Method, primer and kit for identifying sex of conopomorpha sinensis

Abstract

The invention discloses a sex identification method, primer and kit for conopomorpha sinensis. The invention relates to a conopomorpha sinensis male and female identification primer pair, which is characterized in that the conopomorpha sinensis male and female identification primer pair comprises a forward primer sequence of 5-GTGGAATGCGCGAGATGTTC-3, and a reverse primer sequence of 5-TTTGAAGACGAGAGGGTCGC-3. By utilizing a molecular biological technology, efficient, accurate, rapid and stable sex identification of conopomorpha sinensis is realized, and reliable reference is provided for scientific research and prevention and control technology development of conopomorpha sinensis.

Description

Technical Field

[0001] The present invention relates to the field of pest control, and particularly to a method, primers and a kit for differentiating male and female Conopomorpha sinensis Bradley. Background Art

[0002] Litchi and longan are famous subtropical fruits native to China. In 2023, the planting area of litchi in China was 7.9012 million mu, and the planting area of longan was 4.188 million mu, both ranking first in the world in terms of planting scale. During the growth process of litchi and longan, the warm and humid climate environment and the characteristic of not shedding leaves throughout the year make them vulnerable to various pests and diseases.

[0003] Conopomorpha sinensis Bradley belongs to the Lepidoptera and Gracillariidae, and is one of the most important pests on litchi and longan. The larvae of this pest bore into and damage the fruits, flower spikes, tender shoots and young leaves of litchi and longan, especially preferring the fruits. In general years, the fruit infestation rate of litchi and longan is 10%-20%, and in severe years, it reaches 60%-90%, causing huge losses to the production of litchi and longan. At present, the main method for controlling this pest in orchards is the use of chemical agents. Its hidden damage mode results in low control efficiency. In addition, the overuse of chemical pesticides for many years has not only hindered the development of pollution-free litchi and longan products, but also caused great damage to the ecological environment and led to the emergence of drug resistance. Therefore, while using pesticides scientifically, it is urgent to research and develop other green and sustainable prevention and control measures. At present, many scholars have reported a lot on the biology, ecology and integrated control technology of Conopomorpha sinensis Bradley, but there are few reports on the differentiation of male and female. The differentiation of male and female insects is crucial for prevention and control research based on their biology, ecology, etc. Therefore, exploring the differences between male and female individuals of Conopomorpha sinensis Bradley and constructing an accurate, rapid and stable differentiation method are of great significance for the sustainable management of this pest.

[0004] Currently, the gender identification of insects is mainly judged by the morphological differences in the pupal and adult stages. Most of the Conopomorpha sinensis Bradley collected in orchards are in the late larval stage. The morphological characteristics of males and females are not obvious. The pupae and adults are small in size, and the adults have strong activity ability. Identifying by the observation method requires the operator to have rich experience, which has a certain degree of subjectivity, is prone to misjudgment, and has low accuracy and instability. Therefore, it is very necessary to develop an accurate, rapid and stable gender identification technology for all life stages of Conopomorpha sinensis Bradley. Summary of the Invention

[0005] The purpose of the present invention is to provide a sequence for differentiating male and female Conopomorpha sinensis Bradley.

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

[0007] The second object of the present invention is to provide a primer pair for identifying the sexes of Conopomorpha sinensis Bradley, the forward primer sequence of which is: 5

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

[0009] The second object of the present invention is to provide a kit for identifying the sexes of Conopomorpha sinensis Bradley, which contains the above primer pair.

[0010] The third object of the present invention is to provide a method for identifying the sexes of Conopomorpha sinensis Bradley, which comprises the following steps:

[0011] Extract the RNA of Conopomorpha sinensis Bradley, then reverse transcribe it into cDNA, then perform PCR amplification using the above primer pair, and then perform agarose gel electrophoresis. Those with a band at the 334 bp position are male Conopomorpha sinensis Bradley, while those without a band are female Conopomorpha sinensis Bradley.

[0012] Preferably, the Conopomorpha sinensis Bradley can be Conopomorpha sinensis Bradley in various insect states, such as pupae, adults or mature larvae.

[0013] Preferably, for the PCR amplification, the reaction system is: 2×Taq HiFi PCR mix 10 μL, 10 μM forward primer 0.5 μL, 10 μM reverse primer 0.5 μL, cDNA template 1 μL, and RNase Free Water is added to 20 μL.

[0014] Preferably, for the PCR amplification, the reaction procedure is: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 25 s; annealing at 62

[0015] °C for 25 s; extension at 72°C for 8 s; denaturation-annealing-extension is repeated for 34 cycles; extension at 72°C for 5 min.

[0016] The present invention uses molecular biology techniques to achieve efficient, accurate, rapid and stable identification of the sexes of Conopomorpha sinensis Bradley, providing a reliable reference for the scientific research and prevention and control technology development of this insect. Description of the Drawings

[0017] Figure 1 It is a schematic diagram for identifying the sexes of mature larvae, pupae and adults of Conopomorpha sinensis Bradley, 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 It is an analysis of the relative expression levels of different insect states of male Conopomorpha sinensis Bradley. Detailed implementation manners

[0019] The following embodiments are further descriptions of the present invention rather than limitations thereof.

[0020] Embodiment 1:

[0021] I. Experimental method

[0022] 1. Primer design and specificity detection

[0023] A gene differentially expressed between males and females was screened from the transcriptome of mature larvae of Conopomorpha sinensis using the DEseq2 software. Annotated as hypothetical protein KGM_202694 in the NR database, it showed differential expression between male and female mature larvae.and obtained its coding sequence as: 5`-ATGGATGACGAAGAAAATACAACGAAAAAGAAAAAGAAGAGTT CCAAATATAACTACGCTTTTGGTGATCTTTATCCAGGTGTTACGATTGGACATAAGACATGCAATCCTTTCATGGTACCTGGCCTCCCTGTGCCAGCCGACATGGGGTGGTTGTGGAATGCGCGAGATGTTCCTGGGATAAAGTATCGTCGAGGCTGGCAGCCAGGCATCATCGGCAAGAGTGTTGCAAAAGTGATGACATTTAAGACACCACGAGTTGGACCCGATAAAGACAAGAACAAAAAGAAGAAGAAAAAGGGGGCGAAGCCTGGCGAGGCGTTGGGAGGAGGTGACTCCGAACCTGAAGAGCCGTTGGAACCGAAACCCGCTTTGAAGGTTCAGAGGAAAGGAGACGTTTTTACTATACAGGTTAACCCCTTGAAAGACCTCACTGAAATCCATCCAAATGAAGATCCTTACGTGGACTGCGACCCTCTCGTCTTCAAAATCATCAAAAAGAGGAGTCCAGAAGAACAGGCTAAAGTTGAAGCCAGGAGAATGGTGAAACTAAAGAAAAAGAGAGACGCTGAAATTCGCAATGCTTTAGCCGAAGCCACAGAAGACATCTGCAAGTGTGCTTATATGGATGTCTTTTGCAACGACTTAACGGCCATCGACAAAGTGATTGAAAAGTGTCCTACTTTCAAAGATCCAGAGTGTATTTGTCAGTCGGAATCGTTGAGTTCGCTTTCCAGCAACGCGACATGGGACATAGAGTACACGCCTCCATTCGGCTGCTTCGATCTGGCTCCTAGAAAACGCATAAACTATATCCACGTAGAGACACAGTACCTTGCTACTGACGCTGGCATTCCGGACACGCCGAAGGTAAAGTGTAAAAAGTCTACAGCGTCTATAAGGAAGAGTTCGCTGAAGAAGTCCTCGTGTAAGAAATGTTGCAGTCCATGCTTTTGA-3`(SEQ ID NO.1).

[0024] Primer 5 software was used for primer design. After screening, a pair of primers with good specificity was obtained and entrusted to Guangzhou Tianyi Huayu Biotechnology Co., Ltd. for synthesis. The forward primer sequence is: 5`-GTGGAATGCGCGAGATGTTC-3`, and the reverse primer sequence is: 5`-TTTGAAGACGAGAGGGTCGC-3`. The nucleotide sequence of the amplification product is: 5`-GTGG AATGCGCGAGATGTTCCTGGGATAAAGTATCGTCGAGGCTGGCAGCCAGGCATCATCGGCAAGAGTGTTGCAAAAGTGATGACATTTAAGACACCACGAGTTGGACCCGATAAAGACAAGAACAAAAAGAAGAAGAAAAAGGGGGCGAAGCCTGGCGAGGCGTTGGGAGGAGGTGACTCCGAACCTGAAGAGCCGTTGGAACCGAAACCCGCTTTGAAGGTTCAGAGGAAAGGAGACGTTTTTACTATACAGGTTAACCCCTTGAAAGACCTCACTGAAATCCATCCAAATGAAGATCCTTACGTGGACTGCGACCCTCTCGTCTTCAAA-3` (SEQ ID NO.2).

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

[0026] 2. RNA Extraction from Litchi Stem Borer Samples

[0027] The fallen fruits were collected from the litchi orchard and brought back to the laboratory. The insect sources for the experiment were collected with corrugated paper. On the morning of the second day, the corrugated paper was checked for mature larvae, and some mature larvae were collected separately, waiting for the remaining mature larvae on the corrugated paper to pupate, and some male and female pupae were collected separately 4 days later. After the remaining pupae emerged, the male and female were identified by the external genitalia and collected separately. The collected mature larvae, pupae and adults were washed twice with 1×PBS buffer and placed in 1.5mL centrifuge tubes. The RNA of each sample was extracted using a micro RNA extraction kit (Adlai Bio, Beijing). The integrity and purity of the sample RNA were detected by 1.5% agarose gel electrophoresis and an enzyme marker, and stored at -80℃ for later use.

[0028] 3. Synthesis of cDNA Library of Conopomorpha sinensis Bradley Samples

[0029] The RNA of the above-extracted Conopomorpha sinensis Bradley samples was reverse-transcribed into cDNA by using the Evo M-MLV Reverse Transcription Premix Kit. The reverse transcription system was as follows:

[0030]

[0031]

[0032] The reaction conditions were: 37°C for 15 min; 85°C for 5 s; stored at 4°C.

[0033] 4. PCR Amplification of Conopomorpha sinensis Bradley Samples

[0034] According to the primer sets designed for the mature larvae of Conopomorpha sinensis Bradley, the obtained cDNA diluted 30-fold was used as the template for PCR amplification, and the PCR products corresponding to different Conopomorpha sinensis Bradley samples were obtained respectively. The amplification system was as follows:

[0035]

[0036] The reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 25 s; annealing at 62°C for 25 s; extension at 72°C for 7 s; denaturation-annealing-extension repeated for 34 cycles; extension at 72°C for 5 min; stored at 4°C.

[0037] According to the aforementioned designed primer sets, the obtained cDNA of male insects diluted 40-fold was used as the template for fluorescence quantitative PCR (qRT-PCR) amplification, and the Ct values of two genes corresponding to different Conopomorpha sinensis Bradley samples were obtained respectively. The amplification system was as follows:

[0038]

[0039]

[0040] The reaction conditions were: pre-denaturation at 94°C for 20 s; denaturation at 94°C for 10 s; annealing-extension at 60°C for 20 s; denaturation-annealing-extension for 40 cycles; the melting curve was automatically generated by the machine.

[0041] After the experiment, the data was processed by using the 2 -ΔΔCt method. Taking the Ct values of galactose mutarotase-like variant X1 in each insect stage as the reference, the relative expression levels of hypothetical protein KGM_202694 and galactose mutarotase-like variant X1 in different Conopomorpha sinensis Bradley samples were compared. And the independent samples t-test was used to analyze the difference in the expression levels of the two genes in the same insect stage.

[0042] 5. Agarose Gel Detection of PCR Products

[0043] 6. Prepare a gel with 1.5% agarose concentration. 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 genders of mature larvae, pupae, and adults of Conopomorpha sinensis through the gel imaging results. Among them, for the primer pair, if a band can be amplified between 250 - 500 bp, it is a male Conopomorpha sinensis; if no band can be amplified, it is a female Conopomorpha sinensis.

[0044] II. Experimental Results

[0045] As Figure 1 can be seen, specific bands of 334 bp can be amplified from mature larva samples 1 - 2, pupa samples 5 - 6, and adult samples 9 - 10. Therefore, 1 - 2, 5 - 6, and 9 - 10 are male Conopomorpha sinensis; specific bands cannot be amplified from mature larva samples 3 - 4, pupa samples 7 - 8, and adult samples 11 - 12. Therefore, 3 - 4, 7 - 8, and 11 - 12 are female Conopomorpha sinensis. This is consistent with the actual male and female situations of the samples. This detection method has good sensitivity.

[0046] As Figure 2 can be seen, in the samples of mature larvae, pupae, and adults of male insects, the expression levels of hypothetical protein KGM_202694 are 5.12 ± 1.25, 4.67 ± 1.20, and 4.97 ± 0.41 times that of galactose mutarotase-like variant X1. The statistical results of the data are all significantly different (P < 0.01), indicating that the expression abundance of the hypothetical protein KGM_202694 gene in the three insect stages is significantly higher than that of the galactose mutarotase-like variant X1 gene, and it has better detection sensitivity and lower detection limit when performing male and female discrimination.

Claims

1. A sequence for distinguishing male and female of litchi peduncle borer, characterized in that: The male and female identification sequence is shown in SEQ ID NO.

1.

2. A primer pair for distinguishing male and female litchi peduncle borers, characterized in that: The forward primer sequence is: 5`-GTGGAATGCGCGAGATGTTC-3`, and the reverse primer sequence is: 5`-TTTGAAGACGAGAGGGTCGC-3`.

3. A kit for distinguishing the sexes of litchi peduncle borers, characterized in that: Containing the primer pair according to claim 2.

4. A method for distinguishing the sexes of litchi peduncle borers, characterized in that: The following steps are involved: The RNA of the litchi pedicle borer is extracted and then reverse transcribed into cDNA, and then PCR amplification is performed using the primer pair described in claim 2, and then agarose electrophoresis is performed. The one with a band at the 334 bp position is a male litchi pedicle borer, and the one without a band is a female litchi pedicle borer.

5. The identification method according to claim 4, characterized in that: The litchi stalk borer is a litchi stalk borer in various insect stages, preferably a pupa, an adult or a mature larva.

6. The identification method according to claim 4, characterized in that: The PCR amplification reaction system is: 2×Taq HiFi PCR mix 10 μL, 10 μM forward primer 0.5 μL, 10 μM reverse primer 0.5 μL, cDNA template 1 μL, and RNase Free Water added to 20 μL.

7. The identification method according to claim 4, characterized in that: The PCR amplification reaction procedure is as follows: pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 25 seconds; annealing at 62°C for 25 seconds; extension at 72°C for 8 seconds; denaturation-annealing-extension repeated for 34 cycles; and extension at 72°C for 5 minutes.

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