CN113234804B - Internal reference gene for detecting milRNA of banana fusarium wilt and application thereof - Google Patents

Abstract

The invention discloses an internal reference gene for detecting milRNA of banana fusarium wilt and application thereof, belonging to the field of biotechnology. The internal reference gene is named milR46, and its nucleotide sequence is shown in SEQ ID NO.1. The internal reference gene provided by the invention greatly improves the accuracy of the standardization process of the milRNAs of the banana Fusarium wilt pathogen, is suitable for the detection of milRNAs in different time periods after inoculation, has high stability, and has a good application prospect.

Description

An internal reference gene and its application for the detection of milRNA of Fusarium wilt of banana

technical field

The invention belongs to the field of biotechnology, and in particular relates to an internal reference gene used for detecting milRNA of Fusarium wilt of banana and application thereof.

Background technique

Banana Fusarium wilt, also known as Panama disease or yellow leaf disease, is a soil-borne vascular disease caused by Fusarium oxysporum f.sp.cubense (Foc). The disease generally presents obvious external symptoms in the middle and late stages of banana growth close to budding. Leaf yellowing and wilting in diseased plants, vascular bundles turn brown, and eventually the whole plant dies. At present, the most widespread and most harmful banana wilt pathogen in the world is TR4 (Tropical Race 4).

microRNA (miRNA) is a non-coding single-stranded small molecule RNA with a length of about 19-24 nucleotides, which is produced in eukaryotic organisms. A small molecule RNA is also found in fungi, and its precursor has a typical stem-loop structure, which is similar to miRNA in animals and plants, so it is called miRNA like small RNA (milRNA), which has been confirmed to be involved in the pathogenic process of plant pathogenic fungi play an important regulatory role.

The real-time fluorescent quantitative PCR (quantitative Real-time PCR, qRT-PCR) method reacts the PCR process in real time through the accumulation of fluorescence intensity, and performs quantitative analysis on the starting template. This method has high sensitivity, high specificity, high accuracy and rich information It has been widely used in many research and application fields such as molecular biology, medicine, food testing and environmental monitoring. Among the many factors affecting the accuracy of the experimental results, the selection of internal reference genes used to standardize the experimental results is the premise and key to ensure the accuracy of the experimental results. However, due to the influence of the internal and external environment, the genes that are truly and constantly expressed in cells do not exist, so it is necessary to screen the internal reference genes according to the test objects and test conditions.

To date, the selection of reference genes for the standardized measurement of miRNAs in samples has remained empirical. Some endogenous genes are often used as internal reference genes for tissue/cellular miRNA detection, such as 5SrRNA, 18SrRNA, snRNA (U4 and U6), etc., but because these genes are not miRNA, they cannot represent miRNA, and the extraction, inversion of these genes The efficiency of transcription and PCR amplification may vary with miRNAs. Therefore, these genes are also suboptimal.

Therefore, it is of great significance to select stable, suitable and more adaptable internal reference genes to accurately analyze the results of quantitative PCR of milRNA genes of Fusarium wilt.

Contents of the invention

In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide an internal reference gene for detecting milRNA of Fusarium wilt.

Another object of the present invention is to provide the application of the above-mentioned internal reference gene for detecting milRNA of Fusarium wilt.

The object of the present invention is achieved through the following technical solutions:

An internal reference gene for detecting milRNA of Fusarium wilt of banana, the internal reference gene is named milR46, and its nucleotide sequence is shown in SEQ ID NO.1.

The nucleotide sequence of the precursor of the internal reference gene used for the detection of milRNA of Fusarium wilt of banana is shown in SEQ ID NO.2.

Primers for specifically amplifying the above-mentioned internal reference gene used for the detection of Fusarium wilt milRNA.

Among the primers, the sequence of the forward primer is shown in SEQ ID NO.7.

A PCR kit for detecting milRNA of Fusarium wilt of banana, comprising the above primers.

The kit also contains one or more of PCR reaction buffer, DNA polymerase and dNTP.

The above-mentioned internal reference gene or its precursor for the detection of Fusarium wilt of banana milRNA, or the primers for specifically amplifying the internal reference gene for detection of milRNA of Fusarium wilt of banana, or the above-mentioned PCR kit for detection of milRNA of Fusarium wilt of banana, in Application in gene expression analysis or research of Fusarium wilt pathogen.

Further, the application specifically refers to the application in the expression analysis or research of milRNA in each stage of infection by Fusarium wilt pathogen of banana.

The banana fusarium wilt pathogen is the No. 4 physiological race of Fusarium oxysporum Cuban specialization.

A method for detecting banana fusarium wilt milRNA, comprising the steps of:

Reverse transcription of S1, milRNA

To extract the total RNA of the pathogen of Fusarium wilt of banana to be detected, first, under the action of Poly A polymerase, add poly A tail (PolyA) to the 3' end of milRNAs, and then generate the first RNA corresponding to milRNAs under the action of reverse transcriptase. strand cDNA;

S2, milRNA expression detection

Using the cDNA obtained by reverse transcription in step S1 as a template, specific primers and PCR reagents designed according to the sequence of the target milRNAs were used for quantitative PCR, and the above-mentioned internal reference gene for the detection of Fusarium wilt milRNA was used as an internal reference for expression analysis.

Compared with the prior art, the present invention has the following advantages and effects:

(1) The internal reference gene provided by the present invention greatly improves the accuracy of the standardization process of Fusarium wilt milRNAs;

(2) The internal reference gene provided by the present invention is suitable for the detection of milRNA in different time periods after inoculation;

(3) The internal reference gene provided by the present invention has high stability.

Description of drawings

Figure 1 is a diagram of the primer-specific amplification results of three internal reference genes; wherein, lane M is Marker, lane 1 is milR46, lane 2 is milR137, and lane 3 is milR140.

Figure 2 is the qRT-PCR melting curves of three internal reference genes; wherein, A is milR46, B is milR137, and C is milR140.

Figure 3 is a box-and-whisker plot of the Ct values of five internal reference genes (the analysis box and the upper and lower edges are the original Ct values of all samples, and abnormal values are represented by "dots").

Figure 4 is a graph of the calculation results of the algorithm Comprehensive Ranking.

Figure 5 is the calculation result diagram of the algorithm DeltaCT.

Figure 6 is a diagram of the calculation results of the algorithm BestKeeper.

Figure 7 is the calculation result diagram of the algorithm Normfinder.

Figure 8 is a graph of the calculation results of the algorithm Genorm.

Figure 9 is a graph showing the ddCt expression results of milR123 when milR46 is used as an internal reference gene.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Unless otherwise defined, all scientific and technical terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains.

The acquisition of internal reference gene of embodiment 1

Through high-throughput sequencing, compare the XJZ2 strain of Fusarium wilt of banana (already in the literature "Li Minhui, Xi Pinggen, Jiang Zide, et al. Identification of physiological races of Fusarium wilt of banana in Guangdong [J]. Journal of South China Agricultural University, 2007, 28(2 ): 38-38." Open) the sRNAs expression level after pure culture and inoculation host banana seedling 36h, select the sRNAs of stable high expression under two kinds of conditions as candidate internal reference gene. According to the high-throughput sequencing results, the precursor sequences (about 200bp-300bp in length) of the above-mentioned candidate internal reference sRNAs were intercepted, and the online software (http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite /RNAfold.cgi) predicts the secondary structure of the precursor, screens out the sRNAs that can produce a good stem-loop structure, and obtains its actual sequence through reverse transcription, cloning and sequencing, wherein the sequence length is between 20- 24bp were considered as milRNAs. After screening, one milRNA and two sRNAs were obtained as internal reference genes for the detection of milRNA expression in Fusarium wilt, namely milR46, mi lR137, and milR140. The secondary structures of its precursors all meet the conditions for the formation of milRNA. The sequence information of the above three internal reference genes is shown in Table 1, and these three internal reference genes were used for subsequent evaluation of expression stability. Combined with the research results of the existing technology, the traditional internal reference gene snRNAs (U4 and U6) for milRNA detection were selected as controls, and participated in the expression stability evaluation together with the above three internal reference genes.

Table 1 Sequence information of milRNA as an internal reference gene

Primer Design and Primer Specific Amplification Test of Example 2 Internal Reference Gene

To extract the total RNA of the XJZ2 strain of Fusarium wilt pathogen XJZ2, first add a poly A tail (PolyA) to the 3' end of the sRNA under the action of Poly A polymerase, and then generate the first sRNA corresponding to the sRNA under the action of reverse transcriptase. Strand cDNA.

Primers were designed according to the mature sequences of milR46, milR137, milR140, U4, and U6. The forward primer sequences of each milRNA and control internal reference gene are shown in Table 2 (downstream primers are unified as Up2: 5'-CTACGTAACGGCATGACAGTG-3' (SEQ ID NO.7)). Before the quantitative PCR detection is carried out, conventional PCR technology is used to detect the specific amplification effect of the primers for the target internal reference gene.

Using the cDNA obtained by the above reverse transcription as a template, PCR specific amplification was performed using the primers in Table 2. The reaction system (20 μL) was shown in Table 3. The reaction procedures included: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, Anneal at 56°C for 30min, extend at 72°C for 1min, 30 cycles, and finally extend for 5min.

Table 2 Primer sequences and related information of internal reference gene and control internal reference gene

Table 3 Candidate Internal Reference Gene Primer-specific Amplification Reaction System

Agarose gel electrophoresis (3%) was used to verify the specific amplification results, as shown in Figure 1 , a single band of each amplified product could be seen, and its length was consistent with the expectation. Therefore, the primers of these candidate internal reference genes can be used for subsequent qRT-PCR experimental analysis. Among them, M: Marker 500.

qRT-PCR reaction and stability evaluation of internal reference gene in embodiment 3

In order to evaluate the stability of internal reference gene expression in different experimental conditions, to inoculate 0h, 12h, 24h, 36h and 48h of banana wilt fungus Fusarium oxysporumf. .cubense, FOC) race 4 wild-type strain (XJZ2) as the research object.

The evaluation method of the stability of internal reference gene expression in different experimental conditions is as follows:

1. Extraction of milRNA (CTAB extraction method)

(1) The XJZ2 strain was cultured on a PDA plate at 28°C for 5 days. Use a medium-sized pipette to take a plate of approximately the same age and inoculate it into a conical flask containing 150 mL of YPD. Shake the culture at 28°C and 180 rpm for 2 days, then filter The spores were collected and concentrated by centrifugation to about 10^8 spores/mL.

(2) Take 5-6 leaves of Brazilian plantain (Guangzhou South China Botanical Garden) and soak them in MM culture medium containing 10^8 spores/mL in a tissue culture bottle, and culture them under light at 28°C and 75rpm for 0h and 12h, respectively. , 24h, 36h and 48h, the banana roots were collected and freeze-dried.

(3) Weigh 0.1-0.2g of freeze-dried banana root into a pre-cooled mortar, add liquid nitrogen and grind to powder, then quickly transfer it to a 15mL RNase-free centrifuge tube and add 6mL of RNA extraction buffer in advance (preheated at 65°C) and 20 μL of mercaptoethanol solution, vortexed to mix, bathed in a water bath at 65°C for 10 minutes, and centrifuged at 7000 rpm and 4°C for 15 minutes.

(4) Take the supernatant in a new 2mL RNase-free centrifuge tube, add 1/3 volume of 5MKAC solution, mix well, and ice-bath for 30min, then add 700μL chloroform isoamyl alcohol (24:1) and mix well, then 12000rpm, 4 Centrifuge for 10 min.

(5) Take the supernatant and add 500 μL water-saturated phenol to a new 2 mL RNase-free centrifuge tube, mix well, then add 500 μL chloroform isoamyl alcohol, vortex to mix, and centrifuge at 12000 rpm and 4 °C for 10 min.

(6) Take the supernatant and add an equal volume of chloroform isoamyl alcohol to a new 2 mL RNase-free centrifuge tube, and centrifuge at 12000 rpm at 4°C for 5 min.

(7) Take the supernatant into a new 1.5 mL RNase tube, add an equal volume of isopropanol, mix slowly, place on ice for 30 min, and centrifuge at 12,000 rpm at 4°C for 10 min.

(8) Discard the supernatant and add 800 μL of pre-cooled 75% absolute ethanol, carefully wash the precipitate twice, and centrifuge at 12,000 rpm and 4° C. for 5 min.

(9) Remove the supernatant and turn it upside down on filter paper, air-dry the precipitate, add 20-50 μL of DEPC water to fully dissolve the RNA precipitate, and store at -80°C.

2. Construct a sequencing library, submit it to Novogene to complete the sequence determination, and normalize the sequence number obtained from the statistics to the TPM value.

3. Small RNA plus A tail and reverse transcription

(1) First use E.coli Poly(A) polymerase to add A tail, put the PCR tube on ice during the whole operation, and add the system (Table 4):

Table 4 small RNA plus A tail system

After brief centrifugation and mixing, PCR amplification: 37°C for 30 minutes, 60°C for 20 minutes, and rapid cooling on ice for 5 minutes.

(2) Perform reverse transcription according to the following system (Table 5):

Table 5 small RNA reverse transcription system

After slow mixing and rapid centrifugation, PCR amplification: 5min at 65°C, rapidly cooled on ice for 5min.

(3) Add 4 μL of 5x primerScript II buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase to the reaction solution configured in the above PCR tube, perform PCR amplification at 42°C for 60 minutes, and at 70°C for 15 minutes, rapidly cool on ice for 5 minutes, and place in -20 Store cDNA at °C.

4.qRT-PCR analysis

Take the same amount of pure culture, 0h, 12h, 24h, 36h and 48h after inoculation sRNA samples of Fusarium wilt as template for reverse transcription, use the primers designed in Table 2 as fluorescent quantitative PCR primers, and use GeneCopoeia's All-in-One ^TM miRNAqRT-PCR Detection System User Manual Kit. Reaction system: SYBR Premin Ex TaqTM (2x) 10 μL, template cDNA 2 μL (100 μg/μL), upstream primer 1 μL (10 μM), downstream primer 1 μL (10 μM), ddH ₂ O 6 μL. The reaction program is: 95°C for 30s; 95°C for 30s, 60°C for 30s, for 40 cycles. The data were automatically read by a real-time fluorescent quantitative PCR instrument.

5. Stability evaluation of internal reference genes

Figure 2 shows the qRT-PCR melting curves of three internal reference genes, where A: milR46; B: milR137; C: milR140; the melting curves of the three internal reference genes all showed a significant single peak, indicating that the primers can specifically amplify the target fragment , and the repeatability of the amplification curve of each sample is good, indicating that the qRT-PCR results are more accurate.

The Ct value of qRT-PCR can be used to evaluate the expression abundance of genes. Typically, lower Ct values indicate higher expression levels, and higher Ct values indicate lower expression levels. Therefore, we used the Ct value to evaluate the expression abundance of the five internal reference genes under pure culture conditions and at different time periods after inoculation. The box-and-whisker plot can well show the dispersion and distribution characteristics of the data. We plotted all raw Ct values as box-and-whisker plots, as shown in Figure 3. From the figure, we can observe that the Ct values of all candidate internal reference genes are between 13.42 and 25.16. Among them, the expression of U4 showed great variability, the standard deviation (SD) was as high as 4.39, and the Ct value also changed greatly between 18.67 and 25.16. However, the Ct values of milR46 (excluding outliers) fluctuated between 19.23 and 21.92, and the Ct values of milR137 fluctuated between 17.00 and 19.98. The expression level changes minimally.

Using the data analysis webpage (http://www.ciidirsinaloa.com.mx/RefFinder-master/?type=reference) to analyze the obtained fluorescent quantitative PCR data, and screen out the stably expressed internal reference genes. Five different algorithms, namely Comprehensive Ranking, Delta CT, BestKeeper, Normfinder, and Genorm, are used for calculation, and the results are shown in Figure 4 to Figure 8.

From the above algorithm, it can be seen that the stability of the internal reference genes milR137 and milR46 is higher than that of the traditional internal reference genes U4 and U6. Comprehensive evaluation, milR46 is a suitable internal reference gene selection.

Example 4 Application of internal reference genes in the detection of pathogenicity-related milRNA expression

In order to further verify the application effect of milR46 as an internal reference in qRT-PCR detection, milR123 (a pathogenicity-related milRNA of Fusarium wilt of banana, its precursor, structure and application (has been described in the patent "a pathogenicity-related milRNAs of and its precursors and applications", disclosed in 202010641713.1), as the detection object, using milR46 as an internal reference, to analyze the relative expression level of milR123, and use the experimental results as verification. The verification process is as follows:

1. qRT-PCR analysis

Take the same amount of pure culture, Fusarium wilt sRNA samples 0h, 12h, 24h, 36h and 48h after inoculation as the template for reverse transcription, using GeneCopoeia's All-in-One ^TM miRNA qRT-PCR Detection System User Manual kit. Reaction system: SYBR Premin Ex TaqTM (2x) 10 μL, template cDNA 2 μL (100 μg/μL), upstream primer 1 μL (10 μM), downstream primer 1 μL (10 μM), ddH ₂ O 6 μL. The reaction program is: 95°C for 30s; 95°C for 30s, 60°C for 30s, for 40 cycles. The data were automatically read by a real-time fluorescent quantitative PCR instrument. Among them, the primer sequence of milR123 is as follows (the downstream primer is Up2):

milR123-F: 5'-TGCACTGTATGTCTTGGTATGA-3', Tm = 54.0°C (SEQ ID NO. 13).

2. Experimental results

For all test samples and calibration samples, use the Ct value of the internal reference gene to normalize the Ct value of the target gene, and then use the dCt value of the calibration sample to normalize the dCt value of the test sample to obtain the double ddCt value (this step is performed by real-time fluorescence Quantitative PCR instrument completes automatically.The result is as shown in Figure 9.

The results showed that when milR46 was used as an internal reference gene, the expression pattern of milR123 showed a slow increase from 0h to 36h, and a significant increase after 48h. The results indicate that milR46 can be used as an internal reference gene in the detection of milRNA of Fusarium wilt, and it can be used to judge the relative expression level and change trend of milRNA in each stage of pathogen parasitism, and has a better standardization effect.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

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Claims (4)

1. The application of the milR46 serving as an internal reference gene in quantitatively detecting the expression level of the milRNA gene in each stage of infection of banana fusarium wilt pathogen is characterized in that:

   the banana vascular wilt pathogen is fusarium oxysporum cubeba specialized No. 4 physiological race;

   the nucleotide sequence of the milR46 is shown as SEQ ID NO. 1.
2. 2. Use according to claim 1, characterized in that:

   the nucleotide sequence of the precursor of the milR46 is shown as SEQ ID NO. 2.
3. 3. Use according to claim 1, characterized in that:

   the sequence of a forward primer in the primers for specifically amplifying the milR46 is shown as SEQ ID NO. 10.
4. 4. A method for detecting the expression level of milRNA genes in each stage of infection of banana vascular wilt pathogens is characterized by comprising the following steps:

   the method comprises the following steps:

   reverse transcription of S1, milRNA

   Extracting total RNA of banana vascular wilt germs to be detected, firstly adding Poly A tail to the 3' end of the milRNAs under the action of Poly A polymerase, and then generating first chain cDNA corresponding to the milRNAs under the action of reverse transcriptase; the banana vascular wilt pathogen is fusarium oxysporum cubeba specialized No. 4 physiological race;

   s2, detection of expression level of milRNA

   Performing quantitative PCR by using cDNA obtained by reverse transcription in the step S1 as a template and specific primers and PCR reagents designed according to sequences of target milRNAs, and performing expression analysis by using the milR46 as an internal reference of the quantitative PCR in claim 1.

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