Application of hermetia illucens cyp311a1 gene and dsrna in growth regulation

Abstract

The present application belongs to the field of biotechnology, and particularly relates to Hermetia illucens Cyp311a1 gene, dsRNA and application thereof. In order to regulate the growth and development of Hermetia illucens, the Hermetia illucens Cyp311a1 gene is obtained: the target gene is obtained through nested PCR, the target gene is connected with a T vector, is transferred into competent cells, white spots are picked to LB liquid medium, and bacterial liquid PCR is carried out, and bacterial liquid with a target band is sent for sequencing. The dsRNA synthesized by the Hermetia illucens Cyp311a1 gene and the application in insect growth and development: upstream primers and downstream primers with T7 promoters are designed, PCR amplification is carried out, and the dsRNA template is obtained by cutting and recovering the gel, the dsRNA is synthesized by in vitro transcription, after the Hermetia illucens larvae are injected with the dsRNA, the growth and development of the larvae are delayed.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the black soldier fly Cyp311a1 gene and its dsRNA in growth regulation. Background Technology

[0002] Black soldier fly (Hermetia illucens), belonging to the family Hermetidae in the order Diptera, is a resource insect. Black soldier fly larvae have a wide diet, consuming various organic wastes and converting them into their own nutrients. The converted larvae have high nutritional value, thus finding wide application in the harmless treatment of organic waste and animal husbandry. Lipids and proteins are the two products with the greatest development potential. Black soldier fly protein has advantages such as high protein content, balanced amino acid composition, rapid reproduction rate, and large resource potential, possessing the potential to replace traditional protein raw materials and receiving extensive research in the feed industry.

[0003] Black soldier flies have significant applications in the resource utilization of organic waste and the production of protein feed. However, in large-scale farming, the asynchronous growth and development of larvae is a prominent issue, leading to mixed larval stages, low harvesting efficiency, and unstable product quality. Currently, the methods for regulating the growth and development of black soldier flies mainly rely on indirect regulation of environmental or nutritional conditions, lacking precise and specific intervention methods. RNA interference technology, which can specifically inhibit the expression of target genes by introducing double-stranded RNA (dsRNA), has been widely used in insect gene function research and pest control. Summary of the Invention

[0004] This invention addresses the above-mentioned problems by providing the black soldier fly Cyp311a1 gene, its dsRNA, and its applications.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] This invention provides a black soldier fly larvae gene Cyp311a1, the nucleotide sequence of which is shown in SEQ ID NO.1 and the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] This invention also provides a method for obtaining the Cyp311a1 gene from the black soldier fly, comprising the following steps:

[0008] Two pairs of primers were designed. The upstream primer sequence of the first pair is shown in SEQ ID NO. 3, and the downstream primer sequence is shown in SEQ ID NO. 4. The upstream primer sequence of the second pair is shown in SEQ ID NO. 5, and the downstream primer sequence is shown in SEQ ID NO. 6.

[0009] Total RNA was extracted and reverse transcribed into cDNA to obtain the template required for PCR amplification.

[0010] The target gene was obtained by nested PCR amplification, the target gene was ligated to the T vector and transformed into competent cells. White spots were picked and placed in LB liquid medium for bacterial PCR. Bacterial solutions with the target band were sent for sequencing. Strains with correct sequencing results were stored at -80℃.

[0011] The present invention also provides dsRNA synthesized based on the Cyp311a1 gene fragment of black soldier fly, the nucleotide sequence of which is shown in SEQ ID NO.7 and the amino acid sequence of which is shown in SEQ ID NO.8.

[0012] The present invention also provides a method for synthesizing the dsRNA described herein, comprising the following steps:

[0013] Design primer sequences for synthesizing dsRNA, with the upstream primer sequence shown in SEQ ID NO.9 and the downstream primer sequence shown in SEQ ID NO.10;

[0014] Using the designed primers, PCR amplification was performed with the Cyp311a1 gene fragment of black soldier fly as a template, and the desired template was obtained by gel extraction and recovery.

[0015] The obtained template was used to synthesize dsRNA through in vitro transcription using the kit.

[0016] The present invention also provides the application of the dsRNA in regulating the growth and development of black soldier fly larvae.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] This invention provides for the first time the dsRNA of the Cyp311a1 gene of black soldier fly. After injection of dsRNA, black soldier flies exhibit a large number of delayed larval development phenotypes, providing technical support for the synchronization of black soldier fly farming and having important significance for optimizing and precision farming. Attached Figure Description

[0019] Figure 1 The image shows the PCR amplification detection of the cDNA of the Cyp311a1 gene from the black soldier fly larvae. M represents the DL1000 DNA Marker, and 1 represents the Cyp311a1 gene fragment from the black soldier fly larvae.

[0020] Figure 2 This is a graph showing the gene silencing efficiency. dsGFP is the control group and dsCyp311a1 is the experimental group.

[0021] Figure 3 To investigate the effect of injecting synthetic dsRNA into third-instar larvae on larval development and to statistically analyze developmental status, the third-instar larvae injected with dsCyp311a1 showed significantly slower development compared to the control group.

[0022] Figure 4 To investigate the effect of injecting synthetic dsRNA into fourth-instar larvae on larval development and to measure body length and weight, it was found that larvae injected with dsCyp311a1 showed significantly delayed growth and development in the early stages of third-instar larval development. Detailed Implementation

[0023] To further illustrate the technical solution of the present invention, the present invention will be further described below through embodiments.

[0024] Example 1

[0025] A black soldier fly gene Cyp311a1, the nucleotide sequence of which is shown in SEQ ID NO.1 and the amino acid sequence of which is shown in SEQ ID NO.2.

[0026] The method for obtaining the black soldier fly Cyp311a1 gene includes the following steps:

[0027] (1) Primer design: Two pairs of primers were designed using SnapGene as follows:

[0028] Black soldier fly Cyp311a1 gene: The first pair of upstream primer sequences is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.4; the second pair of upstream primer sequences is shown in SEQ ID NO.5, and the downstream primer sequence is shown in SEQ ID NO.6;

[0029] (2) Template preparation: Total RNA was extracted using the TaKaRa Trizol kit and then reverse transcribed into cDNA to obtain the template required for PCR amplification;

[0030] (3) PCR amplification: The target gene is obtained by PCR amplification, and detection is performed using agarose gel electrophoresis. Figure 1 As shown, the target gene was ligated to the T vector and transformed into competent cells. White spots were picked and placed in LB liquid medium for bacterial PCR. Strains with correct sequencing results were stored at -80℃.

[0031] Example 2

[0032] The dsRNA synthesized from the Cyp311a1 gene fragment of the black soldier fly has the nucleotide sequence shown in SEQ ID NO.7 and the amino acid sequence shown in SEQ ID NO.8.

[0033] The method for synthesizing the dsRNA includes the following steps:

[0034] (1) Primer design: The primer sequences for Cyp311a1 to synthesize dsRNA are as follows:

[0035] The upstream primer sequence is shown in SEQ ID NO.9, and the downstream primer is shown in SEQ ID NO.10 (with the T7 promoter).

[0036] (2) Template preparation: Using the primers designed in step (1), PCR amplification was performed with the black soldier fly gene fragment as a template, and the required template was obtained by gel extraction and recovery.

[0037] (3) sRNA synthesis: The template obtained in step (2) was transcribed into dsRNA in vitro using the T7RiboMAX™ ExpressRNAiSystem (Promega) kit. The dsRNA was quantified using NaNoDrop2000 (Thermoscientific) to achieve a final concentration of 3.2 μg / μL for subsequent microinjection. The dsRNA was stored at -80°C.

[0038] Example 3

[0039] Effects of black soldier fly gene fragment synthesis dsRNA on the growth and development of black soldier fly larvae

[0040] 1. Injection of dsRNA

[0041] Normally developing third-instar larvae (0h) were selected for microinjection, specifically dsGFP and dsRNA (dsHiCyp311a1). The injection volume of dsRNA was 350 nL. The dsGFP-injected control group and the dsHiCyp311a1-injected experimental group were designated as experimental groups. After injection, the larvae were observed and cultured in feed.

[0042] 2. Gene silencing efficiency detection

[0043] Third-instar larvae of black soldier flies were injected on day 0, and the silencing efficiency was assessed 72 hours later. RNA was extracted using a TaKaRaTrizol kit and reverse transcribed into cDNA. The silencing efficiency was detected by RT-qPCR, with RP49 as the internal reference gene. The results showed that, compared with the control group, the mRNA expression level of the target gene was significantly reduced in the experimental group. Figure 2 ).

[0044] 3. Effects of RNAi on the growth and development of black soldier fly larvae

[0045] Synthetic dsGFP and dsHiCyp311a1 were injected into third- and fourth-instar larvae of black soldier fly larvae, respectively. The results showed that Cyp311a1 did not significantly affect the body length and weight of all third-instar larvae after injection. From day 4 post-injection, the body length and weight gain of the experimental group larvae were significantly slower than those of the control group, indicating stunted growth and development. Continued monitoring until day 10 showed that the development of the experimental group larvae remained significantly slower than that of the control group, indicating that dsHiCyp311a1 has a good regulatory effect on larval growth and development. Figure 3 (A~B)

[0046] After injection of fourth-instar larvae, there were no significant differences in body length and weight among all fourth-instar larvae. From the 4th day after injection, the growth in body length and weight of larvae in the experimental group was significantly slower than that in the control group. Figure 4 (A~B)

[0047] The foregoing has shown and described the main features and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0048] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A Cyp311a1 gene from the black soldier fly larvae, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. The method for obtaining the Cyp311a1 gene of the black soldier fly as described in claim 1, characterized in that, Includes the following steps: Two pairs of primers were designed. The upstream primer sequence of the first pair is shown in SEQ ID NO. 3, and the downstream primer sequence is shown in SEQ ID NO.

4. The upstream primer sequence of the second pair is shown in SEQ ID NO. 5, and the downstream primer sequence is shown in SEQ ID NO.

6. Total RNA was extracted and reverse transcribed into cDNA to obtain the template required for PCR amplification. The target gene was obtained by nested PCR amplification, the target gene was ligated to the T vector and transformed into competent cells. White spots were picked and placed in LB liquid medium for bacterial PCR. Bacterial solutions with the target band were sent for sequencing. Strains with correct sequencing results were stored at -80℃.

3. A dsRNA synthesized based on the Cyp311a1 gene fragment of the black soldier fly, characterized in that, The nucleotide sequence of the dsRNA is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.

8.

4. The method for synthesizing dsRNA according to claim 3, characterized in that, Includes the following steps: Design primer sequences for synthesizing dsRNA, with the upstream primer sequence shown in SEQ ID NO.9 and the downstream primer sequence shown in SEQ ID NO.10; Using the designed primers, PCR amplification was performed with the black soldier fly Cyp311a1 gene fragment as a template, and the desired template was obtained by gel extraction and recovery. The obtained template was used to synthesize dsRNA through in vitro transcription using the kit.

5. The application of the dsRNA according to claim 3 in regulating the growth and development of black soldier fly larvae.

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