技术领域Technical Field
本发明属于基因编辑技术领域，具体涉及一种将外源核酸片段导入双壳贝类精子中的方法。The present invention belongs to the technical field of gene editing, and in particular relates to a method for introducing exogenous nucleic acid fragments into bivalve sperm.
背景技术Background technique
基因编辑技术是近年来探索基因功能和解析性状直接有效的方法，其中CRISPR/Cas9基因编辑技术具有操作简单、靶点选择广、成本低、效率高等优点，为重要水产物种经济性状的遗传解析和良种培育提供了有力工具。自2014年以来，随着cas9技术的日渐成熟，其应用也越来越广泛。而目前最热门且应用最为广泛的CRISPR/Cas9系统，则是以Cas9蛋白和sgRNA为核心组成。Cas9含有在氨基末端的RuvC和蛋白质中部的HNH两个活性位点，在crRNA成熟和双链DNA剪切中发挥作用，可引起DNA双链断裂。当DNA断裂后，细胞核内同时存在与损伤DNA同源的DNA片段，则可通过同源介导的双链DNA修复在目的位点引入外源DNA片段，从而达到片段敲入或编辑的效果。CRISPR/Cas9技术的不断发展为生物学及医学领域的研究带来了革命性的突破,利用该技术构建基因突变模式生物有助于基因功能的研究,对于遗传疾病的治疗等具有重要的参考价值,同时还可以从基因组水平上有效改善经济动物的生产性能,提高抗病能力等。Gene editing technology is a direct and effective method for exploring gene functions and analyzing traits in recent years. Among them, CRISPR/Cas9 gene editing technology has the advantages of simple operation, wide target selection, low cost and high efficiency, providing a powerful tool for genetic analysis and breeding of economic traits of important aquatic species. Since 2014, with the increasing maturity of cas9 technology, its application has become more and more extensive. The most popular and widely used CRISPR/Cas9 system is composed of Cas9 protein and sgRNA as the core. Cas9 contains two active sites, RuvC at the amino terminus and HNH in the middle of the protein, which play a role in crRNA maturation and double-stranded DNA shearing, and can cause double-stranded DNA breaks. When the DNA is broken, there are DNA fragments homologous to the damaged DNA in the cell nucleus at the same time, and the exogenous DNA fragments can be introduced at the target site through homology-mediated double-stranded DNA repair, thereby achieving the effect of fragment knock-in or editing. The continuous development of CRISPR/Cas9 technology has brought revolutionary breakthroughs in research in the fields of biology and medicine. Using this technology to construct gene mutation model organisms is helpful for the study of gene function and has important reference value for the treatment of genetic diseases. It can also effectively improve the production performance of economic animals and enhance their disease resistance at the genomic level.
在CRISPR/Cas系统基因编辑导入方法中，主要分为三大类，即生物转染(慢病毒、腺病毒等)、化学转染(脂质体、阳离子聚合物)、物理转染(电转染、显微注射、基因枪)。对比上述导入方法，生物转染的方法相对操作难度较低，然而存在细胞毒素残留可能；化学转染方法最突出的优点是操作简便，无需复杂技术要求，也不存在细胞毒素问题，然而其最大弊端是稳定性难以保证，转染效率相对较低；物理转染很大程度上降低此类毒素风险。Among the gene editing introduction methods of the CRISPR/Cas system, there are three main categories, namely biological transfection (lentivirus, adenovirus, etc.), chemical transfection (liposomes, cationic polymers), and physical transfection (electroporation, microinjection, gene gun). Compared with the above introduction methods, the biological transfection method is relatively easy to operate, but there is a possibility of residual cytotoxins; the most prominent advantage of the chemical transfection method is that it is easy to operate, does not require complex technical requirements, and does not have cytotoxin problems, but its biggest disadvantage is that stability is difficult to guarantee and the transfection efficiency is relatively low; physical transfection greatly reduces the risk of such toxins.
海洋双壳贝类是我国主要的食用水产品，而与水产鱼类相比，基因编辑育种技术在贝类中应用尚处于起步阶段。同时贝类卵径小、显微操作难、幼虫死亡率高、间接发育时间长以及在获得可遗传纯系方面难度大、成本高、耗时长的难题也阻碍了基因组编辑技术在海洋双壳贝类上的发展。Marine bivalve molluscs are the main edible aquatic products in my country. Compared with aquatic fish, the application of gene editing breeding technology in shellfish is still in its infancy. At the same time, the small diameter of shellfish eggs, the difficulty of micromanipulation, the high mortality rate of larvae, the long indirect development time, and the difficulty, high cost and long time in obtaining heritable pure lines have also hindered the development of genome editing technology in marine bivalve molluscs.
在实际操作中，由于海洋双壳贝类(包括泥蚶)的卵径小，并且注射后存活率低，显微注射和基因枪等操作难以在泥蚶上实现。另外，CN114164232.A公开了一种电转染双壳贝类基因编辑的方法,但该方法需要在受精卵-8细胞阶段内进行操作，超过8阶段后，细胞过于成熟，难以达到编辑效果，且电场脉冲对易胚胎造成损伤，影响受精卵正常发育，致畸率高，同时电转染方法对设备要求高，操作繁琐，不易服务于贝类繁育一线生产作业。因此亟待一种操作简易，效率高，无细胞毒性，可批量操作的质粒导入方法来解决泥蚶基因编辑的种种难题。In actual operation, due to the small egg diameter of marine bivalve molluscs (including mud clams) and the low survival rate after injection, operations such as microinjection and gene guns are difficult to implement on mud clams. In addition, CN114164232.A discloses a method for electrotransfection gene editing of bivalve molluscs, but the method needs to be operated within the fertilized egg-8 cell stage. After exceeding the 8th stage, the cells are too mature and it is difficult to achieve the editing effect, and the electric field pulse damages the embryo, affects the normal development of the fertilized egg, and has a high teratogenicity rate. At the same time, the electrotransfection method has high requirements for equipment, cumbersome operation, and is not easy to serve the production operations of shellfish breeding. Therefore, it is urgent to have a simple operation, high efficiency, no cytotoxicity, and a batch-operated plasmid introduction method to solve the various problems of mud clams gene editing.
精子介导基因转移法是利用动物精子具有自发结合和内化转运外源DNA能力的特点,并使其在受精时导入卵细胞。然而传统的精子介导法方法价高繁琐且并不适用于水生动物，因为水生生物在精子排放后遇水激活，且经过处理后会大量降低水生动物精子活力甚至造成死亡。因此传统的精子介导方法并不能实现将将外源核酸片段，例如sgRNA有效的导入泥蚶精子中。因此，构建一种简易高效的质粒导入方法已经成为了发展贝类基因编辑的首要任务。Sperm-mediated gene transfer utilizes the ability of animal sperm to spontaneously bind and internalize exogenous DNA, and introduces it into the egg cell during fertilization. However, the traditional sperm-mediated method is expensive and cumbersome and is not suitable for aquatic animals, because aquatic organisms are activated by water after sperm release, and after treatment, the sperm motility of aquatic animals is greatly reduced or even causes death. Therefore, the traditional sperm-mediated method cannot effectively introduce exogenous nucleic acid fragments, such as sgRNA, into mud clam sperm. Therefore, constructing a simple and efficient plasmid introduction method has become the primary task in the development of shellfish gene editing.
发明内容Summary of the invention
本发明的目的是提供一种将外源核酸片段导入双壳贝类精子中的方法，针对现有贝类基因编辑难度大，操作繁琐，成本高，效率低的缺点，提供了一种高效，简易，价廉，无细胞毒性，且可批量操作的双壳贝类体内sgRNA重组表达质粒的导入方法。The purpose of the present invention is to provide a method for introducing exogenous nucleic acid fragments into bivalve sperm. In view of the shortcomings of existing shellfish gene editing, such as great difficulty, cumbersome operation, high cost and low efficiency, a method for introducing sgRNA recombinant expression plasmids into bivalve bodies that is efficient, simple, inexpensive, non-cytotoxic and can be operated in batches is provided.
本发明所提供的将外源核酸片段导入双壳贝类精子中的方法，包括如下的步骤：The method for introducing exogenous nucleic acid fragments into bivalve sperm provided by the present invention comprises the following steps:
1)将收集的双壳贝类的精子离心去除上清后，加入浓度为0.9％的NaCl溶液混匀，再3000rpm离心后去除上清；1) After the collected bivalve mollusc sperm is centrifuged and the supernatant is removed, a 0.9% NaCl solution is added and mixed, and then centrifuged at 3000 rpm and the supernatant is removed;
2)将步骤1)中去除上清的沉淀加入浓度为0.01％的CaCl2溶液后吹打混匀；再加入外源核酸片段后进行冰水浴；2) adding a 0.01%CaCl2 solution to the precipitate after removing the supernatant in step 1) and mixing by pipetting; then adding the exogenous nucleic acid fragment and placing in an ice water bath;
所述的外源核酸片段，作为实施例的具体记载，为表达sgRNA的重组质粒；The exogenous nucleic acid fragment, as specifically described in the embodiment, is a recombinant plasmid expressing sgRNA;
3)将步骤2)冰浴结束后的溶液立即在42℃水浴条件下热激45秒；3) Immediately heat shock the solution after the ice bath in step 2) in a 42° C. water bath for 45 seconds;
4)将步骤3)转化完成后的溶液随后再次冰水浴2分钟，完成外源片段导入精子的操作。4) The solution after transformation in step 3) is then placed in an ice water bath for 2 minutes to complete the operation of introducing the exogenous fragment into the sperm.
实验证明，采用本发明的方法导入sgRNA重组表达质粒，受精成功后泥蚶胚胎体内导入率可达47％～70％。与现有技术相比，本发明的导入方法操作简单，导入成本低，操作时间短，可以实现批量化作业，满足贝类产卵量大的现实需求。Experiments have shown that the introduction rate of sgRNA recombinant expression plasmids into the embryos of mud clams after successful fertilization can reach 47% to 70%. Compared with the prior art, the introduction method of the present invention is simple to operate, has low introduction cost, short operation time, can realize batch operation, and meet the actual demand for large spawning of shellfish.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1：tdTomato sgRNA重组质粒图谱，Figure 1: tdTomato sgRNA recombinant plasmid map,
图2：泥蚶D型幼虫时期体内tdTomato sgRNA重组质粒表达图，Figure 2: Expression diagram of tdTomato sgRNA recombinant plasmid in D-type larvae of mud clam.
图3：泥蚶tdTomato sgRNA重组质粒导入率统计图。Figure 3: Statistical graph of the introduction rate of tdTomato sgRNA recombinant plasmid.
具体实施方式Detailed ways
以下结合实施例对本发明作进一步详细描述。The present invention is further described in detail below with reference to the embodiments.
实施例1：泥蚶体内tdTomato sgRNA重组质粒的转化导入Example 1: Transformation and introduction of tdTomato sgRNA recombinant plasmid into mud clams
2022年7月在浙江省海洋水产养殖研究所清江基地和浙江大学进行相关的实验。Relevant experiments were conducted at the Qingjiang Base of Zhejiang Institute of Marine Fisheries and Zhejiang University in July 2022.
将催产后去除杂质后的泥蚶精子在0.01％ CaCl2溶液经过30min冰水浴，45s热激以及2min冰水浴稳定后，批量导入tdTomato sgRNA重组表达质粒于泥蚶精子内，并成功完成人工授精，在发育至胚胎阶段后观察重组质粒在泥蚶胚胎体内的表达状况。其具体操作步骤如下：After the impurities were removed after induced spawning, the sperm of the mud clam was placed in a 0.01%CaCl2 solution for 30 minutes in an ice-water bath, 45 seconds of heat shock, and 2 minutes of ice-water bath for stabilization. Then, the tdTomato sgRNA recombinant expression plasmid was introduced into the sperm of the mud clam in batches, and artificial insemination was successfully completed. After the development to the embryonic stage, the expression of the recombinant plasmid in the mud clam embryo was observed. The specific operation steps are as follows:
1)质粒选用：采用带有Cas9序列和tdTomato序列的质粒载体(lentiCRISPR v2)，cas9和sgRNA表达框在同一个载体上，而且Cas9的C端带有FLAG融合标签，载体包含Puromycin抗性基因。在重组质粒成功导入细胞后tdTomato，在细胞内表达为红色荧光，因此选用tdTomato sgRNA重组质粒作为导入成功的报告基因；1) Plasmid selection: A plasmid vector (lentiCRISPR v2) with Cas9 and tdTomato sequences was used. The cas9 and sgRNA expression frames were on the same vector, and the C-terminus of Cas9 had a FLAG fusion tag. The vector contained a Puromycin resistance gene. After the recombinant plasmid was successfully introduced into the cell, tdTomato was expressed as red fluorescence in the cell, so the tdTomato sgRNA recombinant plasmid was selected as the reporter gene for successful introduction;
2)配制溶液：称取一定量NaCl放入玻璃容器中，加水配置为0.9％的NaCl溶液；称取一定量CaCl2放入玻璃容器中，加水配制为浓度为0.01％的CaCl2溶液；2) Prepare the solution: weigh a certain amount of NaCl into a glass container, add water to prepare a 0.9% NaCl solution; weigh a certain amount ofCaCl2 into a glass container, add water to prepare a 0.01%CaCl2 solution;
3)清洗泥蚶：用砂滤海水清洗泥蚶表面污物；3) Cleaning the mud clams: Use sand-filtered seawater to clean the dirt on the surface of the mud clams;
4)精子获取：在模拟环境刺激对泥蚶进行催产后，收取足量优质泥蚶精子进行转化处理。在泥蚶排放精子后立即收集在3000rpm转速下离心3分钟，弃上清后(约100μL沉淀)立即加入1mL 0.9％NaCl溶液混匀，经3000rpm室温离心3分钟后去上清；4) Sperm acquisition: After induced spawning of mud clams by simulated environmental stimulation, sufficient high-quality mud clams sperm were collected for transformation. Immediately after the mud clams released sperm, they were collected and centrifuged at 3000 rpm for 3 minutes. After discarding the supernatant (about 100 μL of precipitate), 1 mL of 0.9% NaCl solution was immediately added and mixed. After centrifugation at 3000 rpm for 3 minutes at room temperature, the supernatant was discarded.
5)精子膜通透：将步骤(4)所得沉淀再加入500μL 0.01％ CaCl2溶液后立即吹打混匀，所得溶液在经3000rpm室温离心3分钟去上清后再加入100μL 0.01％ CaCl2溶液；5) Sperm membrane permeabilization: Add 500 μL of 0.01% CaCl2 solution to the precipitate obtained in step (4) and mix by pipetting immediately. Centrifuge the resulting solution at 3000 rpm for 3 minutes at room temperature to remove the supernatant and then add 100 μL of 0.01% CaCl2 solution;
6)质粒附着：将步骤(5)所得溶液加入10μL sgRNA重组表达质粒(约2μg)并进行冰水浴30分钟(0℃)；6) Plasmid attachment: Add 10 μL of sgRNA recombinant expression plasmid (about 2 μg) to the solution obtained in step (5) and place in an ice water bath for 30 minutes (0°C);
7)热激转化：将步骤(6)冰浴结束后溶液立即在42℃水浴条件下热激45秒；7) Heat shock conversion: After the ice bath in step (6), the solution was immediately heat-shocked in a 42°C water bath for 45 seconds;
8)精子稳定：将步骤(7)转化完成后溶液随后再次冰水浴2分钟(0℃)，整个操作过程保持平稳减少摇晃碰撞；8) Sperm stabilization: After the transformation in step (7) is completed, the solution is then placed in an ice water bath for 2 minutes (0°C). The entire operation process is kept stable to reduce shaking and collision;
泥蚶受精：将步骤(8)转化完成后的泥蚶精子立即进行人工受精，定时更换净化海水。Fertilization of mud cockles: Artificial insemination is immediately performed on the mud cockle sperm after transformation in step (8), and purified seawater is replaced regularly.
将步骤(9)受精后的泥蚶胚胎发育至D型幼虫时期后观察其体内红色荧光表达状态来判断质粒导入表达情况。After the fertilized mud clam embryos in step (9) develop to the D-type larvae stage, the red fluorescence expression status in the body is observed to determine the expression status of the introduced plasmid.
表1是对实施例1的转化导入结果进行统计，质粒转化导入操作三次以减少技术误差并具有生物统计学意义。转化步骤完成后每次重复取20个受精卵进行转化导入率统计，并观察在发育至D型幼虫后胚胎体内红色荧光的表达情况，并进行生物学统计。Table 1 is a statistical analysis of the transformation results of Example 1. The plasmid transformation was performed three times to reduce technical errors and to have biostatistical significance. After the transformation step was completed, 20 fertilized eggs were taken for each repetition to perform transformation and introduction rate statistics, and the expression of red fluorescence in the embryos after development to D-type larvae was observed and biological statistics were performed.
表1：质粒转入率统计表Table 1: Plasmid transfer rate statistics
上述结果表明发明方法的使用可以高效地导入泥蚶sgRNA重组质粒，且导入方法操作简单，导入成本低，操作时间短，可以实现批量化作业，满足贝类产卵量大的现实需求。The above results show that the use of the inventive method can efficiently introduce the mud clam sgRNA recombinant plasmid, and the introduction method is simple to operate, with low introduction cost and short operation time, and can realize batch operation to meet the actual needs of large shellfish spawning.
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