Internal reference gene UBC and its primers and applications under abiotic stress of Centipedegrass

Abstract

The present invention provides an internal reference gene UBC under abiotic stress of centipedegrass and primers and applications thereof, belonging to the field of molecular biology. The UBC gene in the present invention is used as an internal reference gene in detecting the expression amount of the centipedegrass gene under abiotic stress of centipedegrass, and the abiotic stress is salt stress. The present invention also provides specific primers for the UBC internal reference gene, which have strong specificity, can greatly improve the detection efficiency when detecting the expression amount of the centipedegrass gene, and improve the credibility of the detection result.

Description

[0001] This application is a divisional application. The application number of the original application is: 202211536965.3, the application date is 2022.12.02, and the name of the invention is: Internal reference genes and primers and applications of Ervatamia oxyphylla under abiotic stress. Technical Field

[0002] The invention belongs to the field of molecular biology, and in particular relates to an internal reference gene UBC of Ervatamia oxyphylla and primers and applications thereof. Background Art

[0003] Eremochloa ophiuroides (Munro) Hack is a warm-season perennial herb with a C4 structure. It belongs to the genus Eremochloa of the subfamily Panicoideae of the Poaceae family. It spreads quickly, has well-developed stolons, has low plants, requires less water and fertilizer, tolerates extensive management, and has strong disease resistance. It is an ideal lawn grass for urban and rural greening, slope greening, soil and water conservation, and ecological management. It is one of the three major warm-season lawn grasses in the world. At present, the frequent occurrence of extreme weather such as drought and cold, acidic soil in the south and salinity in coastal land have seriously affected the normal growth of Eremochloa ophiuroides, and there is a lack of new varieties resistant to glufosinate ammonium. These have put forward new requirements for the later breeding of Eremochloa ophiuroides. It is very necessary to breed Eremochloa ophiuroides varieties that are resistant to stress, salt, acid, and glufosinate.

[0004] The selection of stress resistance genes such as drought resistance and cold resistance and glufosinate-ammonium resistance genes is of great significance for studying the relevant molecular mechanisms of Ervatamia oxyphylla and promoting the breeding of related new varieties. Real-time fluorescence quantitative PCR (qRT-PCR) is an important experimental method for accurate nucleic acid quantification. It has the characteristics of high sensitivity, good repeatability, strong specificity and high throughput, and is widely used in gene expression research. Stably expressed internal reference genes are an important prerequisite for accurate gene expression analysis results. Ideal internal reference genes should have the characteristics of stable expression in all cells, different growth stages and various physiological states, but the stability of traditional housekeeping genes varies. Therefore, according to specific experimental conditions, screening suitable internal reference genes is the key to ensuring accurate and reliable quantitative expression of target genes.

[0005] At present, there are no reports on the screening of reference genes for cold resistance, drought resistance, salt tolerance, aluminum resistance and glufosinate resistance of Ervatamia oxyphylla. Therefore, it is very necessary to develop a set of reference genes for Ervatamia oxyphylla under low temperature, drought, salt, aluminum and glufosinate stress to ensure the breeding effect of Ervatamia oxyphylla. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a group of internal reference genes and primers and applications of abiotic stress of Eremochloa sutchuenensis. The present invention uses qRT-PCR technology to detect the expression stability of 13 Eremochloa sutchuenensis internal reference genes under low temperature, drought, salt, aluminum and glufosinate-ammonium stress, and takes the gene stability evaluation analyzed by three softwares, geNorm, NormFinder and BestKeeper, as a reference, and takes the gene stability evaluation by RefFinder software as the main basis. According to the comprehensive software analysis results, the internal reference genes stably expressed under each stress are screened out respectively, and two genes are screened for each stress treatment, and a total of 10 internal reference genes are screened out; the current situation that there is no internal reference gene in the quantitative PCR detection of Eremochloa sutchuenensis is solved.

[0007] To achieve the above object, the present invention is implemented by the following technical solutions:

[0008] The application of UBC gene as internal reference gene in detecting the gene expression of Ervatamia oxyphylla under abiotic stress;

[0009] Preferably, the abiotic stress is aluminum stress or low temperature stress.

[0010] The specific primers of the internal reference gene UBC, the forward primer and the reverse primer are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively.

[0011] The application of ANI gene as an internal reference gene in detecting the gene expression of Ervatamia oxyphylla under abiotic stress;

[0012] Preferably, the abiotic stress is glufosinate-ammonium stress.

[0013] The specific primers of the internal reference gene ANI, the forward primer and the reverse primer are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0014] Application of RIP gene as internal reference gene in detecting the gene expression of Ervatamia oxyphylla under abiotic stress;

[0015] Preferably, the abiotic stress is drought stress, glufosinate stress or salt stress.

[0016] The specific primers of the internal reference gene RIP, the forward primer and the reverse primer are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0017] Application of MD gene as internal reference gene in detecting the gene expression of Ervatamia oxyphylla under abiotic stress;

[0018] Preferably, the abiotic stress is salt stress, low temperature stress, drought stress or aluminum stress.

[0019] The specific primers of the internal reference gene MD, the forward primer and the reverse primer are shown as SEQ ID NO.7 and SEQ ID NO.8 respectively.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention excavates a group of reference genes of Pseudo-Eclectic Grass based on the early transcriptome sequencing of Pseudo-Eclectic Grass, and designs primers of each reference gene using the base sequences of these reference genes; the present invention not only solves the current situation that there is no reference gene in the quantitative PCR detection of Pseudo-Eclectic Grass, but also when the primers of the reference genes are used for gene expression analysis under the stress of low temperature, drought, salt, aluminum and glufosinate ammonium of Pseudo-Eclectic Grass, the stability, reliability and repeatability of the gene expression analysis research of Pseudo-Eclectic Grass can be improved. At the same time, the specificity of the primers of the reference genes designed by the present invention is strong, which can greatly improve the detection efficiency when detecting the gene expression amount of Pseudo-Eclectic Grass, and improve the credibility of the detection result. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Primer specificity for 13 candidate internal reference genes (from left to right, UBC, GADPH, ACT, SuS, ANI, ADP, CYP, H3, 50S, RIP, MD, CP, HSP70)

[0023] Figure 2 is the Ct value of each reference gene under different stresses;

[0024] Figure 3 is the expression stability of each reference gene under different stresses;

[0025] Figure 4 is the expression level of the target gene after normalization of the internal reference gene under different stresses;

[0026] Figure 4 a in represents the expression level of the target gene after normalization of different internal reference genes under drought stress;

[0027] Figure 4 b in represents the expression level of the target gene after normalization of different reference genes under salt stress;

[0028] Figure 4 The c in represents the expression level of the target gene after normalization of different internal reference genes under low temperature stress;

[0029] Figure 4 The d in represents the expression level of the target gene after normalization of different internal reference genes under aluminum stress;

[0030] Figure 4 The e in represents the expression level of the target gene after normalization of different reference genes under glufosinate stress. DETAILED DESCRIPTION

[0031] The present invention provides an application of a UBC gene as an internal reference gene in detecting the expression amount of a gene of Ervatamia oxyphylla under abiotic stress of Ervatamia oxyphylla; the abiotic stress is aluminum stress or low temperature stress.

[0032] The present invention provides specific primers for the UBC gene, wherein the forward primer and the reverse primer are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively.

[0033] The present invention provides the application of an ANI gene as an internal reference gene in detecting the expression amount of an Ervatamia oxyphylla gene under an abiotic stress of Ervatamia oxyphylla; the abiotic stress is glufosinate-ammonium stress.

[0034] The present invention provides specific primers for the ANI gene, wherein the forward primer and the reverse primer are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0035] The present invention provides the use of a RIP gene as an internal reference gene in detecting the expression amount of a gene of Centipedegrass under abiotic stress of Centipedegrass; the abiotic stress is drought stress, glufosinate-ammonium stress or salt stress.

[0036] The present invention provides specific primers for the RIP gene, wherein the forward primer and the reverse primer are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0037] The present invention provides an application of an MD gene as an internal reference gene in detecting the expression amount of an Ervatamias sutchuenensis gene under abiotic stress of Ervatamias sutchuenensis; the abiotic stress is salt stress, low temperature stress, drought stress or aluminum stress.

[0038] The present invention provides specific primers for the MD gene, wherein the forward primer and the reverse primer are shown as SEQ ID NO.7 and SEQ ID NO.8 respectively.

[0039] In the present invention, the method for detecting the gene expression amount of Ervatamia oxyphylla under abiotic stress of Ervatamia oxyphylla is a fluorescent quantitative PCR method;

[0040] In the present invention, the nucleotide sequences of the internal reference genes UBC, ANI, RIP and MD are shown in Table 1.

[0041] Table 1 Base sequences of reference genes

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] In the present invention, the specific primers of each internal reference gene are shown in Table 2.

[0048] Table 2 Specific primers for each internal reference gene

[0049]

[0050]

[0051] The F represents a forward primer, and the R represents a reverse primer.

[0052] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] Example 1

[0054] 1. Materials and processing

[0055] The seeds of Ervatamia oxyphylla were soaked in 10% sodium hydroxide for 5 min, then rinsed with sterile distilled water, and sown in 20×15×5 cm plastic pots, 1.3 g per pot, and poured with 50% Hoagland nutrient solution. The temperature was 23°C and 19°C respectively during the day and night, the photoperiod was 12 h, the relative humidity was 75%, and the light intensity was 250 (umol·m -2 ·s -1 ) were cultured in a growth chamber for 90 days, and then subjected to 4°C low temperature stress, 20% PEG-6000 drought stress, 200mM NaCl salt stress, 100μmMAlCl3 aluminum stress, and 6μL / mL glufosinate stress, respectively. Each treatment group was replicated 3 times (i.e., 3 pots). Leaf samples were collected at 0, 0.5, 1.5, 3, 6, 12, 24, 48, and 72h after each treatment, and the collected samples were immediately frozen in liquid nitrogen and stored in a refrigerator at -80°C for subsequent RNA extraction.

[0056] 2. Methods

[0057] 2.1 Total RNA extraction and reverse transcription

[0058] Leaf tissues of Pseudocentema sutchuenensis weighing 0.1 g were crushed with a tissue grinder, and total RNA was extracted using the M5 HiPer Plant Complex Mini Kit (Beijing Polymer Biotech 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 the M5 Superplus QPCR RT kit with gDNA Remover (Beijing Polymer Biotech Co., Ltd.), and stored at -80°C for further analysis.

[0059] 2.2 Design and verification of specific primers

[0060] Based on the transcriptome sequencing of Pseudo-Egg clover, 13 candidate reference genes were selected, namely: UBC (Ubiquitin-conjugating enzyme), GADPH (Glyceraldehyde-3-phosphate dehydrogenase), ACT (Actin), SuS (sucrose synthase), ANI (Alkaline and neutral invertase), ADP (ADP-ribosylation factor), CYP (Cyclophilin), H3 (Histone H3), 50S (50S ribosomal protein L2), RIP (60SRibosomal protein L2), MD (Malate dehydrogenase), CP (Chaperone protein), HSP70 (Heat shock70kDaprotein: heat shock 70k Da protein).

[0061] The specific primers of the 13 internal reference genes were designed by Primer Quest software and synthesized by Youkang Biotechnology Co., Ltd. (Chengdu, China). The specificity of the primers was verified by the melting curve of the RT-qPCR reaction. Figure 1 .

[0062] The primer sequences of candidate internal reference genes in RT-qPCR are shown in Table 3.

[0063] Table 3 Primer sequences of 13 candidate internal reference genes

[0064]

[0065]

[0066] The F is a forward primer, and R is a reverse primer.

[0067] 2.3 Real-time fluorescence quantitative PCR

[0068] Quantitative analysis was performed using 2×M5 HiPer SYBR Premix EsTaq (with Tli RnaseH) and a real-time PCR system (Bio-Rad, USA) produced by Beijing PolyMei Biotechnology Co., Ltd. The experiment was performed in a 10 μL ice bath reaction. The PCR reaction system included: 1 μL cDNA, 0.2 μL Primer, 0.2 μL Primer R, 5 μL 2×M5 HiPer SYBR Premix EsTaq, and 3.6 μL ddH2O.

[0069] The amplification program was as follows: 95°C pre-denaturation for 10 min; 95°C denaturation for 15 s, 55°C annealing for 1 min, 35 cycles, and then 65-95°C melting curve analysis, with each cycle increasing by 0.5°C for 5 s to obtain the melting temperature, collecting the melting curve fluorescence signal, and the Ct value data was automatically read by the CFX96TM Real Time System fluorescence quantitative PCR instrument. At each time point, each stress was repeated three times.

[0070] 2.4 Data Analysis

[0071] The cycle threshold (Ct value) of each reference gene was obtained by RT-qPCR and analyzed by GeNorm, NormFinder, BestKeeper and RefFinder software. When using GeNorm and NormFinder for data analysis, first use the formula Q = 2 -ΔCt (ΔCt=Ct sample -Ct min ), and convert the Ct value into a relative quantitative Q value. sample is the Ct value of the housekeeping gene in each stress treatment; Ct min Indicates that the Ct value of the gene in each stress treatment is the lowest. The GeNorm program is then used to calculate the expression stability measurement (M) value of each candidate reference gene. Best Keeper directly uses the Ct value for stability analysis without additional conversion steps to measure the comparison of the coefficient of variation (CV) and standard deviation (SD). Finally, RefFinder combines the above three methods to calculate the geometric mean and comprehensive stability ranking index for each reference gene. A lower index value indicates that the reference gene is more stable. The pairwise coefficient of variation Vn / V n+1 Determine the optimal number of reference genes. It is generally believed that when V n / V n+1 When the value of is less than 0.15, there is no need to introduce new reference genes; otherwise, the (n+1)th reference gene is required.

[0072] 2.5 Verify the reference gene by the target gene

[0073] This study used different target genes to verify the reference genes under different stresses. PIP1 gene was used for drought stress, PAL gene was used for salt stress, Cor413 gene was used for low temperature stress, ALMT gene was used for aluminum stress, and BAR gene was used for glufosinate stress. To verify the selected reference genes, the two most stable reference genes and the least stable reference gene under each stress were used to analyze the expression levels of the two genes. -ΔΔCt Each treatment was performed with 3 biological replicates and each biological replicate was performed with 3 technical replicates.

[0074] Table 45 target gene base sequences

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] 3. Experimental results

[0082] 3.1 Analysis of Ct values ​​of internal reference genes

[0083] The Ct value of the 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 of the target gene in the sample. The upper and lower limits of the box plot represent the maximum and minimum Ct values, respectively. The lowest value indicates the highest expression. Figure 2 shown.

[0084] Depend on Figure 2 It can be seen that the Ct value of GADPH was the lowest under salt stress, drought stress and aluminum stress, indicating that its expression abundance was the highest.

[0085] 3.2 Evaluation of expression stability of internal reference genes

[0086] 3.2.1geNorm software analysis

[0087] The expression stability of the reference genes was analyzed by geNorm software, and the M value was calculated. The lower the M value, the higher the stability of the reference gene. Figure 3 shown.

[0088] Depend on Figure 3 It can be seen that under salt stress, RIP and MD genes are the most stably expressed genes; under low temperature stress, UBC and MD genes are the most stably expressed genes; under drought stress, RIP and MD genes are the most stably expressed genes; under aluminum stress, RIP and MD genes are the most stably expressed genes, while 50S gene expression is the least stable; in glufosinate stress, RIP and ADP genes are the most stably expressed genes, and 50S gene expression is the worst.

[0089] 3.2.2 NormFinder software analysis

[0090] The stability values ​​of candidate reference genes were calculated by NormFinder software and gene evaluation was performed. The stability value was negatively correlated with the stability of the reference gene. The lower the value, the higher the stability. The stability values ​​of each reference gene under different stresses are shown in Table 5.

[0091] Table 5 Expression stability values ​​of 13 Ervatamia oxyphylla reference genes calculated using NormFinder

[0092]

[0093]

[0094] It can be seen that UBC is the most stable gene 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.

[0095] 3.2.3Bestkeeper Analysis

[0096] The value of CV±SD of the reference gene was calculated. When the value of (CV+SD)-(CV-SD) was the smallest, it was the reference gene with the best expression stability. The stability values ​​of each reference gene under different stresses are shown in Table 6.

[0097] Table 6 Expression stability values ​​of 13 Ervatamia oxyphylla reference genes calculated using BestKeeper

[0098]

[0099]

[0100] As shown in Table 6, RIP is the best expressed gene under aluminum stress and glufosinate stress. In addition, UBC is a well-expressed internal reference gene under low temperature stress, MD is a well-expressed internal reference gene under salt stress, and GADPH is the most stably expressed gene under drought stress.

[0101] 3.2.4 RefFinder analysis

[0102] The online website http: / / blooge.cn / RefFinder / ?type=reference was used to integrate and analyze the geNorm, NormFinder and BestKeeper data rankings and perform a comprehensive ranking. The ranking results are shown in Table 7.

[0103] Table 7 Expression stability values ​​of 13 Ervatamia oxyphylla reference genes calculated using ReFinder

[0104]

[0105]

[0106] As shown in Table 7, MD and RIP are the 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-ammonium stress; the expression stability of HSP70 gene under drought stress and low temperature stress is the worst, H3 has the worst stability under salt stress and glufosinate-ammonium stress; SuS has the worst stability under aluminum stress.

[0107] The optimal number of reference genes was determined by geNorm software analysis using the pairwise coefficient of variation Vn / Vn+1. It is generally believed that when the value of Vn / Vn+1 is less than 0.15, there is no need to introduce new reference genes; otherwise, the (n+1)th reference gene is required. Analysis by geNorm software showed that V2 / V3<0.15, so only two genes were needed for verification.

[0108] In summary, MD and RIP were selected as the internal reference genes under drought stress of Pseudocentropus sutchuenensis, UBC and MD were selected as the internal reference genes under low temperature stress of Pseudocentropus sutchuenensis, MD and RIP were selected as the internal reference genes under salt stress of Pseudocentropus sutchuenensis, UBC and MD were selected as the internal reference genes under aluminum stress of Pseudocentropus sutchuenensis, and RIP and ANI were selected as the internal reference genes under glufosinate-ammonium stress of Pseudocentropus sutchuenensis.

[0109] Example 2 Detection of target gene expression level after normalization of internal reference gene

[0110] In order to verify the reliability of the reference genes in the software program analysis, a target gene was selected for verification under each stress, and the two most stably expressed reference genes and the least stably expressed reference gene in Table 7 of Example 1 were selected for expression pattern analysis. -ΔΔCt The result is as follows: Figure 4 shown. Figure 4 The bar graphs shown in represent the expression levels of the target genes after normalization with different reference genes under different stresses.

[0111] Depend on Figure 4 As shown in a, under drought stress, the PIP1 gene was used for verification. When the best internal reference gene combination (MD and RIP genes) was used for normalization, the expression level of PIP1 showed a trend of rising, falling, rising, and then falling, which was consistent with the expression trend of the most stable genes MD and RIP. When the most unstable gene HSP70 was used for normalization, the expression level of PIP1 showed a trend of rising first and then falling, and the expression reached the maximum value at 6 hours of stress, which was approximately 30 times that of 0 hours.

[0112] Depend on Figure 4 As shown in b, under salt stress, the PAL gene was used for verification. When the most stably expressed gene combination (MD and RIP genes) was used for verification, the expression trend of the PAL gene basically showed a trend of first increasing and then decreasing; however, when the most unstable gene H3 was used, the expression of the PAL gene reached the maximum at 12h of stress, and then gradually decreased;

[0113] Depend on Figure 4 As shown in c, under low temperature stress, the Cor413 gene was used for verification. When the most stable internal reference genes (UBC and MD genes) were used for normalization, it was found that the expression level of the Cor413 gene first increased, then decreased, and then gradually increased; when the most unstable gene HSP70 was used for normalization, the expression level of the Cor413 gene did not have an obvious trend;

[0114] Depend on Figure 4 As shown in d, under aluminum stress, when the ALMT gene was used for verification and the most unstable gene SuS was used for normalization, the expression trend of the ALMT gene did not change significantly and the expression level was extremely high; however, when the most stable expression gene (UBC or MD gene) was used for normalization, the expression level of the ALMT gene was low;

[0115] Depend on Figure 4From e in the figure, we can see that under glufosinate stress, the BAR gene was used for verification. When the most stable internal reference gene combination (RIP and ANI genes) was used for normalization, the expression level of BAR showed a trend of first decreasing and then increasing. At 72h, the expression level of the BAR gene reached its maximum value. When the most unstable internal reference gene H3 was used for normalization, the expression level of the BAR gene showed a trend of gradually decreasing.

[0116] In summary, the internal reference genes screened out in the present invention have certain accuracy and reliability, that is, the expression of MD and RIP genes is most stable under drought stress and salt stress, the expression stability of UBC and MD genes is the best under low temperature stress, the expression stability of UBC and MD genes is the best under aluminum stress, and the expression stability of RIP and ANI genes is the best under glufosinate-ammonium stress.

[0117] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The use of specific primers for detecting internal reference genes in detecting gene expression under abiotic stress of Ervatamia fruticosa, characterized in that: The abiotic stress is salt stress; the internal reference gene is UBC gene; The forward primer and reverse primer of the specific primer of the UBC gene are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively.

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