CN119842750B - A method for improving the growth rate and adductor muscle yield of Chlamys farreri - Google Patents

Abstract

The present invention discloses an HPGD gene sequence related to the growth of scallop adductor muscle and its application, and belongs to the technical field of scallop cultivation. The present invention uses RNA interference (RNAi) technology to inhibit the expression of HPGD gene to increase the yield and growth rate of scallop adductor muscle. The Escherichia coli HT115 bacterial liquid containing a recombinant plasmid of the targeted target gene is fed to the scallop to achieve the inhibition of the expression of the CfHPGD gene. Through the interference of the CfHPGD gene, the present invention reveals for the first time the negative regulatory effects of the CfHPGD‑2 and CfHPGD‑3 genes on the adductor muscle and overall growth of Chlamys farreri. Therefore, in scallop cultivation, the HPGD gene of bivalve shellfish such as scallops is inhibited 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.

Description

Method for improving growth rate and adductor muscle yield of chlamys farreri

Technical Field

The invention belongs to the technical field of scallop cultivation, and particularly relates to HPGD gene sequences related to growth of adductor muscles of scallops and application thereof.

Background

With the rapid development of the global aquaculture industry, scallops are important commercial cultivation objects due to extremely high nutritional value and economic benefit. The adductor muscle is used as a main edible part of scallop, is not only rich in various nutritional components such as protein, mineral substances, vitamins and the like, but also is an ideal model for researching a growth regulation mechanism. In recent years, with the continuous breakthrough of molecular biology techniques such as CRISPR/Cas9 gene editing and RNA interference, genetic improvement strategies for economic characters such as production performance and growth rate of aquaculture animals have evolved from traditional phenotypic selection to molecular design breeding. Under the background, the system identifies and regulates the scallop adductor muscle and the key functional gene and the interaction network of the scallop adductor muscle and the whole growth, has double strategic significance, namely, the system can obviously improve the yield and the growth rate of the adductor muscle and create larger economic value, and secondly, provides important references for analyzing the marine invertebrate muscle and the whole growth regulation network.

Disclosure of Invention

The invention aims to provide HPGD gene sequences related to the growth of scallop adductor muscles and provides specific applications of the sequences so as to overcome the defects of the prior art.

The HPGD gene related studies in invertebrates are still limited, and whether the HPGD gene regulates prostaglandin metabolism through a conserved mechanism, thereby affecting the muscle and overall growth of marine invertebrates such as scallops is not clear. Therefore, there is a need to develop a highly efficient and targeted gene regulation technology to analyze the role of HPGD genes in adductor muscle and overall growth of scallops and to realize trait improvement. The invention discovers that the chlamys farreri has four HPGD genes, and the genes are expressed in each period of embryo development and main tissues of adults, and the genes are used for researching the chlamys farreri adductor muscle and the whole growth regulation mechanism by regulating the metabolism of prostaglandin lipoid molecules, so that important references are provided for breeding a high-muscle-mass and fast-growing scallop strain.

In order to achieve the above object, based on the above findings, the present invention adopts the following technical scheme:

HPGD gene sequence related to the growth of the adductor scallop muscle, which comprises CfHPGD-2 with the sequence shown in SEQ NO.2 and CfHPGD-3 with the sequence shown in SEQ NO. 3.

The gene sequences CfHPGD-2 and CfHPGD-3 are applied to scallop cultivation.

Further, the gene sequences CfHPGD-2 and CfHPGD-3 are applied to the regulation of scallop growth traits.

Furthermore, the gene sequences CfHPGD-2 and CfHPGD-3 are applied to the growth regulation of the adductor scallop muscle.

A method for improving the yield and growth rate of the adductor muscle of scallop based on the gene sequences CfHPGD-2 and CfHPGD-3 is disclosed, which can inhibit the expression of CfHPGD-2 and CfHPGD-3 genes in scallop by RNA interference technology.

Further, the method comprises the following steps:

(1) Obtaining CfHPGD-2 and CfHPGD-3 target gene sequences;

(2) Designing an RNA interference fragment and an interference primer of a target gene, wherein the interference fragment of CfHPGD-2 gene sequences is shown as SEQ ID NO.6 and the interference fragment of CfHPGD-3 gene sequences is shown as SEQ ID NO.7;

(3) Amplifying the interference fragments;

(4) Constructing an L4440 recombinant plasmid containing an interference fragment;

(5) Expression of double-stranded RNA (dsRNA) by IPTG induction;

(6) The expression inhibition of CfHPGD-2 and CfHPGD-3 genes was achieved by feeding chlamys farreri by mixing the cells expressing dsRNA with microalgae.

Further, in the step (2), based on CDS sequences of CfHPGD-2 and CfHPGD-3 genes, an on-line tool is utilized to predict and select high-efficiency siRNA interference fragments, then an on-line tool is utilized to design interference primers containing enzyme cutting sites and protecting bases thereof for each interference fragment, and the sequences of the primers are as follows:

CfHPGD-2-RNAi-F-HindIII	CAAGCTTGGTGATGTCACAGACCACGATCAA
		CfHPGD-2-RNAi-R-XhoI	CCTCGAGGCATCGAGTCAGAACTTGAGCTATCA
CfHPGD-3-RNAi-F-HindIII	CAAGCTTGATTACTCAACAAAGGGGCGAAGATT
		CfHPGD-3-RNAi-R-XhoI	CCTCGAGGGTTCTTTGCATTTGTGGCTTGGAC

In the step (3), scallop cDNA is used as a template, high-fidelity DNA polymerase is used for amplifying the interference fragments of the target CfHPGD-2 and CfHPGD-3 genes, gel recovery and purification are carried out on amplified products after electrophoresis verification, and then the amplified products are connected to a Blunt vector and transformed into DH5 alpha competent cells, and the sequence accuracy of positive clones is verified through colony PCR and sequencing.

Further, in the step (4), blunt plasmids and L4440 empty plasmids with correct sequencing are extracted, interference fragments of target genes are respectively connected with the L4440 plasmids through double enzyme digestion, glue recovery and T4 connection, products are transformed into HT115 (DE 3) competent cells, and the sequence accuracy of positive clones is verified through colony PCR and sequencing again, so that the escherichia coli containing CfHPGD-L4440 recombinant plasmids is obtained.

Further, in the step (5), the properly sequenced E.coli strain is grown in an expanded culture and IPTG is added to induce efficient expression of dsRNA when it enters the exponential growth phase (OD ₆₀₀ = 0.4-0.6).

Further, in the step (6), the bacterial precipitate after the induction of dsRNA is collected centrifugally, and the bacterial precipitate is resuspended and uniformly mixed by using mixed concentrated microalgae liquid, so that a bacterial mixture is obtained and fed to the chlamys farreri as a bait.

The method further comprises the verification steps of (1) CfHPGD-2 and CfHPGD-3 gene interference efficiency detection, namely sampling the outer mantle, gill, striated muscle, smooth muscle and five main tissues of the chlamys farreri after 40 days of continuous feeding interference, and detecting the relative expression level of the corresponding target genes in different tissues in each experimental group by using real-time fluorescence quantitative PCR (qRT-PCR). (2) And (3) detecting the growth characteristics, namely accurately measuring and performing difference significance analysis on key growth characteristics such as shell length, shell width, shell height, body weight, soft tissue weight, adductor muscle weight and the like of the chlamys farreri of each experimental group after continuous interference for 40 days, so as to reflect the influence of CfHPGD-2 and CfHPGD-3 genes on the growth of the chlamys farreri. (3) And (3) detecting the growth characteristics of the adductor muscles, namely carrying out statistics and difference significance analysis on the quantity and the cross section area of the muscle fibers in the adductor muscle tissue cross section of the chlamys farreri of each experimental group after continuous interference for 40 days, so as to reflect the influence of CfHPGD-2 and CfHPGD-3 genes on the adductor muscle growth of the chlamys farreri.

The method can be applied to researches on the closing muscle and integral growth regulation of other scallops and even bivalve shellfish, and particularly can inhibit HPGD gene expression by using an RNA interference technology, so that the promotion of the closing muscle yield and the growth rate of the bivalve shellfish is realized.

Compared with the prior art, the invention has the beneficial effects that:

According to the method for improving the yield and the growth rate of the adductor muscle of the scallop by inhibiting HPGD gene expression through RNA interference (RNAi) technology, the escherichia coli HT115 bacterial liquid containing the target gene recombinant plasmid is fed to the scallop, so that the expression inhibition of CfHPGD gene is realized.

The invention discloses the negative regulation and control effects of CfHPGD-2 and CfHPGD-3 genes on the adductor muscle and the whole growth of the chlamys farreri for the first time through the CfHPGD gene interference. Therefore, the HPGD genes of bivalve shellfish such as scallop are inhibited in the cultivation of scallop to obtain improved variety with high adductor muscle mass and fast growth, and potential application value is provided for the genetic and breeding practice of aquatic organisms.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of the construction of recombinant interfering plasmids of Chlamys farreri CfHPGD-L4440, respectively.

FIG. 2 is a gel electrophoresis detection chart of dsRNA extraction of the CfHPGD genes of Chlamys farreri.

FIG. 3 is a graph showing quantitative results of relative expression in 5 major tissues 40 days after the CfHPGD gene interference of Chlamys farreri.

FIG. 4 is a graph showing the results of statistical analysis of chlamys farreri growth traits 40 days after each CfHPGD gene interference.

FIG. 5 is a graph of the results of a statistical analysis of the chlamys farreri adductor growth trait 40 days after each CfHPGD gene interference.

FIG. 6 is a graph II showing the results of statistical analysis of the growth traits of the adductor muscle of Chlamys farreri 40 days after each CfHPGD gene interference.

Detailed Description

The present invention will be described in further detail with reference to examples below in order to make the objects, technical solutions and gist of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Example 1 construction of recombinant interfering plasmids of Chlamys farreri CfHPGD-L4440 respectively

(1) Homologous sequence screening and analysis of HPGD genes in chlamys farreri:

Homologous comparison is carried out on HPGD protein sequences of other representative species serving as reference sequences and the complete genome of the chlamys farreri, and four HPGD genes, cfHPGD-1 (shown in a sequence table as SEQ ID NO. 1), cfHPGD-2 (shown in a sequence table as SEQ ID NO. 2), cfHPGD-3 (shown in a sequence table as SEQ ID NO. 3) and CfHPGD-4 (shown in a sequence table as SEQ ID NO. 4) of the chlamys farreri are identified. The CfHPGD-1 contains 13 exons, CDS total length 1,686bp and codes 561 amino acids, the CfHPGD-2 gene contains 6 exons, CDS total length 627bp and codes 208 amino acids, the CfHPGD-3 gene contains 4 exons, CDS total length 561bp and codes 186 amino acids, the CfHPGD-4 gene contains 10 exons, CDS total length 987bp and codes 328 amino acids.

(2) Designing RNA interference fragments and interference primers of four target genes:

Based on the CDS sequence of each CfHPGD gene, an on-line tool siDirect 2.0 (http:// sidirect2.Rnai. Jp /) was used to predict the high-efficiency siRNA interference fragments. To ensure targeting specificity, BLAST alignment was used to verify the uniqueness of selected target sequences in the genome, aimed at designing RNA interference fragments with efficient and specific interference functions, minimizing the generation of off-target effects.

The corresponding primers were designed using PRIMER PREMIER 5.0.0 software and the appropriate two restriction endonucleases (not including their cleavage sites within the entire interference fragment) were screened using the online tool NEBcut 3.0 (https:// nc3.Neb. Com/NEBcut /) for the presence of their cleavage sites in the L4440 plasmid. After the XhoI and HindIII cleavage sites are determined, they are added to the 5' end of the primer with the corresponding protecting base. The primers used in this example are shown in Table 1.

TABLE 1 primer sequence listing of RNA interference target fragments

(3) Amplification and sequencing verification of the interference fragments:

The cDNA of chlamys farreri is used as a template, and Phanta Max Super-FIDELITY DNA polymerase is used for amplifying the interference fragments of four CfHPGD genes (the interference fragment of CfHPGD-1 gene sequence is shown as SEQ ID NO.5, the interference fragment of CfHPGD-2 gene sequence is shown as SEQ ID NO.6, the interference fragment of CfHPGD-3 gene sequence is shown as SEQ ID NO.7, and the interference fragment of CfHPGD-4 gene sequence is shown as SEQ ID NO. 8). The specific reaction system is shown in Table 2, and 200. Mu.L was amplified in total. The PCR procedure was set to 95℃pre-denaturation for 5min, 95℃denaturation for 30s,60℃annealing for 30s,72℃extension for 30s for 32 cycles, and 72℃final extension for 10min.

TABLE 2 DNA polymerase reaction System for interfering fragment amplification

After amplification was completed, the band correctness of the PCR product was checked by 1.5% agarose gel electrophoresis. Subsequently, the RNA interference fragments in the gel were recovered using QIAquick PCR Purification Kit gel recovery kit, and the product concentration and quality were detected using a Nanodrop One spectrophotometer. Next, a ligation system (10. Mu.L) containing the interference fragment and Blunt vector was constructed, and after ligation at 25℃for 15min, transformed into DH 5. Alpha. E.coli competent cells by heat shock, and cultured under shaking at 37℃for 40min. Then, the product was spread uniformly on LB solid medium containing ampicillin (Amp ⁺), and cultured upside down at 37℃for 10-12 hours.

The next day, a single colony was picked using a sterilized toothpick, 800 μl of LB liquid medium containing Amp ⁺ and a PCR amplification system were prepared simultaneously (table 3), and after the monoclonal picking was completed, the toothpick was repeatedly shaken in the PCR reaction system, and then the toothpick was placed in the liquid medium (note number corresponds). Subsequently, colony PCR amplification was verified, and the PCR procedure was set to 95℃for 5min, 95℃for 1min,60℃for 30s,72℃for 1min, 25 cycles total, and 72℃for 10min. Meanwhile, the liquid culture inoculated with the single colony was cultured for 4 to 6 hours based on shaking at 37 ℃. After amplification was completed, the amplified product was detected by 1.5% agarose gel electrophoresis, and the culture verified as positive clone was sent to sequencing company (Sangon Biotech) for sequencing.

TABLE 3 DNA polymerase reaction System at the time of colony PCR amplification verification

(4) Construction and sequencing verification of L4440 interfering plasmids:

After obtaining the strain with correct sequence, it is cultivated in an expanded manner and the plasmids contained therein are extracted using a plasmid extraction kit for endotoxin removal. The obtained Blunt plasmid and L4440 blank vector were subjected to double digestion treatment according to the following reaction system (Table 4), and the PCR procedure was set at 37℃for 20min. After completion of the cleavage, 1.5% agarose gel electrophoresis detection, gel recovery and the like were sequentially performed with reference to the previous procedure, and an RNA interference fragment having a cohesive end and an L4440 empty plasmid fragment were obtained and the concentration thereof was measured.

Table 4 double cleavage reaction System

Thereafter, the ligation amount was calculated using an on-line tool NEBioCalculator (https:// Nebioconductor. Neb. Com /), and the RNA interference fragment was ligated with the L4440 empty plasmid fragment using T4 ligase, and the specific ligation system is shown in Table 5. The PCR program was set at 25℃for 10min. After the connection is completed, each CfHPGD-L4440 recombinant plasmid can be obtained, and the structural schematic diagram of the recombinant plasmid is shown in figure 1.

Finally, referring to the previous procedure, the ligation product and L4440 empty plasmid described above were transformed into HT115 (DE 3) E.coli competent cells, respectively, and the product was spread evenly on LB solid medium containing Amp ⁺ and tetracycline (TET ⁺). After inversion culture at 37 ℃ for 10-12 hours, single colonies are picked for colony PCR amplification, and after amplification culture of the strains verified to be positive clones in LB liquid medium containing Amp ⁺ and TET ⁺, sequencing is carried out again.

TABLE 5T4 ligase ligation reaction System

(5) Induction expression and extraction verification of dsRNA:

10 mu L of positive strain with correct sequencing is inoculated into 3-5mL of LB liquid medium containing Amp ⁺ and TET ⁺, and the culture is carried out for 10-12 hours at 37 ℃ in a shaking way. The next day, 1:100 ratio was transferred to fresh LB liquid medium containing Amp ⁺ and TET ⁺ for expansion culture. After a period of incubation, the absorbance (OD ₆₀₀) of the bacterial solution at 600nm wavelength was measured using a spectrophotometer, and when it entered the exponential growth phase (OD ₆₀₀ =0.4-0.6), 0.5mM isopropyl- β -D-thiogalactoside (IPTG) was added to induce efficient expression of dsRNA. After further culturing for 4 hours, centrifugation was carried out at 8,000rpm for 3 minutes, the supernatant was discarded and the bacterial pellet was collected and stored at-80℃for further use.

Total RNA is extracted from the induced bacterial liquid and the uninduced bacterial liquid respectively by the traditional TRIzol method, single-stranded RNA (ssRNA) is digested by RNaseT1, the concentration and quality of the extracted dsRNA are measured by a Nanodrop One spectrophotometer, and the accuracy of dsRNA bands is detected by 1.5% agarose gel electrophoresis. As a result, as shown in FIG. 2, lanes 5, 8, 11 and 14 represent RNA of the strain in which each CfHPGD-L4440 recombinant plasmid was not induced by IPTG, lanes 6, 9, 12 and 15 represent RNA of the strain in which each CfHPGD-L4440 recombinant plasmid was induced by IPTG, lane 2 represents RNA of the strain in which the L4440 empty plasmid was not induced by IPTG, lane 3 represents RNA of the strain in which the L4440 empty plasmid was induced by IPTG, and lanes 1, 4, 7, 10 and 13 represent 100bp DNA ladders. This result shows that after IPTG induction, e.coli HT115 successfully expressed the target dsRNA fragments targeting each CfHPGD gene (indicated by white arrows), while verifying the necessity of IPTG induction and that the L4440 empty plasmid could be used as a negative control group to exclude false positives.

Example 2 RNA interference of CfHPGD Gene of Chlamys Vibrio by Mixed feeding with Chlorella

(1) Experimental animals:

250 live chlamys farreri of the first age, which are more uniform in individuals, from the same population were randomly selected and their initial growth traits, 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) were systematically determined. Then, the chlamys farreri was randomly divided into five groups, namely, control group, RNAi-CfHPGD-1 group, RNAi-CfHPGD-2 group, RNAi-CfHPGD-3 group and RNAi-CfHPGD-4 group, each group containing 50 individuals. Finally, each group of chlamys farreri was placed in an independent and strictly sterilized 500L tank (filled with about 400L of 20 ℃ thermostatically filtered seawater, changed once a day) and continuously oxygenated into the tank to maintain proper survival conditions.

(2) Preparation of dsRNA:

dsRNA expression was induced by reference example 1. Then, 200mL of coliform HT115 bacterial liquid containing L4440 empty load plasmid, cfHPGD-1-L4440, cfHPGD-2-L4440, cfHPGD-3-L4440 and CfHPGD-4-L4440 recombinant plasmid are respectively taken, supernatant is removed by centrifugation, bacterial precipitate is collected, and 20-30mL of mixed concentrated algae liquid of Chlorella pyrenoidosa (Chlorella pyrenoidesa) and Chaetoceros muelleri (Chaetoceros muelleri) is used for re-suspending the bacterial precipitate, so that bacterial algae mixture is obtained as bait.

(3) The feeding scheme is as follows:

During the RNA interference period, equal amounts of mixed concentrated algae solution are fed to the chlamys farreri groups in four times a day (9 hours, 13 hours, 17 hours and 21 hours), and the algae cell concentration in the water body is kept to be about 5-10 multiplied by 10 ⁵/mL when each feeding is carried out. Wherein, the mixture of the algae is used in the second and fourth feeding, the mixture of the algae containing L4440 empty plasmid is fed by the Control group, the mixture of the algae containing CfHPGD-1-L4440 recombinant plasmid is fed by the RNAi-CfHPGD-1 group, the mixture of the algae containing CfHPGD-2-L4440 recombinant plasmid is fed by the RNAi-CfHPGD-2 group, the mixture of the algae containing CfHPGD-3-L4440 recombinant plasmid is fed by the RNAi-CfHPGD-3 group, and the mixture of the algae containing CfHPGD-4-L4440 recombinant plasmid is fed by the RNAi-CfHPGD-4 group.

The technical effects of the present invention will be described in detail with reference to experimental results.

(1) Detection of expression level after each CfHPGD gene was interfered:

qRT-PCR primers for each CfHPGD genes were designed, taking care to avoid RNA interference fragments to ensure accuracy in quantification of the target genes. The specific primer sequences for the reference gene, eF1α, were as shown in Table 6.

TABLE 6 quantitative primer sequence listing

After 40 days of feeding, 30 chlamys farreri were randomly selected for dissection in each experimental group, and their five main tissues, i.e., mantle, gill, striated muscle, smooth muscle and foot were collected. The above tissue was cut into small pieces of about 1cm in diameter, frozen with liquid nitrogen, and stored at-80 ℃. Total RNA of each main tissue of each group of chlamys farreri is extracted respectively, and cDNA is obtained through reverse transcription and diluted to 10 ng/. Mu.L to be used as a quantitative template.

Afterwards, the experimental design is performed in advance, 3 technical replicates are set for each sample, and 5 biological replicates are set for each group, so as to reduce experimental errors as much as possible. Subsequently, a qRT-PCR reaction system (Table 7) was constructed and placed inIn 480 apparatus, the procedure was set to 50℃pre-denaturation for 2min,94℃pre-denaturation for 10min, 94℃denaturation for 15s,60℃annealing for 1min, 40 cycles total, and finally cooling to 55℃at 0.5℃every 10s and 1min at 55 ℃. After the reaction was completed, the specificity of the amplification was verified according to the dissociation curve, and the relative expression level of each CfHPGD was calculated by the method of 2 ^-△△Ct. Thereafter, a significant analysis of the difference in the expression level of the target gene between each RNA interference group and the control group was performed by t-test (Student's t-test), where P <0.050 is represented and P <0.01 is represented.

TABLE 7qRT-PCR reaction System

The results of the analysis of the expression level of the corresponding CfHPGD genes in the outer mantle, gill, striated muscle, smooth muscle, and foot 5 major tissues of the Chlamys farreri in each RNA interference group and the differences between the control groups are shown in FIG. 3. After the RNA interference is continuously carried out for 40 days, compared with a control group, the expression quantity of CfHPGD-1 genes in RNAi-CfHPGD-1 group mantle, gill, striated muscle, smooth muscle and foot is obviously reduced by 71.03%, 51.09%, 66.40%, 60.93% and 30.74%, the expression quantity of CfHPGD-2 genes in RNAi-CfHPGD-2 group mantle, gill, striated muscle, smooth muscle and foot is obviously reduced by 50.55%, 66.55%, 57.57%, 57.10% and 54.92%, and the expression quantity of CfHPGD-3 genes in RNAi-CfHPGD-3 group mantle, gill, striated muscle, smooth muscle and foot is obviously reduced by 87.99%, 84.89%, 47.00%, 58.03%, 42.94%, and the expression quantity of CfHPGD-4 genes in RNAi-CfHPGD-4 group mantle, gill, striated muscle, smooth muscle and foot is obviously reduced by 60.89%, 55.90%, 84.99%, 86.72% and 66.17%. The quantitative result shows that the RNA interference has high-efficiency and systematic inhibition effect on the CfHPGD genes of the chlamys farreri.

(2) Detecting the growth traits of the chlamys farreri:

After continuous interference for 40 days, the critical growth traits such as shell length, shell width, shell height, body weight, soft tissue weight, adductor muscle weight and the like of the chlamys farreri of each experimental group are accurately measured and the difference significance is analyzed. As a result, as shown in FIG. 4, there was no significant change in the respective growth traits of Chlamys in RNAi-CfHPGD-1 group (P > 0.05), there was a significant increase in the average shell length, shell width, shell height, body weight, soft tissue weight and adductor muscle weight of Chlamys in RNAi-CfHPGD-2 group (P < 0.01), 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), respectively, in RNAi-CfHPGD-3 group (P > 3), there was a significant increase in the average shell length, shell width, shell height, body weight, soft tissue weight and adductor muscle weight of Chlamys in RNAi-CfHPGD-2 group (P < 0.01), respectively, by 2.98% (P < 0.01), 6.77% (P < 0.01), 2.90% (P < 0.05), 18.02% (P < 0.01), 35.24% (P < 0.01), and 15.38% (P < 0.05), respectively, whereas there was no significant change in the average growth traits of Chlamys in RNAi-CfHPGD-3 group (P > 0.05). The statistical result shows that CfHPGD-2 and CfHPGD-3 genes have the function of negatively regulating the integral growth of the chlamys farreri, and when the expression of the two genes is inhibited, the growth rate of the chlamys farreri is obviously accelerated. Whereas targeted inhibition of CfHPGD-1 and CfHPGD-4 genes did not result in a systemic change in growth phenotype, suggesting that they are not involved in core regulatory functions in chlamys growth and are not key regulatory genes that directly affect growth phenotype. Notably, the RNAi-CfHPGD-2 and RNAi-CfHPGD-3 groups exhibited more significant growth promoting effects in terms of the weight of the adductor muscle and soft tissue, indicating that the CfHPGD-2 and CfHPGD-3 genes were closely related to the growth of the tissues such as the adductor muscle of Chlamys farreri.

(3) Detection of the growth traits of the adductor muscle of chlamys farreri:

After the continuous interference for 40 days, the quantity and the cross-sectional area of the myofibers in the closed shell muscle tissue cross section of the chlamys farreri of each experimental group are subjected to statistics and difference significance analysis by using histomorphometric research methods such as paraffin embedding, tissue section, hematoxylin-eosin (HE) staining and the like. As a result, as shown in FIGS. 5 and 6, compared with the control group, the number of striated muscle fibers of Chlamys in RNAi-CfHPGD-2 group was increased by 19.43% (P < 0.01), the number of smooth muscle fibers was increased by 5.72% (P < 0.01), the cross-sectional areas of both types of striated muscle fibers did not significantly change (P > 0.05), the number of striated muscle fibers of Chlamys in RNAi-CfHPGD-3 group was increased by 27.89% (P < 0.01), the number of smooth muscle fibers was increased by 10.10% (P < 0.01), the cross-sectional areas of both types of striated muscle fibers did not significantly change (P > 0.05), and the numbers and cross-sectional areas of both types of striated muscle fibers of Chlamys in RNAi-CfHPGD-1 and RNAi-CfHPGD-4 group were not significantly changed (P > 0.05). The statistical result shows that CfHPGD-2 and CfHPGD-3 genes have the function of negatively regulating the growth of the adductor muscle of the chlamys farreri, and when the expression of the two genes is inhibited, the striated muscle and smooth muscle fibers of the scallop are obviously proliferated.

Based on the RNA interference technology, the invention realizes the systematic inhibition of 15-hydroxy prostaglandin dehydrogenase encoding gene (HPGD) in chlamys farreri by feeding escherichia coli which can induce the generation of target gene specific dsRNA for 40 days. By combining with the measurement of the growth traits of the chlamys farreri, the integral and adductor muscle growth rate of the chlamys farreri after CfHPGD-2 and CfHPGD-3 genes are interfered is obviously improved, and muscle fibers are obviously proliferated, so that the negative regulation and control effects of the two genes on the adductor muscle and integral growth of the chlamys farreri are reflected. Therefore, the invention provides a method for promoting the yield and the growth rate of the adductor scallop muscle, namely, the RNA interference technology fed with dsRNA is used for inhibiting HPGD gene expression, so that the growth of soft tissues such as the adductor scallop muscle and the like and the whole body is promoted.

While the foregoing embodiments have been described in some detail for purposes of clarity of understanding, it will be understood that the invention is not limited to the details of the disclosed embodiments, but is capable of modification, equivalents, and improvements within the spirit and principles of the present disclosure.

Claims (7)

1. HPGD genes related to the growth of adductor scallop muscle, which are characterized in that the genes comprise CfHPGD-2 shown in SEQ NO.2 and CfHPGD-3 shown in SEQ NO. 3.

2. The use of the gene CfHPGD-2 and CfHPGD-3 in promoting the growth of the adductor muscle of chlamys farreri according to claim 1, wherein the silencing method is to inhibit the expression of the CfHPGD-2 and CfHPGD-3 genes in chlamys farreri by RNA interference technology, the interference fragment of the CfHPGD-2 gene is shown as SEQ ID NO.6, and the interference fragment of the CfHPGD-3 gene is shown as SEQ ID NO. 7.

3. A method for increasing the yield and growth rate of adductor scallops, comprising the steps of:

(1) Obtaining CfHPGD-2 and CfHPGD-3 target gene sequences;

(2) Designing an RNA interference fragment and an interference primer of a target gene respectively, wherein the interference fragment of CfHPGD-2 gene is shown as SEQ ID NO.6, and the interference fragment of CfHPGD-3 gene is shown as SEQ ID NO. 7;

(3) Amplifying the interference fragments;

(4) Constructing an L4440 recombinant plasmid containing an interference fragment;

(5) Induction of double-stranded RNA expression by IPTG;

(6) The expression inhibition of CfHPGD-2 and CfHPGD-3 genes was achieved by feeding chlamys farreri by mixing the cells expressing dsRNA with microalgae.

4. The method of claim 3, wherein in the step (2), the interfering fragments of siRNA are predicted and selected by using an on-line tool based on CDS sequences of CfHPGD-2 and CfHPGD-3 genes, and then an interfering primer containing an enzyme cutting site and a protecting base thereof is designed for each interfering fragment by using the on-line tool, wherein the primer sequences are as follows:

。

5. the method of claim 3, wherein in step (3), the interference fragments of the CfHPGD-2 and CfHPGD-3 genes targeted by using scallop cDNA as a template are amplified by using high-fidelity DNA polymerase, and the amplified products are subjected to gel recovery purification after electrophoresis verification, then connected to a Blunt vector and transformed into DH5 alpha competent cells, and the sequence accuracy of positive clones is verified by colony PCR and sequencing.

6. The method according to claim 3, wherein in the step (4), the correctly sequenced Blunt plasmid and L4440 empty plasmid are extracted, the interference fragments of the target genes are respectively connected with the L4440 plasmid by double enzyme digestion, glue recovery and T4 connection, the products are transformed into HT115 (DE 3) competent cells, and the sequence accuracy of the positive clones is verified by colony PCR and sequencing again to obtain the E.coli containing the CfHPGD-L4440 recombinant plasmids.

7. The method of claim 3, wherein in the step (5), the properly sequenced E.coli strain is grown in an expanded manner and IPTG is added to induce efficient expression of dsRNA when the strain enters an exponential growth phase, and in the step (6), the bacterial pellet after dsRNA induction is collected centrifugally, resuspended and mixed uniformly by using a mixed concentrated microalgae solution to obtain a microalgae mixture, and the microalgae mixture is fed to scallops as a bait.