CN116396865A - Chlamydomonas reinhardtii mutant strain capable of stably and highly expressing exogenous genes and construction method thereof - Google Patents
Chlamydomonas reinhardtii mutant strain capable of stably and highly expressing exogenous genes and construction method thereofDownload PDFInfo
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Abstract
Description
Translated fromChinese技术领域technical field
本发明涉及微藻基因工程领域，尤其涉及一株稳定高表达外源基因的莱茵衣藻突变株及其构建方法。The invention relates to the field of microalgae genetic engineering, in particular to a mutant strain of Chlamydomonas reinhardtii stably and highly expressing foreign genes and a construction method thereof.
背景技术Background technique
莱茵衣藻是单细胞植物，是分子生物学研究的模式生物之一。莱茵衣藻表达系统具有诸多优势。在法规方面，莱茵衣藻属于安全级微藻，经进入我国的食品名录，作为一种淡水微藻，它同时能够作为鱼、虾、蟹等水生动物的饵料；在培养工艺方面，莱茵衣藻光合效率高于高等植物，可以根据应用需求选择自养、异养或兼养的培养模式，培养规模可以根据需求调整。莱茵衣藻生长速度快、培养方式简单、培养基材料来源广，廉价易得，成本低。在分子生物学方面，莱茵衣藻基因组测序已经完成，遗传背景清晰，遗传转化操作成熟。Chlamydomonas reinhardtii is a single-celled plant and one of the model organisms for molecular biology research. The Chlamydomonas reinhardtii expression system has many advantages. In terms of laws and regulations, Chlamydomonas reinhardtii belongs to the safety grade microalgae, which has entered my country's food list. As a freshwater microalgae, it can also be used as bait for aquatic animals such as fish, shrimp, and crabs; The photosynthetic efficiency is higher than that of higher plants, and the cultivation mode of autotrophic, heterotrophic or combined cultivation can be selected according to the application requirements, and the cultivation scale can be adjusted according to the requirements. Chlamydomonas reinhardtii has a fast growth rate, a simple culture method, a wide source of culture medium materials, cheap and easy to obtain, and low cost. In terms of molecular biology, the genome sequencing of Chlamydomonas reinhardtii has been completed, the genetic background is clear, and the genetic transformation operation is mature.
随着分子生物学技术的发展，转基因技术逐渐被应用到医药、农业等多个领域，转基因技术的重点在于外源基因在底盘细胞中的表达。已有多个文献报道，利用莱茵衣藻细胞核表达外源蛋白。但是，莱茵衣藻基因表达调控机制尤其是外源基因的沉默机制还没有完全被研究清楚。外源基因在莱茵衣藻中的表达水平常常较低且遗传不稳定(无法稳定高表达外源基因)，常出现基因沉默或传代后丢失的现象。With the development of molecular biology technology, transgenic technology has been gradually applied to many fields such as medicine and agriculture. The key point of transgenic technology is the expression of foreign genes in the chassis cells. There have been many reports in the literature, using the nucleus of Chlamydomonas reinhardtii to express foreign proteins. However, the regulatory mechanism of Chlamydomonas reinhardtii gene expression, especially the silencing mechanism of foreign genes has not been fully studied. The expression level of exogenous genes in Chlamydomonas reinhardtii is often low and genetically unstable (unable to stably express exogenous genes at high levels), and gene silencing or loss after passage often occurs.
已有正向遗传学研究报道，莱茵衣藻中的表观组蛋白去乙酰化酶SRTA通过表观修饰抑制外源基因的表达，因此可通过敲除SRTA提高外源基因在底盘细胞中的表达。然而，已有的SRTA突变藻株存在多个问题：无鞭毛，不能与其他藻种杂交；生长速度慢，生产成本高；存在细胞壁缺陷，不耐剪切力，难以进行下游放大培养(如高密度发酵罐异养培养)。此外，Crispr/cas9技术出现后，衣藻的定点基因编辑也成为可能。但是由于衣藻的GC含量较高(62％)，而Cas9蛋白识别的PAM中富含GC序列，因此容易造成Cas9蛋白的脱靶，并且对衣藻具有潜在的毒性。衣藻DNA双链断裂主要依赖非同源重组修复，同源重组修复效率较低，因此供体片段的设计不能依照目前已成熟的方法设计得到。Forward genetics studies have reported that the epihistone deacetylase SRTA in Chlamydomonas reinhardtii inhibits the expression of foreign genes through epigenetic modification, so the expression of foreign genes in chassis cells can be improved by knocking out SRTA . However, the existing SRTA mutant algal strains have many problems: no flagella, can not hybridize with other algal species; slow growth, high production costs; cell wall defects, not resistant to shear stress, difficult to scale up downstream (such as high density fermenter heterotrophic culture). In addition, after the emergence of Crispr/cas9 technology, site-specific gene editing of Chlamydomonas has also become possible. However, since the GC content of Chlamydomonas is high (62%), and the PAM recognized by Cas9 protein is rich in GC sequences, it is easy to cause off-target of Cas9 protein and has potential toxicity to Chlamydomonas. Chlamydomonas DNA double-strand breaks are mainly repaired by non-homologous recombination, and the repair efficiency of homologous recombination is low, so the design of the donor fragment cannot be designed according to the current mature methods.
综上所述，现有的SRTA突变藻株存在诸多不足，而对莱茵衣藻中SRTA实施精准敲除时也存在较大的执行困难。To sum up, there are many deficiencies in the existing SRTA mutant strains, and there are also great difficulties in implementing precise knockout of SRTA in Chlamydomonas reinhardtii.
发明内容Contents of the invention
(一)要解决的技术问题(1) Technical problems to be solved
鉴于现有技术的上述缺点、不足，本发明提供一株稳定高表达外源基因的莱茵衣藻突变株，该藻株是通过Crispr/cas9基因编辑技术改造莱茵衣藻藻株，并提供含有终止子DNA的片段的供体序列用于双链的修复，终止SRTA的翻译，通过抗性筛选得到的转基因藻株。In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a mutant strain of Chlamydomonas reinhardtii that stably and highly expresses exogenous genes. The donor sequence of the sub-DNA fragment is used for double-strand repair, terminating the translation of SRTA, and the transgenic algae strain obtained by resistance screening.
(二)技术方案(2) Technical solutions
为了达到上述目的，本发明采用的主要技术方案包括：In order to achieve the above object, the main technical solutions adopted in the present invention include:
第一方面，本发明提供一株稳定高表达外源基因的莱茵衣藻突变株，为敲除了莱茵衣藻出发株中SRTA(Cre10.g462200)基因后得到的突变藻株。In the first aspect, the present invention provides a mutant strain of Chlamydomonas reinhardtii that stably and highly expresses foreign genes, which is a mutant strain obtained by knocking out the SRTA (Cre10.g462200) gene in the starting strain of Chlamydomonas reinhardtii.
优选地，所述莱茵衣藻为莱茵衣藻出发株的SRTA基因被错义替换为包含三个终止密码子的核苷酸序列，该序列为：Preferably, the Chlamydomonas reinhardtii is the SRTA gene of the origin strain of Chlamydomonas reinhardtii replaced by a nucleotide sequence containing three stop codons, the sequence is:
5’-TGACGCTAGGGCTGAGCCTCGA-3’。5'-TGACGCTAGGGCTGAGCCTCGA-3'.
优选地，所述莱茵衣藻出发株为任何SRTA可正常表达的藻株，包括但不限于野生型莱茵衣藻藻株、莱茵衣藻突变株(人工诱变或自然突变)或莱茵衣藻转基因工程藻株。Preferably, the starting strain of Chlamydomonas reinhardtii is any algal strain that can normally express SRTA, including but not limited to wild-type Chlamydomonas reinhardtii algal strains, mutant strains of Chlamydomonas reinhardtii (artificial mutagenesis or natural mutation) or Chlamydomonas reinhardtii transgenic Engineered strains.
第二方面，本发明还提供一株稳定高表达外源基因的莱茵衣藻突变株的构建方法，所述方法包括如下步骤：In a second aspect, the present invention also provides a method for constructing a mutant strain of Chlamydomonas reinhardtii that stably and highly expresses an exogenous gene, said method comprising the following steps:
S1、制备靶向莱茵衣藻SRTA基因的gRNA，所述gRNA由如下引物进行PCR扩增得到：S1. Prepare the gRNA targeting the Chlamydomonas reinhardtii SRTA gene, which is obtained by PCR amplification with the following primers:
gRNA-F：gRNA-F:
5’-TAATACGACTCACTATAGGGAAGCGCGTGTTCGTCTT5'-TAATACGACTCACTATAGGGAAGCGCGTGTTCGTCTT
TACGTTTTAGAGCTAGAA-3’；TACGTTTTAGAGCTAGAA-3';
gRNA-R：5’-AAAAAAGCACCGACTCGGTG-3’；gRNA-R: 5'-AAAAAAAGCACCGACTCGGTG-3';
S2、体外组装gRNA/cas9复合体；S2. Assembly of gRNA/cas9 complex in vitro;
S3、制备修复莱茵衣藻双链的供体片段，供体片段由两条单链互补配对形成，其中一条单链上具有TGACGCTAGGGCTGAGCCTCGA，在该序列两端分别连接包含30-40个碱基的同源臂；S3. Prepare a donor fragment for repairing the double-strand of Chlamydomonas reinhardtii. The donor fragment is formed by complementary pairing of two single strands, one of which has TGACGCTAGGGCTGAGCCTCGA, and the two ends of the sequence are respectively connected with homogeneous sequences containing 30-40 bases. source arm;
S4、电转化S4. Electric transformation
将准备好的莱茵衣藻出发株细胞进行热激处理，将莱茵衣藻出发株细胞与gRNA/cas9复合体、供体片段以及抗性片段充分混合，电击导入，之后采用含抗生素的培养基进行抗性筛选，选出长出藻落的藻株，进行PCR鉴定，选出转化子。The prepared Chlamydomonas reinhardtii origin strain cells were subjected to heat shock treatment, the cells of Chlamydomonas reinhardtii origin strain cells were fully mixed with gRNA/cas9 complex, donor fragment and resistance fragment, introduced by electric shock, and then cultured with antibiotic-containing medium For resistance screening, select algal strains that grow algal colonies, perform PCR identification, and select transformants.
优选地，步骤S3中，所述供体片段是由如下单链经退火形成的双链：Preferably, in step S3, the donor fragment is a double strand formed by annealing the following single strand:
donor-F：donor-F:
5’-GCCGCTACTGCTGCAGGTCCGCGACGCCAAGCGCG5'-GCCGCTACTGCTGCAGGTCCGCGACGCCAAGCGCG
TGTGACGCTAGGGCTGAGCCTCGATCTCCACCGCCTGCTGTGACGCTAGGGCTGAGCCTCGATCTCCACCGCCTGC
GGCATCCCTGACTTTCG-3’；GGCATCCCTGACTTTCG-3';
donor-R：donor-R:
5’-CGAAAGTCAGGGATGCCGCAGGCGGTGGAGATCG5'-CGAAAGTCAGGGATGCCGCAGGCGGTGGAGATCG
AGGCTCAGCCCTAGCGTCACACGCGCTTGGCGTCGCGGAGGCTCACGCCCTAGCGTCACACGCGCTTGGCGTCGCGG
ACCTGCAGCAGTAGCGGC-3’。ACCTGCAGCAGTAGCGGC-3'.
优选地，步骤S4中，所述抗性片段为巴龙霉素抗性片段或潮霉素抗性片段；所述抗生素为巴龙霉素或潮霉素。巴龙霉素抗性片段为aphⅧ抗性基因片段。Preferably, in step S4, the resistance fragment is a paromomycin resistance fragment or a hygromycin resistance fragment; and the antibiotic is paromomycin or hygromycin. The paromomycin resistance fragment is aphⅧ resistance gene fragment.
优选地，所述莱茵衣藻出发株为野生型莱茵衣藻藻株、莱茵衣藻突变株(人工诱变或自然突变)或莱茵衣藻转基因工程藻株。Preferably, the starting strain of Chlamydomonas reinhardtii is a wild-type Chlamydomonas reinhardtii strain, a mutant strain of Chlamydomonas reinhardtii (artificially induced or naturally mutated), or a genetically engineered strain of Chlamydomonas reinhardtii.
优选地，步骤S3中，退火是在annealing Buffer(100mM Tris-HCl(pH8.0)，500mMCH3COOK，10mM EDTA)存在条件下进行的。Preferably, in step S3, the annealing is performed in the presence of annealing Buffer (100 mM Tris-HCl (pH8.0), 500 mM CH3COOK, 10 mM EDTA).
优选地，步骤S4中，每5ⅹ105cells添加4-6μL的gRNA/cas9复合体；所述gRNA/cas9复合体的组装方法如下：Preferably, in step S4, add 4-6 μL of gRNA/cas9 complex per 5ⅹ105 cells; the assembly method of the gRNA/cas9 complex is as follows:
在超净台内用无RNA污染的枪头和管子按照下述加样量加样，于37℃孵育10-15min或常温孵育30min；加样量为：In the ultra-clean bench, use the pipette tip and tube without RNA pollution to add the sample according to the following sample volume, and incubate at 37°C for 10-15min or at room temperature for 30min; the sample volume is:
gRNA 7.5-75μg、Cas9蛋白25-50μg、10×reaction Buffer 2-3μL，ddH2O补足到20-30μL；gRNA 7.5-75μg, Cas9 protein 25-50μg, 10×reaction Buffer 2-3μL, add ddH2O to 20-30μL;
电击参数为：Bio-RAD电穿孔仪，参数：电压600V，电容50μF，电阻∞，杯子：4mm；Electric shock parameters are: Bio-RAD electroporation instrument, parameters: voltage 600V, capacitance 50μF, resistance ∞, cup: 4mm;
电击后迅速向电击杯中加入TAP-Suc并轻柔混匀。Immediately after shocking, add TAP-Suc to the shock cup and mix gently.
通过电穿孔方法和控制gRNA/cas9复合体相对衣藻细胞的浓度，精确控制瞬时转入的Cas9蛋白的含量来达到编辑的效果，避免Cas9蛋白对衣藻潜在的毒性。By electroporation and controlling the concentration of the gRNA/cas9 complex relative to the Chlamydomonas cells, the content of the transiently transferred Cas9 protein is precisely controlled to achieve the editing effect and avoid the potential toxicity of the Cas9 protein to Chlamydomonas.
(三)有益效果(3) Beneficial effects
本发明的有益效果是：The beneficial effects of the present invention are:
(1)本发明采用Crispr/cas9技术敲除了莱茵衣藻出发株表达组蛋白去乙酰化酶基因SRTA，得到一株可稳定高表达外源基因的莱茵衣藻突变株，该藻株为外源蛋白的表达提供优良的底盘藻株。该莱茵衣藻出发株可为莱茵衣藻(Chlamydomonas reinhardtii)野生型、突变藻或转基因工程株。(1) The present invention uses Crispr/cas9 technology to knock out the histone deacetylase gene SRTA expressed by the origin strain of Chlamydomonas reinhardtii, and obtain a mutant strain of Chlamydomonas reinhardtii that can stably and highly express exogenous genes. Protein expression provides excellent chassis strains. The starting strain of Chlamydomonas reinhardtii can be wild type, mutant algae or transgenic engineered strain of Chlamydomonas reinhardtii.
(2)本发明还提供一种稳定高表达外源基因的莱茵衣藻突变株的构建方法，所述方法主要是通过Crispr/cas9基因编辑技术改造莱茵衣藻出发株，并提供含有终止子DNA的片段的供体序列用于双链的修复，使突变株的SRTA基因被含有三个终止密码子的核苷酸序列错义替换，终止SRTA的翻译达到敲除SRTA基因以提高外源基因表达量和表达稳定性的目的。(2) The present invention also provides a method for constructing a mutant strain of Chlamydomonas reinhardtii that stably and highly expresses foreign genes. The donor sequence of the fragment is used for double-strand repair, so that the SRTA gene of the mutant strain is replaced by a nucleotide sequence missense containing three stop codons, and the translation of SRTA is terminated to knock out the SRTA gene to improve the expression of foreign genes Quantity and expression stability purposes.
(3)本发明的方法包括设计高效靶向莱茵衣藻SRTA基因的gRNA、体外组装gRNA/cas9复合体、制备用于修复莱茵衣藻双链的donor片段。以本发明设计的gRNA与Cas9蛋白组装成复合体，可有效减小Cas9蛋白的脱靶率，提高转化率。本发明合成的donor片段包含3个终止子，且对莱茵衣藻的DNA双链具有较高的修复效率。采用PCR鉴定，确定所构建的突变株基因序列符合预期。(3) The method of the present invention includes designing a gRNA efficiently targeting the SRTA gene of Chlamydomonas reinhardtii, assembling a gRNA/cas9 complex in vitro, and preparing a donor fragment for repairing the double strand of Chlamydomonas reinhardtii. The gRNA designed in the present invention is assembled into a complex with the Cas9 protein, which can effectively reduce the off-target rate of the Cas9 protein and increase the conversion rate. The donor fragment synthesized by the invention contains three terminators, and has higher repair efficiency for the DNA double strand of Chlamydomonas reinhardtii. PCR identification was used to confirm that the gene sequence of the constructed mutant strains was in line with expectations.
(4)本发明通过转基因功能验证，该株藻可稳定高表达外源基因。通过实验验证，本发明构建的突变藻株与其出发株相比，光照培养生长速率不变，解决已有的SRTA突变藻株存在的生长速度慢，生产成本高的问题。通过向突变藻株中转入外源基因发现，阳性转化子数目高于其出发株315％，蛋白免疫印迹标明阳性转化子的目标蛋白表达量平均值是其出发株的238.96％。此外，经多次传代培养后的蛋白免疫印迹分析表明，外源基因的表达水平随藻株的传代次数基本无变化，这说明本发明构建的突变株可稳定表达其携带的外源基因，解决了现有技术中莱茵衣藻外源蛋白表达水平低，外源基因容易丢失的问题。(4) The present invention has passed the transgenic function verification, and the strain of algae can stably and highly express the exogenous gene. It is verified by experiments that the mutant algae strain constructed by the present invention has the same growth rate in light culture compared with the original strain, which solves the problems of slow growth rate and high production cost in the existing SRTA mutant algae strains. By transferring exogenous genes into the mutant algae strains, it was found that the number of positive transformants was 315% higher than that of the original strain, and Western blot indicated that the average expression level of the target protein of the positive transformants was 238.96% of the original strain. In addition, Western blot analysis after multiple subcultures showed that the expression level of exogenous genes basically did not change with the passage times of algae strains, which indicated that the mutant strains constructed by the present invention could stably express the exogenous genes they carried, solving the problem of The problem of low expression level of exogenous protein of Chlamydomonas reinhardtii and easy loss of exogenous gene in the prior art is solved.
(5)本发明构建的敲除SRTA的莱茵衣藻突变藻株具有鞭毛，能与其他藻种杂交，生长速度与出发株无异，无细胞壁缺陷，可进行下游放大培养(如高密度发酵罐异养培养)。本发明采用电穿孔方法，精确控制瞬时转入的Cas9蛋白的含量来达到编辑效果，避免Cas9蛋白对衣藻潜在的毒性。(5) The SRTA knockout Chlamydomonas reinhardtii mutant algal strain constructed by the present invention has flagella, can hybridize with other algae species, has the same growth rate as the starting strain, has no cell wall defects, and can carry out downstream scale-up culture (such as high-density fermentation tanks) Heterotrophic culture). The present invention uses an electroporation method to precisely control the content of the instantaneously transferred Cas9 protein to achieve the editing effect and avoid the potential toxicity of the Cas9 protein to Chlamydomonas.
附图说明Description of drawings
图1为SRTA基因敲除示意图。Figure 1 is a schematic diagram of SRTA gene knockout.
图2为Crsipr/cas9敲除SRTA基因后得到YYAE001的测序结果与莱茵衣藻出发株的基因序列比对图。Figure 2 is a graph comparing the sequencing results of YYAE001 obtained after Crsipr/cas9 knockout of the SRTA gene with the gene sequence of the origin strain of Chlamydomonas reinhardtii.
图3为敲除SRTA基因的突变株YYAE001与野生型的纤毛长度比较。Fig. 3 is a comparison of the length of cilia between the mutant strain YYAE001 knocking out the SRTA gene and the wild type.
图4为敲除SRTA基因的突变株YYAE001与野生型转化后的转化子数目对比和转化子阳性率比对。Figure 4 is a comparison of the number of transformants and the positive rate of transformants between the mutant strain YYAE001 with knockout of the SRTA gene and the wild-type transformation.
图5为敲除SRTA基因的突变株YYAE001与野生型转化子的蛋白免疫印迹图及定量分析。Fig. 5 is the western blot and quantitative analysis of the mutant strain YYAE001 and the wild-type transformant of the knockout SRTA gene.
图6为敲除SRTA基因的突变株YYAE001经3次传代时，每代对其携带的外源基因的表达水平比较。Figure 6 is a comparison of the expression levels of exogenous genes carried by each generation of the SRTA gene knockout mutant strain YYAE001 after three passages.
具体实施方式Detailed ways
为了更好的解释本发明，以便于理解，下面结合附图，通过具体实施方式，对本发明作详细描述。In order to better explain the present invention and facilitate understanding, the present invention will be described in detail below through specific embodiments in conjunction with the accompanying drawings.
在以下具体实施方式中，以莱茵衣藻(Chlamydomonas reinhardtii)野生型藻株作为出发株来说明一株可稳定高表达外源基因的莱茵衣藻突变株的构建方法。需说明的是，所用出发株(或背景藻株)不限于是野生型莱茵衣藻，其还可以是人工诱变或自然突变产生的莱茵衣藻突变株或莱茵衣藻转基因工程藻株。In the following specific embodiments, the wild-type strain of Chlamydomonas reinhardtii is used as the starting strain to illustrate a method for constructing a mutant strain of Chlamydomonas reinhardtii that can stably and highly express foreign genes. It should be noted that the starting strain (or background algae strain) used is not limited to wild-type C. reinhardtii, it can also be a C. reinhardtii mutant strain or a Chlamydomonas reinhardtii transgenic engineering strain produced by artificial mutagenesis or natural mutation.
1、配制莱茵衣藻培养基1. Preparation of Chlamydomonas reinhardtii culture medium
TAP培养基：按照表5配制TAP液体培养基，固体培养基则添加1.5％琼脂，121℃，高温高压灭菌20min，冷却后倒板备用，也可加入终浓度为10μg/mL巴龙霉素或者20μg/mL潮霉素成抗性平板。表5中所需的母液成分表记载在表1-4中，母液配制后4℃保存备用。TAP medium: prepare TAP liquid medium according to Table 5, and add 1.5% agar to the solid medium, sterilize at 121°C for 20 minutes under high temperature and high pressure, pour it back after cooling, and add paromomycin at a final concentration of 10 μg/mL Or 20μg/mL hygromycin into a resistant plate. The mother liquor composition list required in Table 5 is recorded in Tables 1-4, and the mother liquor is stored at 4°C after preparation.
表1：100×Tris-Acetate母液Table 1: 100×Tris-Acetate stock solution
表2：100×Phosphate Buffer母液Table 2: 100×Phosphate Buffer stock solution
表3：100×Beijerinck’s solution母液Table 3: 100×Beijerinck’s solution mother liquor
表4：1000×Trace elements solution(Hutner trace elements)母液Table 4: 1000×Trace elements solution (Hutner trace elements) mother solution
表5：TAP液体培养基的配置Table 5: Configuration of TAP Liquid Medium
2、莱茵衣藻的培养2. Cultivation of Chlamydomonas reinhardtii
在超净工作台里，从莱茵衣藻培养平板上挑取单一克隆，接种到50mL TAP液体培养基中，25℃，50-100μE m-2s-1连续光照，100rpm摇床培养3-4天，直至达到对数生长期。In the ultra-clean workbench, pick a single clone from the Chlamydomonas reinhardtii culture plate, inoculate it into 50mL TAP liquid medium, 25°C, 50-100μE m-2 s-1 continuous light, 100rpm shaker culture 3-4 days until reaching the logarithmic growth phase.
3、采用Crispr/cas9敲除莱茵衣藻出发株的SRTA基因3. Using Crispr/cas9 to knock out the SRTA gene of the origin strain of Chlamydomonas reinhardtii
(1)gRNA的设计(1) Design of gRNA
设计出靶向莱茵衣藻SRTA基因的引物SRTA-sg-F和sg-R并合成。使用引物SRTA-sg-F和sg-R进行PCR扩增sgDNA，PCR产物通过DNA电泳和胶回收纯化获得片段SRTA-sgDNA。Primers SRTA-sg-F and sg-R targeting Chlamydomonas reinhardtii SRTA gene were designed and synthesized. The sgDNA was amplified by PCR using primers SRTA-sg-F and sg-R, and the PCR product was purified by DNA electrophoresis and gel recovery to obtain fragment SRTA-sgDNA.
gRNA-F：gRNA-F:
5’-TAATACGACTCACTATAGGGAAGCGCGTGTTCGTCTT5'-TAATACGACTCACTATAGGGAAGCGCGTGTTCGTCTT
TACGTTTTAGAGCTAGAA-3’；(SEQ ID No.1)TACGTTTTAGAGCTAGAA-3'; (SEQ ID No. 1)
gRNA-R：5’-AAAAAAGCACCGACTCGGTG-3’(SEQ ID No.2)。gRNA-R: 5'-AAAAAAAGCACCGACTCGGTG-3' (SEQ ID No. 2).
使用Hiscribe T7quick high yield RNA Synthesis Kit(NEB)试剂盒对SRTA-sgDNA进行37℃体外转录6h以上，再使用RNA清洁纯化试剂盒(庄盟)得到SRTA-sgRNA。Use the Hiscribe T7quick high yield RNA Synthesis Kit (NEB) kit to perform in vitro transcription of SRTA-sgDNA at 37°C for more than 6 hours, and then use the RNA cleaning and purification kit (Zhuangmeng) to obtain SRTA-sgRNA.
(2)donor的设计与制备(2) Design and preparation of donor
将切割靶点附近(涵盖SRTA基因的位点)的部分碱基替换成其他碱基，如图1及图2所示，SRTA基因被22个包含3个终止密码子的碱基对所取代。从靶点切割位置前45个bp到后45个bp为设计的插入供体片段(90bp)。如图1所示，3个终止密码子依次为TGA、TAG和TGA。Part of the bases near the cleavage target (covering the site of the SRTA gene) were replaced with other bases. As shown in Figure 1 and Figure 2, the SRTA gene was replaced by 22 base pairs containing 3 stop codons. The first 45 bp to the last 45 bp of the target cutting position is the designed insert donor fragment (90 bp). As shown in Figure 1, the three stop codons are TGA, TAG and TGA in sequence.
所述供体片段由如下单链经退火形成的双链：The donor fragments are double strands formed by annealing the following single strands:
donor-F：donor-F:
5’-GCCGCTACTGCTGCAGGTCCGCGACGCCAAGCGCG5'-GCCGCTACTGCTGCAGGTCCGCGACGCCAAGCGCG
TGTGACGCTAGGGCTGAGCCTCGATCTCCACCGCCTGCTGTGACGCTAGGGCTGAGCCTCGATCTCCACCGCCTGC
GGCATCCCTGACTTTCG-3’；(SEQ ID No.3)GGCATCCCTGACTTTCG-3'; (SEQ ID No. 3)
donor-R：donor-R:
5’-CGAAAGTCAGGGATGCCGCAGGCGGTGGAGATCGA5'-CGAAAGTCAGGGATGCCGCAGGCGGTGGAGATCGA
GGCTCAGCCCTAGCGTCACACGCGCTTGGCGTCGCGGACGGCTCACGCCCTAGCGTCACACGCGCTTGGCGTCGCGGAC
CTGCAGCAGTAGCGGC-3’；(SEQ ID No.4)CTGCAGCAGTAGCGGC-3'; (SEQ ID No. 4)
分别向1OD的donor-F和donor-R干粉内加入45μL ddH2O，然后混在一起，再加入10μL annealing Buffer(100mM Tris-HCl(pH8.0)，500mM CH3COOK，10mM EDTA)，置于水浴锅中沸水10min，然后将其在该水中自然缓慢退温至常温，可通过DNA电泳检测donor制备效果。Add 45μL ddH2 O to 1OD donor-F and donor-R dry powder respectively, then mix together, then add 10μL annealing Buffer (100mM Tris-HCl (pH8.0), 500mM CH3 COOK, 10mM EDTA), place in Boil water in a water bath for 10 minutes, then slowly cool it down to room temperature naturally in the water, and the effect of donor preparation can be detected by DNA electrophoresis.
(3)体外组装gRNA/cas9复合体(3) Assembly of gRNA/cas9 complex in vitro
在超净台内用RNA-free枪头和管子根据下表的取样量加样，然后37℃孵育10-15min(可常温30min)。Use RNA-free tips and tubes to add samples according to the sampling volume in the table below in a clean bench, and then incubate at 37°C for 10-15min (or 30min at room temperature).
表6：Table 6:
4、电转化4. Electric conversion
(1)将莱茵衣藻细胞的收集：当衣藻密度在1-3ⅹ106cells/mL时，2000g室温离心5min收集藻液。每管加入10mL含40mM蔗糖的TAP-Suc清洗细胞一遍，加1-2mL TAP-Suc重悬，使藻细胞浓度在为108cells/mL。(1) Collection of Chlamydomonas reinhardtii cells: when the density of Chlamydomonas is 1-3ⅹ106 cells/mL, centrifuge at 2000 g for 5 minutes at room temperature to collect the algae liquid. Add 10mL TAP-Suc containing 40mM sucrose to each tube to wash the cells once, add 1-2mL TAP-Suc to resuspend, so that the algal cell concentration is 108 cells/mL.
(2)衣藻细胞热激处理：108cells/mL的细胞悬液在金属浴40℃、350rpm热激30min。(2) Heat-shock treatment of Chlamydomonas cells: 108 cells/mL cell suspension was heat-shocked in a metal bath at 40° C. and 350 rpm for 30 minutes.
(3)取约5ⅹ105cells(10-15μL)细胞于1.5mL离心管中，添加4-6μl组装好的gRNA/cas9复合体，5μl donor片段，500ng巴龙霉素抗性片段(aphⅧ抗性片段)，在离心管内充分混合。将离心管内混合物转移至于2mm的电穿孔杯中。(3) Take about 5ⅹ105 cells (10-15μL) in a 1.5mL centrifuge tube, add 4-6μl assembled gRNA/cas9 complex, 5μl donor fragment, 500ng paromomycin resistance fragment (aphⅧ resistance fragments) and mix thoroughly in a centrifuge tube. Transfer the mixture in the centrifuge tube to a 2mm electroporation cuvette.
(4)轻敲电穿孔杯后放入杯池室，开始电击，记录实际电击时间。(4) Tap the electroporation cup and put it into the cup pool chamber, start the electric shock, and record the actual electric shock time.
(5)Bio-RAD电穿孔仪，参数：电压600V，电容50μF，电阻∞，杯子：4mm。(5) Bio-RAD electroporation instrument, parameters: voltage 600V, capacitance 50μF, resistance ∞, cup: 4mm.
(6)迅速向电击杯中加入750μL TAP-Suc，轻柔的用移液枪混匀藻细胞后，将其转移至无菌的15mL离心管。搅拌使混合物悬浮，转移250μL混合物至5mL离心管，再加750μLTAP培养基，弱光照培养箱中孵育16h以上。(6) Quickly add 750 μL TAP-Suc to the electric shock cup, gently mix the algae cells with a pipette gun, and then transfer them to a sterile 15 mL centrifuge tube. Stir to suspend the mixture, transfer 250 μL of the mixture to a 5 mL centrifuge tube, add 750 μL of TAP medium, and incubate in a weak light incubator for more than 16 h.
(7)将转化后的藻液涂布在含有10ug/mL巴龙霉素的TAP平板上，光照培养至藻落长出。(7) Spread the transformed algae solution on a TAP plate containing 10 ug/mL paromomycin, and culture under light until algal colonies grow.
5、转基因莱茵衣藻的PCR鉴定5. PCR identification of transgenic Chlamydomonas reinhardtii
向约20mg藻细胞种加入50μL 10mM EDTA(pH8.0)，室温涡旋30s，重悬细胞；98～100℃孵育8～10min，随即立即冰浴1min。5000rpm室温离心2min，收集上清得到基因组DNA粗提取样品；利用2×Taq PCR mix(北京聚合酶生物有限公司，货号：MF019)以及合适的引物进行转基因衣藻的PCR鉴定。根据表8的DNA-PCR反应体系依次加入到0.2mL PCR管中瞬时离心混匀，将PCR管按照下表PCR程序在PCR仪上进行反应：98℃3min，98℃30s，Tm 60℃(退火温度)30s，72℃2min，30个循环，72℃5min。PCR鉴定转基因衣藻所用到的引物如表7所示。Add 50 μL of 10 mM EDTA (pH 8.0) to about 20 mg of algae cell species, vortex at room temperature for 30 s, and resuspend the cells; incubate at 98-100 °C for 8-10 min, then immediately ice-bath for 1 min. Centrifuge at room temperature at 5000rpm for 2min, collect the supernatant to obtain a crude genomic DNA sample; use 2×Taq PCR mix (Beijing Polymerase Biological Co., Ltd., catalog number: MF019) and appropriate primers for PCR identification of the transgenic Chlamydomonas. Add the DNA-PCR reaction system according to Table 8 into 0.2mL PCR tubes and centrifuge for a short time to mix, and then react the PCR tubes on the PCR machine according to the PCR program in the following table: 98°C for 3min, 98°C for 30s,
表7：PCR鉴定转基因衣藻所用到的引物Table 7: Primers used for PCR identification of transgenic Chlamydomonas
表8：衣藻基因组DNA-PCR反应体系Table 8: Chlamydomonas Genomic DNA-PCR Reaction System
反应结束后取5μL在1.5％琼脂糖凝胶中进行电泳分析，电泳片段大小符合预期时送测序。如图2所示为测序结果，方框标出的部分为替换的序列片段。SRTA基因原始序列TTTGTGTTCACGGGCGCCGGCA中有15个碱基被替换，最终变成TGACGCTAGGGCTGAGCCTCGA(SEQID No.5)，其中TGA、TAG和TGA为终止密码子。至此，获得一株敲除SRTA基因的莱茵衣藻突变株YYAE001。After the reaction, 5 μL was taken for electrophoresis analysis in 1.5% agarose gel, and the electrophoresis fragments were sent for sequencing when the size of the electrophoresis fragment met the expectation. As shown in Figure 2, the sequencing results are shown, and the part marked by the box is the replaced sequence fragment. 15 bases were replaced in the original sequence of SRTA gene TTTGTGTTCACGGGCGCCGGCA, and finally became TGACGCTAGGGCTGAGCCTCGA (SEQID No.5), wherein TGA, TAG and TGA are stop codons. So far, a Chlamydomonas reinhardtii mutant strain YYAE001 with SRTA gene knockout was obtained.
6、向已敲除SRTA基因的莱茵衣藻突变株YYAE001转入外源基因YFP，考察转化子与莱茵衣藻(Chlamydomonas reinhardtii)野生型藻株的生长特性，步骤如下：6. Transfer the exogenous gene YFP to the Chlamydomonas reinhardtii mutant strain YYAE001 that has knocked out the SRTA gene, and investigate the growth characteristics of the transformant and the wild-type strain of Chlamydomonas reinhardtii. The steps are as follows:
(1)衣藻细胞的收集：当衣藻密度在1-3ⅹ106cells/mL时，2000g室温离心5min收集藻液。每管加入10mL含40mM蔗糖的TAP-Suc清洗细胞一遍，加1-2mL TAP-Suc重悬，使藻细胞浓度在为2×108cells/mL。冰上静置10min。(1) Collection of Chlamydomonas cells: When the density of Chlamydomonas is 1-3ⅹ106 cells/mL, centrifuge at 2000g for 5 minutes at room temperature to collect the algae liquid. Add 10mL TAP-Suc containing 40mM sucrose to each tube to wash the cells once, add 1-2mL TAP-Suc to resuspend, so that the algal cell concentration is 2×108 cells/mL. Let stand on ice for 10 minutes.
(2)取约2ⅹ108cells/mL(250μL)细胞于4mm的电穿孔杯中，加入500ng PSAD-YFP片段(该片段是选用PSAD的启动子和PSAD的3'UTR，中间连接YFP基因，串联上hygromycin抗性基因)。(2) Take about 2ⅹ108 cells/mL (250μL) cells into a 4mm electroporation cuvette, add 500ng PSAD-YFP fragment (this fragment is the promoter of PSAD and the 3'UTR of PSAD, the YFP gene is connected in the middle, and the tandem upper hygromycin resistance gene).
(3)轻敲电穿孔杯后放入杯池室，开始电击，记录实际电击时间。Bio-RAD电穿孔仪，参数：电压800V，电容50μF，电阻1575Ω。(3) Tap the electroporation cup and put it into the cup pool chamber, start the electric shock, and record the actual electric shock time. Bio-RAD electroporation instrument, parameters: voltage 800V, capacitance 50μF, resistance 1575Ω.
(6)电击后迅速将电击杯转移到冰上静置10min。(6) After the electric shock, quickly transfer the electric shock cup to ice for 10 minutes.
(7)向电击杯中加入750μL TAP-Suc，轻柔的用移液枪混匀藻细胞后，将其转移至无菌的50mL离心管，补充TAP-Suc至10mL，弱光照培养箱中孵育16h以上。(7) Add 750 μL TAP-Suc to the electric shock cup, gently mix the algal cells with a pipette gun, transfer them to a sterile 50 mL centrifuge tube, add TAP-Suc to 10 mL, and incubate in a weak light incubator for 16 hours above.
(8)将转化后的藻液涂布在含有20μg/mL潮霉素的TAP平板上，光照培养至藻落长出。(8) Spread the transformed algae solution on a TAP plate containing 20 μg/mL hygromycin, and culture under light until algal colonies grow.
(9)待藻落长出，比较莱茵衣藻野生型与突变株YYAE001的纤毛长度，其结果如图3所示。与野生型WT相比，突变株YYAE001的纤毛平均长度分布更集中且略大于野生型。由此说明，本发明构建的SRTA-KO型突变株同样具有纤毛，能与其他藻种杂交。此外，观察突变株YYAE001的生长速度与野生型无异，说明突变株无细胞壁缺陷，可进行下游放大培养。(9) After the algal colony grows, compare the cilia length of the wild type Chlamydomonas reinhardtii and the mutant strain YYAE001, and the results are shown in Figure 3 . Compared with wild-type WT, the average length distribution of cilia in mutant YYAE001 was more concentrated and slightly larger than that of wild-type. This shows that the SRTA-KO mutant strain constructed by the present invention also has cilia and can hybridize with other algal species. In addition, it was observed that the growth rate of the mutant strain YYAE001 was no different from that of the wild type, indicating that the mutant strain had no cell wall defects and could be used for downstream scale-up culture.
(10)统计莱茵衣藻野生型与突变株YYAE001各自长出的转化子数目，并进行比较。统计及比较结果如图4所示。(10) The number of transformants grown from the wild type Chlamydomonas reinhardtii and the mutant strain YYAE001 were counted and compared. The statistical and comparative results are shown in Figure 4.
由图4的左图可看出，携带外源基因YFP的突变株YYAE001阳性转化子数目绝对值是野生型4倍多，相对提高315％；由图4的右图可看出，携带外源基因YFP的突变株YYAE001阳性转化率相对野生型提高178％。It can be seen from the left figure of Figure 4 that the absolute value of the number of positive transformants of the mutant strain YYAE001 carrying the foreign gene YFP is more than 4 times that of the wild type, a relative increase of 315%. The positive transformation rate of the YFP mutant strain YYAE001 was 178% higher than that of the wild type.
7、莱茵衣藻蛋白免疫印迹分析，比较携带YFP的突变株YYAE001阳性转化子与野生型藻株对外源基因YFP的表达水平，步骤如下：7. Western blotting analysis of Chlamydomonas reinhardtii protein, comparing the expression level of the exogenous gene YFP of the mutant strain YYAE001 positive transformants carrying YFP and the wild-type algae strain, the steps are as follows:
(1)衣藻胞内总蛋白的提取(1) Extraction of total intracellular protein of Chlamydomonas
在超净工作台上，吸取1mL TAP液体培养基到1.5mL离心管中，挑取少许藻落到培养基中，25℃，100μE m-2s-1连续光照，培养1周。室温下，5000rpm离心5min，舍弃上清，收集藻细胞沉淀，直到所有细胞收集完毕。加入100μL总蛋白提取缓冲液(酷莱博)。On the ultra-clean workbench, draw 1 mL of TAP liquid medium into a 1.5 mL centrifuge tube, pick a little algae and drop it into the medium, and culture it for 1 week under 100 μE m-2 s-1 continuous light at 25 °C. Centrifuge at 5000rpm for 5min at room temperature, discard the supernatant, and collect the algae cell pellet until all the cells are collected. Add 100 µL of total protein extraction buffer (Cool Lab).
(2)室温下1200rpm涡旋10min，充分重悬藻细胞。将样品在液氮中冷冻处理3min，然后在水浴中进行解冻。该步骤重复操作两次。然后在4℃，14000×g离心10min，绿色上清则为衣藻总蛋白样品。最后根据Bradford试剂盒说明书进行蛋白浓度测定，最后样品保存于-20℃。(2) Vortex at 1200 rpm for 10 minutes at room temperature to fully resuspend the algal cells. The samples were frozen in liquid nitrogen for 3 min and then thawed in a water bath. This step is repeated twice. Then centrifuge at 14000×g for 10 min at 4°C, and the green supernatant is the total protein sample of Chlamydomonas. Finally, the protein concentration was determined according to the instructions of the Bradford kit, and the final samples were stored at -20°C.
(3)衣藻蛋白的Western blot分析(3) Western blot analysis of Chlamydomonas protein
将蛋白样品冰浴解冻，吸取20-100μg总蛋白样品(视蛋白表达量而定)，加入5μL 5×M5SDS-PAGE loading buffer，85℃孵育2～3min，瞬时离心30s。Thaw the protein sample in an ice bath, draw 20-100 μg of total protein sample (depending on the amount of protein expression), add 5 μL of 5×M5 SDS-PAGE loading buffer, incubate at 85°C for 2-3 minutes, and centrifuge briefly for 30 seconds.
安装好蛋白电泳槽，加入蛋白电泳缓冲液，使缓冲液没过梳子，然后垂直拔出梳子，并吸取处理后的蛋白质上清样品进行上样，Marker上样5μL，120V恒压电泳约2小时，直到溴酚蓝到达底部时停止电泳。Install the protein electrophoresis tank, add the protein electrophoresis buffer, make the buffer submerge the comb, then pull out the comb vertically, and draw the processed protein supernatant sample to load the sample, load 5 μL of the marker, and electrophoresis at 120V constant voltage for about 2 hours , until the bromophenol blue reached the bottom to stop the electrophoresis.
从电泳槽中取出蛋白胶，将浓缩胶部分切除后，分离胶部分浸泡在转移电泳缓冲液中3～5min，期间裁剪大小相当的0.2μm的PVDF膜，用100％甲醇浸泡30s后，浸泡在转移电泳缓冲液中。Take out the protein gel from the electrophoresis tank, cut off the part of the stacking gel, soak the part of the separating gel in the transfer electrophoresis buffer for 3 to 5 minutes, and cut out a 0.2 μm PVDF membrane with a similar size, soak it in 100% methanol for 30 seconds, and soak it in Transfer to electrophoresis buffer.
准备好转膜夹，将海绵充分浸泡在转移电泳缓冲液中，并按照负极-海绵-蛋白胶-PVDF膜-海绵-正极的顺序组装好三明治结构，利用滚筒赶走相互之间的气泡。Prepare the transfer clip, fully soak the sponge in the transfer electrophoresis buffer, and assemble the sandwich structure in the order of negative electrode-sponge-protein glue-PVDF membrane-sponge-positive electrode, and use the roller to drive away the air bubbles between each other.
转膜夹组装完毕后，安装到电泳槽中，倒入转膜电泳缓冲液，300mA恒流电泳1小时。并利用冰浴对转膜装置进行降温，以防温度过高导致蛋白质降解。After the assembly of the transfer clip is completed, install it in the electrophoresis tank, pour the transfer electrophoresis buffer, and perform electrophoresis at a constant current of 300mA for 1 hour. And use an ice bath to cool down the transfer device to prevent protein degradation caused by excessive temperature.
转膜结束后，取出转膜夹中的PVDF膜，将其浸泡在ddH2O中，涮洗除去残留的蛋白胶。将膜浸泡在5％脱脂奶粉(伊利，中国)中，室温水平摇床孵育至少1小时。After transferring the membrane, take out the PVDF membrane in the transfer holder, soak it in ddH2 O, and rinse to remove the residual protein glue. Soak the membrane in 5% skimmed milk powder (Yili, China) and incubate on a horizontal shaker at room temperature for at least 1 hour.
孵育结束后，用3％脱脂牛奶来配置一抗溶液。对于含有6×His标签的蛋白，一抗选用M5mouse anti-His Tag antibody(北京聚合美，MF082)，稀释比为1：2000；对于含有3×HA标签的蛋白，一抗选用mouse anti-HA Tag antibody(Biolegend，901513)，稀释比为1：2000。利用一抗溶液孵育PVDF膜，条件为4℃孵育过夜；对于α-tubulin，一抗选用mouseanti-α-tubulin antibody(Sigma，B30271)；对于YFP，一抗使用mouse anti-GFP antibody(Abmart，M20004)。After the incubation, use 3% skim milk to prepare the primary antibody solution. For proteins containing 6×His tags, use M5 mouse anti-His Tag antibody (Beijing Polymer, MF082) as the primary antibody at a dilution ratio of 1:2000; for proteins containing 3×HA tags, use mouse anti-HA Tag as the primary antibody Antibody (Biolegend, 901513), the dilution ratio is 1:2000. The PVDF membrane was incubated with the primary antibody solution at 4°C overnight; for α-tubulin, the primary antibody was mouseanti-α-tubulin antibody (Sigma, B30271); for YFP, the primary antibody was mouse anti-GFP antibody (Abmart, M20004 ).
孵育结束后，回收一抗溶液，将膜浸泡在TBST溶液中，水平摇床洗涤10min，重复3次。期间使用3％脱脂牛奶配置二抗溶液，二抗为Goat anti-mouse IgG antibody(北京博奥龙，BF03001)，稀释比为1：10000；After the incubation, the primary antibody solution was recovered, the membrane was soaked in TBST solution, washed on a horizontal shaker for 10 min, and repeated 3 times. During this period, 3% skimmed milk was used to prepare the secondary antibody solution, the secondary antibody was Goat anti-mouse IgG antibody (Beijing Boaolong, BF03001), and the dilution ratio was 1:10000;
将膜在二抗溶液中室温水平摇床孵育至少1小时。孵育结束后，回收二抗溶液，将膜在TBST中水平摇床洗涤10min，重复3次。Incubate the membrane in the secondary antibody solution for at least 1 hour at room temperature on a horizontal shaker. After the incubation, the secondary antibody solution was recovered, and the membrane was washed on a horizontal shaker in TBST for 10 min, and repeated 3 times.
利用ECL Western HRP Substrate(北京聚合美，货号：MF074)进行目的条带的检测。按照说明书的要求提前将ECL试剂的A液和B液混合。吸取2-3mL ECL试剂到膜上，室温孵育3min，然后利用显影机曝光30s，拍照并保存结果用于分析。ECL Western HRP Substrate (Beijing Jumei, product number: MF074) was used to detect the target band. Mix liquid A and liquid B of the ECL reagent in advance according to the instructions. Pipette 2-3mL of ECL reagent onto the membrane, incubate at room temperature for 3min, and then use a developing machine to expose for 30s, take pictures and save the results for analysis.
利用Image J分析出目的条带的灰度值，利用标准曲线计算出重组蛋白的含量。重组蛋白表达水平(％)＝重组蛋白含量(μg)/蛋白上样量(μg)×100％。实验结果如图5所示，携带YFP的突变株YYAE001阳性转化子中YFP表达量的灰度值是野生型转化子的2倍以上。The gray value of the target band was analyzed by Image J, and the content of the recombinant protein was calculated using the standard curve. Recombinant protein expression level (%)=recombinant protein content (μg)/protein loading amount (μg)×100%. The experimental results are shown in Figure 5, the gray value of YFP expression in the YYAE001 positive transformants carrying the YFP mutant strain is more than 2 times that of the wild-type transformants.
8、验证突变株YYAE001表达外源基因的稳定性，方法如下：8. To verify the stability of exogenous gene expression in the mutant strain YYAE001, the method is as follows:
(1)选择一株高表达外源基因YFP的藻株YYAE001，培养至3-5×106cells/mL，为S0代。(1) Select an algae strain YYAE001 that highly expresses the exogenous gene YFP, and culture it to 3-5×106 cells/mL, which is the S0 generation.
(2)在超净台内吸取部分藻液转移至新鲜的培养基中进行传代培养，为S1代。同时收集S0代的1×107cells至1.5mL EP管中，12000rpm离心3min，液氮冷冻后转移至-80℃冰箱待后续使用。(2) Absorb part of the algae liquid in the ultra-clean bench and transfer it to a fresh medium for subculture, which is the S1 generation. At the same time, 1×107 cells of the S0 generation were collected into a 1.5 mL EP tube, centrifuged at 12,000 rpm for 3 min, frozen in liquid nitrogen, and transferred to a -80°C refrigerator for subsequent use.
(3)待S1代培养至3-5×106cells/mL，重复步骤2，转接获得S2代，收集S1样品存于-80℃。(3) After the S1 generation was cultured to 3-5×106 cells/mL, repeat
(4)待S2代培养至3-5×106cells/mL，收集S2样品存于-80℃，后续进行样品处理和蛋白免疫印迹分析，确定多次传代外源基因YFP的表达情况。(4) After the S2 subculture was cultured to 3-5×106 cells/mL, the S2 samples were collected and stored at -80°C, followed by sample processing and western blot analysis to determine the expression of the exogenous gene YFP in multiple passages .
实验结果如图6所示，S0、S1、S2随着传代次数的增加，蛋白条带灰度没有明显变化，这说明本发明构建的突变株可稳定表达其所携带的外源基因，解决了现有技术中莱茵衣藻外源蛋白表达水平低，外源基因容易丢失的问题。The experimental results are shown in Figure 6. With the increase of passage times, the gray scale of protein bands of S0 , S1 , and S2 has no obvious change, which shows that the mutant strains constructed in the present invention can stably express the foreign genes carried by them. The method solves the problem that the exogenous protein expression level of Chlamydomonas reinhardtii is low and the exogenous gene is easily lost in the prior art.
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. scope.
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