A method, primers, and reagent kit for sexing litchi fruit borers.

Abstract

This invention discloses a method, primers, and kit for sexing the litchi fruit borer. The sexing primer pair for the litchi fruit borer is characterized by comprising the following sequences: forward primer sequence: 5`-GTGGAATGCGCGAGATGTTC-3`, and reverse primer sequence: 5`-TTTGAAGACGAGAGGGTCGC-3`. This invention utilizes molecular biology techniques to achieve efficient, accurate, rapid, and stable sexing of the litchi fruit borer, providing a reliable reference for scientific research and the development of control technologies for this insect.

Description

Technical Field

[0001] This invention relates to the field of pest control, specifically to a method, primers, and reagent kit for sexing litchi fruit borers. Background Technology

[0002] Lychee and longan are subtropical fruits native to my country. In 2023, my country had 7.9012 million mu of lychee cultivation area and 4.188 million mu of longan cultivation area, making it the world's largest producer. The warm and humid climate and the fact that lychee and longan trees never shed their leaves make them susceptible to various pests and diseases during their growth.

[0003] The litchi fruit borer (Conopomorpha sinensis Bradley), belonging to the order Lepidoptera and family Achyranthes, is one of the most significant pests of litchi and longan. The larvae bore into the fruit, flower spikes, tender shoots, and young leaves of litchi and longan, with a particular preference for fruit. In normal years, the infestation rate on litchi and longan fruit is 10%-20%, reaching 60%-90% in severe years, causing substantial losses to litchi and longan production. Currently, orchard control of this pest mainly relies on chemical pesticides, but their covert feeding methods result in low control effectiveness. Furthermore, the long-term overuse of chemical pesticides not only hinders the development of pollution-free litchi and longan products but also causes significant damage to the ecological environment and leads to pesticide resistance. Therefore, while using pesticides scientifically, it is urgent to research and develop other green and sustainable control measures. Currently, many scholars have reported extensively on the biology, ecology, and integrated management techniques of the litchi fruit borer, but reports on sex identification are relatively scarce. Sex identification of insects is crucial for control research based on their biology and ecology. Therefore, exploring the differences between male and female litchi fruit borers and constructing accurate, rapid and stable identification methods are of great significance for the sustainable management of this insect.

[0004] Currently, insect sex identification mainly relies on morphological differences between pupal and adult stages. However, litchi fruit borers collected from orchards are mostly in their mature larval stage, with little difference in morphological characteristics between males and females. Pupae and adults are small, and adults are highly mobile. Identification through observation requires extensive experience, is subjective, prone to misjudgment, and suffers from low accuracy and inconsistency. Therefore, developing an accurate, rapid, and stable technique for sex identification of litchi fruit borers at different developmental stages is essential. Summary of the Invention

[0005] The purpose of this invention is to provide a sex identification sequence for litchi fruit borers.

[0006] The male and female identification sequence is as follows: 5`-ATGGATGACGAAGAAAATACAACGAAAAAGAAA AAGAAGAGTTCCAAATATAACTACGCTTTTGGTGATCTTTATCCAGGTGTTACGATTGGACATAAGACATGCAATCCTTTCATGGTACCTGGCCTCCCTGTGCCAGCCGACATGGGGTGGTTGTGGAATGCGCGAGATGTTCCTGGGATAAAGTATCGTCGAGGCTGGCAGCCAGGCATCATCGGCAAGAGTGTTGCAAAAGTGATGACATTTAAGACACCACGAGTTGGACCCGATAAAGACAAGAACAAAAAGAAGAAGAAAAAGGGGGCGAAGCCTGGCGAGGCGTTGGGAGGAGGTGACTCCGAACCTGAAGAGCCGTTGGAACCGAAACCCGCTTTGAAGGTTCAGAGGAAAGGAGACGTTTTTACTATACAGGTTAACCCCTTGAAAGACCTCACTGAAATCCATCCAAATGAAGATCCTTACGTGGACTGCGACCCTCTCGTCTTCAAAATCATCAAAAAGAGGAGTCCAGAAGAACAGGCTAAAGTTGAAGCCAGGAGAATGGTGAAACTAAAGAAAAAGAGAGACGCTGAAATTCGCAATGCTTTAGCCGAAGCCACAGAAGACATCTGCAAGTGTGCTTATATGGATGTCTTTTGCAACGACTTAACGGCCATCGACAAAGTGATTGAAAAGTGTCCTACTTTCAAAGATCCAGAGTGTATTTGTCAGTCGGAATCGTTGAGTTCGCTTTCCAGCAACGCGACATGGGACATAGAGTACACGCCTCCATTCGGCTGCTTCGATCTGGCTCCTAGAAAACGCATAAACTATATCCACGTAGAGACACAGTACCTTGCTACTGACGCTGGCATTCCGGACACGCCGAAGGTAAAGTGTAAAAAGTCTACAGCGTCTATAAGGAAGAGTTCGCTGAAGAAGTCCTCGTGTAAGAAATGTTGCAGTCCATGCTTTTGA-3`(SEQ ID NO.1).

[0007] The second objective of this invention is to provide a primer pair for sex identification of the litchi fruit borer, comprising the following forward primer sequence: 5

[0008] The reverse primer sequence is `-GTGGAATGCGCGAGATGTTC-3`, and the reverse primer sequence is: 5`-TTTGAAGACGAGAGGGTC GC-3`.

[0009] The second objective of this invention is to provide a kit for identifying the sex of litchi fruit borers, which contains the aforementioned primer pairs.

[0010] The third objective of this invention is to provide a method for identifying the sex of litchi fruit borers, which includes the following steps:

[0011] RNA was extracted from litchi fruit borers and then reverse transcribed into cDNA. The cDNA was then amplified by PCR using the primer pair described above, followed by agarose gel electrophoresis. The presence of a band at the 334bp position indicated male litchi fruit borers, while the absence of a band indicated female litchi fruit borers.

[0012] Preferably, the litchi fruit borer can be litchi fruit borer in various stages, such as pupa, adult or mature larva.

[0013] Preferably, the PCR amplification reaction system is as follows: 10 μL of 2×Taq HiFi PCR mix, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 1 μL of cDNA template, and RNase-free water added to 20 μL.

[0014] Preferably, the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 25 s; 62 ...92℃ pre-denaturation for 3 min; 94℃ denaturation for 25 s;

[0015] Anneal at ℃ for 25s; extend at 72℃ for 8s; repeat the denaturation-annealing-extension cycle for 34 cycles; extend at 72℃ for 5min.

[0016] This invention utilizes molecular biology techniques to achieve efficient, accurate, rapid, and stable sex identification of the litchi fruit borer, providing a reliable reference for scientific research and the development of control technologies for this insect. Attached Figure Description

[0017] Figure 1 This is a schematic diagram for sex identification of mature larvae, pupae, and adults of the litchi fruit borer. M: DL2000 Marker; Lanes 1-4: Mature larvae 1-4; Lanes 5-8: Pupae 1-4; Lanes 9-12: Adults 1-4.

[0018] Figure 2 This study analyzes the relative expression levels of male litchi fruit borers in different life stages. Detailed Implementation

[0019] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0020] Example 1:

[0021] I. Experimental Methods

[0022] 1. Primer design and specific detection

[0023] Using DEseq2 software, a gene differentially expressed between male and female litchi fruit borer larvae was screened in the transcriptome. Annotated in the NR database as hypothetical protein KGM_202694, it showed differential expression in both male and female mature larvae.And obtained its coding sequence as: 5`-ATGGATGACGAAGAAAATACAACGAAAAAGAAAAAGAAGAGTT CCAAATATAACTACGCTTTTGGTGATCTTTATCCAGGTGTTACGATTGGACATAAGACATGCAATCCTTTCATGGTACCTGGCCTCCCTGTGCCAGCCGACATGGGGTGGTTGTGGAATGCGCGAGATGTTCCTGGGATAAAGTATCGTCGAGGCTGGCAGCCAGGCATCATCGGCAAGAGTGTTGCAAAAGTGATGACATTTAAGACACCACGAGTTGGACCCGATAAAGACAAGAACAAAAAGAAGAAGAAAAAGGGGGCGAAGCCTGGCGAGGCGTTGGGAGGAGGTGACTCCGAACCTGAAGAGCCGTTGGAACCGAAACCCGCTTTGAAGGTTCAGAGGAAAGGAGACGTTTTTACTATACAGGTTAACCCCTTGAAAGACCTCACTGAAATCCATCCAAATGAAGATCCTTACGTGGACTGCGACCCTCTCGTCTTCAAAATCATCAAAAAGAGGAGTCCAGAAGAACAGGCTAAAGTTGAAGCCAGGAGAATGGTGAAACTAAAGAAAAAGAGAGACGCTGAAATTCGCAATGCTTTAGCCGAAGCCACAGAAGACATCTGCAAGTGTGCTTATATGGATGTCTTTTGCAACGACTTAACGGCCATCGACAAAGTGATTGAAAAGTGTCCTACTTTCAAAGATCCAGAGTGTATTTGTCAGTCGGAATCGTTGAGTTCGCTTTCCAGCAACGCGACATGGGACATAGAGTACACGCCTCCATTCGGCTGCTTCGATCTGGCTCCTAGAAAACGCATAAACTATATCCACGTAGAGACACAGTACCTTGCTACTGACGCTGGCATTCCGGACACGCCGAAGGTAAAGTGTAAAAAGTCTACAGCGTCTATAAGGAAGAGTTCGCTGAAGAAGTCCTCGTGTAAGAAATGTTGCAGTCCATGCTTTTGA-3`(SEQ ID NO.1).

[0024] Primers were designed using Primer 5 software, and a pair of primers with good specificity were obtained after screening. These primers were then synthesized by Guangzhou Tianyi Huayu Biotechnology Co., Ltd. The forward primer sequence is 5`-GTGGAATGCGCGAGATGTTC-3`, and the reverse primer sequence is 5`-TTTGAAGACGAGAGGGTCGC-3`. The nucleotide sequence of the amplified product is: 5`-GTGG AATGCGCGAGATGTTCCTGGGATAAAGTATCGTCGAGGCTGGCAGCCAGGCATCGGCAAGAGTGTTGCAAAAGTGATGACATTTAAGACACCACGAGTTGGACCCGATAAAGACAAGAACAAAAAGAAGAAGAAAAAGGGGGCGAAGCCTGGCGAGGCGTTGGGA GGAGGTGACTCCGAACCTGAAGAGCCGTTGGAACCGAAACCCGCTTTGAAGGTTCAGAGGAAAGGAGACGTTTTTACTATACAGGTTAACCCCTTGAAAGACCTCACTGAAATCCATCCAAATGAAGATCCTTACGTGGACTGCGACCCTCTCGTCTTCAAA-3`(SEQ ID NO.2).

[0025] To compare the detection sensitivity differences between hypothetical protein KGM_202694 and the previously patented galactosylmutase variant X1, primers were designed using the NCBI website. After screening, one pair of primers with good specificity and high amplification efficiency was obtained for each gene, and their synthesis was commissioned to Guangzhou Tianyi Huayu Biotechnology Co., Ltd. The forward primer sequence for hypothetical protein KGM_202694 is: 5`-GACATGGGGTGGTTGTGGAA-3`, and the reverse primer sequence is: 5`-GGT CCAACTCGTGGTGTCTT-3`; the forward primer sequence for galactosylmutase variant X1 is: 5`-CCTACCGATGACGAAGACGG-3`, and the reverse primer sequence is: 5`-GGCAGGAGACACTTGAGCAT-3`. The forward primer for the internal reference gene Actin is 5`-TGGACTTCGAGCAGGAGATG-3`, and the reverse primer is 5`-CAGGAAGGAAGGCTG GAACA-3`. The amplification product of the hypothetical protein KGM_202694 gene is 5`-GACATGGGGTGGTT GTGGAATGCGCGAGATGTTCCTGGGATAAAGTATCGTCGAGGCTGGCAGCCAGGCATCA TCGGCAAGAGTGTTGCAAAAGTGATGACATTTAAGACACCACGAGTTGGACC-3`; the amplification product of the galactosylmutase variant X1 gene is 5`-CCTACCGATGACGAAGACGGACTTGTTACAGGA GAGCGTAAAGTAGTCGGCGGCACGGCATTTGACATGCGTGTACCGCGGATGCTCAAGTGTCTCCTGCC-3`.

[0026] 2. RNA extraction from litchi fruit borer samples

[0027] Fallen fruit was collected from the lychee orchard and brought back to the laboratory. The insects were collected using corrugated cardboard. On the morning of the second day, the cardboard was checked for mature larvae, and a portion of the mature larvae were collected individually. The remaining mature larvae on the cardboard were allowed to pupate. Four days later, a portion of the male and female pupae were collected individually. After the remaining pupae emerged, their sexes were identified by their external genitalia, and they were collected individually. The collected mature larvae, pupae, and adults were washed twice with 1×PBS buffer and placed in 1.5mL centrifuge tubes. RNA was extracted from each sample using a microRNA extraction kit (Adley Biotechnology, Beijing). The integrity and purity of the RNA samples were detected using 1.5% agarose gel electrophoresis and a microplate reader, and the samples were stored at -80℃ for later use.

[0028] 3. Synthesis of cDNA library from litchi fruit borer samples

[0029] The RNA extracted from the litchi fruit borer samples was reverse transcribed into cDNA using the Evo M-MLV reverse transcription premix kit. The reverse transcription system was as follows:

[0030]

[0031]

[0032] The reaction conditions were: 37℃ for 15 min; 85℃ for 5 s; and 4℃ for storage.

[0033] 4. PCR amplification of litchi stem borer samples

[0034] Primers designed based on mature larvae of the litchi fruit borer were used to amplify PCR using cDNA diluted 30-fold as templates, yielding PCR products for different litchi fruit borer samples. The amplification system was as follows:

[0035]

[0036] The reaction conditions were as follows: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 25 s; 62℃ annealing for 25 s; 72℃ extension for 7 s; repeat the denaturation-annealing-extension cycle for 34 cycles; 72℃ extension for 5 min; store at 4℃.

[0037] Based on the aforementioned primer set, the obtained male insect cDNA was diluted 40-fold as template for quantitative real-time PCR (qRT-PCR) amplification, and the Ct values ​​of the two genes corresponding to different litchi fruit borer samples were obtained. The amplification system was as follows:

[0038]

[0039]

[0040] The reaction conditions were: 94℃ pre-denaturation for 20s; 94℃ denaturation for 10s; 60℃ annealing-extension for 20s; denaturation-annealing-extension for 40 cycles; and the machine had its own melting curve.

[0041] After the experiment, using 2 -ΔΔCt Data processing was performed using the Ct values ​​of galactosylmutase variant X1 at each insect developmental stage as a reference. The relative expression levels of hypothetical protein KGM_202694 and galactosylmutase variant X1 were compared in different litchi fruit borer samples. Independent samples t-tests were used to analyze the differences in expression levels of the two genes at the same insect developmental stage.

[0042] 5. Agarose gel assay of PCR products

[0043] 6. Prepare an agarose gel with a concentration of 1.5%. Take 2.5 μL of the PCR product from the previous step for electrophoresis. Stop when the indicator dye bromophenol blue migrates to 3 / 4 of the gel. Identify the sex of mature larvae, pupae, and adults of the litchi fruit borer by gel imaging results. If the primer pair can amplify a band between 250-500 bp, it is a male litchi fruit borer; if no band is amplified, it is a female litchi fruit borer.

[0044] II. Experimental Results

[0045] Depend on Figure 1 It was found that mature larvae samples 1-2, pupae samples 5-6, and adult samples 9-10 amplified a specific band of 334 bp, therefore samples 1-2, 5-6, and 9-10 are male litchi fruit borers. Mature larvae 3-4, pupae samples 7-8, and adult samples 11-12 did not amplify a specific band, therefore samples 3-4, 7-8, and 11-12 are female litchi fruit borers. This is consistent with the sex distribution of the actual samples. This detection method has excellent sensitivity.

[0046] Depend on Figure 2 It was found that the expression level of hypothetical protein KGM_202694 in mature larvae, pupae, and adults of male insects was 5.12±1.25, 4.67±1.20, and 4.97±0.41 times that of galactosylmutase variant X1, respectively. The statistical results showed significant differences (P<0.01), indicating that the expression abundance of hypothetical protein KGM_202694 gene in the three insect developmental stages was significantly higher than that of galactosylmutase variant X1 gene, and it has better detection sensitivity and lower detection limit when identifying sexes.

Claims

1. A primer pair for sex identification of the litchi fruit borer, characterized in that, The forward primer sequence is 5`-GTGGAATGCGCGAGATGTTC-3`, and the reverse primer sequence is 5`-TTTGAAGACGAGAGGGTCGC-3`.

2. A kit for identifying the sex of litchi fruit borers, characterized in that, Contains the primer pair as described in claim 1.

3. A method for identifying the sex of litchi stem borers, characterized in that, Includes the following steps: RNA was extracted from litchi fruit borers and then reverse transcribed into cDNA. PCR amplification was then performed using the primer pair described in claim 1, followed by agarose gel electrophoresis. A band at the 334 bp position indicated male litchi fruit borers, while no band indicated female litchi fruit borers.

4. The identification method according to claim 3, characterized in that, The litchi fruit borers mentioned refer to litchi fruit borers in various stages, including pupae, adults, or mature larvae.

5. The identification method according to claim 3, characterized in that, The PCR amplification reaction system is as follows: 10 μL of 2×Taq HiFi PCR mix, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 1 μL of cDNA template, and RNase-free water added to 20 μL.

6. The identification method according to claim 3, characterized in that, The PCR amplification procedure is as follows: 95 °C pre-denaturation for 3 min; 94 °C denaturation for 25 s; 62 °C annealing for 25 s; 72 °C extension for 8 s; repeat denaturation-annealing-extension for 34 cycles; 72 °C extension for 5 min.

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