A rapid and efficient shell separation method for shellfish larvae

Abstract

The invention provides a rapid and efficient shell separation method for shellfish larvae, which relates to the technical field of shell separation for shellfish larvae, and comprises the following steps: S1, obtaining scallop larvae, and washing with 3x PBS solution; S2, dissociating larvae with lysis solution, and separating shells and soft tissues, wherein the lysis solution contains the following components: guanidine isothiocyanate, sodium lauroyl sarcosinate, and sodium citrate; S3, collecting and washing larval shells; S4, detecting the degree of separation of larval shells. By adopting the rapid and efficient shell separation method for shellfish larvae of the invention, the separation time is greatly shortened, the damage to the structure and components inside the shell is reduced, and the soft tissue of the shell after separation is less polluted. In addition, the larval shell separated by the method can extract complete DNA, and the high-throughput sequencing library is qualified. The invention effectively solves the technical problem of shell separation for shellfish larvae, and provides a basis for related research on genomics, transcriptomics, proteomics, metabolomics, etc. of larval shells.

Description

Technical Field

[0001] The invention relates to the technical field of shell separation in the larval stage of shellfish, and in particular to a rapid and efficient shell separation method in the larval stage of shellfish. Background Art

[0002] Since the Precambrian period, animals have gradually evolved a rich variety of biomineralized structures with different biological functions, which is considered to be one of the major innovations in the history of animal evolution. Mollusk shells are one of the main types of biomineralized skeletons, and their formation process is also one of the most representative biomineralization processes, and are considered to be an ideal model for studying mineralized skeletons.

[0003] A comprehensive understanding of the shell formation process will help us gain a deeper understanding of the phylogenetic relationships of shelled molluscs and help improve existing biomineralization models. Compared with larval shells, adult shells are easy to observe and can be separated from soft tissues by anatomical means. The experimental operation is simple, so current shell-related research is mainly focused on adult shells. For example, research on the organic components and matrix proteins of adult shells of molluscs, as well as high-throughput DNA sequencing and metagenomics of adult shells, etc.

[0004] The formation of larval shells is the basis of mollusk shell formation. However, since mollusks are tiny in size (mostly at the micrometer level) during the initial stage of larval shell formation, it is impossible to use anatomical methods to perform experimental operations such as separation of shells and soft tissues, so relevant research on larval shells is extremely scarce. Therefore, how to face the challenges at the technical level and successfully separate a sufficient amount of larval shells with as little soft tissue contamination as possible is the basis for studying the initial formation of larval shells of molluscs. In pearl oysters and oysters, researchers used 1M NaOH solution as a lysis solution to perform preliminary separation of their larval shells, but this method requires overnight treatment, and the long treatment process may destroy the structure and composition inside the shell, and the soft tissue residues on the shells treated by this method are relatively serious.

[0005] Therefore, in order to solve the above problems, a rapid and efficient shell separation method for shellfish larvae was proposed. Summary of the invention

[0006] The purpose of the present invention is to establish a rapid and efficient shell separation method for shellfish larvae, shorten the dissociation time, reduce the damage to the structure and components inside the shell, and reduce the soft tissue contamination of the shell after separation, so as to make up for the deficiencies of the prior art and effectively solve the technical problems of shell separation in the shellfish larvae stage.

[0007] In order to achieve the above object, the present invention is specifically implemented through the following technical solutions:

[0008] A method for quickly and efficiently separating shells of shellfish larvae comprises the following steps:

[0009] S1. Take scallop larvae, place them in a centrifuge tube, wash the larvae, and remove the seawater and other residues;

[0010] S2, after washing, adding the lysis solution into a centrifuge tube, rotating and lysing, and separating the shell and soft tissue; the lysis solution is prepared using guanidine isothiocyanate, sodium lauroyl sarcosinate and sodium citrate solution, and the concentration of guanidine isothiocyanate in the lysis solution is 3.5-4.5 mol / L, and the concentration of sodium lauroyl sarcosinate is 15-20 mmol / L;

[0011] S3, larval shell collection and washing;

[0012] S4. Detection of the degree of larval shell separation.

[0013] Furthermore, the concentration of the sodium citrate solution is 0.8-1.2 mol / L, and 20-30 ml of the sodium citrate solution is added to each liter of lysate.

[0014] Furthermore, the concentration of the sodium citrate solution is 1 mol / L, and 25 ml of the sodium citrate solution is added to each liter of lysate.

[0015] Furthermore, the concentration of guanidine isothiocyanate in the lysate is 4 mol / L, and the concentration of sodium lauroyl sarcosinate is 17 mmol / L.

[0016] Furthermore, in step S2, the lysis time is 2-4 hours, and the lysis temperature is 3-5° C. Furthermore, the lysis is performed at 4° C. with rotation for 3 hours.

[0017] Furthermore, the volume ratio of scallop larvae to lysis solution is 50-100ul:1-2ml.

[0018] Furthermore, the volume of the centrifuge tube is 2-50 ml.

[0019] Furthermore, the specific operation in step S3 is: let the centrifuge tube containing the larval shells stand for 5-10 minutes, and the separated shells are distributed in the middle layer; use a pipette to remove the upper layer of liquid, and suck the middle layer of larval shells into a new centrifuge tube, and wash the larval shells upside down with 3x PBS solution; place the centrifuge tube vertically and let it stand for 5-10 minutes, the larval shells gather at the bottom of the centrifuge tube, use a pipette to remove the upper layer of washing liquid, and repeat the washing 3-5 times.

[0020] Furthermore, in step S4, the washed larval shells are observed under a microscope to detect the degree of separation of the larval shells and confirm that no soft tissue remains on the shells.

[0021] Furthermore, in step S1, the membrane was rinsed 2-3 times with 3×PBS solution.

[0022] The advantages and beneficial effects of the present invention are:

[0023] The present invention develops a rapid and efficient shell separation method for shellfish larvae, which greatly shortens the separation time, reduces the damage to the structure and components inside the shell, and reduces the soft tissue contamination of the shell after separation. In addition, the larval shell separated by the present method can extract complete DNA, and the high-throughput sequencing library is qualified. The present invention effectively solves the technical difficulties of shell separation in the larval stage of shellfish, provides a basis for related research on genomics, transcriptomics, proteomics and metabolomics of larval shells, and is of great significance for understanding the formation of mollusk shells and phylogenetic relationships. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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 or the description of the prior art 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 labor.

[0025] Figure 1 The shells of the D-type larvae of the Yesso scallop separated by the method of the present invention (A) and the untreated D-type larvae of the Yesso scallop (B).

[0026] Figure 2 The larval stage shell of the scallop shell top separated by the method of the present invention (A) and the untreated larvae of the scallop shell top (B).

[0027] Figure 3 The figure is the DNA agarose gel electrophoresis diagram of the D-type larval stage shell and the shell top larval stage shell of the Yezo scallop separated by the present invention.

[0028] Figure 4 , Figure 5 , Figure 6 , Figure 7 All of them are peak graphs of high-throughput sequencing libraries, among which, Figure 4 , Figure 5 The shell of the D-type larvae and the shell of the top larvae of the No. 1 individual of the Yesso scallop; Figure 6 , Figure 7 These are the shells of the D-type larvae and the shell-top larvae of individual No. 2 Yezo scallop.

[0029] Figure 8 The shells of the D-type larvae of dwarf clams separated by the method of the present invention (A) and the untreated D-type larvae of dwarf clams (B).

[0030] Fig. 9 The shells of the larval stage of the dwarf clam shell top separated by the method of the present invention (A) and the untreated larvae of the dwarf clam shell top (B).

[0031] Fig.10 The figure is the DNA agarose gel electrophoresis diagram of the shell of the D-type larvae stage and the shell of the top larvae stage of the dwarf clam separated by the present invention.

[0032] Fig.11 , Fig.12 , Fig.13 , Fig.14 All of them are peak graphs of high-throughput sequencing libraries, among which, Fig.11 , Fig.12 The shells of the D-type larvae and the shell-top larvae of the dwarf clam No. 1; Fig.13 , Fig.14 These are the shells of the D-type larvae and the shell-top larvae of individual No. 2 dwarf clam.

[0033] Fig.15 The shells of the D-type larvae (A) and the top larvae (B) of the Yesso scallop separated using 1M NaOH lysis solution.

[0034] Fig.16 Comparative Example 1: The larval stage shell of the scallop shell top separated by lysis solution.

[0035] Fig.17 This is the larval stage shell of the top of the scallop shell separated by the lysis solution of Comparative Example 2.

[0036] Fig.18 This is the larval stage shell of the top of the scallop shell separated by the lysis solution of comparative example 3.

[0037] Fig.19 This is the larval stage shell of the top of the scallop shell separated by the lysis solution of Comparative Example 4.

[0038] Fig. 20 This is the larval stage shell of the scallop shell top separated by the lysis solution of comparative example 5.

[0039] Figure 3 , Fig.10 The Chinese meaning of “marker” is: DNA molecular weight standard. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and key points of the present invention more clearly understood, 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 disclose the present invention and are not used to limit the present invention.

[0041] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0042] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0043] The abbreviation or English name corresponds to the Chinese meaning:

[0044] EDTA: ethylenediaminetetraacetic acid;

[0045] SDS: sodium dodecyl sulfate;

[0046] STE: STE buffer consists of 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 1 mM EDTA (pH 8.0), referred to as STE;

[0047] Tris balanced phenol, Tris: tris(hydroxymethyl)aminomethane;

[0048] NaAc: sodium acetate buffer;

[0049] RNase: ribonuclease;

[0050] Illumina HiSeq sequencing: Illumina high-throughput sequencing platform;

[0051] Reads: short read sequences generated by sequencing;

[0052] Clean reads: the amount of valid data after filtering;

[0053] Effective: The coverage of the alignment to the reference genome.

[0054] Example 1

[0055] The shells of the D-type larvae and the shell-top larvae of the Japanese scallop were separated as an example, and their DNA was extracted to evaluate the quality of the shells obtained by the separation method. The steps are as follows:

[0056] 1) Take 100ul of D-type larvae and shell-top larvae of the Japanese scallop respectively into a 2ml centrifuge tube. If the larvae cannot sink completely to the bottom of the tube, centrifuge at 3000rpm for 5min, use a pipette to remove as much liquid residue as possible, wash the larvae with 3x PBS solution 2-3 times, and then add 1.5-1.8ml of lysis buffer. The lysis buffer is prepared by using 1 mol / L sodium citrate solution, guanidine isothiocyanate, and sodium lauroyl sarcosinate. The final concentration is 4 mol / L guanidine isothiocyanate, the final concentration is 17 mmol / L sodium lauroyl sarcosinate, and the volume percentage is 2.5% sodium citrate solution (that is, add 25ml of 1 mol / L sodium citrate solution per liter of lysis buffer), and rotate and mix at 4℃ for 3h;

[0057] 2) Place the centrifuge tube in the centrifuge tube rack and let it stand for 5-10 minutes. The lysate will be separated into layers. The separated shells will be in the middle layer. Use a pipette to slowly remove the upper layer of liquid.

[0058] 3) Pipette the middle layer of larval shells into a 5 ml tube, wash the larval shells upside down with 3x PBS solution, then place the centrifuge tube vertically and let it stand for 5-10 minutes. The larval shells will gather at the bottom of the centrifuge tube. Slowly remove the upper layer of washing solution with a pipette, and repeat the washing 3-5 times;

[0059] 4) Use a pipette to take an appropriate amount of washed shells and observe them under a microscope to confirm that there is no soft tissue residue on the shells. If there is, repeat step (3) until the shells are clean.

[0060] 5) Pipette the cleaned shells into a 2 mL centrifuge tube, add 500 µL EDTA (0.5 M, pH 8.0), 500 µL STE, 60 µL 10% SDS, 3.5 µL proteinase K (20 mg / mL), and rotate at 56°C for 3 h to lyse;

[0061] 6) Centrifuge at 12000 rpm for 10 min at room temperature, aspirate the supernatant, add 300 µl Tris-balanced phenol and 300 µl chloroform:isoamyl alcohol (24:1, v / v), rotate and shake for 15 min, and centrifuge at 11000 rpm for 10 min;

[0062] 7) Draw out the supernatant, add 600 µl of chloroform:isoamyl alcohol (24:1, v / v), rotate and shake for 15 min, and centrifuge at 8000 rpm for 10 min;

[0063] 8) Draw out the supernatant, add 50µl NaAc and 1mL ice-cold anhydrous ethanol, shake slowly for 1-2min, and place at -20℃ to precipitate for 40min;

[0064] 9) Centrifuge at 4°C, 12000rpm for 10min to obtain DNA precipitate, discard the supernatant, wash twice with 1mL 70% ice ethanol, centrifuge at 4°C, 8000rpm for 5min; discard the supernatant, blow dry the alcohol in a fume hood, and dry the DNA precipitate;

[0065] 10) Add different amounts of sterile ultrapure water and RNase with a final concentration of 2.0 µg / µL according to the amount of precipitation, and place at 37°C for 2 hours to digest RNA;

[0066] 11) Measure the OD value and concentration using a Qubit® 2.0 fluorometer and perform agarose gel electrophoresis.

[0067] Figure 1 The two are the shells of the D-stage larvae of the scallop obtained by this separation method and the untreated D-stage larvae of the scallop. The comparison shows that there is almost no soft tissue left in the shells after separation, and the hardness of the shells at this stage is relatively small; Figure 2The shell of the larval stage of the shell top of the Yezoa scallop obtained by this separation method is compared with the untreated larval stage of the shell top of the Yezoa scallop. The comparison shows that there is almost no soft tissue remaining in the shell after separation. The shell hardness becomes greater during this period, and secondary shells begin to be generated, and there are pigments and growth lines.

[0068] In order to evaluate the quality of the larval shells of the scallops separated by the present invention, we extracted DNA, constructed high-throughput sequencing libraries, and performed whole-genome resequencing on the separated D-type larval shells and shell-top larval shells. Figure 3 The agarose gel electrophoresis diagram shows that the main band is clearly visible, and the obtained DNA is complete and effective. The library was constructed using the VAHTSTMUniversal DNA Library Prep Kit for Illumina, and the library was qualified. The library detection peak diagram is shown in Figure 4 , Figure 5 , Figure 6 , Figure 7 The library was then sequenced by Illumina HiSeq, and the clear reads obtained after removing low-quality reads and the coverage of alignment to the reference genome of the scallop are shown in Table 1. The scallop larval shells separated by this method can also be used for corresponding proteomics, metagenomics and other studies.

[0069] Table 1 Whole genome resequencing data of the shells of the D-type larvae and the top larvae of the Yesso scallop

[0070]

[0071] Example 2

[0072] In order to verify the universal applicability of the present invention, the D-type larvae and the shell-top larvae of dwarf clams were used as examples to separate the shells, and their DNA was extracted to evaluate the quality of the shells obtained by the separation method. The steps were the same as those in the above-mentioned Example 1.

[0073] in, Figure 8 These are the shells of the D-stage larvae of the dwarf clam obtained by this separation method and the untreated D-stage larvae of the dwarf clam as a control. The comparison shows that there is almost no soft tissue left in the shells after separation, and the hardness of the shells at this stage is relatively small; Fig. 9 The shells of the larval stage of the dwarf clam shell top obtained by this separation method are compared with the untreated dwarf clam shell top larvae. The comparison shows that there is almost no soft tissue remaining in the separated shells. The shells at this stage become harder, secondary shells begin to be generated, and there are pigments and growth lines.

[0074] In order to evaluate the quality of the larval shells of the dwarf clam separated by the present invention, we extracted DNA, constructed high-throughput sequencing libraries and performed whole-genome resequencing on the separated D-type larval shells and shell-top larval shells. Fig.10 The agarose gel electrophoresis diagram shows that the main band is clearly visible, and the obtained DNA is complete and effective. The library was constructed using the VAHTSTMUniversal DNA Library Prep Kit for Illumina, and the library was qualified. The library detection peak diagram is shown in Fig.11 , Fig.12 , Fig.13 , Fig.14 The library was then sequenced by Illumina HiSeq, and the clear reads obtained after removing low-quality reads and the coverage of alignment to the reference genome of the scallop are shown in Table 2. The larval shells of the dwarf clam separated by this method can also be used for corresponding proteomics, metagenomics and other studies.

[0075] Table 2 Whole genome resequencing data of the shells of the D-type larvae and the shell-top larvae of the dwarf clam

[0076]

[0077] Example 3

[0078] In order to verify the superiority of the present invention, the larval shells were separated by a published separation method and compared with the method of the present invention, that is, 1M NaOH solution was used as a lysis solution to separate the shells of the D-type larvae and the shell-top larvae of Yezo, and the steps were as follows:

[0079] 1) Take 3 ml of larvae in a 50 ml test tube, add 1M NaOH (35-40 ml) and incubate overnight;

[0080] 2) Wash with deionized water for 5 times and observe under a microscope. Repeat the cleaning process until soft tissue and contaminants are completely removed.

[0081] Fig.15 The shells of the D-type larvae and the shell-top larvae of the scallop separated by 1M NaOH lysis solution are respectively the shells of the D-type larvae and the shell-top larvae of the scallop obtained by the separation method of the present invention ( Figure 1 , Figure 2 ) It can be seen from the comparison that after separation with 1M NaOH lysis solution, there are still obvious soft tissue residues in the shell. In addition, this separation method requires overnight treatment, which is time-consuming, and the long treatment process may destroy the structure and composition of the shell, which is not conducive to the subsequent experiments.

[0082] Comparative Example 1

[0083] Based on Example 1, guanidine isothiocyanate in the lysate was replaced with guanidine hydrochloride. The results showed that the shells separated by the lysate of Comparative Example 1 clearly had soft tissue residues, such as Fig.16 shown.

[0084] Compared with guanidine hydrochloride, guanidine isothiocyanate in the lysis solution of Example 1 of the present invention is a stronger protein denaturant. The lysis solution prepared in Example 1 of the present invention can not only effectively destroy the cell structure and denature the protein, but also inhibit the activity of nucleases and protect RNA from degradation.

[0085] Comparative Example 2

[0086] Based on Example 1, sodium lauroyl sarcosinate in the lysate was replaced with sodium lauroyl glutamate. The results showed that the shells separated by the lysate in Comparative Example 2 still had soft tissue residues, such as Fig.17 shown.

[0087] Comparative Example 3

[0088] Based on Example 1, sodium citrate in the lysate was replaced with sodium acetate. The results showed that the shells separated by the lysate in Comparative Example 3 still had soft tissue residues, such as Fig.18 shown.

[0089] Sodium citrate and sodium acetate both have certain effects in chelating metal ions, but their chelating abilities may differ. The addition of sodium citrate to the lysate of Example 1 of the present invention may have a stronger chelating ability, and can better control the ionic strength in the reaction system, not only playing a role in regulating pH, but also maintaining a more stable buffer system, which is conducive to the lysate to function.

[0090] Comparative Example 4

[0091] Based on Example 1, the concentrations of the components of the lysate were adjusted to 5 M guanidine isothiocyanate and 1 mM sodium lauroyl sarcosinate. The results showed that the shells separated by the lysate of Comparative Example 4 still had soft tissue residues, such as Fig.19 shown.

[0092] Comparative Example 5

[0093] Based on Example 1, the concentrations of the components of the lysate were adjusted to 1 M guanidine isothiocyanate and 20 mM sodium lauroyl sarcosinate. The results showed that the shells separated by the lysate of Comparative Example 5 still had obvious soft tissue residues, such as Fig. 20 shown.

[0094] In summary, the embodiments of the present invention achieve rapid and efficient shell separation in the larval stage of shellfish, and prove that it is applicable to different species; the shells dissociated using this method can extract complete DNA and meet the needs of subsequent sequencing analysis. The present invention is of great significance for promoting relevant research on shells in the larval stage of shellfish and understanding the formation and phylogenetic relationship of mollusk shells.

[0095] The above description is only a preferred 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. A method for rapid and efficient shell separation of shellfish larvae, characterized in that: The following steps are involved: S1. Take scallop larvae and wash them; S2, after washing, adding a lysis solution, wherein the lysis solution is prepared using guanidine isothiocyanate, sodium lauroyl sarcosinate and sodium citrate solution, wherein the concentration of guanidine isothiocyanate in the lysis solution is 3.5-4.5 mol / L, and the concentration of sodium lauroyl sarcosinate in the lysis solution is 15-20 mmol / L, and the shell and soft tissue are separated; S3, larval shell collection and washing; S4, detection of larval shell separation degree; In step S2, the concentration of the sodium citrate solution is 0.8-1.2 mol / L, and 20-30 ml of the sodium citrate solution is added per liter of lysate; the lysis time is 2-4 h, and the lysis temperature is 3-5°C.

2. A method for rapid and efficient shell separation of shellfish larvae according to claim 1, characterized in that: In step S2, the concentration of the sodium citrate solution is 1 mol / L, and 25 ml of the sodium citrate solution is added per liter of lysate.

3. A method for rapid and efficient shell separation of shellfish larvae according to claim 1, characterized in that: In step S2, the concentration of guanidine isothiocyanate in the lysate is 4 mol / L, and the concentration of sodium lauroyl sarcosinate is 17 mmol / L.

4. A method for rapid and efficient shell separation of shellfish larvae according to claim 1, characterized in that: In step S2, the specific operation of the lysis is: rotating lysis at 4°C for 3 hours.

5. A method for rapid and efficient shell separation of shellfish larvae according to claim 1, characterized in that: The volume ratio of scallop larvae to lysis buffer is 50-100ul:1-2ml.

6. A method for rapid and efficient shell separation of shellfish larvae according to claim 1, characterized in that: The specific operation in step S3 is as follows: the centrifuge tube containing the larval shells is allowed to stand for 5-10 minutes, and the separated shells are distributed in the middle layer; the upper layer liquid is sucked out with a pipette, the larval shells in the middle layer are sucked into a new centrifuge tube, and the larval shells are washed upside down with a 3x PBS solution; Place the centrifuge tube vertically and let it stand for 5-10 minutes. The larval shells will gather at the bottom of the centrifuge tube. Use a pipette to remove the upper washing solution and repeat the washing process 3-5 times.

7. A method for rapid and efficient shell separation of shellfish larvae according to claim 1, characterized in that: In step S4, the washed larval shells are observed under a microscope to detect the degree of separation of the larval shells.

8. A method for rapid and efficient shell separation of shellfish larvae according to claim 1, characterized in that: In step S1, the larvae are washed by washing the larvae with 3x PBS solution.

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