit is rich in superoxide dismutase (SOD), and has good functions of scavenging free radicals and resisting oxidation; the root bark is rich in saponin, tannin, linolenic acid, various vitamins, etc. The fruits and roots can be used as medicines, and have the effects of enhancing the immunity of human bodies, dispelling wind-damp and strengthening muscles and bones. Acanthopanax senticosus is mainly distributed in Heilongjiang, Jilin, Liaoning, Hebei and Shanxi, and the planting area is gradually enlarged in recent years. In the cultivation and popularization of acanthopanax, the quality of acanthopanax fruits is uneven, a molecular identification method of excellent germplasm is urgently needed, and a scientific basis is provided for screening of acanthopanax fine varieties with high biological activity.
Microsatellite markers (SSR) are widely distributed on different target genes in plant genomes, and have good repeatability and stability. SSR markers have been applied to plant genetic map construction, diversity analysis, molecular marker-assisted breeding and other aspects. In the past, EST sequences are often adopted in the development of SSR molecular markers for plants without reference genomes, SSR markers linked with characters need to be screened and verified from a large number of primers, and the method is high in workload and uncertain and blind. At present, reports on developing the SSR molecular marker of the acanthopanax target gene based on the RNA-seq technology are not found.
Disclosure of Invention
In order to overcome the defects of large workload, uncertainty and blindness of the existing RNA-seq SSR molecular marker screening method, the invention provides a screening method of an acanthopanax senticosus target gene SSR molecular marker.
The above purpose of the invention is realized by the following technical scheme:
1. respectively extracting total RNA of fruits, roots, stems and leaves of acanthopanax senticosus, and equivalently mixing the total RNA of different tissues and organs;
2. carrying out reverse transcription on the total RNA sample, then carrying out RNA-seq sequencing, and carrying out unigene assembly by using Trinity software;
3. mainly utilizing gene function annotation of unigene to screen unigene related to synthesis and accumulation of eleutheroside, SOD, vitamins, sugar, acid, fatty acid and the like, and utilizing MicroSatellite software to screen SSR sites on the unigene;
4. designing primers by using Primer3.0 software according to complementary sequences at two ends of a target gene SSR locus, and synthesizing;
5. carrying out PCR amplification by taking genomic DNA of acanthopanax leaf as a template;
and 6, carrying out electrophoresis detection on the PCR product, and screening the SSR primer of the target gene, which is consistent with the size of the expected product and has clear bands.
Further, the unigene annotation in the step 3 comprises the functional annotations of the Nr public database, the Swiss-Prot public database, the KEGG public database and the COG public database. Mapping unigene to KEGG database using KAAS function to obtain metabolic pathway information. According to the complicated and more unigene annotation information, unigene (target gene) related to the target character and containing SSR sites is screened out, and then target gene SSR primers related to the target character are designed and developed in a more targeted manner, so that candidate SSR molecular markers are provided for character correlation analysis of acanthopanax germplasm.
Further, the method for developing SSR markers of target genes in the step 6 comprises the following steps: (1) preliminarily screening the availability of the SSR primer according to the agarose gel electrophoresis result; (2) and (3) adopting polyacrylamide gel electrophoresis detection to analyze the accuracy and the applicability of the target gene SSR primer amplification band.
Compared with the prior art, the invention has the beneficial effects that: the invention develops the SSR molecular marker of the acanthopanax target gene based on the RNA-Seq technology, organically combines the characteristics of unigene with gene function annotation and SSR marker distribution for the first time, develops the SSR primers related to the synthesis and accumulation of acanthopanax saponin, SOD, vitamins, sugar, acid, fatty acid and the like in a targeted manner, and has the effective amplification rate of 52.3 percent. The obtained SSR marker of the target gene can be used as a candidate functional marker for genetic diversity analysis, fingerprint construction and character association analysis, supplements the rare number of the SSR markers of the acanthopanax senticosus and provides an effective marker for molecular identification of excellent acanthopanax senticosus germplasm resources. Overcomes the defects of uncertainty and blindness of screening SSR markers in the prior art, and has important significance for early identification of excellent acanthopanax germplasm resources and acceleration of breeding process.
Detailed Description
The invention is described in more detail below with reference to specific examples, without limiting the scope of the invention. Unless otherwise specified, the experimental methods adopted by the invention are all conventional methods, and experimental equipment, materials, reagents and the like used in the experimental method can be obtained from commercial sources.
Examples
1. Extracting total RNA of acanthopanax fruit, root, stem and leaf
Taking 1 acanthopanax germplasm SL1 of Suizhizu county in Heilongjiang province as a material. Extracting total RNA of different tissues and organs of acanthopanax according to a recommended method of the Shanghai plant RNA extraction kit. The total RNA concentration of each tissue organ was 200-400 ng/. mu.L, and the cells were stored at-80 ℃.
2. RNA-seq data analysis of fruits, roots, stems and leaves of Acanthopanax senticosus
The transcriptome library was constructed by reverse transcription using a sample of 4 tissue organ total RNA equal mixtures as a template. And removing the joint from raw reads obtained by sequencing, obtaining clean reads with the N proportion of more than 0.1% and low quality, and splicing the clean reads by using Trinity software to obtain unigene. Unigene was compared to known functional genes in public databases (Nr, Swiss-Prot, KEGG, COG, etc.). Mapping unigene to KEGG database using KAAS function to obtain metabolic pathway information.
3. Screening unigene (containing SSR locus) related to synthesis of saponin, SOD, vitamin, sugar, acid, fatty acid and the like
Screening unigene related to synthesis of eleutheroside, SOD, vitamins, sugar, acid, fatty acid and the like according to unigene annotation, metabolic pathway information and related literature reports. The target genes selected comprise SS (quaternary synthase), HMGR (3-hydroxy-3-methylglutaryl coenzyme A), SE (quaternary epoxide), DXPS (1-deoxy-D-xylulose-5-phosphate synthase) and OSCs (epoxy quaternary cyclase) groups related to saponin synthesis; acyl-coA carboxylase, 3-keto-ACP-synthsase II, 3-keto-ACP-synthsase III, fat acyl-ACP thioesterase B, fat acyl-ACP thioesterase a, stearoyl-coA desaturase, late delta-12desaturase, glycerol-3-phosphate acyl transferase, diacylglycerol acyl transferase, mitochondril ns-2-enol-coA reductase, 3-keto-coA synthsase and phosphate acid phosphate genes associated with lipid synthesis; the genes of free-biphosphate aldolase, citrate synthase, malate dehydrogenase, phosphoenolpyruvate carboxylase, alpha-amyrase, glucosidase, sucrose synthase, neutral invertase, pectinasterase and polylactanase related to the synthesis of sugar acids; TC (tocopherol cyclase), GDP-mannose phosphorylase and Myo-inositol oxidase genes involved in vitamin synthesis; and SOD genes. The SSR sites of these unigene were detected using MicroSatellite software.
4. PCR amplification with Acanthopanax senticosus leaf genome DNA as template
Extracting the genomic DNA of the acanthopanax leaf according to an improved method of a plant genomic DNA extraction kit of Tiangen company, wherein the specific method comprises the following steps: fully grinding leaves (20-40 mg), then moving the leaves into a centrifuge tube, adding 400-600 mu L of buffer solution LP1 and 4-6 mu L of RNase A, and then carrying out vortex oscillation for 1 min; adding 100-200 mu L of buffer solution LP2, and then carrying out vortex oscillation for 1 min; ③ centrifuging at 12000rpm for 5min, and transferring supernatant; adding buffer solution LP3 with the volume of 1.5 times of the volume of the buffer solution, and then shaking and mixing the mixture evenly for 15 s; fifthly, transferring all the solution into the center of an adsorption column CB3, standing for 10min, centrifuging at 12000rpm for 2min, and discarding the waste liquid; sixthly, 600 mu L of rinsing liquid PW is added into the adsorption column CB3, centrifugation is carried out at 13000rpm for 1.5min, and waste liquid is discarded; seventhly, repeating the step; placing the adsorption column CB3 back into the collection tube, centrifuging at 12000rpm for 2min, and standing CB3 at room temperature for 15 min; ninthly, moving the adsorption column CB3 into a new centrifugal tube, hanging and dripping 36-50 mu L of TE solution to the central part of the adsorption film, standing at room temperature for 5min, and centrifuging at 12000rpm for 2 min; dripping the DNA solution into the center of the adsorption membrane again, standing at room temperature for 2min, and centrifuging at 12000rpm for 2min to obtain the genomic DNA solution of the leaves of Acanthopanax senticosus.
According to the target gene sequence containing SSR locus in the step 3, 107 pairs of primers are designed, the genomic DNA of acanthopanax leaf is taken as a template for PCR amplification, and the total reaction system is 20 mu L: 2. mu.L of 10 XPCR buffer, 0.4. mu.L of 10mmol/L dNTP, 1. mu.L of each of 10. mu.mol/L upstream and downstream primers, 0.2. mu.L of 20 ng/. mu.L template DNA 2. mu. L, Taq enzyme, ddH2O13.4 μ L; the reaction procedure is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extending for 10min at 72 ℃; 4Storing at deg.C.
5. Electrophoresis detection of PCR product and development of SSR molecular marker of target gene
Firstly, 2.5% agarose gel electrophoresis is utilized to detect PCR products of 107 pairs of SSR primers, and 56 pairs of SSR markers suitable for acanthopanax are obtained by preliminary screening. Then, these 56 PCR amplification products were further detected by 8% polyacrylamide gel vertical plate electrophoresis (Table 1), and a clear band of the expected product size was also obtained.
TABLE 1 SSR primers of Acanthopanax senticosus target gene developed based on RNA-Seq
Claims (7)
1. A screening method of an acanthopanax senticosus target gene SSR molecular marker is characterized in that 1 acanthopanax senticosus germplasm SL1 in Suizhizu county of Heilongjiang province is taken as a material; the screening method comprises the following steps:
step 1, respectively extracting total RNA of acanthopanax fruit, root, stem and leaf, and equivalently mixing the total RNA of different tissues and organs;
step 2, performing reverse transcription on the total RNA sample in the step 1, performing RNA-seq sequencing, and performing unigene assembly by using Trinity software;
and 3, screening unigene related to synthesis of saponin, SOD, vitamins, sugar, acid, fatty acid and the like by utilizing the characteristic that unigene has gene function annotation: and contains an SSR site; screening unigene related to synthesis of eleutheroside, SOD, vitamins, sugar, acid, fatty acid and the like according to unigene annotation and metabolic pathway information; the target genes selected comprise SS (quaternary synthase), HMGR (3-hydroxy-3-methylglutaryl coenzyme A), SE (quaternary epoxide), DXPS (1-deoxy-D-xylulose-5-phosphate synthase) and OSCs (epoxy quaternary cyclase) groups related to saponin synthesis; acyl-coA carboxylase, 3-keto-ACP-synthsase II, 3-keto-ACP-synthsase III, fat acyl-ACP thioesterase B, fat acyl-ACP thioesterase a, stearoyl-coA desaturase, late delta-12desaturase, glycerol-3-phosphate acyl transferase, diacylglycerol acyl transferase, mitochondril ns-2-enol-coA reductase, 3-keto-coA synthsase and phosphate acid phosphate genes associated with lipid synthesis; the genes of free-biphosphate aldolase, citrate synthase, malate dehydrogenase, phosphoenolpyruvate carboxylase, alpha-amyrase, glucosidase, sucrose synthase, neutral invertase, pectinasterase and polylactanase related to the synthesis of sugar acids; TC (tocopherol cyclase), GDP-mannose phosphorylase and Myo-inositol oxidase genes involved in vitamin synthesis; screening SSR loci on unigene by using MicroSatellite software;
step 4, designing primers by using Primer3.0 software according to complementary sequences at two ends of the SSR locus of the target gene, and synthesizing;
step 5, performing PCR amplification by using the genomic DNA of the acanthopanax leaf as a template; the method for extracting the genomic DNA of the acanthopanax leaf comprises the following steps: weighing 20-40 mg of leaves, fully grinding, transferring into a centrifuge tube, adding 400-600 mu L of buffer solution LP1 and 4-6 mu L of RNase A, and carrying out vortex oscillation for 1 min; adding 100-200 mu L of buffer solution LP2, and then carrying out vortex oscillation for 1 min; ③ centrifuging at 12000rpm for 5min, and transferring supernatant; adding buffer solution LP3 with the volume of 1.5 times of the volume of the buffer solution, and then shaking and mixing the mixture evenly for 15 s; fifthly, transferring all the solution into the center of an adsorption column CB3, standing for 10min, centrifuging at 12000rpm for 2min, and discarding the waste liquid; sixthly, 600 mu L of rinsing liquid PW is added into the adsorption column CB3, centrifugation is carried out at 13000rpm for 1.5min, and waste liquid is discarded; seventhly, repeating the step; placing the adsorption column CB3 back into the collection tube, centrifuging at 12000rpm for 2min, and standing CB3 at room temperature for 15 min; ninthly, moving the adsorption column CB3 into a new centrifugal tube, hanging and dripping 36-50 mu L of TE solution to the central part of the adsorption film, standing at room temperature for 5min, and centrifuging at 12000rpm for 2 min; dripping the DNA solution into the center of the adsorption membrane again, standing at room temperature for 2min, and centrifuging at 12000rpm for 2min to obtain radix Acanthopanacis Senticosi leaf genome DNA solution;
step 6, carrying out electrophoresis detection on the PCR product, and screening the SSR primer of the target gene which accords with the size of the expected product and has clear bands; the 56 primer groups developed by the acanthopanax senticosus target gene SSR molecular marker screening method comprise the following primer groups:
2. the screening method according to claim 1, wherein the total RNA concentration of Acanthopanax senticosus tissue/organ in step 1 is 200 ng/μ L and 400ng/μ L, and the tissue/organ is stored at-80 ℃.
3. The screening method according to claim 1, wherein in the step 2, clean reads are obtained after the sequencing obtained raw reads are removed of the linker, the proportion of N is greater than 0.1% and the low quality reads are obtained, the clean reads are spliced by using Trinity software to obtain unigene, the unigene is compared with the known functional genes in the public database, and the unigene is mapped to the KEGG database by using the KAAS function to obtain the metabolic pathway information.
4. The screening method according to claim 1, wherein in the step 3, according to complex and large amount of unigene annotation information, unigene related to synthesis and accumulation of acanthopanax senticosus functional active ingredients is screened out, SSR sites of the acanthopanax senticosus unigene are detected by adopting MicroSatellite software, and then target gene SSR primers related to target traits are designed and developed in a more targeted manner, so that candidate SSR molecular markers are provided for trait association analysis of acanthopanax senticosus germplasm.
5. The screening method according to claim 1, wherein in the step 5, the leaf genomic DNA is used as a template for PCR amplification, and the total reaction volume is 20 μ L: 2. mu.L of 10 XPCR buffer, 0.4. mu.L of 10mmol/L dNTP, 1. mu.L of each of 10. mu.mol/L upstream and downstream primers, 0.2. mu.L of 20 ng/. mu.L template DNA 2. mu. L, Taq enzyme, ddH2O13.4 μ L; the reaction procedure is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extending for 10min at 72 ℃; storing at 4 ℃.
6. The screening method according to claim 1, wherein in the step 6, the method for developing SSR markers of interest genes comprises: (1) primarily screening the availability of the SSR primers by using an agarose gel electrophoresis result; (2) and (3) adopting polyacrylamide gel electrophoresis detection to analyze the accuracy and the applicability of the target gene SSR primer amplification band.
7. The application of the SSR molecular marker of the acanthopanax senticosus target gene obtained by the screening method according to any one of claims 1-6 in diversity analysis and trait association analysis of acanthopanax senticosus germplasm resources and early screening of excellent germplasm resources.
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