Application of pacific oyster mineralization carbon sequestration related gene

Abstract

The invention discloses application of a pacific oyster mineralization carbon sequestration related gene, and belongs to the technical field of gene engineering. The nucleotide sequence of the pacific oyster mineralization carbon sequestration related gene Gigin-6 disclosed by the invention is as shown in SEQ ID NO. 1. The invention finds that the Gigin-6 gene participates in the oyster mineralization carbon sequestration process, the specific dsRNA of the Gigin-6 gene is designed according to the Gigin-6 gene, the growth of oyster shells can be remarkably promoted by interfering the transcription expression of the gene, the mineralization carbon sequestration capability of crassostrea gigas can be improved, and a new technical thought is provided for promoting high-quality development and green low-carbon transformation of fishery.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to the application of genes related to mineralization and carbon sequestration in the Pacific oyster (Crassostrea gigas). Background Art

[0002] Global carbon emissions have been 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 natural disasters like typhoons, high temperatures, heavy rains, debris flows, and droughts. 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 characteristics such as zero bait input and long-term carbon sequestration. Currently, the related research on the carbon sink function of shellfish at home and abroad is still in its infancy. The research mainly focuses on carbon sink benefit assessment, carbon footprint calculation, and the carbon cycle process of the ecosystem. There are no relevant methods and technologies for improving the carbon sequestration ability by modifying oysters themselves.

[0003] The Pacific oyster (Crassostrea gigas) is the main oyster species cultured in the north. The shell accounts for about 70%-95% of the total weight of the oyster. Oysters use carbon in the ocean through the process of biological calcification to achieve mineralization and carbon sequestration, which can achieve long-term carbon sequestration. It is estimated that about 6 million tons of oysters produced annually can remove nearly 5 million tons of carbon from seawater through mineralization and carbon sequestration, which is equivalent to afforesting about 800,000 hectares. Therefore, there is great potential to achieve "negative emissions" in the ocean through oyster mineralization and carbon sequestration. It is urgent to develop effective methods to improve the mineralization and carbon sequestration ability of oysters, enhance the ecological service function of carbon sink fisheries, and accelerate the high-quality development and green and low-carbon transformation and upgrading of fisheries. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of genes related to mineralization and carbon sequestration in the Pacific oyster (Crassostrea gigas) to solve the problems existing in the above-mentioned prior art. The present invention discovers that the Gigasin-6 gene is involved in the process of oyster mineralization and carbon sequestration. By designing its specific dsRNA according to the Gigasin-6 gene and interfering with the transcriptional expression of this gene, it can significantly promote the growth of oyster shells and enhance the mineralization and carbon sequestration ability of the Pacific oyster (Crassostrea gigas), providing a new technical idea for promoting the high-quality development and green and low-carbon transformation and upgrading of fisheries.

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

[0006] The present invention provides the application of the gene Gigasin-6 related to mineralization and carbon sequestration in the Pacific oyster (Crassostrea gigas) in regulating the formation of oyster shells. The nucleotide sequence of the gene Gigasin-6 related to mineralization and carbon sequestration in the Pacific oyster (Crassostrea gigas) is shown in SEQ ID NO.1.

[0007] Furthermore, the CDS sequence of the gene Gigasin-6 related to mineralization and carbon sequestration in the Pacific oyster (Crassostrea gigas) 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 oyster Crassostrea gigas mineralization and carbon sequestration-related gene Gigasin-6. The CDS sequence of the oyster Crassostrea gigas mineralization and carbon sequestration-related gene Gigasin-6 is shown in SEQ ID NO.2; the sense strand of the dsRNA is shown in SEQ ID NO.3, and the antisense strand is shown in SEQ ID NO.4.

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

[0011] The present invention also provides the use of the above dsRNA in the preparation of a product for improving the oyster mineralization and carbon sequestration ability.

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

[0013] The present invention also provides a method for improving the oyster mineralization and carbon sequestration ability, including the step of introducing the above dsRNA into oysters.

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

[0015] Optionally, the oyster is Crassostrea gigas.

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

[0017] The present invention for the first time discovers that the Gigasin-6 gene is involved in the oyster Crassostrea gigas mineralization and carbon sequestration process, which can negatively regulate shell formation. By inhibiting or interfering with the expression of Gigasin-6, the growth of the shell can be significantly promoted. The Gigasin-6 gene can be used as an important molecular target for cultivating high-carbon sequestration oyster strains, and its interfering sequence can be used as a related product for enhancing the oyster Crassostrea gigas mineralization and carbon sequestration ability, providing technical support for the cultivation of high-carbon sequestration oyster strains and new ideas for promoting the high-quality development of fisheries and the transformation and upgrading of the marine green and low-carbon industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0019] Figure 1Detection result graph of Gigasin-6 gene expression levels in Crassostrea gigas at 2, 5, and 8 days after injection for different treatment groups; * indicates significant difference p < 0.05, ** indicates significant difference p < 0.01, *** indicates significant difference p < 0.001;

[0020] Figure 2 Detection result graph of shell growth length in Crassostrea gigas at 2, 5, and 8 days after injection for different treatment groups; * indicates significant difference p < 0.05, ** indicates significant difference p < 0.01, *** indicates significant difference p < 0.001. Detailed implementation manners

[0021] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation to the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0023] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0024] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0025] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

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

[0027] Example 1 Obtaining of Gigasin-6 gene

[0028] By jointly analyzing the genome of the Pacific oyster (NCBI accession number: PRJNA598006) and the matrix protein database of the Pacific oyster (ProteomeXchange accession number: PXD053708), integrating analysis methods such as gene structure prediction, genome annotation, and bioinformatics, the Gigasin-6 gene with potential mineralization function was identified. The Gigasin-6 gene is located on the 9th chromosome of the Pacific oyster, at the fragment 28027417-28030899, with a length of 3483 bp. Its full-length sequence is shown in SEQ ID NO.1, and the length of its CDS sequence is 1689 bp, as shown in SEQ ID NO.2. Through in vitro isolation and purification of the Gigasin-6 protein for in vitro calcium carbonate crystallization, calcium carbonate crystallization rate, and calcium carbonate binding experiments, it was found that the Gigasin-6 protein can significantly inhibit the crystallization and nucleation of calcium carbonate 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 found that the Gigasin-6 gene has a potential regulatory effect on inhibiting shell formation. Therefore, in this example, the regulatory effect of the Gigasin-6 gene on the shell growth of the Pacific oyster was verified by interfering with the Gigasin-6 gene. The specific process is as follows:

[0035] 1. According to the CDS region sequence of the Gigasin-6 gene (SEQ ID NO.2), a specific Gigasin-6 dsRNA sequence was designed, and 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 the negative control sequence were submitted to Suzhou GenePharma Co., Ltd. for in vitro artificial chemical synthesis.

[0036] Table 1 dsRNA Sequences

[0037]

[0038] 2. Animal Experiment

[0039] Adult wild Pacific oysters with consistent growth were selected for the RNAi experiment. The specific operation is as follows: The wild Pacific oysters were thoroughly cleaned and then placed in pre-prepared seawater containing MgCl 2 for anesthesia. After the oysters opened, they were randomly divided into three groups, named the DEPC group, the NC group, and the siRNA group. DEPC water (DEPC group), negative control dsRNA (NC group), and Gigasin-6 dsRNA (siRNA group) were respectively injected into the adductor muscle of the Pacific oyster using a microsyringe, and the injection dose was 66 μg per oyster. Subsequently, the shells of the three groups of oysters were cracked: A grinding wheel was used to cut a notch of about 4 cm at the edge of the oyster shell to stimulate the Pacific oyster to secrete substances quickly to form shells, so as to compare the shell formation ability. On the 2nd, 5th, and 8th days after injection, the mantle was sampled to detect the expression level of the Gigasin-6 gene; and the shells of the Pacific oysters in the DEPC group, the NC group, and the siRNA group were taken, and a vernier caliper was used to accurately measure the cracked parts of the shells of the three groups of Pacific oysters, accurate to 0.01 mm. One-way ANOVA combined with Tukey multiple tests were used to compare and analyze the shell growth of the three groups of Pacific oysters to evaluate the effect of Gigasin-6 gene interference on shell formation.

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

[0041] According to the CDS sequence of the Gigasin-6 gene shown in SEQ ID NO.2, qRT-PCR primers were designed using Primer Premier5 software. Total RNA was extracted from the mantle of the above-mentioned Crassostrea gigas, and after reverse transcription, the expression levels of the Gigasin-6 gene and the housekeeping gene EF1α were detected by real-time quantitative PCR (qRT-PCR). The primers are shown in Table 2. The qRT-PCR reaction system was 20 μL, and each reaction included 2 μL of cDNA, 7.2 μL of DEPC water, 10 μL of 2 × Taq Pro Universal SYBR qPCR Master Mix, and 0.4 μL each of the upstream and downstream primers (10 μM). The amplification program was: 30 seconds at 95 °C; 5 seconds at 95 °C, 30 seconds at 60 °C, for a total of 40 cycles.

[0042] Table 2 Primers for detecting qRT-PCR

[0043]

[0044] The detection results of the Gigasin-6 gene expression level are as Figure 1 shown. It can be seen that the expression level of the Gigasin-6 gene in the siRNA group of C. gigas decreased significantly, about 0.12 - 0.35 of the other two groups, and there was no significant difference between the other two groups, indicating that Gigasin-6 dsRNA can successfully interfere with the Gigasin-6 gene in C. gigas.

[0045] The detection results of the growth length of the oyster shell are as Figure 2 shown. It can be seen that in the siRNA group injected with Gigasin-6 dsRNA, the growth amount of the oyster shell was significantly higher than that of the other two groups, indicating that the interference of the Gigasin-6 gene significantly promoted the growth of the oyster shell, and interfering with the Gigasin-6 gene can significantly improve the ability of C. gigas to mineralize and fix carbon.

[0046] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of Gigasin-6, a gene related to mineralization and carbon fixation in oysters, in regulating oyster shell formation, characterized in that: The nucleotide sequence of the oyster mineralization and carbon fixation related gene Gigasin-6 is shown in SEQ ID NO.

1.

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. The use according to claim 1, characterized in that: The regulation of oyster shell formation refers to promoting oyster shell formation by knocking out, inhibiting or interfering with Gigasin-6 expression.

4. A dsRNA targeting Gigasin-6, a gene related to mineralization and carbon fixation in Crassostrea gigas, 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 sense 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 the dsRNA according to claim 4 in preparing a product for promoting the growth of oyster shells.

6. Use of the dsRNA according to claim 4 in the preparation of a product for improving the mineralization and carbon fixation capacity of oysters.

7. Use of the dsRNA according to claim 4 in cultivating a high carbon-fixing oyster strain.

8. A method for improving the mineralization and carbon fixation capacity of oysters, characterized in that: The method comprises the step of introducing the dsRNA according to claim 4 into the oyster.

9. The method according to claim 8, characterized in that The method of introduction is injection, and the amount of introduction is 60-70 μg / mouse.

10. The use according to any one of claims 1 to 3, the use according to any one of claims 5 to 7, or the method according to claim 8 or 9, characterized in that: The oyster is a long oyster.

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