Reference Gene ACT, Its Primers and Application under Abiotic Stress in Eremochloa ophiuroides

Abstract

The present invention provides an internal reference gene ACT of Eremochloa ophiuroides under abiotic stress, its primers and applications, belonging to the field of molecular biology. The ACT gene in the present invention is used as an internal reference gene in the application of detecting the gene expression level of Eremochloa ophiuroides under abiotic stress, and the abiotic stress is glufosinate stress. The present invention also provides specific primers for the ACT internal reference gene. The specificity of the specific primers is strong, which can greatly improve the detection efficiency when detecting the gene expression level of Eremochloa ophiuroides and improve the credibility of the detection results.

Description

[0001] This application is a divisional application. The application number of the original application is: 202211536965.3, the application date is December 2, 2022, and the invention title is: Reference Genes, Primers Thereof and Applications under Abiotic Stresses of Eremochloa ophiuroides Technical Field

[0002] The present invention belongs to the field of molecular biology, and particularly relates to the reference gene ACT of Eremochloa ophiuroides, primers thereof and applications Background Art

[0003] Eremochloa ophiuroides (Munro) Hack is a warm-season perennial herb with a C4 structure, belonging to the genus Eremochloa in the subfamily Panicoideae of the family Poaceae. It has a fast spreading speed and well-developed stolons, and has the characteristics of low plant height, low water and fertilizer requirements, tolerance to extensive management, and strong disease resistance. It is an ideal turfgrass for urban and rural greening, slope greening, soil and water conservation and ecological governance, and is one of the three major warm-season turfgrasses in the world. At present, extreme weather such as drought and cold occurs frequently, and factors such as acidic soil in the south and soil salinity in coastal areas seriously affect the normal growth of Eremochloa ophiuroides. There is a lack of new varieties resistant to glufosinate, which puts forward new requirements for the later breeding of Eremochloa ophiuroides. Therefore, it is very necessary to breed Eremochloa ophiuroides varieties resistant to stress, salt, acid and glufosinate

[0004] Selecting stress-resistant genes such as drought resistance and cold resistance and glufosinate-resistant genes is of great significance for studying the related molecular mechanisms of Eremochloa ophiuroides 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, with the characteristics of high sensitivity, good repeatability, strong specificity and high throughput, and is widely used in gene expression research. Stable expression of reference genes is an important prerequisite for accurate results of gene expression analysis. An ideal reference gene should be stably expressed in all cells, at different growth stages and under various physiological states. However, the stability of traditional housekeeping genes varies. Therefore, according to specific experimental conditions, screening suitable reference genes is the key to ensuring accurate and reliable quantification of target gene expression

[0005] At present, there is no report on the screening of reference genes for cold resistance, drought resistance, salt tolerance, aluminum tolerance and glufosinate tolerance of Eremochloa ophiuroides. Therefore, it is very necessary to develop a set of reference genes of Eremochloa ophiuroides under low temperature, drought, salt, aluminum and glufosinate stresses to ensure the breeding effect of Eremochloa ophiuroides Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a set of reference genes and their primers and applications for Eremochloa ophiuroides under abiotic stress. The present invention uses qRT-PCR technology to detect the expression stability of 13 reference genes of Eremochloa ophiuroides under low temperature, drought, salt, aluminum and glufosinate stress. Taking the gene stability evaluation analyzed by three software, namely geNorm, NormFinder and BestKeeper, as a reference and the gene stability evaluation by RefFinder software as the main basis, and comprehensively analyzing the results of the software, reference genes stably expressed under each stress are respectively screened. Two genes are screened for each stress treatment, and a total of 10 reference genes are screened, solving the current situation that there is no reference gene in the quantitative PCR detection of Eremochloa ophiuroides.

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

[0008] Application of the UBC gene as a reference gene in detecting the expression level of Eremochloa ophiuroides genes under abiotic stress of Eremochloa ophiuroides;

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

[0010] Specific primers for the reference gene UBC, the forward primer and the reverse primer are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

[0011] Application of the ANI gene as a reference gene in detecting the expression level of Eremochloa ophiuroides genes under abiotic stress of Eremochloa ophiuroides;

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

[0013] Specific primers for the reference gene ANI, the forward primer and the reverse primer are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0014] Application of the RIP gene as a reference gene in detecting the expression level of Eremochloa ophiuroides genes under abiotic stress of Eremochloa ophiuroides;

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

[0016] Specific primers for the reference gene RIP, the forward primer and the reverse primer are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0017] Application of the MD gene as a reference gene in detecting the expression level of Eremochloa ophiuroides genes under abiotic stress of Eremochloa ophiuroides;

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

[0019] Specific primers for the internal reference gene MD, the forward primer and the reverse primer are shown in 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] Based on the previous transcriptome sequencing of Eremochloa ophiuroides, the present invention has mined a group of internal reference genes of Eremochloa ophiuroides, and designed primers for each internal reference gene using the base sequences of these internal reference genes; the present invention not only solves the current situation that there is no internal reference gene in the quantitative PCR detection of existing Eremochloa ophiuroides, but also can improve the stability, reliability and repeatability of the gene expression analysis research of Eremochloa ophiuroides when the primers of the internal reference genes are respectively used for the gene expression analysis under low temperature, drought, salt, aluminum and glufosinate stress of Eremochloa ophiuroides. At the same time, the primers of the internal reference genes designed by the present invention have strong specificity, can greatly improve the detection efficiency when detecting the gene expression level of Eremochloa ophiuroides, and improve the credibility of the detection results. Brief Description of the Drawings

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

[0023] Figure 2 For the Ct values of each internal reference gene under different stresses;

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

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

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

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

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

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

[0030] Figure 4 In which e represents the expression level of the target gene after normalization of different internal reference genes under glufosinate stress. Specific embodiments

[0031] The present invention provides the application of the UBC gene as a reference gene in detecting the gene expression level of Eremochloa ophiuroides under abiotic stress; the abiotic stress is aluminum stress or low temperature stress.

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

[0033] The present invention provides the application of the ANI gene as a reference gene in detecting the gene expression level of Eremochloa ophiuroides under abiotic stress; the abiotic stress is glufosinate stress.

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

[0035] The present invention provides the application of the RIP gene as a reference gene in detecting the gene expression level of Eremochloa ophiuroides under abiotic stress; the abiotic stress is drought stress, glufosinate stress or salt stress.

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

[0037] The present invention provides the application of the MD gene as a reference gene in detecting the gene expression level of Eremochloa ophiuroides under abiotic stress; 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, and the forward primer and the reverse primer are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively.

[0039] In the present invention, the method for detecting the gene expression level of Eremochloa ophiuroides under abiotic stress is fluorescence quantitative PCR.

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

[0041] Table 1 Base sequences of each reference gene

[0042]

[0043]

[0044]

[0045]

[0046]

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

[0048] Table 2 Specific primers for each reference gene

[0049]

[0050]

[0051] wherein F represents the forward primer and R represents the reverse primer.

[0052] The technical solutions provided by the present invention will be 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 treatments

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

[0056] 2. Methods

[0057] 2.1 Extraction and reverse transcription of total RNA

[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 (60S Ribosomal 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] Wherein, F is the forward primer and R is the 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) produced by Beijing Polymer Beauty Biotechnology Co., Ltd. and a real-time PCR system (Bio-Rad, USA). The experiment was carried out in an ice-bath reaction with a volume of 10 μL. Among them, the PCR reaction system included: 1 μL of cDNA, 0.2 μL of Primer, 0.2 μL of Primer R, 5 μL of 2×M5 HiPer SYBR Premix EsTaq, and 3.6 μL of ddH 2 O.

[0069] The amplification program was: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s, annealing at 55°C for 1 min, 35 cycles, and then melting curve analysis was performed at 65 - 95°C, increasing by 0.5°C per cycle for 5 s to obtain the melting temperature, and the fluorescence signal of the melting curve was collected. The Ct value data was automatically read by the CFX96TM Real Time System fluorescence quantitative PCR instrument. At each time point, 3 technical replicates were performed for each stress.

[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 performing data analysis with GeNorm and NormFinder, first, through the formula Q = 2 -ΔCt (ΔCt = Ct sample -Ct min ), the Ct value was converted into the relative quantification Q value. Ct sample is the Ct value of the housekeeping gene in each stress treatment; Ct minIndicates the lowest Ct value of this gene in each stress treatment. Then, the expression stability measurement (M) value of each candidate reference gene was calculated using the GeNorm program. 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 of each reference gene. A lower index value indicates higher stability of the reference gene. Using the pairwise variation coefficient V n / V n+1 to determine the optimal number of reference genes. Generally, when the value of V n / V n+1 is less than 0.15, no new reference gene needs to be introduced; otherwise, the (n + 1)th reference gene is required.

[0072] 2.5 Verification of reference genes by target genes

[0073] In this study, different target genes were used to verify the reference genes under different stresses. The PIP1 gene was used for drought stress, the PAL gene was used for salt stress, the Cor413 gene was used for low-temperature stress, the ALMT gene was used for aluminum stress, and the BAR gene was used for glufosinate stress. To verify the selected reference genes, the expression levels of two genes were analyzed using the two most stable reference genes and the least stable reference gene under each stress, and the 2 -ΔΔCt method was used for calculation. There were 3 biological replicates for each treatment, and 3 technical replicates were performed for each biological replicate.

[0074] Table 4 Base sequences of 5 target genes

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] 3. Experimental results

[0082] 3.1 Analysis of Ct values of 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 level of the target gene in the sample. The upper and lower limits of the box plot represent the maximum and minimum Ct values respectively, and the shortest one indicates the highest expression level. The Ct values of each reference gene under different stresses are as Figure 2 shown.

[0084] As Figure 2 can be seen, GADPH has the lowest Ct value under salt stress, drought stress and aluminum stress, indicating that its expression abundance is the highest.

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

[0086] 3.2.1 geNorm software analysis

[0087] The expression stability of 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. The expression stability of each reference gene under different stresses is as Figure 3 shown.

[0088] As Figure 3 can be seen, under salt stress, RIP and MD genes are the most stable genes; under low temperature stress, UBC and MD genes are the most stable; under drought stress, RIP and MD genes are the most stable genes; under aluminum stress, RIP and MD genes are the most stable genes, while the 50S gene is the least stable; under glufosinate stress, RIP and ADP genes are the most stable genes, and the 50S gene has the worst expression stability.

[0089] 3.2.2 NormFinder software analysis

[0090] The stability values of candidate reference genes were calculated by NormFinder software and gene evaluation was carried out. The stability value is 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 Eremochloa ophiuroides reference genes calculated by 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] 3.2.3 Bestkeeper analysis

[0096] Calculate the CV±SD value of the reference gene. When the value of (CV + SD)-(CV - SD) is the smallest, it is 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 Eremochloa ophiuroides reference genes calculated by BestKeeper

[0098]

[0099]

[0100] As can be seen from Table 6, RIP is the gene with the best expression under aluminum stress and glufosinate stress. In addition, UBC is a reference gene with good expression under low temperature stress, MD is a reference gene with good expression under salt stress, and GADPH is the gene with the most stable expression under drought stress.

[0101] 3.2.4 RefFinder analysis

[0102] Use the online website http: / / blooge.cn / RefFinder / ?type=reference to integrate and analyze the data sorting of geNorm, NormFinder and BestKeeper and conduct a comprehensive sorting. The sorting results are shown in Table 7.

[0103] Table 7 Expression stability values of 13 Eremochloa ophiuroides reference genes calculated by ReFinder

[0104]

[0105]

[0106] As can be seen from 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 stress; The expression stability of the HSP70 gene is the worst under drought stress and low temperature stress, and the stability of H3 is the worst under salt stress and glufosinate stress; The stability of SuS is the worst under aluminum stress.

[0107] Analysis by geNorm software was used to determine the optimal number of reference genes based on the pairwise variation coefficient Vn / Vn+1. Generally, when the value of Vn / Vn+1 is less than 0.15, there is no need to introduce a new reference gene; otherwise, the (n + 1)th reference gene is required. Analysis by geNorm software showed that V2 / V3 < 0.15, so only two genes are needed for verification.

[0108] In summary, MD and RIP were selected as reference genes under drought stress in Eremochloa ophiuroides, UBC and MD were selected as reference genes under low temperature stress in Eremochloa ophiuroides, MD and RIP were selected as reference genes under salt stress in Eremochloa ophiuroides, UBC and MD were selected as reference genes under aluminum stress in Eremochloa ophiuroides, and RIP and ANI were selected as reference genes under glufosinate stress in Eremochloa ophiuroides.

[0109] Detection of the expression level of target genes after normalization of reference genes in Example 2

[0110] To verify the reliability of the reference genes in the software program analysis, one target gene was selected for verification under each stress. Two of the most stable reference genes and one of the most unstable reference genes in Table 7 of Example 1 were selected for expression pattern analysis, and the results were calculated using the 2 -ΔΔCt method. The results are as Figure 4 shown. Figure 4 The bar graphs shown represent the expression levels of target genes normalized by different reference genes under different stresses.

[0111] As can be seen from Figure 4 a in, under drought stress, the PIP1 gene was used for verification. When normalized using the optimal reference gene combination (MD and RIP genes), the expression level of PIP1 showed a trend of rising, decreasing, rising again, and then decreasing again, which was consistent with the expression trends of the most stable genes MD and RIP; when normalized using the most unstable gene HSP70, the expression level of PIP1 showed a trend of rising first and then decreasing, reaching the maximum value at 6 h of stress, which was approximately 30 times that at 0 h;

[0112] As can be seen from Figure 4 b in, under salt stress, the PAL gene was used for verification. When verified using the most stable gene combination (MD and RIP genes), the expression level trend of the PAL gene basically showed a trend of increasing first and then decreasing; however, when using the most unstable gene H3, the expression level of the PAL gene reached the maximum at 12 h of stress and then gradually decreased;

[0113] As can be seen from Figure 4As can be seen from c in [reference], under low-temperature stress, when the Cor413 gene was verified and normalized using the most stable reference genes (UBC and MD genes), it was found that the expression level of the Cor413 gene first increased, then decreased, and then gradually increased; when normalized using the gene HSP70 with the most unstable expression, the expression level of the Cor413 gene did not show an obvious trend.

[0114] As can be seen from Figure 4 d in [reference], under aluminum stress, when the ALMT gene was verified and normalized using the most unstable gene SuS, the expression trend of the ALMT gene did not change significantly and the expression level was extremely high; while when normalized using the most stable expression genes (UBC or MD genes), the expression level of the ALMT gene was relatively low.

[0115] As can be seen from Figure 4 e in [reference], under glufosinate stress, when the BAR gene was verified and normalized using the combination of the most stable reference genes (RIP and ANI genes), the expression level of BAR first decreased and then increased, and at 72 h, the expression level of the BAR gene reached the maximum value; when normalized using the most unstable reference gene H3, the expression level of the BAR gene showed a gradually decreasing trend.

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

[0117] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a primer pair for detecting a reference gene in detecting the expression level of Eremochloa ophiuroides genes under abiotic stress of Eremochloa ophiuroides, characterized in that, the abiotic stress is glufosinate stress; the reference gene is the ACT gene; the sequences of the forward primer and the reverse primer of the primer pair are shown in SEQ ID NO.17 and SEQ ID NO.18 respectively.

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