Primers and method for detecting expression characteristics of papaya Pleurotus geesteranus transaminase gene

Abstract

The invention provides a primer and a method for detecting the expression characteristic of a papaya Pleurotus geesteranus transaminase gene. The sequences of the primers are as shown in SEQ ID NO. 1-2. Based on the primer, the invention establishes a fluorescent quantitative PCR method for detecting the change of the expression quantity of the transaminase gene after the papaya Pleurotus geesteranus eats two different hosts, namely papaya and potato. Through transcriptome sequencing verification, the gene expression trend obtained by the fluorescent quantitative PCR is consistent with the transcriptome analysis result. The invention lays an important foundation for exploring the influence of host conversion on the defense mechanism and transaminase gene expression characteristics of the papaya Pleurotus geesteranus.

Description

Technical Field

[0001] This invention relates to primers and methods for detecting the expression characteristics of transaminase genes in papaya mealybugs, belonging to the field of biotechnology. Background Technology

[0002] The papaya mealybug belongs to the order Hemiptera, superfamily Mealyroidea, and genus Mealybug in the family Mealyroidea. It has a wide host range and is characterized by strong dispersal ability, vigorous vitality, and high reproductive rate, making it an important potential economic pest. The first specimens of the papaya mealybug were collected in Mexico in 1955. The papaya mealybug prefers to inhabit relatively concealed locations such as leaf veins, stem and leaf growing points, uneven or overlapping areas of leaves and flowers, and depressions or grooves on the surface of fruits. It primarily feeds on the sap of the stems, leaves, flowers, and fruits of its host plants as nymphs and adult females. Symptoms of damage include leaf curling and deformity, the appearance of rosette-like structures, chlorosis, yellowing, wilting, and leaf drop; it can also lead to branch dieback, stunted or stagnant plants, weak or deformed fruits, and flower and fruit drop; in severe cases, it can cause the death of the entire plant. Since the first report of papaya mealybug damage in San Martin in 1995, the insect has spread to many regions worldwide, including Central America, North America, Pacific islands, Africa, and Asia, causing severe economic losses to the papaya industry. Currently, the papaya mealybug has also invaded my country, posing a significant potential threat to papaya and related industries such as floriculture.

[0003] Current research on the papaya mealybug mainly focuses on its morphological characteristics, biochemical mechanisms, and chemical control. Chen Qing et al. (2018) measured the differences in enzyme activity in the papaya mealybug under different temperature feeding conditions, and found that both high and low temperatures could induce an increase in the activity of protective enzymes. Wang Yaru et al. (2018) found that the activity of protective enzymes in papaya mealybugs that fed on cassava was significantly reduced, and their feeding ability was also reduced. Chen Qian et al. (2020) found that the consumption of certain cassava varieties by the papaya mealybug could inhibit the activity of antioxidant enzymes in its body. It is worth noting that there are currently no reports, either domestically or internationally, on the expression characteristics of transaminase genes in the papaya mealybug under different host feeding conditions.

[0004] Transaminases (EC2.6.1), also known as aminotransferases, are a class of enzymes widely distributed in organisms, playing a crucial catalytic role in amino acid metabolism. These enzymes can transfer amino groups from α-amino acids to α-keto acids, thereby generating new amino acids and realizing the synthesis and degradation of amino acids. Amino acid transaminases belong to the class of enzymes that use pyridoxal 5'-phosphate (PLP) as a coenzyme, catalyzing reversible reactions. The specific reaction process is as follows: First, the amino group of the substrate, the amino donor, is transferred to PLP and covalently binds to it, generating pyridoxamine (PMP); subsequently, the amino group on PMP is transferred to the carboxyl group of the substrate, completing the catalytic action, while PLP is regenerated. Based on their structural characteristics and evolutionary relationships, transaminases can be divided into four subfamilies: Subfamily I includes aspartate transaminase, alanine transaminase, tyrosine transaminase, histidine phosphate transaminase, and phenylalanine transaminase; Subfamily II includes acetylornithine transaminase, ornithine transaminase, 4-aminobutyrate transaminase, and diaminononanoate aminotransferase; Subfamily III includes D-alanine transaminase and branched-chain amino acid transaminase; and Subfamily IV includes serine transaminase and phosphoserine transaminase.

[0005] Comparative analysis of the genomes of aphids and several other model insects revealed that the aphid genome contains 10 transaminases with known functions. Nine of these are also widely present in the genomes of the Drosophila melanogaster and most other insects. Transaminases with unclear functions exist in both aphids and the Red Flounder (EC 2.6.1). The Italian honeybee genome contains two copies of this gene, while all known Diptera insects specifically lack this transaminase. Most insect genomes contain one aspartate transaminase gene (EC 2.6.1.1) and one alanine-glyoxylate transaminase gene (EC 2.6.1.44). However, in aphids, the aspartate transaminase 1 (GOT1) gene has three copies, and the alanine-glyoxylate transaminase gene has two copies. Aspartate transaminases play an important role in metabolism: they catalyze the transamination reaction of aspartic acid (Asp) with α-ketoglutarate to produce oxaloacetic acid (OAA) and glutamate (Glu). Oxaloacetate is an intermediate metabolite in the tricarboxylic acid cycle, while aspartic acid is an intermediate metabolite in the synthesis of other amino acids, nucleotides, and other metabolic processes. Therefore, aspartic acid and glutamate are metabolic precursors for the synthesis of all protein amino acids, and the interconversion between aspartic acid and oxaloacetate plays a crucial role in basal metabolism.

[0006] There are currently no systematic research reports on the expression characteristics of transaminase genes in the papaya mealybug. This invention uses real-time quantitative PCR to study the expression characteristics of transaminase genes in papaya mealybugs under different host treatments. The results show that the expression level of transaminase genes varies significantly with different host species, and this trend is consistent with the trend of transcriptome expression levels, indicating that host switching can affect the expression regulation process of transaminase genes. Using such key genes as targets for pest control may open up new directions in the molecular regulation of pests and provide important theoretical basis for the development of green pest control technologies in agriculture. Summary of the Invention

[0007] The purpose of this invention is to design different host treatments to comprehensively understand the expression characteristics of the transaminase gene of *Papaya mealybug*, and to provide primers and methods for detecting the expression characteristics of the transaminase gene of *Papaya mealybug*.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Twenty Papaya mealybugs feeding on different hosts were collected into Eppendorf tubes and divided into three biological replicates, for a total of six samples.

[0009] Design a pair of primers for detecting the expression characteristics of transaminase genes in Papaya mealybug, including upstream and downstream primers that conform to the characteristics of quantitative real-time PCR: Upstream primer (transaminase-F): 5'-CTATTCACGGAGCAAGGATTGTAG-3', Downstream primer (transaminase-R): 5'-GTTCGCATGGAGATTATTCTGTCA-3'; And the upstream and downstream primers for the internal reference gene Tubulin: Upstream primer (Tubulin-F): 5'-CTTCACTTCTTCATGCCTGG-3', Downstream primer (Tubulin-R): 5'-TTTGTTCGTCAACTTCTTTC-3'.

[0010] The quantitative real-time PCR method for detecting the expression characteristics of transaminase gene in *Papaya mealybug* as described in this invention is performed according to the following steps: (1) cDNA synthesis: After extracting and purifying total RNA from the sample, the purified RNA was used as a template to perform reverse transcription reaction using a reverse transcription kit to obtain cDNA; (2) Using the cDNA obtained in step (1) as a template, perform routine PCR detection using the following primers: Upstream and downstream primers for the transaminase gene of *Papaya mealybug*: Upstream primer (transaminase-F): 5'-CTATTCACGGAGCAAGGATTGTAG-3', Downstream primer (transaminase-R): 5'-GTTCGCATGGAGATTATTCTGTCA-3'; upstream and downstream primers for the internal reference gene Tubulin: Upstream primer (Tubulin-F): 5'-CTTCACTTCTTCATGCCTGG-3', Downstream primer (Tubulin-R): 5'-TTTGTTCGTCAACTTCTTTC-3'; The standard PCR reaction system is as follows: 10 µL of 2× Taq Plus Master Mix, 0.8 µL (5 µM) of upstream primer, 0.8 µL (5 µM) of downstream primer, 1.0 µL of 100 ng / µL cDNA template, and 7.4 µL of water; The standard PCR reaction procedure is as follows: 94℃ for 5 min; 95℃ for 30 s, 50℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 10 min. (3) Real-time quantitative PCR reaction: Using the cDNA obtained in step (1) as a template, add the primers in step (2) to perform a real-time PCR reaction. Set up 3 biological replicates for each sample and take the average value after amplification. The reaction system for real-time quantitative PCR was as follows: 10 µL of 2× ChamQ SYBR Color qPCR Master Mix, 0.8 µL (5 μM) of upstream primer, 0.8 µL (5 μM) of downstream primer, 0.4 µL of 50× ROX Reference Dye 2, 2.0 µL of 100 ng / µL cDNA template, and water to a final volume of 20 µL. The reaction program for real-time quantitative PCR is as follows: 95℃ for 5 min; 95℃ for 5 s, 50℃ for 30 s, 72℃ for 40 s, for 40 cycles.

[0011] The more detailed experimental methods of this invention are as follows: The real-time quantitative PCR detection method for the transaminase gene of Papaya mealybug can be achieved through the following steps: (1) Primer design: Based on the sequence of the transaminase gene of Papaya mealybug obtained from transcriptome sequencing, specific primers suitable for real-time PCR detection were designed using DNAMAN software. The primer sequences are as follows: Upstream primer (transaminase-F): 5'-CTATTCACGGAGCAAGGATTGTAG-3', Downstream primer (transaminase-R): 5'-GTTCGCATGGAGATTATTCTGTCA-3'; Meanwhile, based on the sequence of the tuberulin gene obtained from the transcriptome sequencing results, primers for the internal control in quantitative real-time PCR were designed. The primer sequences are as follows: Upstream primer (Tubulin-F): 5'-CTTCACTTCTTCATGCCTGG-3', Downstream primer (Tubulin-R): 5'-TTTGTTCGTCAACTTCTTTC-3'; (2) Papaya mealybug treatment experiment: Papaya seedlings and potato seedlings were cultivated in artificial climate chambers to feed papaya mealybugs. Twenty insects from each were collected into Eppendorf tubes and divided into three biological replicates, for a total of six groups of samples. After treatment, the insect samples were quickly fixed in liquid nitrogen and stored at -80℃ for later use. (3) cDNA synthesis: Refer to TransGen Biotech's method. TransZol TMTotal RNA was extracted using the Up Plus RNA Kit, and cDNA was synthesized according to the instructions of the Novizan HiScriptQ RT SuperMix for qPCR (+gDNA wiper) kit. (4) Real-time quantitative PCR reaction: Real-time quantitative PCR was performed using the ChamQ SYBR Color qPCR Master Mix (2X) kit from Nanjing Novizan Biotechnology Co., Ltd. Using the synthesized cDNA as a template, and the transaminase-F, transaminase-R, Tubulin-F, and Tubulin-R as specific primers, quantitative PCR was performed. Each sample was tested in triplicate, and the average Ct value of the parallel replicates was taken after amplification. The reaction system for real-time quantitative PCR was as follows: 10 µL of 2× ChamQ SYBR Color qPCR Master Mix, 0.8 µL (5 µM) of upstream primer, 0.8 µL (5 µM) of downstream primer, 0.4 µL of 50× ROX Reference Dye 2, 2.0 µL of 100 ng / µL cDNA template, and water to a final volume of 20 µL. The reaction program for real-time quantitative PCR is as follows: 95℃ for 5 min; 95℃ for 5 s, 50℃ for 30 s, 72℃ for 40 s, for 40 cycles; (5) The formula for calculating the expression level of the papaya mealybug transaminase gene relative to the tubulin gene is: relative mRNA expression = 2 -ΔΔCt ×100%, where Ct value = target gene Ct value - internal reference gene Ct value.

[0012] Figure 3 The differential expression of transaminase genes in *Papaver somniferum* after feeding on different hosts was investigated. The results showed that different host species led to significant differences in transaminase gene expression levels, and this trend was consistent with the expression trends obtained from transcriptome sequencing. These results provide important basic data for further research on transaminase genes in *Papaver somniferum*.

[0013] Compared with the prior art, the present invention has the following significant advantages and technical effects: (1) The different host treatment schemes used in this invention are applicable to most transaminase genes in insects, which is of great significance for a comprehensive and in-depth study of the expression characteristics of such genes.

[0014] (2) The efficient and rapid fluorescence quantitative PCR method disclosed in this invention (including a dedicated fluorescence quantitative PCR program, etc.) takes only about 50 minutes in actual use, which significantly shortens the reaction time compared with the prior art and has the technical characteristics of high efficiency and speed.

[0015] (3) The fluorescence quantitative PCR method disclosed in this invention provides a conventional PCR electrophoresis result image simultaneously, which can intuitively and quickly reflect the specificity of the primers. Attached Figure Description

[0016] Figure 1 Electrophoresis results of conventional PCR products for the transaminase gene of *Papaya spp.* (a type of mealybug) using quantitative real-time PCR primers. The product length is 109 bp. The marker bands from top to bottom are: 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp.

[0017] Figure 2 Electrophoresis results of routine PCR products of the tubebulin gene from *Papaya spp.* using quantitative real-time primers. The product length is 202 bp, and the marker bands from top to bottom are: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp.

[0018] Figure 3 Differential expression of transaminase genes in *Papaya mealybug* after feeding on different hosts. The bars and left axis represent expression levels from quantitative real-time PCR, while the scatter plots and right axis represent transcriptome expression levels. MF6 represents *Papaya mealybug* feeding on papaya, and TF6 represents *Papaya mealybug* feeding on potato. Detailed Implementation Plan

[0019] The present invention will be further described below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments described are only for explaining the present invention and do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, simplifications, or combinations made without departing from the spirit and principle of the present invention should be considered equivalent alternatives and included within the scope of protection of the present invention.

[0020] Example 1: Preparation of test materials: Papaya seedlings and potato seedlings were cultured separately in artificial climate chambers to rear the papaya mealybug. Twenty papaya mealybugs were collected from each host plant and placed in Eppendorf tubes, with three biological replicates, resulting in a total of six samples. After collection, the insect samples were rapidly fixed in liquid nitrogen and then transferred to -80°C for storage.

[0021] Example 2: Primer design: (1) Primer design: Based on the sequence of the transaminase gene of Papaya mealybug obtained from transcriptome sequencing, specific primers suitable for real-time PCR detection were designed using DNAMAN software. The primer sequences are as follows: Upstream primer (transaminase-F): 5'-CTATTCACGGAGCAAGGATTGTAG-3', Downstream primer (transaminase-R): 5'-GTTCGCATGGAGATTATTCTGTCA-3'.

[0022] The primers for real-time quantitative PCR of the Papaya mealybug transaminase gene produced a product of 109 bp in conventional PCR. Agarose gel electrophoresis showed that the amplified products were of uniform length and formed a single band, indicating that the designed primers have high specificity and are suitable for real-time quantitative PCR detection. The agarose gel electrophoresis results are as follows: Figure 1 As shown.

[0023] (2) Simultaneously, based on the sequence of the tubeulin gene obtained from transcriptome sequencing, primers for the internal control in quantitative real-time PCR were designed. The primer sequences are as follows: Upstream primer (Tubulin-F): 5'-CTTCACTTCTTCATGCCTGG-3', Downstream primer (Tubulin-R): 5'-TTTGTTCGTCAACTTCTTTC-3'.

[0024] The primers for real-time quantitative PCR of the tuberulin gene in *Papaya spp.* produced a product of 202 bp in conventional PCR. Agarose gel electrophoresis showed that the amplified products were of uniform length and formed a single band, indicating that the designed primers have high specificity and are suitable for real-time quantitative PCR detection. (Agarose gel electrophoresis results are shown below.) Figure 2 As shown.

[0025] The standard PCR reaction system is as follows: 10 µL of 2× Taq Plus Master Mix, 0.8 µL (5 µM) of upstream primer, 0.8 µL (5 µM) of downstream primer, 1.0 µL of 100 ng / µL cDNA template, and 7.4 µL of water; The standard PCR reaction procedure is as follows: 94℃ for 5 min; 95℃ for 30 s, 50℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 10 min.

[0026] Example 3: Total RNA extraction: Referencing Quanshijin Company TransZol TM The Up Plus RNA Kit instructions describe the extraction of total RNA. The specific steps are as follows: First, grind the insect body into powder using liquid nitrogen, then add 1 ml of... TransZol TMUp, transfer to a 1.5 ml centrifuge tube and incubate at room temperature for 5 min; then add 200 μL of chloroform (per 1 ml). TransZol TM (Up corresponds), shake vigorously for 30 seconds, incubate at room temperature for 3 minutes; after centrifugation at 4℃ and 10000g for 15 minutes, transfer the upper aqueous phase (generally <80%) to a new centrifuge tube, add 1 / 3 volume of anhydrous ethanol, and gently invert to mix. Place the RNA centrifuge column into a 2ml collection tube. Transfer the entire mixture from the previous step into the centrifuge column and centrifuge at 12000g for 30-60s. Discard the mobile phase and reuse the collection tube. Add 500μL of CB9 and centrifuge at 12000g for 30s at room temperature. Discard the mobile phase. Add another 500μL of CB9 and centrifuge at 12000g for 30s at room temperature. Discard the mobile phase. Add 500μL of diluted WB9 and centrifuge at 12000g for 30s. Discard the mobile phase. Add another 500μL of diluted WB9 and centrifuge at 12000g for 30s. Discard the mobile phase. Centrifuge at 12000g for 2min to completely remove residual ethanol. Let the column air dry at room temperature for several minutes. Transfer the centrifuge column to an RNase-free centrifuge tube. Add 50μL of RNase-free water to the center of the column membrane. Let stand at room temperature for 1min, then centrifuge at 12000g for 1min to elute the RNA. Store the obtained RNA at -80℃ for later use.

[0027] Example 4: cDNA synthesis: Following the instructions of the Novozymes HiScript Q RT SuperMix for qPCR (+gDNAwiper) kit, cDNA was generated by reverse transcription using the RNA obtained in Example 3 as a template. The specific steps are as follows: (1) Removal reaction of genomic DNA Reaction system: 4× gDNA wiper Mix 4.0 μl, template RNA 1 μg, RNase-free ddH2O to a final volume of 16 μl.

[0028] (2) Preparation of reverse transcription reaction system Reaction system: 5× qRT SuperMix II 4.0μl, (1) reaction solution 16.0μl.

[0029] (3) Perform reverse transcription reaction, incubate at 50℃ for 15 min, heat at 85℃ for 2 min, and store the product at -20℃ or -80℃.

[0030] Example 5: Real-time quantitative PCR was performed. The ChamQ SYBR Color qPCR Master Mix (2×) kit from Nanjing Novizan Biotechnology Co., Ltd. was used. Using the cDNA from Example 4 as a template, real-time quantitative PCR was performed with the primers from Example 2. Each sample was tested in triplicate, and the average Ct value from each replicate was taken after amplification.

[0031] (1) The reaction system for real-time quantitative PCR is as follows: 10µL of 2× ChamQ SYBR Color qPCR Master Mix, 0.8µL (5µM) of upstream primer, 0.8µL (5µM) of downstream primer, 0.4µL of 50× ROX Reference Dye 2, 2.0µL of 100ng / µL cDNA template, and water to 20µL. (2) The reaction program for real-time quantitative PCR is: 95℃ for 5 min; 95℃ for 5 s, 50℃ for 30 s, 72℃ for 40 s, for 40 cycles.

[0032] (3) After real-time quantitative PCR is completed, the ratio of relative expression levels under different treatment conditions is calculated based on the Ct value. -ΔΔCt The formula for calculating the expression level of the papaya mealybug transaminase gene relative to the tubulin gene is: relative mRNA expression = 2. -ΔΔCt ×100%, where Ct value = target gene Ct value - internal reference gene Ct value.

[0033] Table 1. C(T) values, mean values, standard deviations, quantitative fluorescence expression levels, and transcriptome expression levels of transaminase genes after *Papaya mealybug* fed on different hosts. (4) Figure 3 The differential expression of transaminase genes in *Papaver somniferum* after feeding on different hosts was investigated. The results showed that different host species led to significant differences in transaminase gene expression levels, and this trend was consistent with the expression trends obtained from transcriptome sequencing. These results provide important basic data for further research on transaminase genes in *Papaver somniferum*.

Claims

1. A real-time quantitative PCR primer for detecting the expression characteristics of transaminase gene in Papaya mealybug, characterized in that: The quantitative PCR primers consist of an upstream primer and a downstream primer, with the following specific sequences: Upstream primer: 5'-CTATTCACGGAGCAAGGATTGTAG-3', Downstream primer: 5'-GTTCGCATGGAGATTATTCTGTCA-3'.

2. A method for detecting the expression characteristics of transaminase genes in Papaya mealybug, characterized in that: Includes the following steps: (1) cDNA synthesis: After extracting and purifying total RNA from the sample, the purified RNA was used as a template to perform reverse transcription reaction using a reverse transcription kit to obtain cDNA; (2) Using the cDNA obtained in step (1) as a template, perform routine PCR detection using the following primers: Upstream and downstream primers for the transaminase gene of *Papaya spp.* mealybug: Upstream primer: 5'-CTATTCACGGAGCAAGGATTGTAG-3', Downstream primer: 5'-GTTCGCATGGAGATTATTCTGTCA-3'; upstream and downstream primers for the internal reference gene Tubulin: Upstream primer: 5'-CTTCACTTCTTCATGCCTGG-3', Downstream primer: 5'-TTTGTTCGTCAACTTCTTTC-3'; (3) Real-time quantitative PCR reaction: Using the cDNA obtained in step (1) as a template, the primers in step (2) were added to perform a real-time PCR reaction. Three biological replicates were set up for each sample, and the average value was taken after amplification.

3. The method according to claim 2, characterized in that: The conventional PCR reaction system consisted of: 10 µL of 2× Taq Plus Master Mix, 0.8 µL of 5 µM upstream primer, 0.8 µL of 5 µM downstream primer, 1.0 µL of 100 ng / µL cDNA template, and 7.4 µL of water.

4. The method according to claim 2, characterized in that: The conventional PCR reaction procedure is as follows: 94℃ for 5 min; 95℃ for 3 s, 50℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 10 min.

5. The method according to claim 2, characterized in that: The reaction system for the real-time quantitative PCR was as follows: 10 µL of 2×ChamQ SYBR Color qPCR Master Mix, 0.8 µL of 5 µM upstream primer, 0.8 µL of 5 µM downstream primer, 0.4 µL of 50× ROX Reference Dye 2, 2.0 µL of 100 ng / µL cDNA template, and water to a final volume of 20 µL.

6. The method according to claim 2, characterized in that: The reaction program for the real-time quantitative PCR is as follows: 95℃ for 5 min; 95℃ for 5 s, 50℃ for 30 s, 72℃ for 40 s, for 40 cycles.

Images