Ebony gene regulating body color and adult eclosion of gray tea geometrid, application and preparation method of dsRNA

Abstract

The present invention relates to the fields of biotechnology and pest control, and discloses an ebony gene, dsRNA application, and preparation method for regulating the body color and adult eclosion of a gray tea geometrid. The ebony gene for gray tea geometrid is first cloned and sequenced to obtain the nucleotide sequence of the ebony gene for gray tea geometrid, as shown in SEQ ID NO: 1. A gene fragment with the sequence of SEQ ID NO: 3 is then obtained, and dsRNA is synthesized from the fragment. After the synthesized dsRNA is introduced into the body cavity of gray tea geometrid larvae, the body color of the larvae and pupae becomes black, the pupal shell becomes harder, and the adults cannot eclode normally, resulting in their complete death. The gray tea geometrid ebony gene screened and obtained by the present invention can be used as an important molecular target for insect control, providing a new approach for the development of safe, efficient, and environmentally friendly pest control methods.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and pest control, and in particular to an ebony gene for regulating the body color and adult eclosion of a gray tea geometrid, and the application and preparation method of dsRNA. Background Art

[0002] The gray tea looper (Ectropis truncatus) is a major pest in Chinese tea gardens, found across all tea-growing regions. Its larvae primarily feed on young and mature leaves, and in large outbreaks, it can consume entire tea leaves, new shoots, tender bark, and young fruit. Currently, control of this insect relies primarily on chemical pesticides. However, the widespread use of these pesticides has led to increased resistance and severely impacted tea quality and safety, as well as the ecological security of tea gardens. Therefore, the development of safe, non-toxic, and environmentally friendly alternatives is a key issue that needs to be addressed.

[0003] The numerous issues with chemical pest control technologies have compelled researchers to develop novel pest control methods. RNA interference (RNAi), awarded the Nobel Prize in 2006, is a highly efficient and specific silencing technology for homologous RNA, mediated by double-stranded RNA molecules. Since its emergence, RNAi has demonstrated significant potential due to its specificity and efficiency in silencing target genes. It has opened up new avenues for research on gene function, human disease treatment, and crop pest control. Pest control based on RNAi technology has the following advantages: 1) It is specific, selectively interfering with genes specific to pests while having no killing effect on non-target organisms; 2) RNA is easily degraded in nature, leaving no residue; and 3) it is environmentally non-toxic and relatively safe. Therefore, RNAi technology is an ideal system for species-specific pest control.

[0004] The larvae of the gray tea geometre (Ectropis truncatus) can mimic their surroundings through their body color and morphology to avoid predators. Melanin is one of the most important pigments in insect body color. The ebony gene plays a key role in insect melanin synthesis, catalyzing dopamine to produce N-β-alanyldopamine, which lightens the insect's body color. Furthermore, genes involved in melanin synthesis are associated with insect growth and development. Loss of some genes can affect the growth and development of insect embryos and larvae, leading to insect death. Therefore, understanding the molecular mechanism by which gray tea geometre larvae use color to achieve mimicry and identifying lethal genes in gray tea geometre has important theoretical and practical implications for the development of new pest control technologies. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an ebony gene, dsRNA application and preparation method for regulating the body color and adult emergence of the gray tea geometrid. By introducing the synthesized dsRNA into the body cavity of the gray tea geometrid larvae, the gray tea geometrid ebony gene can be efficiently silenced, and the expression of the ebony gene in the gray tea geometrid larvae is significantly reduced, resulting in a zero emergence rate of the gray tea geometrid adults, thereby causing all the death.

[0006] In order to achieve the above-mentioned purpose, the present invention provides the following technical solutions:

[0007] In a first aspect, an ebony gene that regulates the body color and adult emergence of the gray tea looper has a nucleotide sequence as shown in SEQ ID NO.1.

[0008] Furthermore, an ebony gene that regulates the body color and adult emergence of the gray tea looper has an amino acid sequence as shown in SEQ ID NO.2.

[0009] The acquisition method includes the following steps: based on the transcriptome database of the gray tea looper, the ebony gene is retrieved using a bioinformatics method, and after sequence analysis and alignment, the nucleotide sequence of the ebony gene SEQ ID NO.1 is obtained, and upstream and downstream primers (whose sequences are SEQ ID NO.4 and SEQ ID NO.5, respectively) are designed. The ebony gene is obtained by PCR amplification and named ebony. Its length is 2583bp and encodes 860 amino acids.

[0010] Furthermore, the nucleotide sequence of an ebony gene fragment that regulates the body color and adult eclosion of the gray tea looper is the sequence of SEQ ID NO.3.

[0011] Furthermore, a dsRNA is synthesized from the ebony gene fragment SEQ ID NO.3 that regulates the body color and adult eclosion of the gray tea looper.

[0012] According to the sequence of SEQ ID NO.1, PCR amplification was performed using the upstream primer sequence SEQ ID NO.6 and the downstream primer sequence SEQ ID NO.7, and the dsRNA of the ebony gene was further synthesized using a related kit.

[0013] In a second aspect, the present invention also provides an application of the synthesized dsRNA, which is to introduce the dsRNA into the body cavity of the gray tea looper larvae.

[0014] Furthermore, the application of the synthesized dsRNA is achieved by injecting the dsRNA into the body cavity of the gray tea looper larvae by injection, or by feeding the gray tea looper larvae.

[0015] In addition to injection, the present invention can also be used through other methods. For example, but not limited to, introduction into the insect body through feeding, including direct dsRNA feeding and feeding with bacterial liquid expressing the dsRNA. Alternatively, the dsRNA can be introduced into tea leaves to produce plant-derived expressed dsRNA.

[0016] In a third aspect, the present invention further provides a method for preparing dsRNA, comprising the following steps:

[0017] S1. Total RNA of Geometridae cinerea was collected and first-strand cDNA was obtained by RT-PCR.

[0018] S2, designed upstream primer SEQ ID NO.3 and downstream primer SEQ ID NO.4, using the first-strand cDNA obtained in step S1 as a template, amplified the full-length ebony gene of Geometridae glauca by PCR, and verified the nucleotide sequence and amino acid sequence of the ebony gene. The nucleotide sequence is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO.2;

[0019] S3. Based on SEQ ID NO.1, upstream primer SEQ ID NO.6 and downstream primer SEQ ID NO.7 were designed to amplify the ebony gene fragment by PCR, the nucleotide sequence of which is SEQ ID NO.3, and purified;

[0020] S4, based on the ebony gene fragment SEQ ID NO.3 obtained in step S3, using T7RiboMAX TM dsRNA was prepared using the Express RNAi System (Promega) kit.

[0021] Beneficial effects of the present invention:

[0022] (1) The dsRNA of the ebony gene of the gray tea looper provided by the present invention can significantly inhibit the expression of the insect ebony gene, thereby causing the body color of the gray tea looper larvae to become black and the hardness of the pupal shell to become stronger, resulting in the gray tea looper adult being unable to break through the pupal shell and normally emerge and die.

[0023] (2) The dsRNA of the ebony gene of the gray tea looper provided by the present invention is specific and highly effective for controlling the gray tea looper. It is lethal only to the gray tea looper and does not cause harm to other non-target organisms. Moreover, it is easily degraded in nature and is safe for the ecological environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the electrophoresis diagram of PCR amplification of dsRNA of the ebony gene fragment of the gray tea looper;

[0025] Figure 2 is the silencing efficiency of the ebony gene by injecting dsRNA containing the ebony gene fragment of the gray tea looper;

[0026] Figure 3 The body color and growth and development after injection of dsRNA of the ebony gene fragment of the gray tea looper;

[0027] Figure 4 The shear force of pupae injected with dsRNA encoding the ebony gene fragment of the gray tea looper.

[0028] Figure 5 This is the cross-section structure of the pupa shell injected with dsRNA of the ebony gene fragment of the gray tea looper;

[0029] Figure 6 The figure shows the emergence rate of pupae injected with dsRNA of the ebony gene fragment of the gray tea looper.

[0030] Among them, "dsebony" represents the treatment group injected with ebony gene fragment dsRNA, and "dsGFP" represents the control group injected with GFP gene dsRNA. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to the embodiments and accompanying drawings:

[0032] Example 1: Cloning of the ebony gene of Geometridae grisea

[0033] (1) Obtaining the ebony gene from the gray tea looper

[0034] Bioinformatics methods were used to analyze the transcriptome data of Geometridae grisea and retrieve the ebony gene.

[0035] (2) Obtaining total RNA from the gray tea looper

[0036] Ten third-instar larvae of the gray tea looper were selected and frozen in liquid nitrogen. Total RNA was extracted using the Easy RNA Extraction kit (Zhejiang Easyside Biotechnology Co., Ltd.). The specific extraction method was referred to the kit instructions.

[0037] (3) Synthesis of the first-strand cDNA of Geometridae grisea

[0038] PrimeScript reverse transcription kit from Bio-Rad Biotechnology (Beijing) Co., Ltd. TM RT reagent Kit was used to synthesize the first-strand cDNA.

[0039] (4) PCR amplification

[0040] Specific primers were designed using SnapGene software; the upstream primer sequence is SEQ ID NO. 4, and the downstream primer sequence is SEQ ID NO. 5. These primers were synthesized by Nanjing GenScript Biotech Co., Ltd. Using the first-strand cDNA as a template, the full-length ebony gene of the geometrid was amplified by PCR. The PCR product was transformed into Escherichia coli and sequenced at Zhejiang Shangya Biotechnology Co., Ltd. The sequencing results were compared with the transcriptome data to verify the nucleotide and amino acid sequences of the ebony gene. The nucleotide sequence is SEQ ID NO. 1, and the amino acid sequence is SEQ ID NO. 2.

[0041] Example 2: Obtaining the ebony gene fragment of the gray tea looper

[0042] Based on the ebony gene nucleotide sequence obtained in Example 1, specific primers were designed using SnapGene software. The upstream primer sequence is SEQ ID NO. 6, and the downstream primer sequence is SEQ ID NO. 7. The primers were synthesized by Nanjing GenScript Biotech Co., Ltd. Using the ebony gene cloning vector plasmid obtained in Example 1 as a template, an ebony gene fragment was obtained by PCR amplification. The nucleotide sequence of the fragment is SEQ ID NO. 3, and the fragment was purified using the FastPure Gel DNA Extraction Mini Kit from Nanjing Novozymes Biotech Co., Ltd.

[0043] Example 3: Obtaining dsRNA of the Ebony Gene Fragment of the Grey Tea Looper

[0044] Based on the ebony gene fragment of the gray tea looper obtained in Example 2, T7 RiboMAX TM Express RNAiSystem (Promega) kit was used to synthesize dsRNA in vitro according to the instructions, and the obtained dsRNA was further tested for its homogeneity by 1.0% agarose gel electrophoresis. Figure 1 As shown, the concentration of dsRNA was determined using NanoDrop One, and the dsRNA was diluted to a final concentration of 2.5 μg / μl and stored in a -80°C freezer until use.

[0045] Example 4: Observation of the body color of the gray tea looper after injection of dsRNA

[0046] (1) dsRNA injection of the ebony gene fragment of the gray tea looper

[0047] The present invention selects gray tea geometrid larvae of the same age and size as injection targets. Using a 25-microliter microsyringe, the larvae's abdominal junction between segments 2 and 3 is injected, followed by blood flow, at a dose of 4 micrograms per larva. The same dose of dsGFP is administered as a control group. Following injection, the larvae are reared in fruit fly tubes under the following conditions: a temperature of 25±2°C, a humidity of 60%, a light:dark cycle of 14 hours:10 hours, and daily replacement of tea leaves.

[0048] (2) Detection of silencing effect of ebony gene in gray tea looper

[0049] The larvae of the gray tea looper injected with dsGFP and dsRNA were collected, with 10 in each group and 3 biological replicates. Total RNA was extracted using the Easy RNA Extraction kit from Zhejiang Easy Biotechnology Co., Ltd. and the PrimeScript TM The total RNA was reverse transcribed using the RT reagent Kit with gDNAEraser to obtain the first-strand cDNA. The expression levels of the ebony gene relative to the internal reference gene GAPDH were detected by real-time fluorescence quantitative PCR, and the silencing efficiency of the synthesized dsRNA on the ebony gene was calculated. The results showed that Figure 2 As shown, compared with the control group dsGFP, the expression of the ebony gene in the gray tea looper larvae was significantly reduced after injection of dsRNA, and the silencing efficiency was 72.6%.

[0050] (3) Observation of the body color and growth and development of the gray tea looper after dsRNA injection

[0051] like Figure 3 As shown in Figure 2, the body color of the third-instar larvae, fourth-instar larvae, prepupae and pupae of the gray tea looper after injection of dsRNA turned black, but the growth and development of the larvae were not affected; Figure 4 After pupation, the shear force of the pupa increases significantly; Figure 5 As shown in the cross-section of the pupa shell, its basic structure has not changed significantly, but the thickness of its black layer has increased; Figure 6 As shown, the emergence rate of adult G. griseae was zero after dsRNA injection. Therefore, injection of ebony gene dsRNA can significantly increase the strength of the G. griseae pupal case, causing the G. griseae adults to be unable to break through the pupal case and die, with a significant lethal effect.

[0052] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An ebony gene for regulating the body color and adult emergence of the gray tea looper, characterized by: The nucleotide sequence is shown in SEQ ID NO.

1.

2. A dsRNA, characterized in that The dsRNA is obtained by in vitro transcription using the ebony gene fragment of Geometridae griseae as a template; the nucleotide sequence of the ebony gene fragment is SEQ ID NO.

3.

3. The use of the dsRNA in controlling the gray tea looper according to claim 2, characterized in that: The dsRNA is injected into the body cavity of the gray tea looper larvae by injection, or is fed to the gray tea looper larvae.

4. The method for preparing dsRNA according to claim 2, wherein: The following steps are involved: S1. Total RNA of Geometridae cinerea was collected and first-strand cDNA was obtained by RT-PCR. S2. Using the upstream primer SEQ ID NO. 4 and the downstream primer SEQ ID NO: 5, and the first-strand cDNA obtained in step S1 as a template, amplify the full-length ebony gene of the geometridae by PCR, and verify the nucleotide sequence and amino acid sequence of the ebony gene. The nucleotide sequence is SEQ ID NO. 1, and the amino acid sequence is SEQ ID NO. 2; S3. Based on SEQ ID NO.1, upstream primer SEQ ID NO.6 and downstream primer SEQ ID NO.7 were designed to amplify the ebony gene fragment by PCR, the nucleotide sequence of which is SEQ ID NO.3, and purified; S4, based on the ebony gene fragment SEQ ID NO.3 obtained in step S3, using T7 RiboMAX TM dsRNA was prepared using the Express RNAi System kit.

Images