HPGD gene sequence related to growth of adductor muscle of scallop and application of HPGD gene sequence

Abstract

The invention discloses an HPGD gene sequence related to growth of adductor muscles of scallops and application of the HPGD gene sequence, and belongs to the technical field of scallop cultivation. According to the method for improving the yield and the growth rate of the adductor muscle of the scallop by inhibiting the expression of the HPGD gene through an RNA (Ribonucleic Acid Interference) technology, the scallop is fed with an escherichia coli HT115 bacterial solution containing target gene recombinant plasmids, so that the expression inhibition of the CfHPGD gene is realized. According to the invention, through the interference of the CfHPGD gene, the negative regulation effect of the CfHPGD-2 and CfHPGD-3 genes on the adductor muscle and overall growth of the chlamys farreri is disclosed for the first time. Therefore, the HPGD gene of the double-shell shellfish such as the scallop is inhibited in scallop cultivation to obtain an improved variety with high adductor muscle content and fast growth, and potential application value is provided for inheritance and breeding practice of aquatic organisms.

Description

Technical Field

[0001] The invention belongs to the technical field of scallop cultivation, and particularly relates to a method for cultivating scallop adductor muscle. HPGD Gene sequences and their applications. Background Art

[0002] With the rapid development of the global aquaculture industry, scallops have become an important commercial aquaculture target due to their extremely high nutritional value and economic benefits. As the main edible part of scallops, the adductor muscle is not only rich in various nutrients such as protein, minerals and vitamins, but also an ideal model for studying growth regulation mechanisms. In recent years, with the continuous breakthroughs in molecular biology technologies such as CRISPR / Cas9 gene editing and RNA interference, the genetic improvement strategy for economic traits such as production performance and growth rate of aquatic animals has evolved from traditional phenotypic selection to molecular design breeding. In this context, the systematic identification of key functional genes and their interaction networks that regulate the adductor muscle and overall growth of scallops has dual strategic significance: first, it can significantly increase the yield and growth rate of adductor muscles and create greater economic value; second, it provides an important reference for analyzing the muscle and overall growth regulation network of marine invertebrates. Summary of the invention

[0003] The present invention aims to provide a method for the growth of scallop adductor muscle. HPGD Gene sequence, and provide specific applications of the sequence to make up for the shortcomings of the existing technology.

[0004] In invertebrates HPGD Gene-related research is still relatively limited. HPGD It is still unclear whether genes regulate prostaglandin metabolism through conservative mechanisms, thereby affecting the muscle and overall growth of marine invertebrates such as scallops. Therefore, it is urgent to develop an efficient and targeted gene regulation technology to analyze HPGD The role of genes in the adductor muscle and overall growth of scallops and achieve trait improvement. The present invention found that there are four HPGD The gene is expressed in all stages of embryonic development and major adult tissues. Research on the mechanism by which this gene regulates the metabolism of lipid molecules such as prostaglandins to regulate the adductor muscle and overall growth of the scallop provides an important reference for breeding high-muscle and fast-growing scallop strains.

[0005] In order to achieve the above purpose, based on the above research findings, the present invention adopts the following technical solutions: Related to the growth of scallop adductor muscle HPGD Gene sequence, the gene sequence includes the sequence shown in SEQ NO.2 CfHPGD-2 , and the sequence is shown in SEQ NO.3 CfHPGD-3 .

[0006] The gene sequence CfHPGD-2 and CfHPGD-3 Application in scallop cultivation.

[0007] Furthermore, the gene sequence CfHPGD-2 and CfHPGD-3 Application in regulating growth traits of scallops.

[0008] Furthermore, the gene sequence CfHPGD-2 and CfHPGD-3 Application in the regulation of scallop adductor muscle growth.

[0009] Based on the gene sequence CfHPGD-2 and CfHPGD-3 A method for increasing the yield and growth rate of scallop adductor muscle by inhibiting the expression of RNA interference in scallop CfHPGD-2 and CfHPGD-3 Gene expression.

[0010] Furthermore, the method comprises the following steps: (1) Obtain CfHPGD-2 , CfHPGD-3 Target gene sequence; (2) Design RNA interference fragments and interference primers targeting target genes respectively, CfHPGD-2 The interference fragment of the gene sequence is such as SEQ ID NO.6, CfHPGD-3 Interference fragment of gene sequence SEQ ID NO.7; (3) Amplifying the interfering fragment; (4) Construction of L4440 recombinant plasmid containing interference fragment; (5) Expression of double-stranded RNA (dsRNA) by IPTG induction; (6) Mixing the dsRNA-expressing bacteria with microalgae and feeding them to Chlamys farreri to achieve the inhibition of CfHPGD-2 and CfHPGD - 3. Inhibition of gene expression.

[0011] Furthermore, in step (2), based on CfHPGD-2 , CfHPGD-3 The CDS sequence of the gene was predicted and selected using online tools for efficient siRNA interference fragments; and then interference primers containing restriction sites and their protective bases were designed for each interference fragment using online tools; the primer sequences were as follows: .

[0012] Furthermore, in step (3), scallop cDNA is used as a template and a high-fidelity DNA polymerase is used to amplify the target CfHPGD-2 , CfHPGD-3The interference fragment of the gene was amplified and purified by gel recovery after electrophoresis verification, and then connected to the Blunt vector and transformed into DH5α competent cells. The sequence accuracy of the positive clone was verified by colony PCR and sequencing.

[0013] Furthermore, in step (4), the Blunt plasmid and L4440 empty plasmid with correct sequencing were extracted, and the interference fragments of each target gene were connected to the L4440 plasmid by double restriction digestion, gel recovery and T4 ligation, and the products were transformed into HT115 (DE3) competent cells. The sequence accuracy of the positive clones was verified again by colony PCR and sequencing to obtain the cells containing each target gene. CfHPGD -L4440 recombinant plasmid in Escherichia coli.

[0014] Furthermore, in step (5), the E. coli strain with the correct sequencing is expanded and cultured until it enters the exponential growth phase (OD 600 = 0.4-0.6) to induce efficient expression of dsRNA.

[0015] Furthermore, in the step (6), the bacterial precipitate after dsRNA induction is collected by centrifugation, and the mixed concentrated microalgae liquid is used to resuspend and mix it to obtain a bacterial-algae mixture as bait to feed the Chlamys farreri.

[0016] The above method also includes a verification step: (1) CfHPGD-2 , CfHPGD-3 Detection of gene interference efficiency: After 40 days of continuous feeding interference, samples were taken from the mantle, gills, striated muscle, smooth muscle and feet of the Chlamys farreri. The relative expression levels of the corresponding target genes in different tissues of each experimental group were detected using real-time fluorescence quantitative PCR (qRT-PCR). (2) Detection of growth traits: After 40 days of continuous interference, the key growth traits of the Chlamys farreri in each experimental group, such as shell length, shell width, shell height, body weight, soft tissue weight and adductor muscle weight, were accurately measured and analyzed for significant differences, thus reflecting the relationship between the growth and development of the Chlamys farreri. CfHPGD-2 , CfHPGD-3 (3) Detection of adductor muscle growth traits: After 40 days of continuous interference, the number and cross-sectional area of ​​muscle fibers in the cross-section of the adductor muscle tissue of each experimental group were statistically analyzed to reflect the growth of the adductor muscle. CfHPGD-2 , CfHPGD-3 Genetic influence on adductor muscle growth in Chlamys farreri.

[0017] The above method can be applied to the study of other scallops and even bivalve shellfish related to the regulation of shell muscle and overall growth; specifically: inhibiting HPGDThe expression of genes can promote the production and growth rate of adductor muscles of bivalve shellfish.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention inhibits the HPGD The method of increasing the yield and growth rate of scallop adductor muscle by expressing the target gene was achieved by feeding the scallop with Escherichia coli HT115 bacterial solution containing the target gene recombinant plasmid. CfHPGD Inhibition of gene expression.

[0019] The present invention is achieved by CfHPGD Gene interference revealed for the first time CfHPGD-2 and CfHPGD-3 The gene has a negative regulatory effect on the adductor muscle and overall growth of Chlamys farreri. Therefore, in scallop cultivation, by inhibiting the growth of scallops and other bivalve shellfish HPGD Genes are used to obtain high-quality varieties with high adductor muscle mass and fast growth, which provides potential application value for the genetics and breeding practices of aquatic organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0021] Figure 1 Chlamys farreri CfHPGD -Schematic diagram of the L4440 recombinant interference plasmid structure.

[0022] Figure 2 Chlamys farreri CfHPGD Gel electrophoresis detection of gene dsRNA extraction.

[0023] Figure 3 Chlamys farreri CfHPGD Quantitative results of relative expression in five major tissues 40 days after gene interference.

[0024] Figure 4 Yes CfHPGD Statistical analysis results of the growth traits of Chlamys farreri 40 days after gene interference.

[0025] Figure 5 Yes CfHPGD Statistical analysis results of the growth traits of adductor muscle of Chlamys farreri after 40 days of gene interference Figure 1 .

[0026] Figure 6 YesCfHPGD Statistical analysis results of the growth traits of adductor muscle of Chlamys farreri after 40 days of gene interference Figure 1 . DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and key points of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0028] Example 1: Chlamys farreri CfHPGD -Construction of L4440 recombinant interference plasmid (1) HPGD Screening and analysis of homologous sequences of genes in Chlamys farreri: The HPGD protein sequences of other representative species were used as reference sequences and compared with the whole genome of Chlamys farreri. Four HPGD proteins of Chlamys farreri were identified. HPGD Gene, CfHPGD-1 (The sequence list is shown in SEQ ID NO.1), CfHPGD-2 (The sequence list is shown in SEQ ID NO.2), CfHPGD-3 (Sequence listing is shown in SEQ ID NO.3) and CfHPGD-4 (The sequence list is shown in SEQ ID NO.4) gene. CfHPGD-1 It contains 13 exons, with a total CDS length of 1,686 bp, encoding 561 amino acids; CfHPGD-2 The gene contains 6 exons, with a total CDS length of 627 bp, encoding 208 amino acids; CfHPGD-3 The gene contains 4 exons, with a total CDS length of 561 bp, encoding 186 amino acids; CfHPGD-4 The gene contains 10 exons, with a total CDS length of 987 bp, encoding 328 amino acids.

[0029] (2) Design of RNA interference fragments and interference primers targeting four target genes: Based on each CfHPGD The CDS sequence of the gene was used to predict the efficient siRNA interference fragment using the online tool siDirect 2.0. To ensure the targeting specificity, BLAST comparison was used to verify the uniqueness of the selected target sequence in the genome, aiming to design RNA interference fragments with efficient and specific interference functions to minimize the occurrence of off-target effects.

[0030] Primer Premier 5.0 software was used to design the corresponding primers, and the online tool NEBcutter3.0 (https: / / nc3.neb.com / NEBcutter / ) was used to screen two restriction endonucleases that were suitable (their restriction sites were not included in the entire interference fragment) and whose restriction sites existed in the L4440 plasmid. XhoI and HindIII After the restriction site is removed, it and the corresponding protection base are added to the 5' end of the primer. The primers used in this example are shown in Table 1.

[0031] Table 1 Primer sequences of RNA interference target fragments

[0032] (3) Amplification and sequencing verification of interference fragments: Phanta Max Super-Fidelity DNA Polymerase was used to polymerize four CfHPGD The interfering fragment of the gene ( CfHPGD-1 The interference fragment of the gene sequence is shown in SEQ ID NO.5, CfHPGD-2 The interference fragment of the gene sequence is shown in SEQ ID NO.6, CfHPGD-3 The interference fragment of the gene sequence is shown in SEQ ID NO.7, CfHPGD-4 The interference fragment of the gene sequence is shown in SEQ ID NO.8) for amplification. The specific reaction system is shown in Table 2, and a total of 200 μL was amplified. The PCR program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, a total of 32 cycles; 72℃ final extension for 10 min.

[0033] Table 2 DNA polymerase reaction system for interference fragment amplification

[0034] After amplification, the correctness of the PCR product bands was detected by 1.5% agarose gel electrophoresis. Subsequently, the RNA interference fragments in the gel were recovered using the QIAquick PCR Purification Kit gel recovery kit, and the product concentration and quality were detected using the Nanodrop One spectrophotometer. Next, a connection system (10 μL) containing the interference fragment and the Blunt vector was constructed. After connection at 25°C for 15 min, it was transformed into DH5α Escherichia coli competent cells by the heat shock method and cultured at 37°C for 40 min. Then, the product was evenly spread on a plate containing ampicillin (Amp +) on LB solid medium and culture inverted at 37°C for 10-12 h.

[0035] The next day, use a sterilized toothpick to pick a single colony and prepare + 800 μL of LB liquid culture medium and PCR amplification system (Table 3) were added. After the single clone was picked, the toothpick was repeatedly shaken in the PCR reaction system, and then the toothpick was placed in the liquid culture medium (note the corresponding number). Subsequently, the colony PCR amplification verification was performed, and the PCR program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 1 min, 60℃ annealing for 30 s, 72℃ extension for 1 min, a total of 25 cycles; 72℃ final extension for 10 min. At the same time, the liquid culture inoculated with a single colony was shaken and cultured at 37℃ for 4-6 h. After the amplification was completed, it was detected by 1.5% agarose gel electrophoresis, and the culture verified as a positive clone was sent to a sequencing company (Sangon Biotech) for sequence determination.

[0036] Table 3 DNA polymerase reaction system for colony PCR amplification verification

[0037] (4) Construction and sequencing verification of L4440 interference plasmid: After obtaining the strain with the correct sequencing, expand it and use the endotoxin-free plasmid extraction kit to extract the plasmid contained therein. According to the following reaction system (Table 4), the obtained Blunt plasmid and L4440 blank vector were double-digested, and the PCR program was set to 37℃ for 20min. After the digestion was completed, refer to the previous operation, and perform 1.5% agarose gel electrophoresis detection, gel recovery and other steps in sequence to obtain RNA interference fragments with sticky ends and L4440 empty plasmid fragments and measure their concentrations.

[0038] Table 4 Double enzyme digestion reaction system

[0039] Afterwards, the amount of ligation was calculated using the online tool NEBioCalculator (https: / / nebiocalculator.neb.com / ), and the RNA interference fragment was ligated with the L4440 empty plasmid fragment using T4 ligase. The specific ligation system is shown in Table 5. The PCR program was set to 25°C for 10 min. CfHPGD -L4440 recombinant plasmid, the schematic diagram of its structure is shown in Figure 1 shown.

[0040] Finally, referring to the previous operation, the above ligation products and L4440 empty vector plasmid were transformed into HT115 (DE3) Escherichia coli competent cells, and the products were evenly spread on the Amp + and tetracycline (TET + ) on LB solid medium. After inverted culture at 37°C for 10-12 h, single colonies were picked for colony PCR amplification. The strains verified as positive clones were + and TET + After expansion culture in LB liquid medium, sequence determination was performed again.

[0041] Table 5 T4 ligase ligation reaction system

[0042] (5) dsRNA induced expression and extraction verification: Take 10 μL of the above sequenced positive strains and inoculate them in 3-5 mL of Amp + and TET + The culture was shaken at 37℃ for 10-12 h. The next day, the cells were transferred to fresh LB medium containing Amp at a ratio of 1:100. + and TET + After a period of culture, the absorbance of the bacterial solution at a wavelength of 600 nm (OD 600 ), wait until it enters the exponential growth phase (OD 600 = 0.4-0.6), add 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) to induce efficient expression of dsRNA. Continue to culture for 4 h, centrifuge at 8,000 rpm for 3 min, discard the supernatant and collect the bacterial precipitate, and store it at -80℃ for later use.

[0043] Total RNA was extracted from the induced and uninduced bacterial cultures by the traditional TRIzol method. After single-stranded RNA (ssRNA) was digested with RNaseT1, the concentration and quality of the extracted dsRNA were determined using a Nanodrop One spectrophotometer, and the correctness of the dsRNA bands was detected using 1.5% agarose gel electrophoresis. Figure 2 As shown, lanes 5, 8, 11 and 14 represent the various CfHPGD -L4440 recombinant plasmid in the strain RNA; Lane 6, 9, 12 and 15 represent the IPTG-induced CfHPGDLane 1 represents the RNA of the strain containing the L4440 recombinant plasmid; Lane 2 represents the RNA of the strain containing the L4440 empty plasmid without IPTG induction; Lane 3 represents the RNA of the strain containing the L4440 empty plasmid induced by IPTG; Lanes 1, 4, 7, 10 and 13 represent the 100 bp DNA ladder. This result shows that after IPTG induction, E. coli HT115 successfully expressed the target protein. CfHPGD The target dsRNA fragment of the gene (indicated by a white arrow) also verified the necessity of IPTG induction and that the L4440 empty plasmid could be used as a negative control group to exclude false positives.

[0044] Example 2: Effect of mixed feeding of bacteria and algae on the growth of Chlamys farreri CfHPGD RNA interference (1) Experimental animals: 250 one-year-old live scallops from the same population with relatively uniform individuals were randomly selected, and their initial growth traits were systematically measured, including shell length (46.31±3.19 mm), shell width (13.40±1.27 mm), shell height (50.40±2.79 mm), and body weight (12.59±2.45 g). Afterwards, the scallops were randomly divided into five groups: Control, RNAi- CfHPGD-1 Group, RNAi- CfHPGD-2 Group, RNAi- CfHPGD-3 Group, RNAi- CfHPGD-4 Finally, each group of scallops was placed in an independent and strictly disinfected 500 L water tank (filled with about 400 L of 20°C constant temperature filtered seawater, replaced once a day), and oxygen was continuously added to the water tank to maintain suitable living conditions.

[0045] (2) Preparation of dsRNA: The dsRNA expression was induced by referring to Example 1. Then, 200 mL of L4440 empty plasmid, CfHPGD- 1 -L4440, CfHPGD-2 -L4440, CfHPGD-3 -L4440 and CfHPGD-4 -L4440 recombinant plasmid Escherichia coli HT115 culture medium, centrifuge and discard the supernatant, collect the bacterial precipitate, and use 20-30 mL of Chlorella pyrenoidosa ( Chlorella pyrenoidesa ) and Chaetoceros muelleri ( Chaetoceros muelleri ) is mixed with concentrated algae liquid and resuspended and mixed to obtain a bacteria-algae mixture as bait.

[0046] (3) Feeding plan: During the RNA interference period, each group of scallops was fed with an equal amount of mixed concentrated algae solution four times a day (9:00, 13:00, 17:00, and 21:00). The algae cell concentration in the water was maintained at about 5-10×10 5 The second and fourth feedings used a mixture of bacteria and algae. The Control group was fed with a mixture of bacteria and algae containing an empty plasmid of L4440, and the RNAi- CfHPGD-1 Group feeding CfHPGD-1 -L4440 recombinant plasmid bacteria-algae mixture, RNAi- CfHPGD-2 Group feeding CfHPGD-2 -L4440 recombinant plasmid bacteria-algae mixture, RNAi- CfHPGD-3 Group feeding CfHPGD-3 -L4440 recombinant plasmid bacteria-algae mixture, RNAi- CfHPGD-4 Group feeding CfHPGD-4 -Bacteria-algae mixture containing L4440 recombinant plasmid.

[0047] The technical effects of the present invention are described in detail below in conjunction with experimental results.

[0048] (1) Each CfHPGD Expression level detection after gene interference: Design CfHPGD Select the qRT-PCR primers for the target gene, and avoid RNA interference fragments to ensure the accuracy of the quantification of the target gene. ef1α As the internal reference gene, the specific primer sequences are shown in Table 6.

[0049] Table 6 Quantitative primer sequence list

[0050] After 40 days of feeding, 30 scallops were randomly selected from each experimental group for dissection, and five major tissues including mantle, gill, striated muscle, smooth muscle and foot were collected. The above tissues were cut into small pieces with a diameter of about 1 cm, frozen in liquid nitrogen and stored at -80°C. Total RNA was extracted from each major tissue of scallops in each group, and cDNA was obtained by reverse transcription and diluted to 10 ng / μL as a quantitative template.

[0051] After that, the experiment was designed in advance, with 3 technical replicates for each sample and 5 biological replicates for each group to minimize experimental errors. Subsequently, the qRT-PCR reaction system was constructed (Table 7) and placed in the LightCycler®480 instrument. The program was set as follows: 50°C pre-denaturation for 2 min, 94°C pre-denaturation for 10 min; 94°C denaturation for 15 s, 60°C annealing for 1 min, for a total of 40 cycles; finally, the temperature was lowered by 0.5°C every 10 s until 55°C, and then at 55°C for 1 min. After the reaction was completed, the specificity of the amplification was verified according to the dissociation curve, and the results were analyzed by 2 -△△Ct The method of calculating each CfHPGD Afterwards, the Student's t-test was used to analyze the significance of the difference in target gene expression between each RNA interference group and the control group. * represents P < 0.050, ** represents P < 0.01.

[0052] Table 7 qRT-PCR reaction system

[0053] The corresponding expression of each RNA interference group in the five main tissues of Chlamys farreri, including mantle, gill, striated muscle, smooth muscle and foot CfHPGD The results of gene expression level and its difference analysis between the control group are shown in Figure 3 After 40 days of continuous RNA interference, compared with the control group, CfHPGD-1 Genes in RNAi- CfHPGD-1 The expression levels of mantle, gill, striated muscle, smooth muscle, and foot in the group were significantly reduced by 71.03%, 51.09%, 66.40%, 60.93%, and 30.74%, respectively; CfHPGD-2 Genes in RNAi- CfHPGD-2 The expression levels of mantle, gill, striated muscle, smooth muscle, and foot in the group were significantly reduced by 50.55%, 66.55%, 57.57%, 57.10%, and 54.92%, respectively; CfHPGD-3 Genes in RNAi- CfHPGD-3 The expression levels of mantle, gill, striated muscle, smooth muscle, and foot in the group were significantly reduced by 87.99%, 84.89%, 47.00%, 58.03%, and 42.94%, respectively; CfHPGD-4 Genes in RNAi- CfHPGD-4 The expression levels of mantle, gill, striated muscle, smooth muscle and foot in the group were significantly reduced by 60.89%, 55.90%, 84.99%, 86.72% and 66.17%, respectively. This quantitative result shows that the RNA interference has a significant effect on the expression of various genes in Chlamys farreri. CfHPGD Genes were efficiently and systematically inhibited.

[0054] (2) Detection of growth traits of Chlamys farreri: After 40 days of continuous interference, the key growth traits of the scallops in each experimental group, such as shell length, shell width, shell height, body weight, soft tissue weight and adductor muscle weight, were accurately measured and analyzed for significant differences. Figure 4 As shown, compared with the control group, RNAi- CfHPGD-1 There were no significant changes in the growth traits of the scallops in the group ( P > 0.05); RNAi- CfHPGD-2 The average shell length, shell width, shell height, body weight, soft tissue weight and adductor muscle weight of the group C. farreri significantly increased by 2.98% ( P <0.05), 6.77% ( P < 0.01), 2.90% ( P < 0.05)、18.02%( P < 0.01), 20.60% ( P < 0.01) and 37.36% ( P < 0.01); RNAi- CfHPGD-3 The average shell length, shell width, shell height, body weight, soft tissue weight and adductor muscle weight of the group C. farreri significantly increased by 3.99% ( P < 0.01)、4.92%( P < 0.01), 2.96% ( P < 0.05), 22.60% ( P < 0.01), 34.07% ( P < 0.01) and 15.38% ( P < 0.05); RNAi- CfHPGD-4 The average body weight of the scallops in the group increased significantly by 9.53% ( P < 0.05), while other growth traits showed no significant changes ( P > 0.05). This statistical result shows that CfHPGD-2 and CfHPGD-3 The genes have the function of negatively regulating the overall growth of Chlamys farreri. When the expression of these two genes is inhibited, the growth rate of scallops is significantly accelerated. CfHPGD-1 and CfHPGD-4 The knockdown of these genes did not cause systemic changes in growth phenotypes, suggesting that they are not involved in core regulatory functions in the growth of Chlamys farreri and are not key regulatory genes that directly affect growth phenotypes. CfHPGD-2 and RNAi- CfHPGD-3 The two experimental groups showed more significant growth-promoting effects in adductor muscle and soft tissue weight, indicating thatCfHPGD-2 and CfHPGD-3 The gene is closely related to the growth of tissues such as the adductor muscle of the Chlamys farreri.

[0055] (3) Detection of growth traits of adductor muscle of Chlamys farreri: After 40 days of continuous interference, the number and cross-sectional area of ​​muscle fibers in the cross-section of the adductor muscle tissue of the Chlamys farreri in each experimental group were statistically analyzed using paraffin embedding, tissue sectioning, and hematoxylin-eosin (HE) staining and other tissue morphological research methods. Figure 5 and Figure 6 As shown, compared with the control group, RNAi- CfHPGD-2 The number of striated muscle fibers in the group C. farreri increased by 19.43% ( P < 0.01), the number of smooth muscle fibers increased by 5.72% ( P < 0.01), and the cross-sectional area of ​​the adductor muscles did not change significantly ( P > 0.05); RNAi- CfHPGD-3 The number of striated muscle fibers in the group C. farreri increased by 27.89% ( P < 0.01), the number of smooth muscle fibers increased by 10.10% ( P <0.01), and the cross-sectional area of ​​the two adductor muscles did not change significantly ( P > 0.05); RNAi- CfHPGD-1 and RNAi- CfHPGD-4 There was no significant change in the number and cross-sectional area of ​​the two adductor muscles of the scallops in the group ( P > 0.05). This statistical result shows that CfHPGD-2 and CfHPGD-3 The genes have the function of negatively regulating the growth of the adductor muscle of the scallop. When the expression of these two genes is inhibited, the scallop's striated muscle and smooth muscle fibers proliferate significantly.

[0056] The present invention is based on RNA interference technology, and by continuously feeding Escherichia coli that can be induced to produce target gene-specific dsRNA for 40 days, the 15-hydroxyprostaglandin dehydrogenase encoding gene ( HPGD ) systemic inhibition. Combined with the determination of the growth traits of Chlamys farreri, it was found that CfHPGD-2 and CfHPGD-3 After the gene was interfered, the growth rate of the whole body and adductor muscle of the scallop was significantly improved, and the muscle fibers proliferated significantly, reflecting the negative regulatory effect of these two genes on the adductor muscle and even the overall growth of the scallop. Therefore, the present invention provides a method for promoting the yield and growth rate of the adductor muscle of scallops, that is, inhibiting the growth of the adductor muscle of scallops by feeding dsRNA RNA interference technology. HPGDGene expression, thereby promoting the growth of soft tissues such as the scallop adductor muscle and the whole body.

[0057] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects disclosed in the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Related to the growth of scallop adductor muscle HPGD A gene sequence, characterized in that The gene sequence includes the sequence shown in SEQ NO.2 CfHPGD-2 , and the sequence is shown in SEQ NO.3 CfHPGD-3 .

2. The gene sequence of claim 1 CfHPGD-2 and CfHPGD-3 Application in scallop cultivation.

3. The gene sequence of claim 1 CfHPGD-2 and CfHPGD-3 Application in regulating growth traits of scallops.

4. The gene sequence of claim 1 CfHPGD-2 and CfHPGD-3 Application in the regulation of scallop adductor muscle growth.

5. Based on the gene sequence of claim 1 CfHPGD-2 and CfHPGD-3 The method for increasing the yield and growth rate of scallop adductor muscle is characterized in that: This method uses RNA interference technology to inhibit CfHPGD-2 and CfHPGD-3 Gene expression.

6. The method according to claim 5, characterized in that The method comprises the following steps: (1) Obtain CfHPGD-2 , CfHPGD-3 Target gene sequence; (2) Design RNA interference fragments and interference primers targeting target genes respectively, CfHPGD-2 The interference fragment of the gene sequence is such as SEQ ID NO.6, CfHPGD-3 The interference fragment of the gene sequence SEQ ID NO.7; (3) Amplifying the interfering fragment; (4) Construction of L4440 recombinant plasmid containing interference fragment; (5) Expression of double-stranded RNA by IPTG induction; (6) Mixing the dsRNA-expressing bacteria with microalgae and feeding them to Chlamys farreri to achieve CfHPGD-2 and CfHPGD-3 Inhibition of gene expression.

7. The method according to claim 6, characterized in that In step (2), based on CfHPGD-2 , CfHPGD-3 The CDS sequence of the gene was predicted and selected using online tools for efficient siRNA interference fragments; and then interference primers containing restriction sites and their protective bases were designed for each interference fragment using online tools; the primer sequences were as follows: 。 8. The method according to claim 6, characterized in that In the step (3), scallop cDNA is used as a template and a high-fidelity DNA polymerase is used to amplify the target CfHPGD-2 , CfHPGD-3 The interference fragment of the gene was amplified and purified by gel recovery after electrophoresis verification, and then connected to the Blunt vector and transformed into DH5α competent cells. The sequence accuracy of the positive clone was verified by colony PCR and sequencing.

9. The method according to claim 6, characterized in that In step (4), the Blunt plasmid and L4440 empty plasmid with correct sequencing were extracted, and the interference fragments of each target gene were connected to the L4440 plasmid by double restriction digestion, gel recovery and T4 ligation, and the products were transformed into HT115 (DE3) competent cells. The sequence accuracy of the positive clones was verified again by colony PCR and sequencing to obtain the fragments containing each target gene. HkDJ -L4440 recombinant plasmid in Escherichia coli.

10. The method according to claim 6, characterized in that In the step (5), the Escherichia coli strain with correct sequencing is expanded and cultured, and IPTG is added when it enters the exponential growth phase to induce efficient expression of dsRNA; in the step (6), the bacterial precipitate after dsRNA induction is collected by centrifugation, and the mixed concentrated microalgae liquid is used to resuspend and mix it to obtain a bacterial and algae mixture to be fed to scallops as bait.

Images