阅读说明：本技术 一种虾夷扇贝肌原纤维的分离和鉴定方法 (Separation and identification method of patinopecten yessoensis myofibrils ) 是由 孙秀俊 刘志鸿 周丽青 杨爱国 吴彪 田吉腾 于 2019-11-27 设计创作，主要内容包括：本发明涉及一种虾夷扇贝肌原纤维的分离和鉴定方法,属于海洋无脊椎动物的肌肉生物学领域,所述方法的具体步骤如下,用扇贝肌肉专用洗涤液洗带壳闭壳肌组织,加入扇贝肌肉松弛缓冲液,旋转搅拌3天；切取闭壳肌组织,加入扇贝肌肉松弛缓冲液,匀浆直至组织块完全消失；在4℃条件下离心,上清液加入扇贝肌肉松弛缓冲液,4℃条件下离心获得粗肌丝的沉淀物,用扇贝肌肉松弛缓冲液迅速吹打,立即进行涡旋混匀,离心获得上清液；上清液加入的扇贝肌肉松弛缓冲液,离心获得粗肌丝的沉淀物,再用扇贝肌肉松弛缓冲液吹打混匀,离心所得到的上清液即为纯化的粗肌丝悬浮液,可直接用于SDS-PAGE检测及电镜分析；本发明方法能够实现高效分离虾夷扇贝闭壳肌的肌原纤维。(The invention relates to a separation and identification method of comb shell myofibrils, belonging to the field of muscle biology of marine invertebrates, which comprises the following steps of washing the adductor muscle tissue with a special washing liquid for the scallop muscle, adding a relaxation buffer solution for the scallop muscle, and rotating and stirring for 3 days; cutting the adductor muscle tissue, adding scallop muscle relaxation buffer solution, and homogenizing until the tissue mass completely disappears; centrifuging at 4 deg.C, adding scallop muscle relaxation buffer solution into the supernatant, centrifuging at 4 deg.C to obtain precipitate of coarse myofilament, rapidly blowing with scallop muscle relaxation buffer solution, immediately vortex and mixing, and centrifuging to obtain supernatant; adding scallop muscle relaxation buffer solution into the supernatant, centrifuging to obtain precipitate of the coarse myofilaments, blowing and uniformly mixing with the scallop muscle relaxation buffer solution, centrifuging to obtain supernatant which is purified coarse myofilament suspension and can be directly used for SDS-PAGE detection and electron microscope analysis; the method can realize the high-efficiency separation of the myofibrils of the adductor muscles of the patinopecten yessoensis.)

1. A separation and identification method of comb shell myofibrils is characterized by comprising the following specific steps:

taking fresh scallop adductor muscle tissue with shells of a patinopecten yessoensis, washing off impurities including membrane protein by using a special washing solution for the patinopecten yessoensis muscle, adding a freshly prepared patinopecten yessoensis muscle relaxation buffer solution, and rotating and stirring for 3 days;

cutting the adductor muscle tissue, adding a scallop muscle relaxation buffer solution, and operating in a high-speed mode by using a tissue homogenizer until the scallop muscle tissue blocks completely disappear;

centrifuging at 4 ℃ to remove large particles including tissue block residues, adding scallop muscle relaxation buffer solution into the supernatant, centrifuging at 4 ℃ to obtain precipitates of coarse myofilaments, quickly blowing the precipitates by using the scallop muscle relaxation buffer solution, immediately carrying out vortex mixing, and centrifuging to obtain the supernatant;

adding a scallop muscle relaxation buffer solution into the supernatant, centrifuging to obtain a precipitate of the coarse myofilaments, blowing and uniformly mixing the precipitate with the scallop muscle relaxation buffer solution, and centrifuging to obtain a supernatant which is a purified coarse myofilament suspension and can be directly used for SDS-PAGE detection and electron microscope analysis;

and fifthly, sucking 6 mu L of coarse myofilament suspension to be placed in the center of the ultrathin carbon film special for the electron microscope, flushing the metal mesh by using a muscle relaxation cleaning solution, sequentially sucking a uranyl acetate dye solution with the mass ratio of 1%, air and a muscle activation buffer solution into the aseptic gun head, flushing the metal mesh, naturally airing and storing, and observing and taking a picture under the electron microscope.

2. The method for separating and identifying patinopecten yessoensis myofibrils according to claim 1, wherein in the first step, the preparation method of the buffer solution for relaxing the patinopecten yessoensis muscle comprises the following steps: a mixed solution of NaCl 100mM and MgCl containing the following components at final concentrations was prepared₂3mM、EGTA 1mM、Pipes 5mM、NaH₂PO₄5mM、NaN₃1mM and MgATP 5mM, pH value adjusted to 7.0, 0.2 μm microporous membrane filtration, autoclaving.

3. The method for separating and identifying patinopecten yessoensis myofibrils according to claim 1, wherein in the first step, the method for preparing the washing solution special for patinopecten yessoensis muscle comprises the following steps: adding 0.1g saponin into 100ml scallop muscle relaxation buffer solution, and mixing until completely dissolved.

4. The method for separating and identifying patinopecten yessoensis myofibrils according to claim 1, wherein in the fifth step, the muscle relaxation cleaning solution is prepared by the following steps: a mixed solution was prepared containing the following components in final concentrations NaAC100mM, MgCl₂3mM、EGTA 0.2mM、imidazole 2mM、NaN₃1mM and MgATP 1mM, pH adjusted to 7.0, 0.2 μm microporous membrane filtration, autoclaving.

5. The method for separating and identifying patinopecten yessoensis myofibrils according to claim 1, wherein in the step five, the muscle activation buffer is prepared by: 2mg ofCaCl in water₂Adding into muscle relaxation cleaning solution, and mixing until completely dissolving.

Technical Field

The invention belongs to the field of muscle biology of marine invertebrates, and relates to a separation and identification method of comb shell myofibrils.

Background

Regardless of whether it is skeletal muscle of vertebrates or striated muscle of invertebrates, all animal muscles perform the physiological function of muscle contraction through the relative sliding of thick and thin muscle filaments. The key to the understanding of animal muscle contraction and regulation is to establish a high-efficiency separation and purification method of myofibril, observe the structure of myofibril by methods such as X-ray fiber diffraction, electron paramagnetic resonance, electron microscope and the like, and find out the molecular and ultrastructural characteristics of myofibril. At present, the major research on the separation, purification and structural analysis of animal myofibrils focuses mainly on the skeletal muscle of mammals. Compared with the skeletal muscle of the vertebrate, the muscle of the invertebrate has the characteristics of high structural stability, rich functional diversity, high specialization degree of myofilament assembly and the like, and provides important reference data for further analyzing the muscle structure and the function of the vertebrate. However, an effective method for separating and purifying myofibrils of marine invertebrates such as scallops is lacked at present, and research progress of muscle structures and functions of the marine invertebrates such as scallops is restricted.

Patinopecten yessoensis belongs to cold water shellfish, is originally distributed in Japan, Russian far east, Korean and other sea areas, is one of the most excellent breeding varieties in the world scallop family, has various adult tissues and organs, and has unique physiological characteristics of different tissues. The comb shell muscle of the Japanese scallop is fat, fresh and tender, and rich in nutrition, is deeply favored by consumers at home and abroad, and is a high-grade marketable aquatic product in the aquatic product market. Among them, adductor muscle is a main functional organ of scallop for shell opening and closing, body movement and evasion of enemy. The ultramicro structural characteristics of the original muscle fiber of the scallop can be deeply understood, the structure, the function and the molecular regulation mechanism of the muscle of the scallop can be analyzed, the theoretical basis can be established for improving the scallop column yield and the scallop meat quality character and the like, and the scientific basis can be provided. Therefore, the development of the efficient separation and identification method suitable for the scallop myofibrils not only is beneficial to deeply analyzing the muscle structure and function of the marine invertebrate, but also has important theoretical and practical significance for accelerating the genetic improvement process of the scallop meat quality character, and provides important reference data for the research of animal muscle biology and muscle evolution.

Disclosure of Invention

The invention aims to provide a separation and identification method of comb shell muscle fibril, which can efficiently separate comb shell muscle fibril and provide an electron microscope identification method of the ultrastructure of the comb shell muscle fibril, and has important theoretical and application values for analyzing the ultrastructure characteristics of marine shellfish muscle fibril and the molecular mechanism of muscle contraction and regulation thereof.

A separation and identification method of comb shell myofibrils comprises the following specific steps:

taking fresh scallop adductor muscle tissue with shells of a patinopecten yessoensis, washing off impurities including membrane protein by using a special washing solution for the patinopecten yessoensis muscle, adding a freshly prepared patinopecten yessoensis muscle relaxation buffer solution, and rotating and stirring for 3 days;

cutting the adductor muscle tissue, adding a scallop muscle relaxation buffer solution, and operating in a high-speed mode by using a tissue homogenizer until the scallop muscle tissue blocks completely disappear;

centrifuging at 4 ℃ to remove large particles including tissue block residues, adding scallop muscle relaxation buffer solution into the supernatant, centrifuging at 4 ℃ to obtain precipitates of coarse myofilaments, quickly blowing the precipitates by using the scallop muscle relaxation buffer solution, immediately carrying out vortex mixing, and centrifuging to obtain the supernatant;

adding a scallop muscle relaxation buffer solution into the supernatant, centrifuging to obtain a precipitate of the coarse myofilaments, blowing and uniformly mixing the precipitate with the scallop muscle relaxation buffer solution, and centrifuging to obtain a supernatant which is a purified coarse myofilament suspension and can be directly used for SDS-PAGE detection and electron microscope analysis;

and fifthly, absorbing 6 mu L of thick myofilament suspension to be placed in the center of the ultrathin carbon film special for the electron microscope, flushing the metal mesh by using a muscle relaxation cleaning solution, sequentially sucking 1% of UA (uranyl acetate) dye solution, air and a muscle activation buffer solution into the aseptic gun head, flushing the metal mesh, naturally airing and storing, and observing and photographing under the electron microscope.

Further, in the step one, the preparation method of the scallop muscle relaxation buffer solution comprises the following steps: a mixed solution of NaCl 100mM and MgCl containing the following components at final concentrations was prepared₂3mM、EGTA 1mM、Pipes 5mM、NaH₂PO₄5mM、NaN₃1mM and MgATP 5mM, adjusting the pH value to 7.0, filtering with a0.2 μm microporous filter membrane, and autoclaving;

further, in the step one, the preparation method of the washing liquid special for scallop muscle comprises the following steps: adding 0.1g saponin into 100ml scallop muscle relaxation buffer solution, and mixing until completely dissolved.

Further, in the fifth step, the preparation method of the muscle relaxation cleaning solution comprises the following steps: a mixed solution was prepared containing the following components in final concentrations NaAC100mM, MgCl₂3mM、EGTA 0.2mM、imidazole2mM、NaN₃1mM and MgATP 1mM, adjusting Ph to 7.0, filtering with 0.2 μm microporous membrane, and autoclaving;

further, in step five, the muscle activation buffer solution preparation method comprises the following steps: 2mg of anhydrous CaCl₂Adding into muscle relaxation cleaning solution, and mixing until completely dissolving.

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

the invention provides a separation and identification method of comb shell muscle fibril, which can realize the high-efficiency separation of comb shell muscle fibril of comb shell, and provides an electron microscope identification method of the ultra-microstructure of comb shell muscle fibril, which can be applied to the separation and purification of other marine shellfish muscle fibril, has important theoretical and application values for finding out the ultra-microstructure characteristics of marine shellfish muscle fibril, analyzing muscle contraction and the regulated molecular mechanism thereof, and provides important reference data for the biological research of comb shell muscle.

Drawings

FIG. 1 is an electron micrograph of the thick muscle wire of the adductor muscle of Japanese scallop.

Detailed Description

In order to explain the technical scheme of the invention more clearly, the invention is further described in detail with reference to the attached drawings. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.