Primers and methods for detecting the expression characteristics of POD genes in Pseudococcus papayaensis
By designing primers to detect the POD gene of papaya mealybug and using real-time fluorescence quantitative PCR method, the influence of different hosts on POD gene expression was studied, and the gap in the research on the POD gene expression characteristics of papaya mealybug in the existing technology was solved, and the New ideas for pest control and efficient and fast experimental results have been achieved.
Patent Information
- Application Number
- CN202211033365.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-08-26
- Publication Date
- 2024-12-10
- Estimated Expiration
- 2042-08-26
AI Technical Summary
The research on the expression characteristics of POD genes of papaya mealybugs in the prior art has not been reported, and the impact of different hosts on insect gene expression regulation has not been discussed in depth, affecting the development of pest control methods.
Primers were designed to detect the expression characteristics of POD genes of Papaya mealybugs, and real-time fluorescence quantitative PCR was used to study the expression characteristics of POD genes through different host treatments, including designing POD-F and POD-R primers, as well as upstream and downstream of the internal reference gene Tubulin Primers were performed, cDNA synthesis, conventional PCR and real-time fluorescence quantitative PCR reactions, and the expression of POD genes relative to tubulin genes was calculated.
The effect of different hosts on the POD gene expression of papaya mealybugs was successfully revealed, providing basic data for in-depth research, and the method is efficient and fast, shortening the reaction time, and providing routine PCR electrophoresis results to reflect the specificity of primers.
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Figure CN115927656B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to primers and a method for detecting the expression characteristics of POD genes of Pseudococcus papayae mealybugs, and belongs to the field of biotechnology. Background Art
[0002] The papaya mealybug belongs to the order Hemiptera, the superfamily Coccoidea, the genus Mealycosidae. It has a wide host range and is an important potential economic pest with strong diffusion, vitality and reproduction. The insect is generally believed to be native to Mexico or Central America. The first specimens of the papaya mealybug were collected in Mexico in 1955. The papaya mealybug likes to live in hidden parts such as the veins of leaves, the growth points of stems and leaves, the concave and convex or overlapping parts of flowers and leaves, and the depressions or grooves on the surface of fruits. The nymphs and female adults mainly suck the juice of the organ tissues such as the stems, leaves, flowers and fruits of the host plant, causing the leaves to curl and deform, rosette, lose green and yellow, wither and fall off, etc.; it can also cause the branches to dry up, the plants to be short or dormant, the fruits to be weak or deformed, and the flowers and fruits to fall off. In severe cases, it can even cause the death of the entire plant. Since the papaya mealybug was first reported in San Martin in 1995, it has now spread to at least 40 countries and regions in Central America, North America, the Pacific, Africa, Asia, etc., causing serious losses to the papaya industry. The papaya mealybug has also invaded China, posing a huge potential threat to my country's papaya and flower industries.
[0003] Research on papaya mealybugs mainly focuses on their morphological characteristics, biochemical mechanisms, and pesticide control. Chen Qing et al. (2018) determined the differences in enzyme activity in papaya mealybugs under different temperature cultivation conditions, and found that both high and low temperatures can induce an increase in the activity of protective enzymes in papaya mealybugs. Wang Yaru et al. (2018) found that the activity of protective enzymes in papaya mealybugs feeding on cassava was significantly reduced, and their feeding ability was also reduced. Chen Qian et al. (2020) found that papaya mealybugs can inhibit the activity of antioxidant enzymes in the body after feeding on some cassava varieties. There are no reports on the expression characteristics of POD genes in papaya mealybugs fed on different hosts at home and abroad.
[0004] Cytochrome P450 is a gene superfamily composed of multiple gene families, which exists in almost all organisms (Werck-Reichhart, 2000). At present, more than 60 P450 gene families have been reported in insects, including CYP4, CYP6, CYP9 gene families, etc. (Fogleman, 2000). P450 genes have extensive and diverse functions and are involved in the growth and development of insects, including the biosynthesis of endogenous substances such as molting hormones and juvenile hormones, and the metabolism of exogenous substances such as plant secondary metabolites and pesticides (David, 2003; Baldwin, 2009). POD is also involved in physiological processes such as active oxygen metabolism, disease and insect resistance, and drought stress resistance. It is one of the key enzymes for insects to respond to adverse stress. Studies have shown that the CYP6 family is related to the metabolism of exogenous substances. The cytochrome P450 gene CYP681 is involved in the metabolism of furanocoumarins in Drosophila melanogaster (Cohen, 1992); CYP6BG1 is overexpressed in the permethrin-resistant strain of Plutella xylostella (Bautista, 2009). Shen Zhongjian et al. (2016) found that the POD gene of cotton bollworm is not only affected by various adversities but also has a certain effect on resisting nuclear polyhedrosis virus. Park et al. (2014) cloned 10 POD genes of beet armyworm and found that they can regulate cellular immune responses.
[0005] There are no reports on the expression of POD genes in Papaya mealybugs at home and abroad. Therefore, real-time fluorescence quantitative PCR was used to study the expression characteristics of POD genes in Papaya mealybugs under different host treatments. The results showed that the expression levels of POD genes were different in different hosts, and were consistent with the change trend of transcriptome expression, indicating that host conversion can affect the expression regulation of POD genes. If some target genes are used as targets for pest control, a new field of pest molecular regulation can be opened up, providing new methods and ideas for the prevention and control of agricultural pests. Summary of the invention
[0006] The invention aims to provide primers and methods for detecting the expression characteristics of the POD gene of Papaya mealybug, and to design different host treatments for comprehensively understanding the expression characteristics of the POD gene of Papaya mealybug.
[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:
[0008] Twenty papaya mealybugs feeding on papaya and potato were collected into Eppendorf tubes respectively and divided into three biological replicates, for a total of six groups of samples.
[0009] Design a pair of primers for detecting the expression characteristics of the POD gene of Pseudomonas papayaensis, including upstream and downstream primers that conform to the characteristics of fluorescent quantitative PCR reaction:
[0010] POD-F: 5`-TGTGCGTTACTCGGTACACA-3`,
[0011] POD-R:5`-GGCGATCAACTTGACATTTC-3`;
[0012] And the upstream and downstream primers of the internal reference gene Tubulin:
[0013] Tubulin-F: 5`-CTTCACTTCTTCATGCCTGG-3`,
[0014] Tubulin-R: 5`-TTTGTTCGTCAACTTCTTTC-3`.
[0015] The fluorescent quantitative PCR method for detecting the POD gene expression characteristics of Pseudomonas aeruginosa described in the present invention is carried out according to the following steps:
[0016] (1) First-strand cDNA synthesis: Extract total RNA from the sample and purify it, and use a reverse transcription kit to obtain cDNA using the extracted RNA as a template;
[0017] (2) Perform conventional PCR testing using the following primers:
[0018] Fluorescence quantitative upstream and downstream primers of POD gene of Pseudococcus papayaensis:
[0019] POD-F: 5`-TGTGCGTTACTCGGTACACA-3`,
[0020] POD-R:5`-GGCGATCAACTTGACATTTC-3`;
[0021] Upstream and downstream primers of internal reference gene Tubulin:
[0022] Tubulin-F: 5`-CTTCACTTCTTCATGCCTGG-3`,
[0023] Tubulin-R: 5`-TTTGTTCGTCAACTTCTTTC-3`;
[0024] The total volume of the conventional PCR amplification system is 20µL: 2X Taq Plus Master Mix 10µL, upstream primer 0.8µL (5uM), downstream primer 0.8µL (5uM), 100ng / µL cDNA template 1.0µL, water 7.4µL;
[0025] The conventional PCR reaction program was as follows: pre-denaturation at 94°C for 5 min, followed by denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min. This condition was repeated for 35 cycles, and finally, extension was performed at 72°C for another 10 min.
[0026] (3) Real-time fluorescence quantitative PCR reaction:
[0027] Using the cDNA obtained in step (1) as a template, adding the primers described in step (2), performing a fluorescent quantitative PCR reaction, setting 3 replicates for each sample, and taking the average value after amplification;
[0028] The real-time fluorescence quantitative PCR amplification system is: 2X ChamQ SYBR Color qPCR Master Mix 10µL, upstream primer 0.8µL (5uM), downstream primer 0.8µL (5uM), 50X ROX Reference Dye 2 0.4µL, 100 ng / µL cDNA 2.0µL, and water is added to 20µL;
[0029] The real-time fluorescence quantitative PCR reaction program was as follows: pre-denaturation at 95°C for 5 min, followed by denaturation at 95°C for 5 s, annealing at 50°C for 30 s, and extension at 72°C for 40 s, for 40 cycles.
[0030] The more detailed test method of the present invention is as follows:
[0031] The real-time fluorescence quantitative PCR detection method of the POD gene of Pseudomonas papayaensis can be achieved by the following steps:
[0032] (1) Primer design: Based on the sequence of the POD gene of P. papaya obtained from the transcriptome sequencing results, DNAMAN software was used to design specific primers suitable for fluorescence quantitative PCR detection. The primer sequences are as follows:
[0033] POD-F: 5`-TGTGCGTTACTCGGTACACA-3`,
[0034] POD-R:5`-GGCGATCAACTTGACATTTC-3`.
[0035] At the same time, according to the sequence of the papaya mealybug tubulin gene obtained from the transcriptome sequencing results, primers for the internal control of fluorescent quantitative PCR were designed. The primer sequences are as follows:
[0036] Tubulin-F: 5`-CTTCACTTCTTCATGCCTGG-3`,
[0037] Tubulin-R: 5`-TTTGTTCGTCAACTTCTTTC-3`.
[0038] (2) Papaya mealybug treatment experiment: Papaya seedlings and potato seedlings were cultivated in artificial climate chambers to raise papaya mealybugs. Twenty papaya mealybugs were collected from each tube and placed in Eppendorf tubes. The tubes were divided into three biological replicates, for a total of six groups of samples. After the insect samples were treated, they were quickly fixed in liquid nitrogen and stored at -80°C for later use.
[0039] (3) First-strand cDNA synthesis: refer to the method of Quanshijin Company TransZol TM Total RNA was extracted according to the instructions of the Up Plus RNA Kit, and the first-chain cDNA was synthesized according to the instructions of the HiScript Q RT SuperMix for qPCR (+gDNA wiper) kit of Novozymes.
[0040] (4) Real-time fluorescence quantitative PCR reaction: Real-time fluorescence quantitative PCR was performed using the ChamQ SYBR Color qPCR Master Mix (2X) kit of Nanjing Novozyme Biotechnology Co., Ltd. The first-strand cDNA synthesized above was used as a template, and the above-mentioned POD-F, POD-R and Tubulin-F, Tubulin-R were used as specific primers to perform fluorescence quantitative PCR. Three parallel replicates were set for each sample, and the average of the parallel Ct values obtained after amplification was taken.
[0041] The real-time fluorescence quantitative PCR amplification system is: 2X ChamQ SYBR Color qPCR Master Mix 10µL, upstream primer 0.8µL (5uM), downstream primer 0.8µL (5uM), 50X ROX Reference Dye 2 0.4µL, 100 ng / µL cDNA 2.0µL, and water is added to 20µL;
[0042] The real-time fluorescence quantitative PCR reaction program was as follows: pre-denaturation at 95°C for 5 min, followed by denaturation at 95°C for 5 s, annealing at 50°C for 30 s, and extension at 72°C for 40 s, for 40 cycles.
[0043] (5) The expression level of POD gene relative to tubulin gene in P. papaya was calculated as: relative mRNA expression = 2 -ΔΔCt × 100%, where Ct value = target gene Ct value - tubulin Ct value.
[0044] (6) Figure 3The results showed that the expression of POD genes varied with the different hosts, and the expression trend of POD genes was consistent with that of transcriptome, which provided important basic data for our in-depth study of POD genes in Papaya mealybugs.
[0045] Compared with the prior art, the present invention has the following advantages and effects:
[0046] (1) The different host treatment measures in the present invention are applicable to most POD genes of insects, which is of great significance for the comprehensive and in-depth study of gene expression characteristics.
[0047] (2) The efficient and fast fluorescence quantitative PCR method of the present invention, including the fluorescence quantitative PCR program, actually takes 50 minutes, which greatly shortens the reaction time compared with the prior art and has the characteristics of high efficiency and speed.
[0048] (3) The fluorescent quantitative PCR method described in the present invention provides a conventional PCR electrophoresis result diagram, which can intuitively and quickly reflect the specificity of the primers. BRIEF DESCRIPTION OF THE DRAWINGS
[0049] The attached figure shows the results of fluorescence quantitative PCR detection of the transcription level of the POD gene of Pseudococcus papayaensis.
[0050] Figure 1 : Results of conventional PCR product electrophoresis using fluorescent quantitative primers for the POD gene of Pseudomonas papayaensis: The product length was 268 bp.
[0051] The marker bands from top to bottom are: 2000, 1000, 750, 500, 250, 100 bp.
[0052] Figure 2 : Results of conventional PCR products electrophoresis using fluorescent quantitative primers for the tubulin gene of Pseudomonas papayaensis: The product length was 202 bp.
[0053] The marker bands from top to bottom are: 2000, 1000, 750, 500, 250, 100 bp.
[0054] Figure 3 : Differential expression of POD genes after P. papaya feeding on different hosts. The columns and the left axis are the expression levels of fluorescence quantification, and the scatter points and the right axis are the expression levels of the transcriptome. MF6 represents P. papaya feeding on papaya, and TF6 represents P. papaya feeding on potato. Specific implementation plan
[0055] The present invention is explained below in conjunction with specific embodiments and drawings. The schemes of the embodiments described here do not limit the invention. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
[0056] Example 1
[0057] Handling of test materials
[0058] Papaya seedlings and potato seedlings were cultivated in an artificial climate chamber to raise papaya mealybugs. Twenty papaya mealybugs were collected from different hosts and placed in Eppendorf tubes, divided into three biological replicates, for a total of six groups of samples. After the insect samples were collected, they were quickly fixed in liquid nitrogen and stored at -80°C for later use.
[0059] Example 2
[0060] Primer design
[0061] (1) Primer design: Based on the sequence of the POD gene of P. papaya obtained by transcriptome sequencing, specific primers suitable for fluorescence quantitative PCR detection were designed using DNAMAN software. The primer sequences are as follows:
[0062] POD-F: 5`-TGTGCGTTACTCGGTACACA-3`,
[0063] POD-R:5`-GGCGATCAACTTGACATTTC-3`.
[0064] The length of the fluorescent quantitative PCR product of the POD gene of Pseudomonas papayaensis was 268 bp. The results of agarose gel electrophoresis showed that the amplified products were of the same length and were a single band, indicating that the designed primers had strong specificity and were suitable for real-time fluorescent quantitative PCR detection. The results of agarose gel electrophoresis were as follows: Figure 1 shown.
[0065] (2) At the same time, based on the sequence of the papaya mealybug tubulin gene obtained by transcriptome sequencing, primers for internal controls in fluorescent quantitative PCR were designed. The primer sequences are as follows:
[0066] Tubulin-F: 5`-CTTCACTTCTTCATGCCTGG-3`
[0067] Tubulin-R: 5`-TTTGTTCGTCAACTTCTTTC-3`
[0068] The length of the fluorescent quantitative PCR product of the Pseudomonas papayaensis tubulin gene was 202 bp. The results of agarose gel electrophoresis showed that the amplified products were of the same length and were a single band, indicating that the designed primers had strong specificity and were suitable for real-time fluorescent quantitative PCR detection. The results of agarose gel electrophoresis were as follows: Figure 2 shown.
[0069] Example 3
[0070] Extraction of total RNA
[0071] Refer to Quanshijin Company TransZol TM Up Plus RNA Kit Instructions Extract total RNA, grind the insect body into powder with liquid nitrogen, add 1ml TransZol TM Up, transfer to a 1.5 ml centrifuge tube, let stand at room temperature for 5 min, then add 200 μL chloroform / 1 ml TransZol TM Up, shake vigorously for 30 s, incubate at room temperature for 3 min. After centrifugation at 10000 g for 15 min at 4℃, transfer the upper aqueous phase (generally <80%) to a new centrifuge tube, add 1 / 3 volume of anhydrous ethanol, and gently invert to mix. Put the RNA centrifuge column into a 2 ml collection tube, transfer all the mixture in the previous step into the centrifuge column, centrifuge at 12000 g for 30-60 s, discard the mobile phase, and reuse the collection tube. Add 500 μL CB9, centrifuge at 12000 g for 30 s at room temperature, discard the mobile phase, add 500 μL CB9, centrifuge at 12000 g for 30 s at room temperature, discard the mobile phase. Add 500 μL diluted WB9, centrifuge at 12000 g for 30 s, discard the mobile phase, add 500 μL diluted WB9, centrifuge at 12000 g for 30 s, discard the mobile phase. Centrifuge at 12000 g for 2 min to completely remove the residual ethanol, and let it stand at room temperature for several minutes to completely dry the centrifuge column. Place the centrifuge column in an RNase-free tube, add 50 μL RNase-free water to the center of the centrifuge column, let it stand at room temperature for 1 min, and centrifuge at 12000 g for 1 min to elute the RNA. The obtained RNA is stored at -80℃ for later use.
[0072] Example 4
[0073] First-strand cDNA synthesis: According to the instructions of the HiScript Q RT SuperMix for qPCR (+gDNAwiper) kit of Novozymes, the RNA in Example 3 was used as a template for reverse transcription to generate the first-strand cDNA. The specific steps are as follows:
[0074] (1) Removal reaction of genomic DNA.
[0075]
[0076] (2) Prepare the reverse transcription reaction system.
[0077]
[0078] (3) Perform reverse transcription reaction, incubate at 50°C for 15 min, heat at 85°C for 2 min, and store the product at -20°C or -80°C.
[0079] Example 5
[0080] Fluorescence quantitative PCR reaction was performed. Real-time fluorescence quantitative PCR was performed using the ChamQ SYBR Color qPCR Master Mix (2X) kit of Nanjing Novozyme Biotechnology Co., Ltd. The cDNA in Example 4 was used as a template and the primers in Example 2 were used to perform real-time fluorescence quantitative PCR amplification reaction. Three parallel replicates were set for each sample, and the average of the parallel Ct values obtained after amplification was taken.
[0081] (1) The real-time fluorescence quantitative PCR amplification system is as follows:
[0082] The real-time fluorescence quantitative PCR amplification system is: 2X ChamQ SYBR Color qPCR Master Mix 10µL, upstream primer 0.8µL (5uM), downstream primer 0.8µL (5uM), 50X ROX Reference Dye 2 0.4µL, 100 ng / µL cDNA 2.0µL, and water is added to 20µL;
[0083] (2) The real-time fluorescence quantitative PCR reaction program was as follows: pre-denaturation at 95°C for 5 min, followed by denaturation at 95°C for 5 s, annealing at 50°C for 30 s, and extension at 72°C for 40 s, for 40 cycles.
[0084] (3) After the real-time fluorescence quantitative PCR is completed, the ratio of relative expression levels under different treatment conditions is calculated based on the Ct value. -ΔΔCt The expression of POD gene in P. papaya relative to tubulin gene was calculated as: relative mRNA expression = 2 -ΔΔCt × 100%, where Ct value = target gene Ct value - tubulin Ct value.
[0085] Table 1: C(T) values, mean values, standard deviations, fluorescence quantitative expression levels and transcriptome expression levels of POD genes after Pseudomonas papayaensis feeding on different hosts
[0086]
[0087] Note: MF6 refers to the papaya mealybug feeding on papaya, and TF6 refers to the papaya mealybug feeding on potato.
[0088] (4) Figure 3 The results showed that the expression of POD genes varied with the different hosts, and the expression trend of POD genes was consistent with that of transcriptome. This provided important basic data for our in-depth study of POD genes in Papaya mealybugs.
Claims
1. A fluorescent quantitative PCR primer for detecting the expression characteristics of the POD gene of Pseudococcus papayae, characterized in that: Including upstream and downstream primers that meet the characteristics of fluorescent quantitative PCR reaction: POD-F: 5'-TGTGCGTTACTCGGTACACA-3', POD-R: 5'-GGCGATCAACTTGACATTTC-3'.
2. A fluorescent quantitative PCR method for detecting the expression characteristics of the POD gene of Pseudomonas papayaensis using the fluorescent quantitative PCR primers described in claim 1, characterized in that: Follow these steps: (1) First-strand cDNA synthesis: Extract total RNA from the sample and purify it, and use a reverse transcription kit to obtain cDNA using the extracted RNA as a template; (2) Perform conventional PCR detection using the following primers: Fluorescence quantitative upstream and downstream primers of POD gene of Pseudococcus papayaensis: POD-F: 5'-TGTGCGTTACTCGGTACACA-3', POD-R: 5'-GGCGATCAACTTGACATTTC-3'; Upstream and downstream primers of internal reference gene Tubulin: Tubulin-F: 5'-CTTCACTTCTTCATGCCTGG-3', Tubulin-R: 5'-TTTGTTCGTCAACTTCTTTC-3'; The total volume of the conventional PCR amplification system is 20µL: 2X Taq Plus Master Mix 10µL, 5uM upstream primer 0.8µL, 5uM downstream primer 0.8µL, 100ng / µL cDNA template 1.0µL, water 7.4µL; The conventional PCR reaction program was as follows: pre-denaturation at 94°C for 5 min, followed by denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min. This cycle was repeated for 35 cycles, and finally, extension was performed at 72°C for another 10 min. (3) Real-time fluorescence quantitative PCR reaction: Using the cDNA obtained in step (1) as a template, adding the primers described in step (2), performing a fluorescent quantitative PCR reaction, setting 3 replicates for each sample, and taking the average value after amplification; The real-time fluorescence quantitative PCR amplification system is: 2X ChamQ SYBR Color qPCR Master Mix 10µL, 5uM upstream primer 0.8µL, 5uM downstream primer 0.8µL, 50X ROX Reference Dye 2 0.4µL, 100 ng / µL cDNA 2.0µL, and water is added to 20µL; The real-time fluorescence quantitative PCR reaction program was as follows: pre-denaturation at 95°C for 5 min, followed by denaturation at 95°C for 5 s, annealing at 50°C for 30 s, and extension at 72°C for 40 s, for 40 cycles.