Application of a gene related to mineralization and carbon fixation in Crassostrea gigas

Abstract

The present invention discloses an application of a gene related to mineralization and carbon fixation in long oysters, and belongs to the field of genetic engineering technology. The long oyster mineralization and carbon fixation related gene Gigasin‑6 of the present invention has a nucleotide sequence as shown in SEQ ID NO.1. The present invention found that the Gigasin‑6 gene is involved in the mineralization and carbon fixation process of oysters. According to the Gigasin‑6 gene, its specific dsRNA is designed. By interfering with the transcriptional expression of the gene, it can significantly promote the growth of oyster shells and improve the mineralization and carbon fixation capacity of long oysters, providing a new technical idea for promoting high-quality development of fisheries and green and low-carbon transformation.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to the application of a gene related to mineralization and carbon fixation in a giant oyster. Background Art

[0002] Global carbon emissions are increasing year by year. Carbon emissions can damage the ozone layer, exacerbate global warming and the greenhouse effect, and lead to frequent extreme weather events, such as typhoons, high temperatures, heavy rains, mudslides, droughts and other natural disasters. Developing fishery carbon sinks is an important way to achieve "negative emissions" in the ocean. Filter-feeding shellfish play an important role in carbon sink fisheries, with the characteristics of zero feeding and long-term carbon sequestration. At present, research on the carbon sink function of shellfish at home and abroad is still in its infancy. Research mainly focuses on carbon sink benefit assessment, carbon footprint calculation and ecosystem carbon cycle processes. There are no methods and technologies for modifying oysters themselves to enhance their carbon sequestration capacity.

[0003] The long oyster, a major oyster species cultivated in northern China, consists of shells that make up approximately 70%-95% of its total weight. Oysters utilize carbon from the ocean through a biological calcification process, achieving long-term carbon sequestration. With an annual production of approximately 6 million tons of oysters, it is estimated that this mineralization will remove nearly 5 million tons of carbon from seawater annually, equivalent to planting approximately 800,000 hectares of trees. Therefore, there is great potential for achieving "negative emissions" in the ocean through oyster mineralization. There is an urgent need to develop effective methods to enhance the mineralization and carbon sequestration capacity of oysters, enhance the ecological service function of carbon sink fisheries, and accelerate the high-quality development of fisheries and the green, low-carbon transformation and upgrading. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of genes related to mineralization and carbon fixation in long oysters to solve the problems existing in the above-mentioned prior art. The present invention found that the Gigasin-6 gene is involved in the mineralization and carbon fixation process of oysters. According to the Gigasin-6 gene, its specific dsRNA is designed. By interfering with the transcriptional expression of the gene, it can significantly promote the growth of oyster shells and enhance the mineralization and carbon fixation capacity of long oysters, providing new technical ideas for promoting high-quality development of fisheries and green and low-carbon transformation and upgrading.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an application of a gene Gigasin-6 related to mineralization and carbon fixation in the long oyster in regulating the formation of oyster shells. The nucleotide sequence of the gene Gigasin-6 related to mineralization and carbon fixation in the long oyster is shown in SEQ ID NO.1.

[0007] Furthermore, the CDS sequence of the oyster mineralization and carbon fixation related gene Gigasin-6 is shown in SEQ ID NO.2.

[0008] Furthermore, the regulation of oyster shell formation refers to promoting oyster shell formation by knocking out, inhibiting or interfering with Gigasin-6 expression.

[0009] The present invention also provides a dsRNA targeting the long oyster mineralization and carbon fixation-related gene Gigasin-6, the CDS sequence of the long oyster mineralization and carbon fixation-related gene Gigasin-6 is shown as SEQ ID NO.2; the positive strand of the dsRNA is shown as SEQ ID NO.3, and the antisense strand is shown as SEQ ID NO.4.

[0010] The present invention also provides use of the dsRNA in preparing a product for promoting oyster shell growth.

[0011] The present invention also provides the use of the above-mentioned dsRNA in preparing a product for improving the mineralization and carbon fixation capacity of oysters.

[0012] The present invention also provides the use of the above dsRNA in cultivating high carbon-fixing oyster strains.

[0013] The present invention also provides a method for improving the mineralization and carbon fixation capacity of oysters, comprising the step of introducing the above-mentioned dsRNA into the oyster.

[0014] Optionally, the method of introduction is injection, and the amount of introduction is 60-70 μg / mouse.

[0015] Optionally, the oyster is a long oyster.

[0016] The present invention discloses the following technical effects:

[0017] This study discovered for the first time that the Gigasin-6 gene is involved in the mineralization and carbon fixation process of the long oyster. It can negatively regulate shell formation, and by inhibiting or interfering with Gigasin-6 expression, it can significantly promote shell growth. The Gigasin-6 gene can serve as an important molecular target for cultivating high-carbon-fixing oyster strains, and its interference sequence can be used as a related product for improving the mineralization and carbon fixation capacity of the long oyster. This provides technical support for the cultivation of high-carbon-fixing oyster strains and offers new ideas for promoting high-quality development of fisheries and the green, low-carbon transformation and upgrading of the ocean. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1The results of the detection of Gigasin-6 gene expression levels in oysters of different treatment groups at 2, 5, and 8 days after injection; * indicates a significant difference p < 0.05, ** indicates a significant difference p < 0.01, and *** indicates a significant difference p < 0.001;

[0020] Figure 2 The results of shell length test of oysters in different treatment groups at 2, 5, and 8 days after injection; * indicates significant difference p < 0.05, ** indicates significant difference p < 0.01, and *** indicates significant difference p < 0.001. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] Unless otherwise specified, the experimental methods used in the following examples of the present invention are all conventional methods; the materials and reagents used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0027] Example 1 Obtaining Gigasin-6 Gene

[0028] By combining the genome of the Crassostrea gigas (NCBI accession number: PRJNA598006) and the Crassostrea gigas matrix protein database (ProteomeXchange accession number: PXD053708), and integrating gene structure prediction, genome annotation, and bioinformatics analysis, a gene with potential mineralization function, Gigasin-6, was identified. The Gigasin-6 gene is located on chromosome 9 at 28027417-28030899, spanning 3483 bp. Its full-length sequence is shown in SEQ ID NO. 1, and its CDS sequence is 1689 bp long, as shown in SEQ ID NO. 2. The Gigasin-6 protein was isolated and purified in vitro and subjected to in vitro calcium carbonate crystallization, calcium carbonate crystallization rate, and calcium carbonate binding experiments. The results showed that Gigasin-6 significantly inhibited calcium carbonate crystallization and nucleation in vitro, suggesting that the Gigasin-6 gene has a potential regulatory role in inhibiting shell formation.

[0029] SEQ ID NO.1:

[0030]

[0031] SEQ ID NO.2:

[0032]

[0033] Example 2 Application of Gigasin-6 gene

[0034] Previous studies have shown that the Gigasin-6 gene has a potential regulatory effect on inhibiting shell formation. Therefore, this example interferes with the Gigasin-6 gene to verify its regulatory effect on the growth of oyster shells. The specific process is as follows:

[0035] 1. Based on the CDS region sequence of the Gigasin-6 gene (SEQ ID NO. 2), a specific Gigasin-6 dsRNA sequence was designed. A randomly generated nonsense strand negative control dsRNA was used as the negative control (NC) group, and DEPC water was used as the blank control group, as shown in Table 1. The Gigasin-6 interference sequence and negative control sequence were submitted to Suzhou Gene Gene Co., Ltd. for in vitro artificial chemical synthesis.

[0036] Table 1 dsRNA sequences

[0037]

[0038] 2. Animal testing

[0039] Wild adult Crassostrea gigas of uniform growth were selected for RNAi experiments. The specific procedure was as follows: wild Crassostrea gigas were thoroughly cleaned and anesthetized in pre-prepared seawater containing MgCl₂. After the oysters opened, they were randomly divided into three groups: DEPC, NC, and siRNA. DEPC water (DEPC group), negative control dsRNA (NC group), and Gigasin-6 dsRNA (siRNA group) were injected into the adductor muscle of the oysters using a microsyringe, at a dose of 66 μg per oyster. Subsequently, the shells of the three groups were broken: a 4 cm notch was cut into the edge of the shell using a grinder to stimulate the oysters to secrete substances and form shells, thereby comparing their shell formation abilities. On the 2nd, 5th and 8th day after injection, the mantle was sampled to detect the expression level of the Gigasin-6 gene. The shells of the long oysters in the DEPC group, NC group and siRNA group were taken, and the broken shells of the three groups of long oysters were accurately measured using a vernier caliper, with an accuracy of 0.01mm. The one-way analysis of variance combined with Tukey's multiple test was used to compare and analyze the shell growth of the three groups of long oysters to evaluate the effect of Gigasin-6 gene interference on shell formation.

[0040] The detection process of Gigasin-6 gene expression level is as follows:

[0041] qRT-PCR primers were designed using Primer Premier 5 software based on the CDS sequence of the Gigasin-6 gene shown in SEQ ID NO. 2. Total RNA was extracted from the mantle of the oysters described above. After reverse transcription, the expression levels of Gigasin-6 and the housekeeping gene EF1α were detected by real-time quantitative PCR (qRT-PCR). Primers are listed in Table 2. The qRT-PCR reaction system was 20 μL, consisting of 2 μL of cDNA, 7.2 μL of DEPC-free water, 10 μL of 2× Taq Pro Universal SYBR qPCR Master Mix, and 0.4 μL of each upstream and downstream primer (10 μM). The amplification program was as follows: 95°C for 30 seconds, 95°C for 5 seconds, and 60°C for 30 seconds, for a total of 40 cycles.

[0042] Table 2 Primers used for qRT-PCR detection

[0043]

[0044] The results of Gigasin-6 gene expression were as follows: Figure 1 As shown, the expression level of Gigasin-6 gene in the long oysters in the siRNA group was significantly decreased, about 0.12-0.35 of the other two groups, while there was no significant difference between the other two groups, indicating that Gigasin-6 dsRNA can successfully interfere with the Gigasin-6 gene in long oysters.

[0045] The results of oyster shell growth length test are as follows Figure 2 As shown, it can be seen that the oyster shell growth in the siRNA group injected with Gigasin-6 dsRNA was significantly higher than that in the other two groups, indicating that the interference of the Gigasin-6 gene significantly promoted the growth of oyster shells, and interference with the Gigasin-6 gene can significantly enhance the ability of long oysters to mineralize and fix carbon.

[0046] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of Gigasin-6, a gene related to mineralization and carbon fixation in the regulation of oyster shell formation, characterized in that: The nucleotide sequence of the Gigasin-6 gene related to mineralization and carbon fixation in the long oyster is shown in SEQ ID NO. 1; The regulation of oyster shell formation refers to promoting oyster shell formation by knocking out, inhibiting or interfering with Gigasin-6 expression.

2. The use according to claim 1, characterized in that The CDS sequence of the oyster mineralization and carbon fixation-related gene Gigasin-6 is shown in SEQ ID NO.

2.

3. Use of a dsRNA targeting Gigasin-6, a gene related to mineralization and carbon fixation in an oyster, in the preparation of a product that promotes oyster shell growth, characterized in that: The CDS sequence of the oyster mineralization and carbon fixation-related gene Gigasin-6 is shown in SEQ ID NO. 2; the positive strand of the dsRNA is shown in SEQ ID NO. 3, and the antisense strand is shown in SEQ ID NO.

4.

4. Use of a dsRNA targeting Gigasin-6, a gene related to mineralization and carbon fixation in oysters, in the preparation of a product that improves the mineralization and carbon fixation capacity of oysters, characterized in that: The CDS sequence of the oyster mineralization and carbon fixation-related gene Gigasin-6 is shown in SEQ ID NO. 2; the positive strand of the dsRNA is shown in SEQ ID NO. 3, and the antisense strand is shown in SEQ ID NO.

4.

5. Use of a dsRNA targeting the mineralization and carbon fixation-related gene Gigasin-6 in the cultivation of high carbon fixation oyster strains, characterized in that: The CDS sequence of the oyster mineralization and carbon fixation-related gene Gigasin-6 is shown in SEQ ID NO. 2; the positive strand of the dsRNA is shown in SEQ ID NO. 3, and the antisense strand is shown in SEQ ID NO.

4.

6. A method for improving the mineralization and carbon fixation capacity of oysters, characterized in that: The method comprises the steps of introducing dsRNA targeting Gigasin-6, a gene related to mineralization and carbon fixation in long oysters, into oysters; The CDS sequence of the oyster mineralization and carbon fixation-related gene Gigasin-6 is shown in SEQ ID NO. 2; the positive strand of the dsRNA is shown in SEQ ID NO. 3, and the antisense strand is shown in SEQ ID NO.

4.

7. The method according to claim 6, characterized in that The method of introduction is injection, and the amount of introduction is 60-70 μg / mouse.

8. The use according to claim 1 or 2, the use according to any one of claims 3 to 5, or the method according to claim 6 or 7, characterized in that: The oyster is a long oyster.

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