Coral larva RNAi gene editing method and application thereof
By transfecting staghorn cup coral larvae with siRNA using the RNAi method, optimizing the transfection process and qPCR detection, we achieved efficient knockdown of the Calumenin gene, solving the problem of lack of tools for studying coral biological mechanisms and providing an accurate means of verifying gene function.
Patent Information
- Application Number
- CN202511279986.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-09
- Publication Date
- 2025-12-02
AI Technical Summary
There is limited research on the functional characteristics of calcium-binding proteins (CBPs) in marine invertebrates in existing technologies, and there is a lack of effective gene editing tools, making it difficult to elucidate the biological mechanisms of corals.
Using RNA interference (RNAi), siRNA was transfected into staghorn cup coral larvae. Combined with optimized transfection process and qPCR detection method, efficient knockdown and functional verification of the Calumenin gene were achieved.
A successful RNAi gene editing method for coral larvae was constructed, enabling functional verification of the target gene. This provides a powerful tool for elucidating the biological mechanisms of corals. The method is simple to operate, causes minimal damage to coral larvae, has high transfection efficiency, and yields highly accurate results.
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Figure CN121046464A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of gene interference technology, specifically to a method for RNAi gene editing in coral larvae and its application. Background Technology
[0002] As primary calcifiers, reef-building corals form calcium carbonate frameworks, creating the architectural foundation of tropical marine ecosystems and providing complex habitats for over 25% of marine species. Genomic evidence shows a significant expansion of the calcium-binding protein (CBP) gene family in hard-core corals, indicating their crucial role in coral biomineralization. However, few studies have investigated the functional characterization of CBPs in marine invertebrates.
[0003] RNA interference (RNAi) refers to the efficient and specific blocking of specific gene expression in vivo by small double-stranded RNAs (dsRNAs), promoting mRNA degradation, and inducing cells to exhibit a specific gene deletion phenotype. It is an important protective mechanism against external infections in organisms. Because it can serve as a simple and effective genetic tool to replace gene knockout, this invention provides a method for RNAi gene editing in coral larvae and its applications. Summary of the Invention
[0004] The purpose of this invention is to provide a method for RNAi gene editing in coral larvae and its application. Through the successful construction of an RNAi-based gene editing method for staghorn cup coral, the function of the target gene can be verified, providing a powerful tool for elucidating the biological mechanisms of coral.
[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides an RNAi method for knocking down the expression of the coral calcification gene using the above-mentioned RNAi reagent, comprising the following steps: S1. Soak the coral larvae in filtered seawater; S2. Add RNAi reagent for siRNA transfection; S3. Total RNA was extracted from the transfected coral larvae, reverse transcribed into cDNA, and the cDNA was detected by qPCR.
[0006] Preferably, the final concentration of the RNA reagent in the siRNA transfection is 50~70 mg / mL.
[0007] Preferably, the final concentration of the RNA reagent in the siRNA transfection is 60 mg / mL.
[0008] Preferably, the qPCR detection amplification conditions are: pre-denaturation: 95°C, 30s; denaturation: 95°C, 10s; annealing and extension: 65°C, 30s; 40 cycles.
[0009] Preferably, the amplification primers for the qPCR detection include Calu-qP-F with nucleotide sequences as shown in SEQ ID No. 4 and Calu-qP-R with nucleotide sequences as shown in SEQ ID No. 5.
[0010] Preferably, the coral is a staghorn cup-shaped coral.
[0011] This invention also provides an RNAi reagent for interfering with the expression of coral calcification genes. The RNAi reagent comprises Calu siRNA, which consists of a double-stranded region formed by a sense strand and an antisense strand; The sequence of the sense strand of the Calu siRNA is shown in SEQ ID No. 2, and the sequence of the antisense strand of the Calu siRNA is shown in SEQ ID No. 3.
[0012] Preferably, the coral polyp calcification gene is the Calumenin gene with the sequence shown in SEQ ID No. 1.
[0013] The application of the above-mentioned RNAi reagent in the preparation of formulations for controlling coral calcification.
[0014] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention has successfully constructed an RNAi-based gene editing method for staghorn cup corals, which can realize the functional verification of target genes and provide a powerful tool for elucidating the biological mechanisms of corals; 2. This invention optimizes a transfection process for efficiently introducing siRNA into coral larvae. This method is simple to operate (simply soaking), causes little damage to coral larvae, and has high transfection efficiency (confirmed by Cy3 fluorescent labeling). 3. This patent establishes a quantitative efficacy verification method, including specific qPCR primers (SEQ ID No. 4 and SEQ ID No. 5), whose design avoids overlap with siRNA binding sites to prevent false positives; an optimized qPCR reaction program (65°C annealing / extension, 40 cycles); and standardization using the housekeeping gene `HSP70` to ensure the accuracy of the results. Attached Figure Description
[0015] Figure 1 These are fluorescence micrographs of larval sections transfected with Cy3-siRNA; Figure 2 This refers to the gene expression level of Calumenin after RNAi; Figure 3 It is a Calumenin gene knockdown phenotype; Figure 4 This is a quantitative measure of the early calcification area of coral larvae before and after Calumenin gene knockdown (n represents the number of larvae). Detailed Implementation
[0016] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0017] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0019] Example I. Experimental Methods This invention commissioned Sangon Biotech to design and synthesize calumenin siRNA targeting the Calumenin gene sequence shown in SEQ ID NO.1, using random non-targeted siRNA (NC siRNA) as a negative control. Coral larvae were collected into 12-well plates containing 1 mL of filtered seawater (FSW) to establish three experimental groups: an FSW control group, an NC siRNA group, and a calumenin siRNA group (the final concentration of siRNA in the NC siRNA group and the calumenin siRNA group was 60 μg / mL). siRNA transfection was performed using the riboFECT CP transfection kit (RiboBio) according to the manufacturer's protocol. After 10 hours of larval incubation, the solution was replaced with 1 mL of fresh seawater.
[0020] Table 1 Information on siRNA
[0021] Note: RNase-free HPLC refers to high-performance liquid chromatography without RNase.
[0022] The nucleotide sequence of Calumenin is shown in SEQ ID NO.1:
[0023] To verify siRNA uptake, larvae were cryosectioned (10 μm thick), embedded via OCT, and cut on a Leica CM3050S cryostat. Fluorescence was then detected using an Axio Imager Z2 fluorescence microscope. The larvae were cultured at 26°C under light / dark cycles for 10 days, and bilateral calcification areas were imaged every 48 hours using a microscope.
[0024] After 48 hours of siRNA treatment, total RNA was extracted from lysed larvae using TRIzol (Thermo Scientific), followed by chloroform phase separation, isopropanol precipitation, and ethanol washing. DNase-treated RNA was reverse transcribed into cDNA using HiScript III RT SuperMix (Vazyme), and quantitative PCR was performed using ChamQ Universal SYBR qPCRMaster Mix (Vazyme) and specific primers for Calumennin (see Table 1). Amplification conditions were: pre-denaturation: 95°C, 30 s; denaturation: 95°C, 10 s; annealing and extension: 65°C, 30 s; 40 cycles. Primer sequences were designed to avoid overlap with siRNA binding sites to prevent the amplification of residual siRNA. Gene expression levels were normalized to the housekeeping gene HSP70.
[0025] II. Experimental Results Previous research in this invention, based on genomic and transcriptomic data of staghorn cup coral, revealed a significant expansion of its calcification-related gene family (such as Calumenin). To further validate the function of Calumenin, this invention used the early larval calcification process as a model and designed siRNA targeting Calumenin mRNA with a Cy3 fluorescent label. Transfection was then performed using riboFect™ CP transfection reagent. Microscopic observation showed that Cy3 fluorescent signals were detected in coral sections transfected with Cy3-siRNA. Figure 1 This indicates successful siRNA delivery into coral larvae for expression. qPCR results showed that, compared to the control group, the expression level of Calumenin mRNA in the knockdown group was significantly reduced. Figure 2 This indicates that the system successfully achieved effective knockdown of the target gene Calumenin in staghorn cup corals.
[0026] Phenotypic observations showed that, compared with the control group, the growth rate of calcified area in larvae with Calumenin knockout was significantly slower. Statistical results showed that the difference in the rate of increase of calcified area was small from day 1 to 3, but the difference increased significantly from day 3 to 5. Figure 3 and Figure 4Observations of the attachment surface morphology showed that in the control group, the first septum had closed at the center and the second and third septa had grown, while in the knockdown group, the third septum had not formed. These results indicate that calumenin plays an important role in the early calcification process of corals.
[0027] In summary, this invention successfully constructed an RNAi-based gene editing method for staghorn cup corals and achieved functional verification of the target gene Calumenin, providing a powerful tool for elucidating coral biological mechanisms.
[0028] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.
Claims
1. A method for RNAi gene editing in coral larvae and its application, characterized in that, Includes the following steps: S1. Soak the coral larvae in filtered seawater; S2. Add RNAi reagent for siRNA transfection; S3. Total RNA was extracted from transfected coral larvae, reverse transcribed into cDNA, and the cDNA was detected by qPCR; the RNAi reagent included Calu siRNA, which consists of a double-stranded region formed by one sense strand and one antisense strand; The sense strand sequence of the Calu siRNA is shown in SEQ ID No. 2; The sequence of the antisense strand of the Calu siRNA is shown in SEQ ID No. 3; The coral polyp calcification gene is the Calumenin gene with the sequence shown in SEQ ID No.
1.
2. The method for RNAi gene editing in coral larvae according to claim 1 and its application, characterized in that, The final concentration of RNA reagent in the siRNA transfection is 50-70 mg / mL.
3. The method for RNAi gene editing in coral larvae according to claim 1 and its application, characterized in that, The final concentration of the RNA reagent in the siRNA transfection was 60 mg / mL.
4. The method for RNAi gene editing in coral larvae according to claim 1 and its application, characterized in that, The amplification conditions for the qPCR detection were: pre-denaturation: 95°C, 30s; Denaturation: 95°C, 10s; Annealing and extension: 65°C, 30s; 40 cycles.
5. The method for RNAi gene editing in coral larvae according to claim 1 and its application, characterized in that, The amplification primers for the qPCR detection include Calu-qP-F with nucleotide sequences as shown in SEQ ID No. 4 and Calu-qP-R with nucleotide sequences as shown in SEQ ID No.
5.
6. The method for RNAi gene editing of coral larvae according to claim 3 and its application, characterized in that, The coral in question is a staghorn cup-shaped coral.