CN116144822B - Reference gene under abiotic stress of eremochloa ophiuroides, and primers and application thereof - Google Patents

Reference gene under abiotic stress of eremochloa ophiuroides, and primers and application thereof Download PDF

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CN116144822B
CN116144822B CN202211536965.3A CN202211536965A CN116144822B CN 116144822 B CN116144822 B CN 116144822B CN 202211536965 A CN202211536965 A CN 202211536965A CN 116144822 B CN116144822 B CN 116144822B
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CN116144822A (en
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张建波
白史且
马啸
李达旭
王晓耘
刘英杰
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Sichuan Agricultural University
Sichuan Academy of Grassland Science
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Sichuan Academy of Grassland Science
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

The invention provides an internal reference gene of eremochloa ophiuroides under abiotic stress, a primer and application thereof, and belongs to the field of molecular biology. The invention also provides a specific primer of each reference gene, which has strong specificity, can greatly improve the detection efficiency when detecting the gene expression quantity of eremochloa ophiuroides and improve the reliability of the detection result.

Description

Reference gene under abiotic stress of eremochloa ophiuroides, and primers and application thereof
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to an internal reference gene of eremochloa ophiuroides, and a primer and application thereof.
Background
The centipede grass [ Eremochloa ophiuroides (Munro) Hack ] is a warm-season perennial herb plant with a C4 structure, belongs to the genus Centipeda of the subfamily Panzem of the Gramineae, has the characteristics of low plant, low water and fertilizer requirement, rough-put management resistance, strong disease resistance and the like, is an ideal turf grass for urban and rural greening, slope greening, water and soil conservation and ecological management, and is one of the three global warm-season turf grass. At present, factors such as drought, cold and the like frequently occur in extreme weather, the normal growth of the eremochloa ophiuroides is seriously affected by the factors such as acid soil in the south and saline alkali in the coastal lands, and new varieties of glufosinate-resistant are lacking, so that new requirements are provided for the later breeding of the eremochloa ophiuroides, and the breeding of eremochloa ophiuroides varieties with stress resistance, salt resistance, acid resistance and glufosinate-resistant are very necessary.
The selection of stress-resistant genes such as drought resistance, cold resistance and the like and glufosinate-ammonium resistance genes has important significance for researching related molecular mechanisms of eremochloa ophiuroides and promoting the breeding of related new varieties. Real-time fluorescent quantitative PCR (qRT-PCR) is an important test method for accurate nucleic acid quantification, has the characteristics of high sensitivity, good repeatability, strong specificity, high flux and the like, and is widely used in gene expression research. The stable expression of the reference gene is an important precondition for accurate analysis results of gene expression, and the ideal reference gene has the characteristic of stable expression in all cells, different growth stages and various physiological states, but the stability of the traditional housekeeping genes is different, so that the screening of the proper reference gene according to specific experimental conditions is a key for ensuring accurate and reliable quantification of target gene expression.
At present, no report about screening of reference genes of centipede grass for cold resistance, drought resistance, salt resistance, aluminum resistance and glufosinate resistance exists, so that the development of a group of reference genes of centipede grass under the stress of low temperature, drought, salt, aluminum and glufosinate is necessary to ensure the breeding effect of the centipede grass.
Disclosure of Invention
In view of the above, the invention aims to provide a group of reference genes under abiotic stress of eremochloa ophiuroides, and primers and application thereof, the invention utilizes qRT-PCR technology to detect the expression stability of 13 eremochloa ophiuroides reference genes under low temperature, drought, salt, aluminum and glufosinate-ammonium stress, uses the gene stability evaluation analyzed by geNorm, normFinder and BestKeeper3 software as reference, uses refFinder software as main basis to evaluate the gene stability, synthesizes the software analysis result, respectively screens out the reference genes stably expressed under each stress, screens out 2 genes under each stress treatment, and screens out 10 reference genes altogether; solves the current situation that the existing centipede grass quantitative PCR detection has no reference gene.
In order to achieve the above object, the present invention is realized by the following technical scheme:
application of UBC gene as reference gene in detecting gene expression quantity of eremochloa ophiuroides under abiotic stress of eremochloa ophiuroides;
preferably, the abiotic stress is an aluminum stress or a low temperature stress.
The specific primer, forward primer and reverse primer of the reference gene UBC are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively.
The application of ANI gene as reference gene in detecting the gene expression level of eremochloa ophiuroides under abiotic stress;
preferably, the abiotic stress is glufosinate stress.
The specific primer, forward primer and reverse primer of the internal reference gene ANI are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.
Application of RIP gene as reference gene in detecting expression level of eremochloa ophiuroides gene under abiotic stress;
preferably, the abiotic stress is drought stress, glufosinate stress or salt stress.
The specific primer, forward primer and reverse primer of the reference gene RIP are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively.
Application of MD gene as reference gene in detecting gene expression amount of eremochloa ophiuroides under abiotic stress;
preferably, the abiotic stress is a salt stress, a low temperature stress, an drought stress or an aluminum stress.
The specific primer, forward primer and reverse primer of reference gene MD are shown as SEQ ID NO.7 and SEQ ID NO.8 respectively.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a group of centipede grass reference genes based on the sequencing of the centipede grass early transcriptome, and designs primers of each reference gene by utilizing the base sequences of the reference genes; the invention not only solves the current situation that the existing quantitative PCR detection of the centipede grass does not have an internal reference gene, but also can improve the stability, reliability and repeatability of the analysis and research of the gene expression of the centipede grass when the primers of the internal reference gene are respectively used for the analysis of the gene expression of the centipede grass under the stress of low temperature, drought, salt, aluminum and glufosinate. Meanwhile, the primer of the reference gene designed by the invention has strong specificity, can greatly improve the detection efficiency when detecting the gene expression quantity of the eremochloa ophiuroides and improve the reliability of the detection result.
Drawings
FIG. 1 shows the primer specificity of 13 candidate reference genes (UBC, GADPH, ACT, suS, ANI, ADP, CYP, H, 50S, RIP, MD, CP, HSP, respectively, from left to right)
FIG. 2 shows Ct values of various reference genes under different stresses;
FIG. 3 shows the expression stability of each reference gene under different stresses;
FIG. 4a shows the expression levels of the target genes normalized by different reference genes under drought stress;
FIG. 4b shows the expression level of the target gene normalized by different reference genes under salt stress;
FIG. 4c shows the expression level of the target gene normalized by different reference genes under low temperature stress;
FIG. 4d shows the expression level of the target gene normalized by different reference genes under aluminum stress;
FIG. 4e shows the expression level of the target gene normalized by different reference genes under the stress of glufosinate.
Detailed Description
The invention provides an application of UBC gene as an internal reference gene in detecting the gene expression quantity of eremochloa ophiuroides under abiotic stress of eremochloa ophiuroides; the abiotic stress is an aluminum stress or a low temperature stress.
The invention provides a specific primer of the UBC gene, and a forward primer and a reverse primer are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
The invention provides an application of an ANI gene as an internal reference gene in detecting the gene expression quantity of eremochloa ophiuroides under abiotic stress of eremochloa ophiuroides; the abiotic stress is glufosinate stress.
The invention provides a specific primer of the ANI gene, and a forward primer and a reverse primer are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.
The invention provides application of RIP genes as reference genes in detecting the gene expression quantity of eremochloa ophiuroides under abiotic stress of eremochloa ophiuroides; the abiotic stress is drought stress, glufosinate stress or salt stress.
The invention provides a specific primer of the RIP gene, and a forward primer and a reverse primer are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6.
The invention provides application of MD genes as reference genes in detecting the gene expression quantity of eremochloa ophiuroides under abiotic stress of eremochloa ophiuroides; the abiotic stress is a salt stress, a low temperature stress, an drought stress or an aluminum stress.
The invention provides a specific primer of the MD gene, and a forward primer and a reverse primer are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8.
In the invention, the method for detecting the gene expression quantity of the centipede grass under the abiotic stress of the centipede grass is a fluorescent quantitative PCR method;
in the present invention, the nucleotide sequences of the reference genes UBC, ANI, RIP and MD are shown in Table 1.
TABLE 1 base sequences of reference genes
In the present invention, specific primers for the respective reference genes are shown in Table 2,
table 2 specific primers for reference genes
The F represents a forward primer, and the R represents a reverse primer.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Material and treatment
Soaking seed of eremochloa ophiuroides with 10% sodium hydroxide for 5min, washing with sterile distilled water, sowing seed into plastic basin of 20X15X15 cm, 1.3g each basin, pouring 50% Hoagland nutrient solution, respectively at 23deg.C and 19deg.C, photoperiod 12h, relative humidity 75%, and illumination intensity 250 (umol.m) -2 ·s -1 ) After 90 days of culture in the growth chamber of (C), respectively subjecting to low temperature stress at 4deg.C, 20% PEG-6000 drought stress, 200mM NaCl salt stress, and 100. Mu.m MAlCl 3 Aluminum stress, 6 μl/mL glufosinate stress, 3 replicates per treatment group (i.e. 3 flowerpots) were used. 0, 0.5, 1.5 after each treatment,3. Leaf samples were collected at 6, 12, 24, 48 and 72h, and the collected samples were immediately frozen in liquid nitrogen and stored in a-80 ℃ refrigerator for subsequent RNA extraction.
2. Method of
2.1 extraction and reverse transcription of Total RNA
The whole tissue of the centipede grass blades weighing 0.1g was crushed by a tissue grinder, and total RNA was extracted by M5 HiPer Plant Complex Mini Kit (Beijing polymeric Biotechnology Co., ltd.). The quality of RNA was verified by running 1.2% agarose gel electrophoresis. Total RNA (200 ng) was extracted from each sample, reverse transcribed into the first strand of cDNA using M5 Superplus QpcrRT kitwithgDNARemover (Beijing Polymer Biotechnology Co., ltd.) and stored at-80℃for further analysis.
2.2 design and verification of specific primers
On the basis of the centipede grass transcriptome sequencing, 13 candidate internal reference genes are selected, and the candidate internal reference genes are respectively: UBC (Ubiquitin-conjugating enzyme: ubiquitin-binding enzyme), GADPH (glycal de-3-phosphate dehydrogenase: glyceraldehyde-3-phosphate dehydrogenase), ACT (action: actin), suS (sucrose synthase: sucrose synthase), ANI (Alkaline and neutral invertase: alkaline and neutral invertase), ADP (ADP-ribosylation factor: ADP ribosylating factor), CYP (Cyclophilin: cyclophilin), H3 (Histone H3: histone H3), 50S (50S ribosomal protein L2:50S ribosomal protein L2), RIP (60SRibosomal protein L2:60S ribosomal protein L2), MD (Malate dehydrogenase: malate dehydrogenase), CP (Chaperone protein: chaperonin), HSP70 (Heat shock70 kDatagrotein: heat shock70k Da protein).
The specific primers of the 13 reference genes are designed by Primer Quest software and synthesized by Kangshi biotechnology company (Chengdu, china). The specificity of the primers was verified by melting curve of the RT-qPCR reaction, see FIG. 1.
The primer sequences of candidate internal reference genes in RT-qPCR are shown in Table 3.
Table 313 primer sequences of candidate reference genes
F is a forward primer, and R is a reverse primer.
2.3 real-time fluorescent quantitative PCR
Quantitative analysis was performed using a 2×M5 HiPer SYBR Premix EsTaq (with Tli RnaseH) and real-time PCR system (Bio-Rad, USA) manufactured by Beijing Polymer Biotechnology Co. The experiment was performed in an ice bath reaction with a volume of 10 μl. Wherein, the PCR reaction system comprises: 1 μL cDNA, 0.2 μL LPrimer, 0.2 μL Primer R, 5 μL2×M5 HiPer SYBR Premix EsTaq, 3.6 μL ddH 2 O。
The amplification procedure was: pre-denaturation at 95℃for 10min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 1min for 35 cycles, then carrying out dissolution curve analysis at 65-95 ℃, increasing the temperature of 0.5 ℃ for 5s for each cycle to obtain a melting temperature, collecting a dissolution curve fluorescence signal, and automatically reading Ct value data by a CFX96TM Real Time System fluorescence quantitative PCR instrument. At each time point, 3 technical replicates were performed per stress.
2.4 data analysis
The cycle threshold (Ct value) of each reference gene was obtained by RT-qPCR and analyzed by GeNorm, normFinder, bestKeeper and RefFinder software. When data analysis is performed using gemm and normFinder, the data is first analyzed by the formula q=2 -ΔCt (ΔCt＝Ct sample -Ct min ) The Ct value is converted to a relative quantitative Q value. Ct is (Ct) sample Is the Ct value of the housekeeping gene in each stress treatment; ct is (Ct) min The Ct value of this gene was indicated to be the lowest in each stress treatment. The expression stability measurement (M) value of each candidate reference gene was then calculated using the geonorm program. The Best Keeper directly uses Ct values for stability analysis without additional conversion steps to measure Coefficient of Variation (CV) versus Standard Deviation (SD). Finally, the refFinder integrates the 3 methods, and calculates the geometric mean and the stability comprehensive sequencing index of each reference gene. A lower index value indicates a higher stability of the reference gene. By paired coefficient of variation V n /V n+1 The best reference base factor is determined. It is generally considered that when V n /V n+1 When the value of (2) is less than 0.15, no new reference gene need be introduced; otherwise, the (n+1) th reference gene is required.
2.5 verification of reference Gene by target Gene
The study uses different target genes under different stresses to verify reference genes. The PIP1 gene is adopted for drought stress, the PAL gene is adopted for salt stress, the Cor413 gene is adopted for low temperature stress, the ALMT gene is adopted for aluminum stress, and the BAR gene is adopted for glufosinate stress. To verify the selected reference gene, the expression levels of the two genes were analyzed using the two most stable reference genes and the one most unstable reference gene under each stress, using 2 -ΔΔCt The method performs the calculation. Each treatment was performed with 3 biological replicates, each biological replicate was subjected to 3 technical replicates.
Table 45 nucleotide sequences of the target genes
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3. Experimental results
3.1 analysis of Ct value of reference Gene
The Ct value of a reference gene is inversely proportional to the expression level of the gene. The larger the Ct value of the reference gene, the lower the expression level of the target gene in the sample. The highest and lowest limits of the box graph represent the maximum and minimum Ct values, respectively, and the lowest represents the highest expression level. Ct values of the reference genes under different stresses are shown in FIG. 2.
As can be seen from fig. 2, GADPH has the lowest Ct value under salt stress, drought stress and aluminum stress, indicating that its expression abundance is highest.
3.2 evaluation of expression stability of reference Gene
3.2.1GeNorm software analysis
The expression stability of the reference gene was analyzed by gemm software and the M value was calculated, the lower the M value, the higher the stability of the reference gene. The stability of expression of each reference gene under different stresses is shown in FIG. 3.
As can be seen from fig. 3, RIP and MD genes are the most stably expressed genes under salt stress; UBC and MD gene expression are most stable under low temperature stress; RIP and MD genes are the most stable genes under drought stress; RIP and MD genes are the most stable genes under aluminum stress, while 50S genes are the least stable; among glufosinate stresses, RIP and ADP genes are the most stable genes to be expressed, and 50S genes are the least stable to be expressed.
3.2.2NormFinder software analysis
And calculating the stable value of the candidate reference gene by using NormFinder software, and carrying out gene evaluation, wherein the stable value is inversely related to the stability of the reference gene. The lower the value, the higher the stability. The stable values of the reference genes under different stresses are shown in Table 5,
TABLE 5 calculation of expression stability values of 13 reference genes of eremochloa ophiuroides by NormFinder
UBC is known to be the most stable gene expressed under low temperature stress, ANI is the most stable gene under glufosinate stress, and MD is the most stable gene under salt stress, drought stress and aluminum stress.
3.2.3Bestkeeper analysis
And calculating the value of the reference gene CV+ -SD, and when the value of (CV+SD) - (CV-SD) is minimum, obtaining the reference gene with the best expression stability. The stable values of each reference gene under different stresses are shown in Table 6.
TABLE 6 calculation of expression stability values of 13 reference genes of eremochloa ophiuroides by BestKeeper
As shown in table 6, RIP is a gene that is best expressed under aluminum stress and glufosinate stress, UBC is a reference gene that is well expressed under low temperature stress, MD is a reference gene that is well expressed under salt stress, and GADPH is a gene that is most stably expressed under drought stress.
3.2.4RefFinder analysis
With the online website http:// bloom. Cn/RefFinder/? type = reference the geNorm, normFinder and bestrepper data ranking was analyzed in integration and ranked in synthesis. The sorting results are shown in Table 7.
TABLE 7 calculation of expression stability values of 13 reference genes of eremochloa ophiuroides by using Refinder
As can be seen from table 7: MD and RIP are genes with the best expression stability under drought stress; UBC and MD are the genes with the best expression stability under low temperature stress; MD and RIP are the genes with the best expression stability under salt stress; UBC and MD are the genes with the best expression stability under aluminum stress; RIP and ANI are the genes with the best expression stability under glufosinate stress; the expression stability of the HSP70 gene is the worst under drought stress and low temperature stress, and the stability of H3 under salt stress and glufosinate-ammonium stress is the worst; suS has the worst stability under aluminum stress.
The optimal reference base factor is determined by gem software analysis with the paired coefficients of variation Vn/Vn+1. It is generally believed that when the value of Vn/Vn+1 is less than 0.15, no new reference gene need be introduced; otherwise, the (n+1) th reference gene is required. As can be seen from the analysis by the gemm software, V2/V3<0.15, only two genes are required for verification.
In conclusion, MD and RIP are selected as reference genes under the drought stress of eremochloa ophiuroides, UBC and MD are selected as reference genes under the low-temperature stress of eremochloa ophiuroides, MD and RIP are selected as reference genes under the salt stress of eremochloa ophiuroides, UBC and MD are selected as reference genes under the aluminum stress of eremochloa ophiuroides, and RIP and ANI are selected as reference genes under the ammonium phosphine stress of eremochloa ophiuroides.
EXAMPLE 2 detection of target Gene expression level after normalization of reference Gene
In order to verify the reliability of reference genes in software program analysis, one target gene was selected for verification under each stress, and two reference genes with the most stable expression and one reference gene with the least stable expression in Table 7 of example 1 were selected for expression pattern analysis, and the result was 2 -ΔΔCt The method performs the calculation. The results are shown in FIG. 4a, FIG. 4b, FIG. 4c, FIG. 4d, FIG. 4 e. The histograms shown in FIGS. 4a, 4b, 4c, 4d and 4e represent the normalized expression levels of the target genes of different reference genes under different stresses.
As can be seen from fig. 4a, under drought stress, the PIP1 gene was used for verification, and when normalized by using the optimal reference gene combination (MD and RIP genes), the expression level of PIP1 tended to be increased and decreased again, which is consistent with the expression tendencies of the most stable genes MD, RIP; when normalization is performed by using the least stable gene HSP70, the expression level of PIP1 shows a tendency of rising and then falling, and the expression level reaches the maximum value at stress for 6 hours, which is approximately 30 times of 0 hour;
as can be seen from FIG. 4b, under salt stress, the PAL gene was used for verification, and when the verification was performed using the most stably expressed gene combination (MD and RIP genes), the trend of the expression amount of the PAL gene showed a trend of increasing and then decreasing substantially; however, when the least stable gene H3 is used, the expression level of the PAL gene is maximized at stress for 12 hours and then gradually decreased;
as can be seen from fig. 4c, when the normalization is performed using the most stable reference genes (UBC and MD genes) under low temperature stress, it was found that the expression level of the Cor413 gene tended to increase first and then decrease and then gradually increase; when normalization was performed using the gene HSP70 which is the least stably expressed, the expression level of the Cor413 gene did not have a significant trend;
as can be seen from fig. 4d, under aluminum stress, the expression trend of the ALMT gene was not significantly changed and the expression level was extremely high when the normalization was performed using the most unstable gene SuS by using ALMT gene for verification; whereas when normalized using the most stably expressed genes (UBC or MD genes), the expression level of ALMT gene is lower;
as can be seen from fig. 4e, under the stress of glufosinate, the use of the BAR gene for verification, when normalized by the combination of the most stable reference genes (RIP and ANI genes), the expression level of the BAR showed a tendency of decreasing before increasing, and at 72h, the expression level of the BAR gene reached the maximum; when normalization is performed using the most unstable reference gene H3, the BAR gene expression amount tends to gradually decrease.
In conclusion, the reference genes screened by the invention have certain accuracy and reliability, namely, the MD and RIP genes are expressed most stably under drought stress and salt stress, the UBC and MD genes are expressed most stably under low temperature stress, the UBC and MD genes are expressed most stably under aluminum stress, and the RIP and ANI genes are expressed most stably under glufosinate stress.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (8)

  1. The application of UBC gene as reference gene in detecting the gene expression level of eremochloa ophiuroides under abiotic stress of eremochloa ophiuroides is characterized in that the abiotic stress is aluminum stress or low temperature stress; the nucleotide sequence of the UBC gene is shown as SEQ ID NO. 9.
  2. 2. The specific primer of the internal reference gene UBC as claimed in claim 1, wherein the forward primer and the reverse primer are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.
  3. The application of the ANI gene as an internal reference gene in detecting the gene expression quantity of the centipede grass under the abiotic stress of the centipede grass is characterized in that the abiotic stress is glufosinate stress; the nucleotide sequence of the ANI gene is shown as SEQ ID NO. 10.
  4. 4. A specific primer for an internal reference gene ANI according to claim 3, characterized in that the forward primer and the reverse primer are shown in SEQ ID No.3 and SEQ ID No.4, respectively.
  5. The application of RIP genes as reference genes in detecting the expression quantity of eremochloa ophiuroides gene under abiotic stress of eremochloa ophiuroides is characterized in that the abiotic stress is drought stress, glufosinate stress or salt stress; the nucleotide sequence of the RIP gene is shown as SEQ ID NO. 11.
  6. 6. The specific primer of the reference gene RIP as claimed in claim 5, wherein the forward primer and the reverse primer are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively.
  7. The application of MD genes as reference genes in detecting the gene expression quantity of eremochloa ophiuroides under abiotic stress of eremochloa ophiuroides is characterized in that the abiotic stress is salt stress, low-temperature stress, drought stress or aluminum stress; the nucleotide sequence of the MD gene is shown as SEQ ID NO. 12.
  8. 8. The specific primer of the reference gene MD of claim 7, wherein the forward primer and the reverse primer are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively.
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