阅读说明：本技术 蛹虫草子实体发育阶段内参基因、引物、筛选方法及应用 (Cordyceps militaris fruiting body development stage reference gene, primer, screening method and application ) 是由 刘勇男 刘必扬 马酉初 刘高强 于 2019-12-05 设计创作，主要内容包括：蛹虫草子实体发育阶段内参基因、引物、筛选方法及应用,所述内参基因为TUB,核苷酸序列为SEQ ID NO:1。TUB的正向、反向引物的核苷酸序列为SEQ ID NO:13-14。所述筛选方法为：(1)选择12个内参基因为备选内参基因,设计引物；(2)诱导蛹虫草子实体发育,提取不同发育阶段的总RNA,测定RNA浓度,将RNA反转录合成cDNA,以cDNA为模板,利用实时荧光定量PCR对备选内参基因进行分析；(3)实时荧光定量PCR数据进行Ct值分析,筛选出稳定内参基因。所述应用是以TUB为内参基因,检测目的基因不同发育阶段的相对表达量。所述内参基因稳定性好；所述方法简单、快捷、成本低。(An internal reference gene, a primer, a screening method and application in the development stage of a cordyceps militaris sporocarp are disclosed, wherein the internal reference gene is TUB, and the nucleotide sequence is SEQ ID NO. 1. Nucleotide sequences of forward and reverse primers of TUB are SEQ ID NO 13-14. The screening method comprises the following steps: (1) selecting 12 internal reference genes as alternative internal reference genes, and designing primers; (2) inducing the development of cordyceps militaris sporocarp, extracting total RNA at different development stages, determining the concentration of the RNA, carrying out reverse transcription on the RNA to synthesize cDNA, and analyzing the alternative reference gene by using real-time fluorescent 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 takes TUB as an internal reference gene to detect the relative expression quantity of target genes at different development stages. The stability of the reference gene is good; the method is simple, rapid and low in cost.)

1. An internal reference gene of a cordyceps militaris fruiting body in a development stage is characterized in that: the internal reference gene is TUB, and the nucleotide sequence of the TUB is SEQ ID NO: 1.

2. The internal reference gene of the cordyceps militaris fruiting body development stage according to claim 1, wherein the internal reference gene comprises: the development stage comprises a hyphal stage, an primordial stage, an elongation stage and a maturation stage.

3. A primer of the reference gene in the development stage of the fruiting body of Cordyceps militaris as claimed in claim 1 or 2, wherein: the nucleotide sequences of the forward primer and the reverse primer of the TUB are SEQ ID NO 13-14 in sequence.

4. A method for screening the reference gene in the development stage of the fruiting body of Cordyceps militaris as claimed in claim 1 or 2, comprising the steps of:

(1) selecting 12 reference genes TUB, EF-1 α, UBC, GTPB, RPS, ACTIN, FBOX, CYP, GAPDH, PP2A, PGK and UBQ as alternative reference genes, and designing fluorescent quantitative PCR primers thereof;

(2) inducing the development of cordyceps militaris sporocarp, respectively collecting cordyceps militaris hypha samples in hypha stage and cordyceps militaris sporocarp samples in primordial stage, elongation stage and maturation stage, immediately extracting total RNA, determining RNA concentration, carrying out reverse transcription on the RNA to synthesize cDNA, and then analyzing the alternative reference gene by using the primer in the step (1) and real-time fluorescence quantitative PCR (polymerase chain reaction) by using 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 internal reference gene most stably expressed in the development stage of the cordyceps militaris sporocarp.

5. The method for screening the reference gene in the development stage of the fruiting body of Cordyceps militaris as claimed in claim 4, wherein: 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.

6. The method for screening the reference gene in the development stage of the fruiting body of Cordyceps militaris according to claim 4 or 5, wherein the method comprises the following steps: in the step (2), the culture conditions of the cordyceps militaris 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 the rice, the silkworm chrysalis powder and the nutrient solution according to a solid-to-liquid ratio of 30-50: 1: 40-60; the formula of the nutrient solution is as follows: each 1000mL of distilled water contains 15-25 g of glucose and KH ₂PO ₄1～3g、MgSO ₄0.5-1.5 g, 0.5-1.5 g of ammonium citrate, 4-6 g of peptone and 115-25 mg of vitamin B; the temperature of the constant-temperature shading culture is 20-28 ℃, and the time is 20-30 days; the method for inducing the development of the cordyceps militaris sporocarp comprises the following steps: under the circulation induction of temperature and illumination, continuously culturing the cordyceps militaris to a hypha stage, a primordial stage, an elongation stage and a maturation stage; the cyclic induction of temperature and light is as follows: in each development stage, the illumination treatment is carried out for 12-16 h at the temperature of 20-25 ℃ every day, and then the shading treatment is carried out for 8-12 h at the temperature of 15-20 ℃; the hypha period is 28-32 days, the primordial period is 38-42 days, the elongation period is 48-52 days, and the maturation period is 60-70 days; the real-time fluorescent quantitative PCR amplification system consists of cDNA, PCR buffer Mix, a forward primer, a reverse primer and double distilled water in a volume ratio of 1: 8-12: 1:1: 5-10; 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 fluorescence reagent, a forward primer, a reverse primer and double distilled water in a volume ratio of 1: 8-12: 1:1: 5-10; the reaction conditions of the real-time fluorescent quantitative PCR are as follows: pre-denatured at 90-95 ℃ for 25-35 s and denatured at 90-95 ℃ for 5 ℃Annealing at 50-60 ℃ for 25-35 s for 15s, carrying out 30-50 cycles, then carrying out melting curve analysis at 55-95 ℃, and collecting fluorescence signals every 0.5 ℃.

7. The method for screening the reference gene in the development stage of the fruiting body of Cordyceps militaris according to any one of claims 4 to 6, wherein the method comprises the following steps: 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 use of the reference gene in the development stage of the fruiting body of Cordyceps militaris as claimed in claim 1 or 2, wherein: and detecting the relative expression quantity of a target gene related to the development of the fruiting body of the cordyceps militaris at different development stages by using the TUB as an internal reference gene and using real-time fluorescent quantitative PCR.

9. The application of the reference gene in the development stage of the fruiting body of Cordyceps militaris (L.) Link as claimed in claim 8, wherein: the target gene related to fruiting body development is a phospholipase protein gene; the phospholipase protein gene is PLA, PLC or PLD; the nucleotide sequences of the PLA, the PLC and the PLD are SEQ ID NO: 37-39; forward and reverse primers of the PLA, the PLC and the PLD are designed by primer5.0 software; the nucleotide sequences of the forward primers and the reverse primers of the PLA, the PLC and the PLD are SEQ ID NO:40-45 in sequence.

Technical Field

The invention relates to a cordyceps militaris reference gene, a primer, a screening method and application, and particularly relates to a cordyceps militaris reference gene, a primer, a screening method and application in a development stage of a fruiting body of cordyceps militaris.

Background

Cordyceps militaris is a famous Chinese medicine which is a famous Chinese medicine and has been used as a health food in China for a long time. The Cordyceps militaris fruiting body has antiinflammatory, antibacterial, antitumor, antiaging and immunity regulating effects. At present, insects (such as silkworm chrysalis), rice and the like are used as main raw materials of a culture medium, and a commercialized production method for planting cordyceps militaris sporocarp is established. However, there has been little research on the molecular mechanism of pupa worm seed entity formation, which is a fundamental biological problem.

The development of cordyceps militaris from mycelium to fruiting body shows a simple to complex multicellular transition, which proceeds through four typical stages: the sporophore development of various mushrooms in the hypha stage, primordial stage, elongation stage and maturation stage is all subjected to the four stages, such as flammulina velutipes, pleurotus eryngii, agrocybe cylindracea, pholiota nameko and the like. However, the molecular mechanism of the development of mushroom mycelia into fruiting bodies has been rarely studied, which will severely restrict the further development of commercial production of mushroom fruiting bodies.

Real-time fluorescence 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, the selection of a gene that is relatively stable under the action of the processing factors as an internal reference gene or internal reference standard is critical to the success of real-time fluorescence quantitative PCR.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects in the prior art and providing the reference gene with good stability in the development stage of the cordyceps militaris sporocarp.

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 in the development stage of the cordyceps militaris sporocarp, which has high amplification efficiency.

The invention further aims to solve the technical problem of overcoming the defects in the prior art and providing a simple, quick and low-cost method for screening the reference genes in the development stage of the cordyceps militaris sporocarp.

The invention further solves the technical problem of overcoming the defects in the prior art and provides the application of the reference gene in the development stage of the cordyceps militaris sporocarp, wherein the expression quantity of the target gene is stable and accurate.

The technical scheme adopted by the invention for solving the technical problems is as follows: an internal reference gene of the cordyceps militaris fruiting body in a development stage is TUB, and the nucleotide sequence of the TUB is SEQ ID NO. 1. TUB is known as: tubulin, Tubulin.

Preferably, the developmental stages include a hyphal stage, an primordial stage, an elongation stage, and a maturation stage.

The technical scheme adopted for further solving the technical problems is as follows: the nucleotide sequences of forward and reverse primers of the primer of the reference gene in the development stage of the cordyceps militaris sporocarp 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 reference gene in the development stage of the cordyceps militaris sporocarp comprises the following steps:

(1) selecting 12 reference genes TUB, EF-1 α, UBC, GTPB, RPS, ACTIN, FBOX, CYP, GAPDH, PP2A, PGK and UBQ as alternative reference genes, and designing fluorescent quantitative PCR primers thereof;

(2) inducing the development of cordyceps militaris sporocarp, respectively collecting cordyceps militaris hypha samples in hypha stage and cordyceps militaris sporocarp samples in primordial stage, elongation stage and maturation stage, immediately extracting total RNA, determining RNA concentration, carrying out reverse transcription on the RNA to synthesize cDNA, and then analyzing the alternative reference gene by using the primer in the step (1) and real-time fluorescence quantitative PCR (polymerase chain reaction) by using 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 internal reference gene most stably expressed in the development stage of the cordyceps militaris sporocarp.

Preferably, in step (1), the nucleotide sequences of the 12 internal reference genes are sequentially SEQ ID NO: 1-12. all of the 12 internal reference genes are called Tubulin (TUB), Elongation factor-1 α (electrophoresis factor-1 α -1 α), Ubiquitin-binding enzyme (Ubiquitin-conjugating enzyme, UBC), GTP-binding protein (GTP-binding protein, GTPB), Ribosomal protein S25 (Ribosoman S25, RPS), ACTIN-skeleton protein (Actinytosenton protein, ACTIN), F-box protein (F-box protein, FBOX), Cyclophilin (CYP), Glyceraldehyde-3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate, Phosphoglycerate kinase, PDH 2A), Polyubiquitin-phosphate kinase (PGQ 2P), Polyubiquitin-phosphate kinase (P2, Pp).

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 sequentially SEQ ID NO: 13-36.

Preferably, in the step (2), the culture conditions of the cordyceps militaris 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: 1: 40-60.

Preferably, the formula of the nutrient solution is as follows: each 1000mL of distilled water contains 15-25 g of glucose and KH ₂PO ₄1～3g、MgSO ₄0.5-1.5 g, 0.5-1.5 g ammonium citrate, 4-6 g peptone and 115-25 mg vitamin B.

Preferably, the temperature of the constant-temperature shading culture is 20-28 ℃, and the time is 20-30 days.

Preferably, in the step (2), the method for inducing development of cordyceps militaris fruiting body comprises: under the circulation induction of temperature and illumination, the cordyceps militaris is continuously cultured to a hypha stage, an primordial stage, an elongation stage and a maturation stage.

Preferably, the cyclic induction of temperature and light means: in each development stage, the illumination treatment is carried out for 12-16 h at the temperature of 20-25 ℃ and the shading treatment is carried out for 8-12 h at the temperature of 15-20 ℃ every day. The purpose of the cycle induction is to mimic the changes in natural conditions.

Preferably, the hyphal stage is 28-32 days, the primordial stage is 38-42 days, the elongation stage is 48-52 days, and the maturation stage is 60-70 days.

Preferably, in the step (2), the real-time fluorescence quantitative PCR amplification system consists of cDNA, PCR buffer Mix, forward primer, reverse primer and double distilled water in a volume ratio of 1: 8-12: 1:1: 5-10.

Preferably, in the step (2), the cDNA is diluted into 4-8 dilutions with different concentrations according to a 10-fold equal ratio as a template.

Preferably, in the step (2), 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 in a volume ratio of 1: 8-12: 1:1: 5-10.

Preferably, in the step (2), the reaction conditions of the real-time fluorescence quantitative PCR are as follows: pre-denaturation at 90-95 ℃ for 25-35 s, denaturation at 90-95 ℃ for 5-15 s, annealing at 50-60 ℃ for 25-35 s for 30-50 cycles, and then performing melting curve analysis at 55-95 ℃ to collect fluorescence signals every 0.5 ℃. Each sample was set for more than or equal to 3 technical replicates.

Preferably, in the step (2), the induced cordyceps militaris fruiting body development under the same condition 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 or fruiting body, 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 mu L of RNA sample, and 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 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. During operation, the original Ct value data is input into an Excel table of BestKeeper software, and the operated reference gene and 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 in the development stage of the cordyceps militaris sporocarp is to detect the relative expression quantity of a target gene related to the development of the sporocarp in different development stages of the cordyceps militaris sporocarp by using the TUB as the internal reference gene and using real-time fluorescent quantitative PCR. The application can analyze the expression rule of target gene related to fruiting body development in Cordyceps militaris body at different development stages.

Preferably, the objective gene related to fruiting body development is a phospholipase protein gene.

Preferably, the phospholipase protein gene is PLA, PLC, PLD. PLA, PLC and PLD are key regulating genes in the growth and development stages of the cordyceps militaris sporocarp.

Preferably, the nucleotide sequences of the PLA, the PLC and the PLD are SEQ ID NO: 37-39.

Preferably, the forward and reverse primers of PLA, PLC and PLD are designed by using primer5.0 software.

Preferably, the nucleotide sequences of the forward primers and the reverse primers of the PLA, the PLC and the PLD 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 at the development stage of the cordyceps militaris sporocarp, has good stability, provides stable internal reference gene reference for the analysis of the gene expression profile in the cordyceps militaris at each sporocarp development stage, and provides reference for researching the regulation and control mechanism of each development stage of the cordyceps militaris sporocarp;

(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 levels of 3 phospholipase protein genes in different development stages of the cordyceps militaris sporocarp, and establishes a method for detecting each gene in the cordyceps militaris by using real-time fluorescent quantitative PCR (polymerase chain reaction) in different development stages of the cordyceps militaris sporocarp.

Drawings

FIG. 1 shows Ct values of alternative reference genes in 4 developmental stages of Cordyceps militaris fruiting body in accordance with the present invention;

FIG. 2 shows the stability of the expression of each candidate reference gene analyzed by GeNorm software in 4 developmental stages of the fruiting body of Cordyceps militaris in accordance with an embodiment of the present invention;

FIG. 3 shows the number of reference genes with the most suitable expression of the reference genes in 4 developmental stages of fruiting bodies of Cordyceps militaris according to an embodiment of the present invention;

FIG. 4 shows the relative quantitative expression level of the PLA gene in vivo at the development stage of the fruiting body of Cordyceps militaris with TUB as the reference gene in the embodiment of the present invention;

FIG. 5 shows the relative quantitative expression level of the PLC gene in vivo at the development stage of the fruiting body of Cordyceps militaris with TUB as the reference gene in the embodiment of the present invention;

FIG. 6 shows the relative quantitative expression level of the PLD gene in vivo at the stage of fruiting body development of Cordyceps militaris with TUB as the reference gene in the example of 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 forward and reverse primers of PLA, PLC and PLD are synthesized by 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 are as follows: inoculating Cordyceps militaris mycelium in a glass bottle containing rice culture medium at a clean bench, and culturing at 25 deg.C under constant temperature and light shielding for 25 days until the mycelium is full of riceCulturing to obtain culture medium; the formula of the rice culture medium is as follows: uniformly mixing 20g of rice, 0.5g of silkworm chrysalis meal and 25mL of nutrient solution; the formula of the nutrient solution is as follows: the distilled water per 1000mL contains glucose 20g and KH ₂PO ₄2g、MgSO ₄1g, 1g ammonium citrate, 5g peptone and vitamin B120 mg.