技术领域technical field
本发明属于基因载体技术领域，尤其涉及一种MC1R基因载体及其构建方法。The invention belongs to the technical field of gene carriers, and in particular relates to an MClR gene carrier and a construction method thereof.
背景技术Background technique
动物的毛色或羽色作为一个重要的经济性状，在确定品种纯度和亲缘关系方面发挥着重要的作用。研究发现，动物毛色或羽色之所以不同，是因为黑色素的种类和分布不同造成的。黑素皮质素受体1(melanocortin 1 recptor，MC1R)，是控制动物黑色素形成的主要基因，由一个外显子组成，在黑色素细胞中表达，与天然配体a-MSH相互作用，通过由G蛋白耦合的cAMP信号通路，来调节黑色素细胞内的一系列级联反应，最终经多巴或多巴铬合成各自的衍生物即褐黑素细胞和真黑色素，从而决定动物的毛色。然而，目前在鸟类上，关于MC1R影响黑色素沉积的分子机理尚不明确，在其基因功能学方面，关于MC1R基因敲除及敲除后对羽色性状的影响等相关研究尚未见报道。CRISPR/Cas9系统是细菌在噬菌体长期的选择压力下进化出来的一种有效抵御外源DNA入侵的免疫机制之一。在菌体内，CRISPR簇在其前导区的调控下转录成precrRNA，并在tracrRNA和Cas9参与下加工成成熟的crRNA，引导crRNA/tracrRNA/Cas9复合体识别结合外源DNA特定序列，剪切DNA双链，从而沉默外源基因的表达。CRISPR/Cas9系统被开发成了一种新型的基因打靶系统。相对于较早的RNAi、ZFN和TALEN系统，这种新型打靶系统具有操作简单、成本低、效率高、可同时沉默任意数量基因等优点。As an important economic trait, the coat or plumage color of animals plays an important role in determining the purity and kinship of breeds. Studies have found that the different fur or feather colors of animals are caused by the different types and distributions of melanin. Melanocortin 1 receptor (MC1R), the main gene controlling the formation of animal melanin, consists of one exon, expressed in melanocytes, and interacts with the natural ligand a-MSH, through G The protein-coupled cAMP signaling pathway regulates a series of cascade reactions in melanocytes, and finally synthesizes their respective derivatives, pheomelanocytes and eumelanin, through dopa or dopachrome, thereby determining the coat color of animals. However, the molecular mechanism of MC1R affecting melanin deposition in birds is still unclear. In terms of gene function, there are no reports on the knockout of MC1R gene and its effect on feather color traits. The CRISPR/Cas9 system is one of the immune mechanisms that bacteria have evolved under the long-term selection pressure of phages to effectively resist the invasion of foreign DNA. In bacteria, the CRISPR cluster is transcribed into precrRNA under the control of its leader region, and processed into mature crRNA with the participation of tracrRNA and Cas9, guiding the crRNA/tracrRNA/Cas9 complex to recognize and bind to a specific sequence of exogenous DNA, and cut the DNA double chain, thereby silencing the expression of foreign genes. The CRISPR/Cas9 system has been developed as a novel gene targeting system. Compared with the earlier RNAi, ZFN and TALEN systems, this new targeting system has the advantages of simple operation, low cost, high efficiency, and the ability to simultaneously silence any number of genes.
目前在鸟类上，关于MC1R影响黑色素沉积的分子机理尚不明确，在其基因功能学方面，关于MC1R基因敲除及敲除后对羽色性状的影响等相关研究尚未见报道。At present, in birds, the molecular mechanism of MC1R affecting melanin deposition is still unclear. In terms of its gene function, there are no reports on the knockout of MC1R gene and its effect on feather color traits.
发明内容Contents of the invention
本发明的目的在于提供一种MC1R基因载体及其构建方法，旨在针对家鸡MC1R基因进行定向敲除，为对家鸡的羽毛毛色进行人工调控奠定基础。The object of the present invention is to provide an MC1R gene carrier and a construction method thereof, aiming at targeted knockout of the chicken MC1R gene, and laying a foundation for artificially regulating the feather coat color of the chicken.
本发明是这样实现的，一种MC1R基因载体，可用于家鸡MC1R的基因定向敲除，适用于鸡羽色等科研领域，进一步可适用于鸡羽色性状的人工控制，所述MC1R基因载体包括MC1R-neo和MC1R-GFP；所述MC1R-neo核苷酸序列为SEQ ID NO：1；所述MC1R-GFP核苷酸序列为SEQ ID NO：2。The present invention is achieved in this way, a MC1R gene carrier can be used for directional knockout of chicken MC1R gene, suitable for scientific research fields such as chicken feather color, and further applicable to artificial control of chicken feather color traits, the MC1R gene carrier It includes MC1R-neo and MC1R-GFP; the nucleotide sequence of MC1R-neo is SEQ ID NO: 1; the nucleotide sequence of MC1R-GFP is SEQ ID NO: 2.
本发明的另一目的在于提供一种所述MC1R基因载体的构建方法，所述构建方法包括以下步骤：Another object of the present invention is to provide a construction method of the MClR gene carrier, the construction method comprising the following steps:
步骤一，引物退火：1ul F-Oligo(100uM)，1ul R-Oligo(100uM)，8ul YSY oligo退火缓冲液，以上溶液混合于PCR管内，在PCR仪中以每分钟1.5℃逐渐从95℃降至22℃；Step 1, primer annealing: 1ul F-Oligo (100uM), 1ul R-Oligo (100uM), 8ul YSY oligo annealing buffer, mix the above solutions in a PCR tube, and gradually lower the temperature from 95°C to 1.5°C per minute in the PCR instrument. to 22°C;
步骤二，连接：0.5ul退火产物，1ul YSY线性化三合一CRISPR/Cas9n质粒，1ul T4连接酶，2ul 5*T4Buffer，5.5ul Milli Q；Step 2, ligation: 0.5ul annealed product, 1ul YSY linearized three-in-one CRISPR/Cas9n plasmid, 1ul T4 ligase, 2ul 5*T4Buffer, 5.5ul Milli Q;
步骤三，转化：室温15min后用pfu≥108的大肠杆菌DH5a感受态细胞进行转化；Step 3, transformation: after 15 minutes at room temperature, transform with Escherichia coli DH5a competent cells with pfu ≥10 8 ;
步骤四，转化子验证：转化涂板后挑取单克隆进行10ul体系菌液PCR验证；Step 4, transformant verification: after transformation and plating, pick a single clone for 10ul system bacterial liquid PCR verification;
步骤五，经PCR初步鉴定后进行测序；Step 5, performing sequencing after preliminary identification by PCR;
步骤六，质粒提取：菌液37℃过夜培养，无内毒素质粒大提试剂盒提取质粒，并采用微量紫外分光光度计测定质粒浓度。Step 6, plasmid extraction: culture the bacterial solution overnight at 37°C, extract the plasmid with an endotoxin-free plasmid extraction kit, and measure the concentration of the plasmid with a micro-ultraviolet spectrophotometer.
进一步，所述转化涂板后挑取单克隆进行10ul体系菌液PCR验证，具体包括：挑取单克隆0.5ul菌液，0.5ul YSY验证正向引物，0.5ul R-Oligo，5ul Mastermix，3.5ulMilliQ；PCR反应条件为：1)：95℃预变性2mim；2)：94℃变性30s；3)：56℃退火30s；4)：72℃延伸30s；步骤2)到步骤4)运行35个循环；5)：72℃再延伸10min；6)：4℃保存。Further, after the transformation plated, a single clone was picked for 10ul system bacterial solution PCR verification, specifically including: picking a single clone 0.5ul bacterial solution, 0.5ul YSY verification forward primer, 0.5ul R-Oligo, 5ul Mastermix, 3.5 ulMilliQ; PCR reaction conditions are: 1): 95°C pre-denaturation 2mim; 2): 94°C denaturation 30s; 3): 56°C annealing 30s; 4): 72°C extension 30s; step 2) to step 4) run 35 times Cycle; 5): extend at 72°C for 10 minutes; 6): store at 4°C.
进一步，所述引物包括：Further, the primers include:
F-MC1R-gRNA-L1：CACCGAAGGAGAGGGAGGACACGA；F-MC1R-gRNA-L1: CACCGAAGGAGAGGGAGGACACGA;
R-MC1R-gRNA-L1：AAACTCGTGTCCTCCCTCTCCTTCC；R-MC1R-gRNA-L1: AAACTCGTGTCCTCCCTCTCCTTCC;
F-MC1R-gRNA-R1：CACCGCTTCCTGGGGGTCATCGCCG；F-MC1R-gRNA-R1: CACCGCTTCCTGGGGGTCATCGCCG;
R-MC1R-gRNA-R1：AAACCGGCGATGACCCCCAGGAAG。R-MC1R-gRNA-R1: AAACCGGCGATGACCCCCAGGAAG.
进一步，所述质粒浓度为：Further, the plasmid concentration is:
pYSY-CMV-Cas9n-U6-MC1R-gRNA-L2-SV40-Neo质粒：4ug浓度:190ng/ul；pYSY-CMV-Cas9n-U6-MC1R-gRNA-L2-SV40-Neo plasmid: 4ug Concentration: 190ng/ul;
pYSY-CMV-Cas9n-U6-MC1R-gRNA-R2-EF1a-eGFP质粒：4ug浓度:184ng/ul。pYSY-CMV-Cas9n-U6-MC1R-gRNA-R2-EF1a-eGFP plasmid: 4ug Concentration: 184ng/ul.
本发明提供的MC1R基因载体及其构建方法采用CRISPR/cas9系统，设计MC1R基因的sgRNA片段，化学合成sgRNA核苷酸序列，构建同时表达sgRNA和Cas9 D10A的质粒PYSY-sgRNA，连接并转化至大肠杆菌DH5α感受态细胞，最后对转化子进行验证；酶切和测序鉴定证明MC1R基因敲除载体构建正确。本发明采用CRISPR/Cas9构建的载体，可以定向敲除鸡MC1R基因，因而可为后续获得鸡MC1R基因缺失型细胞株、生产MC1R基因缺失转基因鸡等相关研究奠定基础。本发明通过构建MC1R基因敲除载体，为后续采用该载体制备MC1R基因缺失细胞株，来进一步研究MC1R基因功能提供基础。The MC1R gene carrier and its construction method provided by the present invention adopt the CRISPR/cas9 system to design the sgRNA fragment of the MC1R gene, chemically synthesize the sgRNA nucleotide sequence, construct the plasmid PYSY-sgRNA that simultaneously expresses sgRNA and Cas9 D10A, connect and transform it into the large intestine Bacillus DH5α competent cells, and finally the transformants were verified; enzyme digestion and sequencing identification proved that the MC1R gene knockout vector was constructed correctly. The present invention uses the vector constructed by CRISPR/Cas9 to knock out the chicken MC1R gene in a targeted manner, thus laying the foundation for subsequent related research on obtaining chicken MC1R gene-deleted cell lines and producing MC1R gene-deleted transgenic chickens. The invention provides a basis for further studying the function of the MC1R gene by constructing the MC1R gene knockout vector for subsequent preparation of the MC1R gene deletion cell line using the vector.
本发明采用CRISPR/Cas9系统构建MC1R基因敲除载体，方法简单快捷，只需针对该基因敲除位点设计一个长约20bp左右的sgRNA，然后连接通用的Cas9基因即可，而采用ZFNs或TALENs进行基因敲除，对每个基因位点编辑都需要设计和组装两个核酸酶，构建技术难度较大、构建组装时间较长。因此，与传统的ZFNs、TALENs等基因敲除技术比较而言，采用CRISPR/Cas9构建基因敲除载体更为简单快捷，便于进一步推广和应用于后续实验。The present invention adopts the CRISPR/Cas9 system to construct the MC1R gene knockout vector. The method is simple and fast. It only needs to design a sgRNA about 20bp long for the gene knockout site, and then connect the general Cas9 gene. ZFNs or TALENs For gene knockout, two nucleases need to be designed and assembled for editing each gene site, which is difficult to construct and takes a long time to construct and assemble. Therefore, compared with traditional gene knockout technologies such as ZFNs and TALENs, the construction of gene knockout vectors using CRISPR/Cas9 is simpler and faster, which is convenient for further promotion and application in subsequent experiments.
与ZFN和TALEN这两种人工核酸酶相比，CRISPR/Cas9系统中的Cas9作为切口酶，具有单链切割活性，可以在特定位置制造单链切口，这样基本不会引起非同源末端连接，从而高效地介导外源基因的定点敲入，或对基因组进行点突变，大大降低了非同源末端连接所带来的风险。Compared with the two artificial nucleases, ZFN and TALEN, Cas9 in the CRISPR/Cas9 system, as a nickase, has single-strand cleavage activity and can make single-strand nicks at specific positions, which basically does not cause non-homologous end joining. Therefore, it can efficiently mediate the site-specific knock-in of exogenous genes, or perform point mutations on the genome, greatly reducing the risk of non-homologous end joining.
本发明利用RNA导向的CRISPR-Cas9系统形成双切口，在未影响靶向切割效率的前提下大大降低了脱靶效应，提高基因敲除效率，相对于传统的单切口敲除载体，其敲除效率可提高20％以上。The invention utilizes the RNA-guided CRISPR-Cas9 system to form double nicks, which greatly reduces the off-target effect and improves the gene knockout efficiency without affecting the targeted cutting efficiency. Compared with the traditional single nick knockout carrier, its knockout efficiency It can be increased by more than 20%.
附图说明Description of drawings
图1是本发明实施例提供的MC1R基因载体的构建方法流程图。Fig. 1 is a flowchart of the construction method of the MClR gene vector provided by the embodiment of the present invention.
图2是本发明实施例提供的PCR验证克隆电泳图片示例示意图；Fig. 2 is a schematic diagram of an example of an electrophoresis picture of a PCR-verified clone provided by an embodiment of the present invention;
图中：Marker：Trans 2K Plus DNA Marker，从下到上依次为100bp，250bp，500bo，750bp，1000bp，3000bp，5000bp；2:阴性对照；3-6:MC1R-gRNA-Rg1克隆。In the figure: Marker: Trans 2K Plus DNA Marker, from bottom to top are 100bp, 250bp, 500bo, 750bp, 1000bp, 3000bp, 5000bp; 2: negative control; 3-6: MC1R-gRNA-Rg1 clone.
图3是本发明实施例提供的PCR验证克隆电泳图片示例示意图；Fig. 3 is a schematic diagram of an example of an electrophoresis picture of a PCR-verified clone provided by an embodiment of the present invention;
图中：Marker：Trans 2K Plus DNA Marker，从下到上依次为100bp，250bp，500bo，750bp，1000bp，3000bp，5000bp；2:阴性对照；3-6:MC1R-gRNA-Lg1克隆。In the figure: Marker: Trans 2K Plus DNA Marker, from bottom to top are 100bp, 250bp, 500bo, 750bp, 1000bp, 3000bp, 5000bp; 2: negative control; 3-6: MC1R-gRNA-Lg1 clone.
具体实施方式detailed description
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.
下面结合附图对本发明的应用原理作详细的描述。The application principle of the present invention will be described in detail below in conjunction with the accompanying drawings.
本发明实施例的MC1R基因载体包括MC1R-neo和MC1R-GFP；所述MC1R-neo核苷酸序列为SEQ ID NO：1；所述MC1R-GFP核苷酸序列为SEQ ID NO：2。The MC1R gene carrier of the embodiment of the present invention includes MC1R-neo and MC1R-GFP; the nucleotide sequence of the MC1R-neo is SEQ ID NO:1; the nucleotide sequence of the MC1R-GFP is SEQ ID NO:2.
SEQ ID NO：1为：SEQ ID NO: 1 is:
TGGCTTTATATCTTGTGGAAAGGACGAAACACCGAAGGAGAGGGAGGACACGAGTTTTAGAGCTAGAA；TGGCTTTATATCTTGTGGAAAGGACGAAACACCGAAGGAGAGGGAGGACACGAGTTTTTAGAGCTAGAA;
SEQ ID NO：2为：SEQ ID NO: 2 is:
TGGCTTTATATCTTGTGGAAAGGACGAAACACCGCTTCCTGGGGGTCATCGCCGGTTTTAGAGCTAGA。TGGCTTTATATCTTGTGGAAAGGACGAAACACCGCTTCCTGGGGGTCATCGCCGGTTTTAGAGCTAGA.
如图1所示，本发明实施例的MC1R基因载体的构建方法包括以下步骤：As shown in Figure 1, the construction method of the MC1R gene carrier of the embodiment of the present invention comprises the following steps:
S101：引物退火：1ul F-Oligo(100uM)，1ul R-Oligo(100uM)，8ul YSY oligo退火缓冲液，以上溶液混合于PCR管内，在PCR仪中以每分钟1.5℃逐渐从95℃降至22℃；S101: Primer annealing: 1ul F-Oligo (100uM), 1ul R-Oligo (100uM), 8ul YSY oligo annealing buffer, the above solutions are mixed in the PCR tube, and gradually drop from 95°C to 1.5°C per minute in the PCR machine. 22°C;
S102：连接：0.5ul退火产物，1ul YSY线性化三合一CRISPR/Cas9n质粒，1ul T4连接酶，2ul 5*T4Buffer，5.5ul Milli Q；S102: Ligation: 0.5ul annealed product, 1ul YSY linearized three-in-one CRISPR/Cas9n plasmid, 1ul T4 ligase, 2ul 5*T4Buffer, 5.5ul Milli Q;
S103：转化：室温15min后用大肠杆菌DH5a感受态细胞(pfu≥108)进行转化；S103: Transformation: transform with Escherichia coli DH5a competent cells (pfu≥108 ) after 15 minutes at room temperature;
S104：转化子验证：转化涂板后挑取单克隆进行10ul体系菌液PCR验证；S104: Transformant verification: pick a single clone after transformation and plating for 10ul system bacterial solution PCR verification;
0.5ul菌液，0.5ul YSY验证正向引物，0.5ul R-Oligo，5ul Mastermix，3.5ulMilliQ；PCR反应条件为：1)：95℃预变性2mim；2)：94℃变性30s；3)：56℃退火30s；4)：72℃延伸30s；步骤2)到步骤4)运行35个循环；5)：72℃再延伸10min；6)：4℃保存；0.5ul bacterial liquid, 0.5ul YSY verified forward primer, 0.5ul R-Oligo, 5ul Mastermix, 3.5ulMilliQ; PCR reaction conditions are: 1): 95°C pre-denaturation 2mim; 2): 94°C denaturation 30s; 3): Anneal at 56°C for 30s; 4): extend at 72°C for 30s; run 35 cycles from step 2) to step 4); 5): extend at 72°C for 10 minutes; 6): store at 4°C;
S105:经PCR初步鉴定后进行测序；S105: Sequencing after preliminary identification by PCR;
S106：质粒提取：菌液37℃过夜培养，无内毒素质粒大提试剂盒提取质粒，并采用微量紫外分光光度计测定质粒浓度。S106: Plasmid extraction: culture the bacterial solution overnight at 37°C, extract the plasmid with an endotoxin-free plasmid extraction kit, and measure the concentration of the plasmid with a micro-ultraviolet spectrophotometer.
下面结合实验对本发明的应用原理作进一步的描述。The application principle of the present invention will be further described in conjunction with experiments below.
1材料和方法1 Materials and methods
1.1材料1.1 Materials
Trans2K Plus DNA Marker购自全式金生物技术有限公司，线性化三合一CRISPR/Cas9n质粒购自南京尧舜禹公司，大肠杆菌DH5a感受态细胞、T4连接酶、无内毒素质粒大提试剂盒均购自天根生化有限公司。Trans2K Plus DNA Marker was purchased from Quanshijin Biotechnology Co., Ltd., and the linearized three-in-one CRISPR/Cas9n plasmid was purchased from Nanjing Yaosunyu Company. Escherichia coli DH5a competent cells, T4 ligase, and endotoxin-free plasmid extraction kit were all purchased from Tiangen Biochemical Co., Ltd.
1.2方法1.2 Method
1.2.1 sgRNA靶点确定和引物设计1.2.1 sgRNA target identification and primer design
根据鸡MC1R基因(GenBank:KF379749.1)，找到CDS序列并分析其结构，根据该基因结构确定敲除位点，选择外显子序列输入到软件中，得到gRNA序列。并根据设计生成的2个sgRNA，设计并合成对应的2对引物Oligo如下：According to the chicken MC1R gene (GenBank: KF379749.1), find the CDS sequence and analyze its structure, determine the knockout site according to the gene structure, select the exon sequence and input it into the software to obtain the gRNA sequence. And according to the 2 sgRNAs generated by the design, design and synthesize the corresponding 2 pairs of primers Oligo as follows:
SEQ ID NO：3 F-MC1R-gRNA-L1：CACCGAAGGAGAGGGAGGACACGA；SEQ ID NO: 3 F-MC1R-gRNA-L1: CACCGAAGGAGAGGGAGGACACGA;
SEQ ID NO：4 R-MC1R-gRNA-L1：AAACTCGTGTCCTCCCTCTCCTTCC；SEQ ID NO: 4 R-MC1R-gRNA-L1: AAACTCGTGTCCTCCCTCTCCTTCC;
SEQ ID NO：5 F-MC1R-gRNA-R1：CACCGCTTCCTGGGGGTCATCGCCG；SEQ ID NO: 5 F-MC1R-gRNA-R1: CACCGCTTCCTGGGGGTCATCGCCG;
SEQ ID NO：6 R-MC1R-gRNA-R1：AAACCGGCGATGACCCCCAGGAAG；SEQ ID NO: 6 R-MC1R-gRNA-R1: AAACCGGCGATGACCCCCAGGAAG;
1.2.2 CRISPR/Cas9基因敲除载体的构建1.2.2 Construction of CRISPR/Cas9 gene knockout vector
引物退火：1ul F-Oligo(100uM)，1ul R-Oligo(100uM)，8ul YSY oligo退火缓冲液，以上溶液混合于PCR管内，在PCR仪中以每分钟1.5℃逐渐从95℃降至22℃。Primer annealing: 1ul F-Oligo (100uM), 1ul R-Oligo (100uM), 8ul YSY oligo annealing buffer, mix the above solutions in a PCR tube, and gradually drop from 95°C to 22°C at 1.5°C per minute in the PCR instrument .
连接：0.5ul退火产物，1ul YSY线性化三合一CRISPR/Cas9n质粒，1ul T4连接酶，2ul 5*T4 Buffer，5.5ul Milli Q。Ligation: 0.5ul annealed product, 1ul YSY linearized three-in-one CRISPR/Cas9n plasmid, 1ul T4 ligase, 2ul 5*T4 Buffer, 5.5ul Milli Q.
转化：室温15min后用大肠杆菌DH5a感受态细胞(pfu≥108)进行转化。Transformation: transform with Escherichia coli DH5a competent cells (pfu≥108 ) after 15 minutes at room temperature.
转化子验证：转化涂板后挑取单克隆进行10ul体系菌液PCR验证。Transformant verification: after the transformation plate, pick a single clone for 10ul system bacteria solution PCR verification.
0.5ul菌液，0.5ul YSY验证正向引物，0.5ul R-Oligo，5ul Mastermix，3.5ulMilliQPCR反应条件：Seg1：95℃预变性2mim；Seg2：94℃变性30s；Seg3：56℃退火30s；Seg4：72℃延伸30s；Seg2to Seg4运行35个循环；Seg5：72℃再延伸10min；Seg6：4℃保存。0.5ul bacterial liquid, 0.5ul YSY verified forward primer, 0.5ul R-Oligo, 5ul Mastermix, 3.5ulMilliQPCR reaction conditions: Seg1: 95°C pre-denaturation 2mim; Seg2: 94°C denaturation 30s; Seg3: 56°C annealing 30s; Seg4 : Extend at 72°C for 30s; run Seg2to Seg4 for 35 cycles; Seg5: extend at 72°C for another 10min; Seg6: store at 4°C.
经PCR初步鉴定为阳性克隆送至南京金斯瑞生物科技有限公司进行测序。After preliminary identification by PCR, the positive clones were sent to Nanjing GenScript Biotechnology Co., Ltd. for sequencing.
1.2.3质粒提取1.2.3 Plasmid extraction
菌液37℃过夜培养，无内毒素质粒大提试剂盒提取质粒，并采用微量紫外分光光度计测定质粒浓度。The bacterial solution was cultured overnight at 37°C, the plasmid was extracted with an endotoxin-free plasmid extraction kit, and the concentration of the plasmid was measured with a micro-volume ultraviolet spectrophotometer.
2.结果2. Results
2.1 sgRNA设计2.1 sgRNA design
将sgRNA识别位置设在7tm_1相对应的位置上(60aa之后或总120aa以后)Set the sgRNA recognition position at the position corresponding to 7tm_1 (after 60aa or after 120aa in total)
在U6启动子驱动下预计的sgRNA转录本RNA序列分别为：The predicted RNA sequences of sgRNA transcripts driven by the U6 promoter are:
SEQ ID NO：7为：MC1R-gRNA-Lg1SEQ ID NO: 7 is: MC1R-gRNA-Lg1
GAAGGAGAGGGAGGACACAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU；GAAGGAGAGGGAGGACACAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
SEQ ID NO：8为：MC1R-gRNA-Rg1SEQ ID NO: 8 is: MC1R-gRNA-Rg1
GCUUCCUGGGGGUCAUCGCCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU。GCUUCCUGGGGGUCAUCGCCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU.
2.2 PCR验证阳性克隆电泳图片如图2和图3所示。2.2 PCR verification The electrophoresis pictures of positive clones are shown in Figure 2 and Figure 3.
2.3送检测序结果2.3 Send the sequence results
将之前验证正确的阳性克隆菌液送至金斯瑞生物技术有限公司测序，测序部分序列比对结果具体如下：The previously verified positive clones were sent to GenScript Biotechnology Co., Ltd. for sequencing. The sequence comparison results of the sequencing part are as follows:
2.3.1 pYSY-CMV-Cas9n-U6-MC1R-gRNA-L2-SV40-Neo2.3.1 pYSY-CMV-Cas9n-U6-MC1R-gRNA-L2-SV40-Neo
通过对比，其同源性达到100％。By comparison, their homology reaches 100%.
2.3.2 pYSY-CMV-Cas9n-U6-MC1R-gRNA-R2-EF1a-Egfp2.3.2 pYSY-CMV-Cas9n-U6-MC1R-gRNA-R2-EF1a-Egfp
其同源性达到100％。因此，通过以上序列比对，可确定目标质粒构建成功。Its homology reaches 100%. Therefore, through the above sequence alignment, it can be determined that the target plasmid was successfully constructed.
2.4鸡MC1R基因的敲除质粒对提取及浓度测定：2.4 Extraction and concentration determination of chicken MC1R gene knockout plasmid pair:
pYSY-CMV-Cas9n-U6-MC1R-gRNA-L2-SV40-Neo质粒：4ug浓度:190ng/ul。pYSY-CMV-Cas9n-U6-MC1R-gRNA-L2-SV40-Neo plasmid: 4ug Concentration: 190ng/ul.
pYSY-CMV-Cas9n-U6-MC1R-gRNA-R2-EF1a-eGFP质粒：4ug浓度:184ng/ul。pYSY-CMV-Cas9n-U6-MC1R-gRNA-R2-EF1a-eGFP plasmid: 4ug Concentration: 184ng/ul.
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.
<110>安徽大学<110> Anhui University
<120> 一种MC1R基因载体的构建方法<120> A method for constructing an MC1R gene vector
<160> 8<160> 8
<210> 1<210> 1
<211> 69<211> 69
<212>DNA<212>DNA
<213> 鸡MC1R<213> Chicken MC1R
<400> 核苷酸序列<400> nucleotide sequence
TGGCTTTATATCTTGTGGAAAGGACGAAACACCGAAGGAGAGGGAGGACACGAGTTTTAGAGCTAGAATGGCTTTATATCTTGTGGAAAGGACGAAACACCGAAGGAGAGGGAGGACACGAGTTTTTAGAGCTAGAA
<210> 2<210> 2
<211> 69<211> 69
<212>DNA<212>DNA
<213> 鸡MC1R<213> Chicken MC1R
<400>核苷酸序列<400> nucleotide sequence
TGGCTTTATATCTTGTGGAAAGGACGAAACACCGCTTCCTGGGGGTCATCGCCGGTTTTAGAGCTAGATGGCTTTATATCTTGTGGAAAGGACGAAACACCGCTTCCTGGGGGTCATCGCCGGTTTTAGAGCTAGA
<210> 3<210> 3
<211> 24<211> 24
<212>RNA<212> RNA
<213> F-MC1R-gRNA-L1<213> F-MC1R-gRNA-L1
<400>核苷酸序列<400> nucleotide sequence
CACCGAAGGAGAGGGAGGACACGACACCGAAGGAGAGGGAGGACACGA
<210> 4<210> 4
<211> 25<211> 25
<212>RNA<212> RNA
<213> R-MC1R-gRNA-L1<213> R-MC1R-gRNA-L1
<400>核苷酸序列<400> nucleotide sequence
AAACTCGTGTCCTCCCTCTCCTTCCAAACTCGTGTCCTCCCTCTCCTTCC
<210> 5<210> 5
<211> 25<211> 25
<212>RNA<212> RNA
<213> F-MC1R-gRNA-R1<213> F-MC1R-gRNA-R1
<400>核苷酸序列<400> nucleotide sequence
CACCGCTTCCTGGGGGTCATCGCCGCACCGCTTCCTGGGGGTCATCGCCG
<210>6<210>6
<211> 24<211> 24
<212>RNA<212> RNA
<213> R-MC1R-gRNA-R1<213> R-MC1R-gRNA-R1
<400>核苷酸序列<400> nucleotide sequence
AAACCGGCGATGACCCCCAGGAAGAAACCGGCGATGACCCCCAGGAAG
<210>7<210>7
<211> 103<211> 103
<212>RNA<212> RNA
<213>鸡MC1R<213> Chicken MC1R
<400>核苷酸序列<400> nucleotide sequence
GAAGGAGAGGGAGGACACAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUGAAGGAGAGGGAGGACACAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU
<210>8<210>8
<211> 103<211> 103
<212>RNA<212> RNA
<213>鸡MC1R<213> Chicken MC1R
<400>核苷酸序列<400> nucleotide sequence
GCUUCCUGGGGGUCAUCGCCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUGCUUCCUGGGGGUCAUCGCCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU
| Application Number | Priority Date | Filing Date | Title |
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| CN201611222716.1ACN106636201A (en) | 2016-12-27 | 2016-12-27 | MC1R (melanocortin 1 receptor) gene carrier and construction method thereof |
| Application Number | Priority Date | Filing Date | Title |
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| CN201611222716.1ACN106636201A (en) | 2016-12-27 | 2016-12-27 | MC1R (melanocortin 1 receptor) gene carrier and construction method thereof |
| Publication Number | Publication Date |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201611222716.1APendingCN106636201A (en) | 2016-12-27 | 2016-12-27 | MC1R (melanocortin 1 receptor) gene carrier and construction method thereof |
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