CN110791585B - Internal reference gene, primer, screening method and application of cordyceps militaris mycelium under cold stress - Google Patents
Internal reference gene, primer, screening method and application of cordyceps militaris mycelium under cold stress Download PDFInfo
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Abstract
An internal reference gene under cold stress of cordyceps militaris mycelium, a primer, a screening method and application thereof, wherein the internal reference gene is UBC, and the nucleotide sequence is SEQ ID NO 1. The nucleotide sequences of the forward primer and the reverse primer of the UBC are SEQ ID NO 13-14 in sequence. The screening method comprises the following steps: (1) Selecting 12 internal reference genes as alternative internal reference genes, and designing primers; (2) Processing cordyceps militaris under cold stress, extracting total RNA of mycelia, measuring the concentration of the RNA, carrying out reverse transcription on the RNA to synthesize cDNA, and analyzing alternative reference genes by using real-time fluorescence quantitative PCR (polymerase chain reaction) by taking the cDNA as a template; (3) And (5) carrying out Ct value analysis on the real-time fluorescence quantitative PCR data, and screening out the stable reference gene. The application uses UBC as an internal reference gene and uses real-time fluorescent quantitative PCR to detect the relative expression quantity of a target gene under cold stress. The stability of the reference gene is good; the method is simple, rapid and low in cost.
Description
Technical Field
The invention relates to an internal reference gene, a primer, a screening method and application, in particular to an internal reference gene, a primer, a screening method and application of cordyceps militaris mycelium under cold stress.
Background
Cordyceps militaris is a famous Chinese medicine which is a health food and has been used for a long time in China, and is a model species of Ascomycota, hypocreales, clavicipitaceae and Cordyceps. The Cordyceps militaris fruiting body has antiinflammatory, antibacterial, antitumor, antiaging and immunity regulating effects. At present, insects (such as silkworm pupae) and rice are used as main raw materials of a culture medium, and a commercial production method for planting cordyceps militaris sporocarp is established. However, there has been little research into the molecular mechanisms of pupa seed entity formation, which is a fundamental biological problem.
Temperature is one of the key factors affecting the formation of fruiting bodies of mushroom mycelia, development of fruiting bodies from mycelia represents a simple to complex transition of cells under temperature differential induction conditions, and fruiting body formation of various types of mushrooms is dependent on low temperature (cold stress) induction. For example, the fruiting body development of Flammulina velutipes requires changing the mycelium culture condition from 25 ℃ to 15 ℃ at low temperature, and the fruiting body development of Pleurotus ostreatus requires reducing the mycelium culture temperature from 20 ℃ to 15 ℃. However, under the condition of cold induction, the molecular mechanism of the development of mushroom mycelium into fruiting body is rarely studied, which will severely restrict the further development of commercial production of mushroom fruiting body.
Real-time fluorescent quantitative PCR has become an important tool for researching complex signal networks of organisms under different stimuli due to high sensitivity, specificity and reliability. However, real-time fluorescent quantitative PCR analysis depends to a large extent on the choice of the reference gene, and will lead to erroneous results due to failure to screen for the appropriate reference gene under specific experimental conditions. Therefore, it is critical to select a gene that is relatively stably expressed under the action of the processing factor as an internal reference gene or internal reference standard for the success of real-time fluorescent quantitative PCR.
In the prior art, no relevant research and disclosure of reference genes under cold stress of cordyceps militaris mycelium exists.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the prior art and providing the reference gene under the cold stress of the cordyceps militaris mycelium, which has good stability.
The invention further aims to solve the technical problem of overcoming the defects in the prior art and providing the primer of the reference gene under the cold stress of the cordyceps militaris mycelium, which has high amplification efficiency.
The invention further aims to solve the technical problem of overcoming the defects in the prior art and provide a simple, quick and low-cost method for screening the reference gene under the cold stress of the cordyceps militaris mycelium.
The invention further solves the technical problem of overcoming the defects in the prior art and provides the application of the reference gene under the cold stress of the cordyceps militaris mycelium, wherein the expression level of the target gene is stable and accurate.
The technical scheme adopted by the invention for solving the technical problems is as follows: the internal reference gene is UBC under cold stress of cordyceps militaris mycelium, and the nucleotide sequence of the UBC is SEQ ID NO. 1. The generic name of UBC is: ubiquitin-conjugating enzyme, ubiquitin-conjugating enzyme. The temperature of the cold stress treatment is 10-20 ℃, and the time is more than 0-8 h.
The technical scheme adopted for further solving the technical problems is as follows: the nucleotide sequences of the forward primer and the reverse primer of the UBC are SEQ ID NO 13-14 in sequence.
The technical scheme adopted by the invention for further solving the technical problems is as follows: the method for screening the internal reference gene under the cold stress of the cordyceps militaris mycelium comprises the following steps:
(1) Selecting 12 reference genes UBC, ACTIN, FBOX, TUB, EF-1 alpha, PGK, GTPB, RPS, UBQ, CYP, GAPDH and PP2A as alternative reference genes, and designing fluorescent quantitative PCR primers thereof;
(2) Carrying out cold stress treatment on cordyceps militaris mycelia, collecting cordyceps militaris mycelia samples after treatment, immediately extracting total RNA, determining RNA concentration, carrying out reverse transcription on the RNA to synthesize cDNA, and analyzing alternative reference genes by using the primers in the step (1) and using real-time fluorescence quantitative PCR (polymerase chain reaction) with the cDNA as a template;
(3) And performing internal reference gene Ct value stability analysis on the real-time fluorescent quantitative PCR data by adopting GeNorm, normFinder and BestKeeper software, thereby screening the most stably expressed internal reference gene in cordyceps militaris under cold stress.
Preferably, in the step (1), the nucleotide sequences of the 12 reference genes are SEQ ID NO 1-12 in sequence. The totality of the 12 reference genes is called: ubiquitin-binding enzyme (UBC), actin-binding enzyme (Actin), F-box protein (FBOX), tubulin (Tubulin, TUB), elongation factor-1 alpha (Electron growth factor-1 alpha, EF-1 alpha), phosphoglycerate kinase (PGK), GTP-binding protein (GTP-binding protein, GTPB), ribosomal protein S25 (Ribosol protein S25, RPS), polyubiquitin protein (Polyubuiquitin, UBQ), cyclophilin (Cyclinin, CYP), glyceraldehyde-3-phosphate dehydrogenase (Glycoledin-3-phosphate dehydrogenase, GAPDH), serine/threonine protein 2A (Serine/threonine/phosphatase 2A, protein 2A, PPDH, 2A). These genes include conventional stable reference genes, such as TUB, UBQ, GAPDH, EF-1 α; stable reference genes analyzed by RNA sequencing and quantitative PCR experiments, such as UBC, CYP and the like; and some new potential reference genes, such as PGK, PP2A, etc.
Preferably, in step (1), the forward and reverse primers of the 12 reference genes are designed by using primer5.0 software.
Preferably, in the step (1), the nucleotide sequences of the forward primers and the reverse primers of the 12 reference genes are SEQ ID NO:13-36 in sequence.
Preferably, in the step (2), the culture conditions of the cordyceps militaris mycelium are as follows: inoculating Cordyceps militaris mycelium into a glass bottle containing rice culture medium, and culturing at constant temperature in shade until the mycelium is full of the rice culture medium.
Preferably, the formula of the rice culture medium is as follows: and uniformly mixing the rice, the silkworm chrysalis meal and the nutrient solution according to a solid-to-liquid ratio (g/g/mL) of 30-50.
Preferably, the formula of the nutrient solution is as follows: each 1000mL of distilled water contains 15-25 g and KH 2 PO 4 1～3g、MgSO 4 0.5-1.5 g, 0.5-1.5 g of ammonium citrate, 4-6 g of peptone and 15-25 mg of vitamin B.
Preferably, the temperature of the constant-temperature shading culture is 20-28 ℃ and the time is 20-30 days.
Preferably, in step (2), the cold stress treatment is performed at a temperature of 10 to 20 ℃ for a time of > 0 to 8 hours (preferably 0.5 to 3.0 hours). The invention selects the reference gene by researching the expression quantity of the key gene of the cordyceps militaris in cell response in a short time under cold stress.
Preferably, in the step (2), the amplification system of the real-time fluorescence quantitative PCR consists of cDNA, PCR buffer Mix, forward primer, reverse primer and double distilled water in a volume ratio of 1:8-12.
Preferably, in step (2), the cDNA is diluted into 4 to 8 dilutions with different concentrations in a 10-fold equal ratio as a template.
Preferably, in the step (2), the reaction system of the real-time fluorescence quantitative PCR comprises cDNA diluent, a fluorescent reagent, a forward primer, a reverse primer and double distilled water in a volume ratio of 1:8-12.
Preferably, in the step (2), the reaction conditions of the real-time fluorescence quantitative PCR are as follows: pre-denaturation at 90-95 ℃ for 25-35s, denaturation at 90-95 ℃ for 5-15s, annealing at 50-60 ℃ for 25-35 s, 30-50 cycles, melting curve analysis at 55-95 ℃ and collection of fluorescence signals at every 0.5 ℃. Each sample was set for more than or equal to 3 technical replicates.
Preferably, in step (2), the cold stress treatment under the same conditions is set to be more than or equal to 3 biological repetitions.
Preferably, in step (2), the RNA is extracted by the following method: taking 0.1-0.3 g of cordyceps militaris hypha sample, adding liquid nitrogen, grinding into fine powder, adding 400-600 mu L of Trizol reagent, and manually homogenizing for 3-8 min, wherein the steps are finished according to the Trizol reagent specification.
The method for detecting the quality and the concentration of the RNA comprises the following steps: taking 1 microliter of RNA sample, detecting OD260/280 and concentration by using NanoDrop2000, wherein the OD260/280 value is between 1.8 and 2.2, and the sample is qualified.
First strand cDNA Synthesis: reverse transcription was performed using 3. Mu.g of RNA, and the procedure was performed according to the Thermo Fisher reverse transcription kit instructions.
Preferably, in the step (3), the GeNorm software obtains variation values V for pairwise comparison through normalization factor pairing difference analysis, and finally selects an appropriate number of reference genes, where when the value of Vn/n +1 is less than the threshold value 0.15, the most appropriate number of reference genes is n, and the first n genes are selected as reference genes. The GeNorm program can be used for screening any number of internal reference genes under any conditions and selecting more than two internal reference genes instead of the traditional method of using a single internal reference gene, which is beneficial to correcting system deviation and obtaining more reliable accurate quantitative results of the genes and has extremely important significance for biological research of slight expression difference.
Preferably, in the step (3), the expression stability value M of each gene is directly calculated by the NormFinder software, and the smaller the M value is, the higher the stability is, according to the sorting of the M values.
Preferably, in the step (3), the BestKeeper software determines the most suitable reference genes according to the original Ct value and the amplification efficiency of the primers, and sorts the reference genes in sequence, wherein the reference genes with a stability value less than 1 are qualified as stable reference genes. BestKeeper can compare the expression levels of multiple reference genes and multiple genes of interest in 100 samples. When in operation, the original Ct value data is input into an Excel table of BestKeeper software, and the operated reference gene and the target gene are independently analyzed. The program generated a correlation coefficient and BestKeeper index for the pairings between each gene, i.e., the geometric mean of Ct values for each candidate gene, which were compared according to their magnitude.
The invention further solves the technical problems by adopting the following technical scheme: the application of the internal reference gene under the cold stress of the cordyceps militaris mycelium takes UBC as the internal reference gene and uses real-time fluorescence quantitative PCR to detect the relative expression quantity of a target gene related to low-temperature adjustment under the cold stress of the cordyceps militaris mycelium. The application can analyze the expression rule of target genes related to low-temperature regulation in cordyceps militaris under cold stress.
Preferably, the gene of interest associated with adaptation to low temperature regulation is a histidine kinase gene.
Preferably, the histidine kinase gene is HK1-3. According to studies of model species such as yeast and Arabidopsis, histidine kinases play a role in signal transduction in response to temperature changes such as cold and heat, but functional studies of HK have been rare in Cordyceps militaris.
Preferably, the nucleotide sequence of the HK1-3 is SEQ ID NO 37-39.
Preferably, the forward and reverse primers of HK1-3 are designed by using primer5.0 software.
Preferably, the nucleotide sequences of the forward and reverse primers of HK1-3 are SEQ ID NO:40-45 in sequence.
The invention has the following beneficial effects:
(1) The internal reference gene meets the stability threshold of three analysis software under the cold stress of cordyceps militaris mycelia, has good stability, provides stable internal reference gene reference for the analysis of gene expression profiles in cordyceps militaris under the cold stress, and provides reference for researching a regulation mechanism for the formation of cordyceps militaris cold inducer entities;
(2) The primer amplification efficiency of the reference gene is high;
(3) The screening method is simple, rapid and low in cost, and provides reference for screening of real-time fluorescence quantitative PCR reference genes of other species under specific experimental conditions;
(4) The invention analyzes the relative expression level of 3 HK genes under cold stress and establishes a method for detecting each gene in cordyceps militaris by using real-time fluorescent quantitative PCR under the cold stress.
Drawings
FIG. 1 shows Ct values (at 0h,0.5h,1h,2h,4h, and 8h) of various alternative internal reference genes in Cordyceps militaris after cold stress in the example of the present invention;
FIG. 2 shows the stability of the expression of each alternative internal reference gene (at 0h,0.5h,1h,2h,4h, 8h) analyzed by GeNorm software in Cordyceps militaris after cold stress in the embodiment of the present invention;
FIG. 3 shows the number of reference genes in Cordyceps militaris with the most suitable expression of reference genes after cold stress according to the embodiment of the present invention;
FIG. 4 shows the relative quantitative expression level of HK1 gene in Cordyceps militaris after cold stress using UBC as reference gene in the present invention;
FIG. 5 shows the relative quantitative expression level of HK2 gene in Cordyceps militaris after cold stress using UBC as reference gene in the present invention;
FIG. 6 shows the relative quantitative expression level of HK3 gene in Cordyceps militaris after cold stress using UBC as reference gene in the present invention.
Detailed Description
The invention is further illustrated by the following examples and figures.
The cordyceps militaris hyphae used in the reference example are purchased from China general microbiological culture collection center with the preservation number of CGMCC 3.14242, and the silkworm chrysalis powder is purchased from Meiyue feed science and technology Limited company in Shandong province; the 12 reference genes and the HK1-3 forward and reverse primers are synthesized by the company Biotechnology engineering (Shanghai) GmbH after being designed; trizol reagent used in the embodiment of the present invention, batch number: 15596026, available from Invitrogen; thermo Fisher reverse transcription kit, lot No.: k1622 available from Themo Fisher; PCR buffer Mix was purchased from near-shore protein novoprotein science and technology ltd; SYBR Green fluorescent reagent was purchased from Bio-Rad; CFX96 instrument was purchased from Bio-Rad; the starting materials or chemicals used in the examples of the present invention are, unless otherwise specified, commercially available in a conventional manner.
The use of the reference gene stability analysis software according to the examples of the present invention was carried out in accordance with the instructions for its use.
Reference example 1
The culture conditions of the cordyceps militaris mycelium are as follows: inoculating Cordyceps militaris mycelium in a glass bottle containing rice culture medium at a superclean bench, and culturing at 25 deg.C in constant temperature and shade for 25 days until the mycelium is full of the rice culture medium; the formula of the rice culture medium is as follows: 20g of rice, 0.5g of silkworm chrysalis powder and 25mL of nutrient solution are mixed uniformly; the formula of the nutrient solution is as follows: the distilled water per 1000mL contains glucose 20g and KH 2 PO 4 2g、MgSO 4 1g, 1g ammonium citrate, 5g peptone and 20mg vitamin B.
Examples
(1) Selecting 12 reference genes UBC, ACTIN, FBOX, TUB, EF-1 alpha, PGK, GTPB, RPS, UBQ, CYP, GAPDH and PP2A as alternative reference genes, and designing fluorescent quantitative PCR primers thereof;
the nucleotide sequences of the 12 reference genes are SEQ ID NO. 1-12 in sequence, and the nucleotide sequences of forward primers and reverse primers of the 12 reference genes are designed by primer5.0 software to be SEQ ID NO. 13-36 in sequence;
(2) Placing the cordyceps militaris mycelia cultured in the reference example 1 in a dark incubator, performing cold stress treatment at 20 ℃, collecting cordyceps militaris mycelia samples with the treatment time of 0h,0.5h,1h,2h,4h and 8h (0 h is a control sample) respectively after treatment, immediately extracting total RNA, determining the concentration of the RNA, performing reverse transcription on the RNA to synthesize cDNA, and analyzing alternative internal reference genes by using the primers in the step (1) and real-time fluorescent quantitative PCR (polymerase chain reaction) by using the cDNA as a template;
the extraction method of the RNA comprises the following steps: taking 0.2g of cordyceps militaris mycelium sample, adding liquid nitrogen, grinding into fine powder, adding 500 mu L of Trizol reagent, and manually homogenizing for 5min, wherein the steps are finished according to the Trizol reagent instruction;
the method for detecting the quality and the concentration of the RNA comprises the following steps: taking 1 mu L of RNA sample, detecting that the OD260/280 value is 2.0 by using NanoDrop2000, and detecting that the concentration is 900 ng/mu L by using NanoDrop 2000;
first strand cDNA Synthesis: reverse transcription was performed using 3. Mu.g of RNA, with reference to the instructions of Thermo Fisher reverse transcription kit;
the real-time fluorescent quantitative PCR amplification system consists of 1 mu L of cDNA, 10 mu L of 2X PCR buffer Mix, 1 mu L of forward primer, 1 mu L of reverse primer and 7 mu L of double distilled water, and the length result of the amplified fragment is shown in Table 1;
the reaction system of the real-time fluorescent quantitative PCR consists of 1 mu L of cDNA (which is respectively diluted into 5 diluents with different concentrations, namely 3 mu g, 0.3 mu g, 0.03 mu g, 0.003 mu g and 0.0003 mu g of total RNA), 10 mu L of 2 XTaq SYBR Green fluorescent reagent, 1 mu L of forward primer, 1 mu L of reverse primer and 7 mu L of double distilled water;
the reaction conditions of the real-time fluorescent quantitative PCR are as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 10s, and annealing at 55 ℃ for 30s for 40 cycles, then performing melting curve analysis at 55-95 ℃, and collecting fluorescence signals once at 0.5 ℃ by a CFX96 instrument; each sample was set for 3 technical replicates;
cold stress treatment under the same conditions set 3 biological replicates;
(3) Performing internal reference gene Ct value stability analysis on real-time fluorescent quantitative PCR data by adopting GeNorm, normFinder and BestKeeper software, and screening the most stably expressed internal reference gene in cordyceps militaris under cold stress, wherein the result is shown in table 2;
the GeNorm software obtains variation values V compared in pairs through standardized factor pairing difference analysis, finally, a proper reference gene number is selected, when the Vn/n +1 value is smaller than a threshold value of 0.15, the most proper gene reference number is n, and the first n genes are selected as reference genes; the NormFinder software directly calculates the expression stability value M of each gene, and the expression stability value M is sorted according to the size of the value M, and the smaller the value M is, the higher the stability is; the BestKeeper software determines the most suitable reference genes through the original Ct value and the primer amplification efficiency, and sequences the most suitable reference genes in sequence, wherein the reference genes with the stability value less than 1 are qualified as stable reference genes.
In the step (2), agarose gel electrophoresis is adopted to detect whether the length of the amplified fragment is consistent with that of the selected target fragment, and then a PCR product is recovered and sequenced for verification.
In the step (2), the amplification efficiency of the primers is given by a CFX96 instrument under different cDNA concentrations, and whether the primers are suitable is judged according to whether the amplification efficiency (%) is between 90 and 110 percent, and the results are shown in Table 1.
TABLE 1 primer sequence, amplification length and amplification efficiency table of Cordyceps militaris in vivo candidate internal reference gene amplified by real-time fluorescence quantitative PCR
As can be seen from Table 1, the primers designed above can effectively amplify target fragments, which are consistent in size with the designed target fragments, and have single band and no specific amplification; the amplification efficiency was 95.5 to 106.2% (between 90 and 110%), indicating that the amplification efficiency of the primer was high.
As shown in FIG. 1, the Ct values of 12 different candidate reference genes obtained by CFX96 apparatus have great variation, with the Ct value being 16.6 at the minimum and 28.4 at the maximum. The Ct value variation is the primary screening condition for whether the internal reference gene can be used, the proper Ct value of the internal reference gene needs to be moderate and is about 15-25, and the gene with over-high expression (the Ct value is small) or over-low expression (the Ct value is large) is not suitable for being used as the internal reference gene. Furthermore, the large Ct changes of different genes indicate that the expression levels of these genes under cold stress are not constant but rather in a changing state.
TABLE 2 stability of expression of various alternative internal reference genes in Cordyceps militaris after cold stress (0h, 0.5h,1h,2h,4h, 8h)
As can be seen from Table 2 and FIG. 2, the GeNorm software analysis shows that the stability of the candidate reference gene is as follows: PGK < ACTIN < GAPDH < TUB < EF-1 α < FBOX < RPS < UBQ < CYP < GTPB < PP2A = UBC, with PP2A and UBC being most stable; as can be seen from Table 2, the analysis of the NormFinder software shows that the stability of the candidate reference genes is from low to high as follows: PGK < TUB < GAPDH < ACTIN < EF-1 alpha < RPS < FBOX < CYP < GTPB < PP2A < UBQ < UBC, wherein UBC is most stable; the stability results analyzed by GeNorm and NormFinder software were combined, with the average ranking being the first internal reference being UBC and the second being PP2A. And the analysis of BestKeeper software shows that the stability values of the reference genes UBC and PP2A of the first two ranked genes are less than 1, which indicates that the stability is good. And (3) integrating the analysis results, and displaying the analysis results of the analysis software: the most stable internal gene under cold stress is UBC.
As shown in FIG. 3, analysis of the number of the most suitable reference genes by GeNorm software revealed V 2/3 Less than 0.15 (when the Vn/n +1 value is less than the threshold value of 0.15, the most suitable gene internal reference number is n), which indicates that when the cordyceps militaris is subjected to fluorescence quantitative PCR detection under cold stress, the most suitable internal reference number is 2, namely UBC and PP2A.
The most suitable internal reference gene of the cordyceps militaris screened out under cold stress by combining the 3 software analysis methods is UBC, the nucleotide sequence of the internal reference gene is SEQ ID NO. 1, and the nucleotide sequences of the forward primer and the reverse primer of the UBC are SEQ ID NO. 13-14 in sequence.
The application of the internal reference gene under the cold stress of the cordyceps militaris mycelium comprises the following steps: using UBC as an internal reference gene, and detecting the relative expression quantity of HK1-3 cultured cordyceps militaris mycelia in reference example 1 under cold stress by using real-time fluorescent quantitative PCR; the nucleotide sequence of the HK1-3 is SEQ ID NO:37-39; designing the nucleotide sequences of the forward primer and the reverse primer of the HK1-3 to be SEQ ID NO 40-45 in sequence by using primer5.0 software; the method for testing the relative expression quantity of the cordyceps militaris mycelia under cold stress is the same as the step (2) of the embodiment; the primer sequence, amplification length and amplification efficiency of HK1-3 in the cordyceps militaris body amplified by real-time fluorescence quantitative PCR are shown in Table 3.
TABLE 3 primer sequence, amplification length and amplification efficiency of HK1-3 in vivo from Cordyceps militaris by real-time fluorescence quantitative PCR amplification
As can be seen from Table 3, the primers designed above can effectively amplify target fragments, which are identical in size to the designed target fragments, single in band and free of specific amplification; the amplification efficiency was 95.9 to 102.6%, indicating that the amplification efficiency of the primer was high.
In order to evaluate the gene expression level of HK1-3, data collected by fluorescent quantitative PCR were processed, and a Δ Ct =ΔCt treatment sample (0.5h, 1h,2h,4h, 8h) Δ Ct control sample (0 h) and a Δ Ct =ΔCt target gene- Δ Ct internal reference gene were obtained. Significance of differences between samples was statistically analyzed using GraphPad Prism 6 software.
As shown in FIGS. 4 to 6, in the examples, the relative expression level of the HK gene of Cordyceps militaris is significantly increased under cold stress; compared with the control sample, after 1h of low-temperature treatment, the relative expression levels of the HK1 and HK2 genes are up-regulated by 5 times, and HK3 is up-regulated by 13 times.
In conclusion, the most stable internal reference gene UBC in the cordyceps militaris body under cold stress is screened, and the expression rule of the cordyceps militaris HK1-3 gene under cold stress is determined by taking the UBC as the internal reference gene and the cordyceps militaris histidine kinase gene HK1-3 as the target gene. The screened reference gene UBC is suitable for the analysis of the expression profile of the target gene in cordyceps militaris under cold stress, and provides reference for the quantitative PCR experiment of the cordyceps militaris gene under cold stress later.
Sequence listing
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cggcctcgag cgcgtctttg gtcagcagaa gcccaagga 219
<210> 3
<211> 224
<212> DNA
<213> Cordyceps militaris (Cordyceps militaris)
<400> 3
ccgatgacaa cgacagcgac gcctccgtct tcaacccgga taacacctcc cctaagaatt 60
cgcgccagct tctgagtccg cgcgagcgtg cacagcgtcg ccaggcccgc gaggaggccg 120
cgacgctctc catgacacca tcaattttcc cgacatggaa gagcatgttc cgccggcgcc 180
cgcgcatccg cttcgacggg tgctacatct ccacggtcaa ctac 224
<210> 4
<211> 208
<212> DNA
<213> Cordyceps militaris (Cordyceps militaris)
<400> 4
atgtcgttcg tcgtgaggcc gaaggctgcg actgccttca gggtttccag atcacccact 60
ctctcggtgg tggtactggt gctggtatgg gtactctgct catctccaag atccgcgaag 120
agtttcccga ccgcatgatg gccaccttct ccgttgtccc ctcccccggc aactccgaca 180
ccgttgtcga accctacaac gccactct 208
<210> 5
<211> 200
<212> DNA
<213> Cordyceps militaris (Cordyceps militaris)
<400> 5
tatcggaact gtgcctgtcg gccgtgtcga gactggtatc atcaagcccg gcatggtcgt 60
cacctttgcc ccctccatgg tcagcactga agtcaagtcc gtcgagatgc accacgagca 120
gctggctcag ggtgttcccg gtgacaacgt cggcttcaac gtgaagaacg tttccgtcaa 180
ggaaatccgt cgtggtaacg 200
<210> 6
<211> 159
<212> DNA
<213> Cordyceps militaris (Cordyceps militaris)
<400> 6
gctcaagccc gtcgtttcgg agctcgagaa gcagctgggc aagtcggtca cgtttgcgcc 60
cgactgcgtc ggccccgagg tggagaagat tgtcaatggt gccgacaccg gcgccgtcat 120
cctgctcgag aacctgcgct tccacattga ggaggaggg 159
<210> 7
<211> 181
<212> DNA
<213> Cordyceps militaris (Cordyceps militaris)
<400> 7
taagaagccc aagaagaaaa aggtgctgct gatgggcaag tcgggctctg gcaagtcgag 60
catgcggagc atcatcttca gcaactacat tgcgcgcgac acgcgccgac tcggcgcaac 120
cattgacatt gacctctcgc acgtcaagtt cctcggcaac ctcacgctga acctgtggga 180
c 181
<210> 8
<211> 182
<212> DNA
<213> Cordyceps militaris (cordyces militaris)
<400> 8
aagtggtcta agggcaaggt caaggacaag gcccagcacg ccgtccttct cgacaagacc 60
accgccgaga agctctacaa ggatgtccag tcctaccgcc tggtcaccgt tgctgttctc 120
gtggaccgta tgaagatcaa cggctccctg gcccgccagt gcattaccga cctggaggag 180
aa 182
<210> 9
<211> 209
<212> DNA
<213> Cordyceps militaris (cordyces militaris)
<400> 9
tcaaagaaga taatggtaac gtgacggcac acacatggtc aatgggcgag cagcagtgga 60
tcaatgttgg cacggtagtg gacgcggtcg gtagtacagg gaagaaggtc gaatacaagg 120
gcaagtccta cgactttgtg tttgacgtgg acattgagga cggtaagcca ccgctgaagt 180
tgccatataa cctttccgag aacccatac 209
<210> 10
<211> 197
<212> DNA
<213> Cordyceps militaris (Cordyceps militaris)
<400> 10
ttttccgcct tattccaccc gaagctcaag cacctcgagc gcggcaccgt cagcatggca 60
acggccccat ccacgacgga cccagatatc cgcgtggccg gctcccagtt catcatcacc 120
ctcggcgagg acacagactt tttagacggc aaggccgcca tctttggcaa ggtcgtcgag 180
ggatttgatg ctctgga 197
<210> 11
<211> 220
<212> DNA
<213> Cordyceps militaris (cordyces militaris)
<400> 11
catccactcc tacactgcta cccagaagac tgtcgatggc ccctctgcca aggattggcg 60
cggtggccgt ggtgccgctc agaacatcat cccctccagc actggtgccg ccaaggctgt 120
cggcaaggtc attcctgagc tcaacggcaa gcttactggc atgtccatgc gtgtccctac 180
cgccaacgtt tccgttgtcg acctgactgt tcgtcttgag 220
<210> 12
<211> 198
<212> DNA
<213> Cordyceps militaris (cordyces militaris)
<400> 12
cctcctacag tcgtcatcag cccgactccc ggccatgtcc cacctccagg tgccgccgag 60
acgatgccgc acgacctggc ccccccaaag tctggccaaa agtcgctcat gattcaccgc 120
ggaatagata accgcgatgc tattcccgag gggcttcgaa cgcccaagcg acaacactca 180
tctcgctttg acatttct 198
<210> 13
<211> 20
<212> DNA
<213> Artificial sequence
<400> 13
accattgaca cgagccagtt 20
<210> 14
<211> 19
<212> DNA
<213> Artificial sequence
<400> 14
gcccatgtaa gcctcctca 19
<210> 15
<211> 19
<212> DNA
<213> Artificial sequence
<400> 15
caacaacttc ctgacgggc 19
<210> 16
<211> 19
<212> DNA
<213> Artificial sequence
<400> 16
tccttgggct tctgctgac 19
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence
<400> 17
ccgatgacaa cgacagcgac 20
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence
<400> 18
gtagttgacc gtggagatgt 20
<210> 19
<211> 18
<212> DNA
<213> Artificial sequence
<400> 19
<210> 20
<211> 18
<212> DNA
<213> Artificial sequence
<400> 20
<210> 21
<211> 18
<212> DNA
<213> Artificial sequence
<400> 21
<210> 22
<211> 18
<212> DNA
<213> Artificial sequence
<400> 22
<210> 23
<211> 18
<212> DNA
<213> Artificial sequence
<400> 23
gctcaagccc gtcgtttc 18
<210> 24
<211> 18
<212> DNA
<213> Artificial sequence
<400> 24
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence
<400> 25
taagaagccc aagaagaaaa 20
<210> 26
<211> 18
<212> DNA
<213> Artificial sequence
<400> 26
<210> 27
<211> 19
<212> DNA
<213> Artificial sequence
<400> 27
aagtggtcta agggcaagg 19
<210> 28
<211> 19
<212> DNA
<213> Artificial sequence
<400> 28
ttctcctcca ggtcggtaa 19
<210> 29
<211> 21
<212> DNA
<213> Artificial sequence
<400> 29
tcaaagaaga taatggtaac g 21
<210> 30
<211> 20
<212> DNA
<213> Artificial sequence
<400> 30
gtatgggttc tcggaaaggt 20
<210> 31
<211> 19
<212> DNA
<213> Artificial sequence
<400> 31
ttttccgcct tattccacc 19
<210> 32
<211> 19
<212> DNA
<213> Artificial sequence
<400> 32
tccagagcat caaatccct 19
<210> 33
<211> 21
<212> DNA
<213> Artificial sequence
<400> 33
catccactcc tacactgcta c 21
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence
<400> 34
ctcaagacga acagtcaggt 20
<210> 35
<211> 21
<212> DNA
<213> Artificial sequence
<400> 35
cctcctacag tcgtcatcag c 21
<210> 36
<211> 18
<212> DNA
<213> Artificial sequence
<400> 36
<210> 37
<211> 202
<212> DNA
<213> Cordyceps militaris (cordyces militaris)
<400> 37
gaggtgacgg agcttacaga agtctacaac aaaatgacgg atgagcttgt gagacagtat 60
acatcactcg atcaacaagt ggaaaaaaga acgcgagagc tagagatcag caagaaggcc 120
gccgaggcgg ccaacgagag caaaactctg tttattgcca acatttccca cgaattaaag 180
acgcctctca atggtatcat gg 202
<210> 38
<211> 211
<212> DNA
<213> Cordyceps militaris (cordyces militaris)
<400> 38
ccttgcggct tacaaatagc agtgtcatgc acgatgccac agaagagtct gaaacggcgc 60
ctgatgggcc tgaagaaggt gtcgcagcac aacatagaga tcttcgtttg acaagcagca 120
acgagccgct cttcgagatc ggcgaggtat cagtctcttc tagcgtcgag gaaagactcg 180
aaatggagag acgtgtatcg cagcatcatc a 211
<210> 39
<211> 229
<212> DNA
<213> Cordyceps militaris (Cordyceps militaris)
<400> 39
ctgcgtcatt gcgtgttttg aggattatca ctacgatatc ccattcgcgt ttttatactc 60
catggatgac gcgaaccaaa taccagatgc agaatcatca acccacagtg ctgccgctac 120
tacgtgtcgt ttccaagggt cccttggcat accggaagga cacattgcca cgcctccaca 180
gctcgacctc aacggtagcc gcggagaggg ctttatccca tcctttcgc 229
<210> 40
<211> 18
<212> DNA
<213> Artificial sequence
<400> 40
<210> 41
<211> 20
<212> DNA
<213> Artificial sequence
<400> 41
ccatgatacc attgagaggc 20
<210> 42
<211> 19
<212> DNA
<213> Artificial sequence
<400> 42
ccttgcggct tacaaatag 19
<210> 43
<211> 19
<212> DNA
<213> Artificial sequence
<400> 43
tgatgatgct gcgatacac 19
<210> 44
<211> 17
<212> DNA
<213> Artificial sequence
<400> 44
ctgcgtcatt gcgtgtt 17
<210> 45
<211> 18
<212> DNA
<213> Artificial sequence
<400> 45
Claims (14)
1. An application of internal reference gene of cordyceps militaris mycelium under cold stress is characterized in that: using UBC as an internal reference gene, and detecting the relative expression quantity of a target gene related to adaptation to low-temperature regulation under the cold stress of cordyceps militaris mycelia by using real-time fluorescent quantitative PCR; the nucleotide sequence of the UBC is SEQ ID NO. 1.
2. The application of the internal reference gene under cold stress of cordyceps militaris mycelium is characterized in that: the nucleotide sequences of the forward primer and the reverse primer of the UBC are SEQ ID NO. 13-14 in sequence.
3. The application of the internal reference gene under cold stress of cordyceps militaris mycelium according to claim 1 or 2, wherein the internal reference gene comprises: the method for screening the internal reference gene under the cold stress of the cordyceps militaris mycelium comprises the following steps:
(1) Selecting 12 reference genes UBC, ACTIN, FBOX, TUB, EF-1 alpha, PGK, GTPB, RPS, UBQ, CYP, GAPDH and PP2A as alternative reference genes, and designing fluorescent quantitative PCR primers thereof;
(2) Carrying out cold stress treatment on cordyceps militaris mycelia, collecting cordyceps militaris mycelia samples after treatment, immediately extracting total RNA, determining RNA concentration, carrying out reverse transcription on the RNA to synthesize cDNA, and analyzing alternative reference genes by using the primers in the step (1) and using real-time fluorescence quantitative PCR (polymerase chain reaction) with the cDNA as a template;
(3) And performing internal reference gene Ct value stability analysis on the real-time fluorescent quantitative PCR data by adopting GeNorm, normFinder and BestKeeper software, thereby screening the most stably expressed internal reference gene in cordyceps militaris under cold stress.
4. The application of the internal reference gene under cold stress of cordyceps militaris mycelium is characterized in that: in the step (1), the nucleotide sequences of the 12 reference genes are SEQ ID NO 1-12 in sequence; the forward primers and the reverse primers of the 12 reference genes are designed by using primer5.0 software; the nucleotide sequences of the forward primers and the reverse primers of the 12 reference genes are SEQ ID NO 13-36 in sequence.
5.The application of the internal reference gene under cold stress of cordyceps militaris mycelium is characterized in that: in the step (2), the culture conditions of the cordyceps militaris mycelium are as follows: inoculating Cordyceps militaris mycelium into a glass bottle containing a rice culture medium, and culturing in a constant-temperature shade until the rice culture medium is full of mycelium; the formula of the rice culture medium is as follows: uniformly mixing rice, silkworm chrysalis powder and nutrient solution according to a solid-to-liquid ratio of 30-50; the formula of the nutrient solution is as follows: each 1000mL of distilled water contains 15-25% of glucose g and KH 2 PO 4 1～3g、MgSO 4 0.5-1.5 g, 0.5-1.5 g of ammonium citrate, 4-6 g of peptone and 15-25 mg of vitamin B; the temperature of the constant-temperature shading culture is 20-28 ℃, and the time is 20-30 days; the temperature of the cold stress treatment is 10-20 ℃, and the time is more than 0-8 h; the real-time fluorescent quantitative PCR amplification system consists of cDNA, PCR buffer Mix, forward primers, reverse primers and double distilled water according to a volume ratio of 1:8-12; diluting the cDNA into 4-8 diluents with different concentrations according to an equal ratio of 10 times as a template; the reaction system of the real-time fluorescence quantitative PCR consists of cDNA diluent, a fluorescent reagent, a forward primer, a reverse primer and double distilled water according to a volume ratio of 1:8-12; the reaction conditions of the real-time fluorescent quantitative PCR are as follows: pre-denaturation at 90-95 ℃ for 25-35s, denaturation at 90-95 ℃ for 5-15s, annealing at 50-60 ℃ for 25-35 s, 30-50 cycles, melting curve analysis at 55-95 ℃ and collection of fluorescence signals at every 0.5 ℃.
6. The application of the internal reference gene under cold stress of cordyceps militaris mycelium, which is disclosed by claim 4, is characterized in that: in the step (2), the culture conditions of the cordyceps militaris mycelium are as follows: inoculating Cordyceps militaris mycelium into a glass bottle containing a rice culture medium, and performing constant-temperature light-shielding culture until the mycelium overgrows the rice culture medium; the formula of the rice culture medium is as follows: uniformly mixing rice, silkworm chrysalis powder and nutrient solution according to a solid-to-liquid ratio of 30-50; the formula of the nutrient solution is as follows: each 1000mL of distilled water contains 15-25% of glucose g and KH 2 PO 4 1～3g、MgSO 4 0.5-1.5 g, 0.5-1.5 g of ammonium citrate, 4-6 g of peptone and 15-25 mg of vitamin B;the temperature of the constant-temperature shading culture is 20-28 ℃, and the time is 20-30 days; the temperature of the cold stress treatment is 10-20 ℃, and the time is more than 0-8 h; the real-time fluorescent quantitative PCR amplification system consists of cDNA, PCR buffer Mix, forward primers, reverse primers and double distilled water according to a volume ratio of 1:8-12; diluting the cDNA into 4-8 diluents with different concentrations according to an equal ratio of 10 times as a template; the reaction system of the real-time fluorescence quantitative PCR consists of cDNA diluent, a fluorescent reagent, a forward primer, a reverse primer and double distilled water according to a volume ratio of 1:8-12; the reaction conditions of the real-time fluorescent quantitative PCR are as follows: pre-denaturation at 90-95 ℃ for 25-35s, denaturation at 90-95 ℃ for 5-15s, annealing at 50-60 ℃ for 25-35 s, 30-50 cycles, melting curve analysis at 55-95 ℃ and collection of fluorescence signals at every 0.5 ℃.
7. The application of the internal reference gene under cold stress of cordyceps militaris mycelium, which is disclosed by claim 3, is characterized in that: in the step (3), the GeNorm software obtains variation values V of pairwise comparison through standardized factor pairing difference analysis, and finally selects the number of the suitable reference genes, when the Vn/n +1 value is smaller than the threshold value of 0.15, the number of the most suitable gene reference is n, and the first n genes are selected as the reference genes; the NormFinder software directly calculates the expression stability value M of each gene, and the expression stability value M is sorted according to the size of the value M, and the smaller the value M is, the higher the stability is; the BestKeeper software determines the most suitable reference genes through the original Ct value and the primer amplification efficiency, and sequences the most suitable reference genes in sequence, wherein the reference genes with the stability value less than 1 are qualified as stable reference genes.
8. The application of the internal reference gene under cold stress of cordyceps militaris mycelium, which is disclosed by claim 4, is characterized in that: in the step (3), the GeNorm software obtains variation values V of pairwise comparison through standardized factor pairing difference analysis, and finally selects the number of the suitable reference genes, when the Vn/n +1 value is smaller than the threshold value of 0.15, the number of the most suitable gene reference is n, and the first n genes are selected as the reference genes; the NormFinder software directly calculates the expression stability value M of each gene, and the expression stability value M is sorted according to the magnitude of the value M, and the smaller the value M is, the higher the stability is; the BestKeeper software determines the most suitable reference genes through the original Ct value and the primer amplification efficiency, and sequences the most suitable reference genes in sequence, wherein the reference genes with the stability value less than 1 are qualified as stable reference genes.
9. The application of the internal reference gene under cold stress of cordyceps militaris mycelium, which is disclosed by claim 5, is characterized in that: in the step (3), the GeNorm software obtains variation values V of pairwise comparison through standardized factor pairing difference analysis, and finally selects the number of the suitable reference genes, when the Vn/n +1 value is smaller than the threshold value of 0.15, the number of the most suitable gene reference is n, and the first n genes are selected as the reference genes; the NormFinder software directly calculates the expression stability value M of each gene, and the expression stability value M is sorted according to the size of the value M, and the smaller the value M is, the higher the stability is; the BestKeeper software determines the most suitable reference genes according to the original Ct value and the primer amplification efficiency, and sequences the most suitable reference genes in sequence, wherein the reference genes with the stability value less than 1 are qualified as stable reference genes.
10. The application of the internal reference gene under cold stress of cordyceps militaris mycelium according to claim 1 or 2, wherein the internal reference gene comprises: the target gene related to the low temperature regulation is histidine kinase gene; the histidine kinase gene is HK1-3; the nucleotide sequence of the HK1-3 is SEQ ID NO:37-39; the forward and reverse primers of HK1-3 are designed by primer5.0 software; the nucleotide sequences of the forward primer and the reverse primer of the HK1-3 are SEQ ID NO:40-45 in sequence.
11. The application of the internal reference gene under cold stress of cordyceps militaris mycelium, which is disclosed by claim 3, is characterized in that: the target gene related to the low-temperature regulation is a histidine kinase gene; the histidine kinase gene is HK1-3; the nucleotide sequence of the HK1-3 is SEQ ID NO:37-39; the forward and reverse primers of HK1-3 are designed by primer5.0 software; the nucleotide sequences of the forward primer and the reverse primer of the HK1-3 are SEQ ID NO:40-45 in sequence.
12. The application of the internal reference gene under cold stress of cordyceps militaris mycelium is characterized in that: the target gene related to the low-temperature regulation is a histidine kinase gene; the histidine kinase gene is HK1-3; the nucleotide sequence of the HK1-3 is SEQ ID NO:37-39; the forward and reverse primers of HK1-3 are designed by primer5.0 software; the nucleotide sequences of the forward primer and the reverse primer of the HK1-3 are SEQ ID NO:40-45 in sequence.
13. The application of the internal reference gene under cold stress of cordyceps militaris mycelium is characterized in that: the target gene related to the low-temperature regulation is a histidine kinase gene; the histidine kinase gene is HK1-3; the nucleotide sequence of the HK1-3 is SEQ ID NO 37-39; the forward and reverse primers of HK1-3 are designed by primer5.0 software; the nucleotide sequences of the forward primer and the reverse primer of the HK1-3 are SEQ ID NO:40-45 in sequence.
14. The application of the internal reference gene under cold stress of cordyceps militaris mycelium, which is disclosed by claim 7, is characterized in that: the target gene related to the low-temperature regulation is a histidine kinase gene; the histidine kinase gene is HK1-3; the nucleotide sequence of the HK1-3 is SEQ ID NO:37-39; the forward and reverse primers of HK1-3 are designed by primer5.0 software; the nucleotide sequences of the forward primer and the reverse primer of the HK1-3 are SEQ ID NO:40-45 in sequence.
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