技术领域technical field
本发明属于植物生物技术领域，具体地说利用FMDV 2A连接肽串联标记基因的方法，通过对串联的基因分别进行验证，以确定该植株为转基因阳性。The invention belongs to the field of plant biotechnology, and specifically utilizes the method of FMDV 2A linking peptide tandem marker genes, and confirms that the plant is transgenic positive by separately verifying the tandem genes.
背景技术Background technique
外源基因转化到植物需要通过不同阶段的筛选标记来确认，不同筛选标记容易获得真实的转基因植株，但是植物基因表达需要启动子和终止子配套，因此多个标记基因构建比较繁琐。在植物基因表达载体构建过程中，往往由于载体表达组件的缺少或多克隆位点酶切位点的稀少而使目的基因植物表达载体构建受到限制。为了在植物表达载体中使用多个标记基因，增加转基因植物的筛选可信度，本项目利用口蹄疫病毒(FMDV)2A/2B连接区分别将潮霉素抗性基因（HPT）与Bar抗草胺膦抗性基因串联构成双筛选标记的融合基因，将潮霉素抗性基因（HPT）、Bar抗草胺膦抗性基因以及绿色荧光蛋白基因(EGFP)串联构成双筛选标记和报告基因融合的3基因系统。The transformation of exogenous genes into plants needs to be confirmed by screening markers at different stages. Different screening markers are easy to obtain true transgenic plants, but plant gene expression requires promoter and terminator matching, so the construction of multiple marker genes is cumbersome. In the process of plant gene expression vector construction, the construction of target gene plant expression vector is often limited due to the lack of vector expression components or the scarcity of multiple cloning site restriction sites. In order to use multiple marker genes in plant expression vectors and increase the screening reliability of transgenic plants, this project uses foot-and-mouth disease virus (FMDV) 2A/2B junction region to separate hygromycin resistance gene (HPT) and Bar resistance The phosphine resistance gene is concatenated to form the fusion gene of the double selectable marker, and the hygromycin resistance gene (HPT), the Bar glufosinate resistance gene and the green fluorescent protein gene (EGFP) are concatenated to form the double selectable marker and the reporter gene fusion. 3 gene system.
目前多基因表达策略主要包括表达融合蛋白，构建多顺反子表达载体等。利用内部核糖体进入位点IRES (internal ribosome entry site)或FMDV 2A 连接两个或多个基因，可以在真核系统中实现基因的共表达。利用IRES介导的多顺反子表达结构较大，其应用常受到载体容量的限制，当IRES被置于相邻基因中间时有助于双顺反子蛋白的表达，但IRES对位于其后的基因翻译起始能力相对较弱。在多顺反子蛋白的表达中可以用FMDV 2A来代替IRES。At present, multi-gene expression strategies mainly include expressing fusion proteins and constructing polycistronic expression vectors. Using the internal ribosome entry site IRES (internal ribosome entry site) or FMDV 2A to link two or more genes, the co-expression of genes can be achieved in eukaryotic systems. The use of IRES-mediated polycistronic expression has a large structure, and its application is often limited by the capacity of the vector. When the IRES is placed in the middle of adjacent genes, it is helpful for the expression of bicistronic proteins, but the IRES pair is located behind it. The gene translation initiation ability is relatively weak. FMDV 2A can be used in place of IRES in the expression of polycistronic proteins.
FMDV2A 蛋白约18 个氨基酸残基，具有自我剪切活性，其共翻译剪切活性是在自己的C端切割FMDV多聚蛋白，通过核糖体跳跃使2A蛋白和它的下游蛋白发生不连续的翻译(Donnelly, J. Gen. Virol, 2001, 82： 1027-1041; J. Gen. Virol, 2001, 82：1013-1025)，2A片段的氨基酸连接到上游蛋白C端，一个脯氨酸连接到下游蛋白的N端(Hasegawa,Stem Cells, 2006, 24: 2649-2660) 。 目前已在哺乳动物细胞、植物和酵母中观察到2A介导的对多聚蛋白的自我剪切作用。2A 介导的相邻基因表达产物通过翻译跳跃来实现分离。The FMDV2A protein has about 18 amino acid residues and has self-cleaving activity. Its co-translational cleavage activity is to cut the FMDV polyprotein at its own C-terminal, and make the 2A protein and its downstream proteins undergo discontinuous translation through ribosome jumping. (Donnelly, J. Gen. Virol, 2001, 82: 1027-1041; J. Gen. Virol, 2001, 82: 1013-1025), the amino acid of the 2A fragment is linked to the C-terminus of the upstream protein, and one proline is linked to the downstream The N-terminus of the protein (Hasegawa, Stem Cells, 2006, 24: 2649-2660). 2A-mediated self-cleavage of polyproteins has been observed in mammalian cells, plants and yeast. 2A-mediated separation of adjacent gene expression products is achieved by translational jumping.
序列分析发现，FMDV2A的氨基酸序列高度保守, 多肽序列中含有的基序为“-DxExNPGP-”,最后三个氨基酸(-NPG-)则完全保守，并且2B蛋白N端第一个氨基酸Pro也完全保守，该保守元件在2A起始剪切中起到关键作用。研究表明2A和2B总是在Gly和Pro之间切开, 动力学和结构模型分析表明，Gly和Pro之间的肽键实际上并未形成(Doronina, Mol.Cell. Biol., 2008, 28：4227- 4239)。在翻译过程中, 2A 的高级结构对核糖体肽基转移酶中心造成空间排阻, 使肽基(2A)-tRNA酯键无法形成。由于空间排阻使Pro-tRNA 氨基氮亲核进攻无法完成, 代之则是肽基(2A)-tRNA 酯键的水解作用, 形成与2A的融合蛋白,同时核糖体能继续翻译下游蛋白2B , 整个过程不需要任何蛋白酶参与(ChinnasamyVirol J， 2006， 3: 14)。可见, 2A 能起到类似蛋白水解酶的作用, 在2A 和2B 位点将其顺式切开，FMDV 2A 独特的剪切机制决定了2A只有在真核翻译系统中有活性, 而在原核翻译系统中无活性。FMDV 基因表达产物中不存在未剪切的多聚蛋白前体, 因此FMDV 2A的天然剪切活力可达到100 %，( Szymczak, Nat Biotechnol, 2004, 22: 589– 594), FMDV2A对体外构建的多顺反子剪切效率也能达到85%-95%。Sequence analysis found that the amino acid sequence of FMDV2A is highly conserved, the motif contained in the polypeptide sequence is "-DxExNPGP-", the last three amino acids (-NPG-) are completely conserved, and the first amino acid Pro at the N-terminal of 2B protein is also completely conserved. Conserved, this conserved element plays a key role in the 2A initiation cleavage. Studies have shown that 2A and 2B are always cleaved between Gly and Pro, and kinetic and structural modeling analysis shows that the peptide bond between Gly and Pro is not actually formed (Doronina, Mol. Cell. Biol., 2008, 28 : 4227-4239). During translation, the higher-order structure of 2A causes steric exclusion at the ribosomal peptidyltransferase center, preventing the formation of a peptidyl(2A)-tRNA ester bond. Due to the steric exclusion, the nucleophilic attack of the amino nitrogen of Pro-tRNA cannot be completed. Instead, the hydrolysis of the peptidyl (2A)-tRNA ester bond forms a fusion protein with 2A. At the same time, the ribosome can continue to translate the downstream protein 2B. The process does not require any protease involvement (Chinnasamy Virol J, 2006, 3: 14). It can be seen that 2A can play a similar role as a proteolytic enzyme, cutting it in cis at the 2A and 2B sites. The unique splicing mechanism of FMDV 2A determines that 2A is only active in the eukaryotic translation system, but in prokaryotic translation. There is no activity in the system. There is no uncleaved polyprotein precursor in the FMDV gene expression product, so the natural cleavage activity of FMDV 2A can reach 100%. The polycistronic shearing efficiency can also reach 85%-95%.
体外构建FMDV 2A 多顺反子的特点是:基因之间以2A序列连接, 同时去除上游基因的终止密码子以形成一个长的开放读码框。翻译时多聚蛋白可在编码2A 区域的C 端被2A切割开, 释放出融合了2A多肽尾巴的上游蛋白, 以及完整的下游蛋白(刘必胜,生物工程学报,2007,23(5):765-769)。目前, FMDV 2A多顺反子系统已被应用于腺病毒(adenovirus)、逆转录病毒(retrovirus)、慢病毒(lentivirus) 、腺相关病毒(adeno-associated virus)等载体的构建(Osborn, Mol Ther, 2005, 12: 569– 574)。The characteristics of the FMDV 2A polycistronic constructed in vitro are: the 2A sequence is connected between genes, and the stop codon of the upstream gene is removed to form a long open reading frame. During translation, the polyprotein can be cleaved by 2A at the C-terminus of the coding region of 2A to release the upstream protein fused with the 2A polypeptide tail, as well as the complete downstream protein (Liu Bisheng, Chinese Journal of Biological Engineering, 2007, 23(5):765- 769). At present, the FMDV 2A polycistronic system has been used in the construction of adenovirus, retrovirus, lentivirus, adeno-associated virus and other vectors (Osborn, Mol Ther , 2005, 12: 569–574).
发明内容SUMMARY OF THE INVENTION
本发明利用FMDV 2A连接肽串联多个筛选标记基因和报告基因。首先按照植物偏爱密码，我们将口蹄疫病毒(FMDV) 含有2A蛋白和2B蛋白区LLNFDLLKLAGDVESNPGP 19个氨基酸重新设计引物合成，合成的核苷酸序列为：CTGTTGAATTTCGATCTTCTTAAGCTTGCTGGTGATGTTGAATCCA ACCCAGGTCCA（SEQ ID NO.2所示）。The present invention utilizes the FMDV 2A connecting peptide to connect multiple selectable marker genes and reporter genes in series. First, according to the plant preference code, we synthesized the 19 amino acid primers of foot-and-mouth disease virus (FMDV) containing 2A protein and 2B protein region LLNFDLLKLAGDVESNPGP. The synthesized nucleotide sequence is: CTGTTGAATTTCGATCTTCTTAAGCTTGCTGGTGATGTTGAATCCA ACCCAGGTCCA (SEQ ID NO.2).
本发明通过连续延伸PCR，利用该合成序列将筛选标记基因潮霉素抗性基因（HPT）、草胺膦抗性基因Bar和GFP报告基因片断串联Hygfpbar。The present invention uses the synthetic sequence to connect the selectable marker gene hygromycin resistance gene (HPT), glufosinate resistance gene Bar and GFP reporter gene fragments in series Hygfpbar through continuous extension PCR.
本发明利用BstEII和BglII 双酶切植物表达载体pCAMBIA1301，切除其中的GUS报告基因，通过DNA末端平滑试剂盒处理后，连接获得植物表达载体pCAMBIA1301-GUS。Xho I酶切融合标记基因Hygfpbar，通过T4 DNA连接酶将融合标记基因Hygfpbar插入植物表达载体pCAMBIA1301-GUS，替换其中的潮霉素抗性基因，获得植物表达载体pHygfpbar。The present invention utilizes BstEII and BglII double enzyme to cut the plant expression vector pCAMBIA1301, excises the GUS reporter gene, and then connects to obtain the plant expression vector pCAMBIA1301-GUS after being processed by DNA end smoothing kit. The fusion marker gene Hygfpbar was digested with Xho I, and the fusion marker gene Hygfpbar was inserted into the plant expression vector pCAMBIA1301-GUS by T4 DNA ligase, and the hygromycin resistance gene was replaced to obtain the plant expression vector pHygfpbar.
本发明利用电击法将质粒导入根癌农杆菌中。农杆菌介导方法将三价基因表达载体转化单子叶模式植物水稻（Clough 1998，植物学杂志）和双子叶模式植物拟南芥，通过检测，获得了转基因植株具有潮霉素和草胺膦抗性，转基因植株组织能表达荧光蛋白。The invention utilizes electric shock method to introduce the plasmid into Agrobacterium tumefaciens. The trivalent gene expression vector was transformed into the monocotyledonous model plant rice (Clough 1998, Botany) and the dicotyledonous model plant Arabidopsis thaliana by Agrobacterium-mediated method, and the transgenic plants with hygromycin and glufosinate resistance were obtained by testing. Sex, transgenic plant tissue can express fluorescent protein.
附图说明Description of drawings
图1三价标记基因融合基因植物表达载体pHygfpbar构建图谱。Fig. 1 Construction map of trivalent marker gene fusion gene plant expression vector pHygfpbar.
图2转基因水稻愈伤组织的潮霉素抗性，水稻幼苗的草胺膦抗性及组织荧光检测。Fig. 2 Hygromycin resistance of transgenic rice callus, glufosinate resistance and tissue fluorescence detection of rice seedlings.
图3转基因拟南芥种子潮霉素抗性、幼苗的草胺膦抗性及组织荧光检测。Figure 3. Hygromycin resistance of transgenic Arabidopsis seeds, glufosinate resistance of seedlings and tissue fluorescence detection.
本发明有益效果Beneficial effects of the present invention
该标记基因转化植物后，具有多重抗性，可以通过愈伤组织、苗期和成熟植株的检测，有利于加快转基因材料的纯合，同时该载体可以用于多基因表达载体构建。After the plant is transformed by the marker gene, it has multiple resistances, and can be detected by callus, seedling stage and mature plants, which is beneficial to speed up the homozygosity of the transgenic material, and the vector can be used for the construction of a multi-gene expression vector.
具体实施方式Detailed ways
实施例1：标记基因Hygfpbar构建Example 1: Construction of marker gene Hygfpbar
利用重叠延伸PCR串联潮霉素抗性基因（HPT）、绿色荧光蛋白基因EGFP和草胺膦抗性基因Bar。设计的引物为：HPTZ1：5’-CCGCTCGAGATG AAAAAGCCTGAACTCACCG-3’（SEQ ID NO.3所示）；HPTF1:5’-ACATCAC CAGCAAGCTTAAGAAGATCGAAATTCAACAGCTATTTCTTTGCCCTCGG ACG-3’(SEQ ID NO.4所示)；GFPZ：5’-CTTAAGCTTGCTGGTGATGTTGA ATCCAACCCAGGTCCAATGGTGAGCAAGGGCGAGGAG-3’(SEQ ID NO.5所示)；GFPF：5’-ACATCACCAGCAAGCTTAAGAAGATCGAAATTCAACAGTCAGATCTCGGTGACGGGCAG-3’(SEQ ID NO.6所示)；BarZ：5’-CTTAA GCTTGCTGGTGATGTTGAATCCAACCCAGGTCCAATGAGCCCAGAACG ACGCCCG-3’(SEQ ID NO.7所示)；BarF：5’-CCGCTCGAGTCAGATCTCGG TGACGGGCAG-3’(SEQ ID NO.8所示)。The hygromycin resistance gene (HPT), the green fluorescent protein gene EGFP and the glufosinate resistance gene Bar were concatenated by overlap extension PCR. The designed primers are: HPTZ1: 5'-CCGCTCGAGATG AAAAAGCCTGAACTCACCG-3' (shown in SEQ ID NO. 3); HPTF1: 5'-ACATCAC CAGCAAGCTTAAGAAGATCGAAATTCAACAGCTATTTCTTTGCCCTCGG ACG-3' (shown in SEQ ID NO. 4); GFPZ: 5' - CTTAAGCTTGCTGGTGATGTTGA ATCCAACCCAGGTCCAATGGTGAGCAAGGGCGAGGAG-3' (shown in SEQ ID NO. 5); GFPF: 5'-ACATCACCAGCAAGCTTAAGAAGATCGAAATTCAACAGTCAGATCTCGGTGACGGGCAG-3' (shown in SEQ ID NO. 6); BarZ: 5'-CTTAA GCTTGCTGGTGATGTTGAATCCAACCCAGGTCCAATGA IDGCCCAGAACG 7); BarF: 5'-CCGCTCGAGTCAGATCTCGG TGACGGGCAG-3' (SEQ ID NO. 8).
在50µl反应体系中，利用引物HPTZ1 和HPTF1 从pCAMBIA1301扩增HPT基因；利用引物BarZ 和BarF从pPZp-RCS2-Bar（Novagen， Madison，美国） 中扩增Bar基因；利用引物GFPZ和GFPF从 pLEGFP-C1（Clontech公司，美国）中扩增EGFP基因，回收上述3个扩增基因片段，然后利用引物HPTZ1和BarF1将3个扩增片断通过重叠延伸方法串联，PCR扩增条件为：94℃ 预热1 min；94℃, 30 s, 50℃, 30 s, 72℃, 150 s，使用的Taq DNA聚合酶为KOD FXtaq酶（Toyobo公司，日本），共25个循环。PCR产物进行1% 琼脂糖胶回收，取10 µl 直接与平端克隆载体相连（大连宝生物公司）。4℃连接过夜，高效转化 DH5α感受态中，获得阳性克隆，测序鉴定获得序列正确的新型标记基因Hygfpbar。In a 50 µl reaction, the HPT gene was amplified from pCAMBIA1301 using primers HPTZ1 and HPTF1; the Bar gene was amplified from pPZp-RCS2-Bar (Novagen, Madison, USA) using primers BarZ and BarF; the Bar gene was amplified using primers GFPZ and GFPF from pLEGFP- The EGFP gene was amplified in C1 (Clontech, USA), the above three amplified gene fragments were recovered, and then the three amplified fragments were concatenated by the overlap extension method using primers HPTZ1 and BarF1. PCR amplification conditions were: 94 ℃ preheating 1 min; 94°C, 30 s, 50°C, 30 s, 72°C, 150 s, the Taq DNA polymerase used was KOD FXtaq enzyme (Toyobo, Japan), a total of 25 cycles. The PCR product was recovered by 1% agarose gel, and 10 µl was directly connected to the blunt-end cloning vector (Dalian Bao Bio Co., Ltd.). After ligation overnight at 4°C, the DH5α competent cells were efficiently transformed to obtain positive clones, and the novel marker gene Hygfpbar with the correct sequence was obtained by sequencing and identification.
实施例2：植物表达载体的构建Example 2: Construction of Plant Expression Vectors
首先将植物表达载体pCAMBIA1301利用BglII和BstEII双酶切，加入末端平滑试剂盒中的T4 DNA聚合酶（Takara 宝生物），然后加入5 U T4DNA 连接酶，通过PCR鉴定载体GUS基因的存在，筛选去除GUS基因的载体作为新型标记基因表达载体。XhoI酶切pCAMBIA1301-GUS载体和Hygfpbar片断，回收载体和片断以1：10以上的摩尔比例连接。利用PCR鉴定插入基因的方向，找到潮霉素抗性基因在启动子一侧的克隆进行进一步鉴定，构建成由CaMV35S启动子控制的新型标记基因植物表达载体pHygfpbar。First, the plant expression vector pCAMBIA1301 was double digested with BglII and BstEII, added with T4 DNA polymerase (Takara Bio) in the end smoothing kit, and then 5 U of T4 DNA ligase was added to identify the presence of the vector GUS gene by PCR, and screened to remove The vector of GUS gene is used as a novel marker gene expression vector. The pCAMBIA1301-GUS vector and the Hygfpbar fragment were digested with XhoI, and the recovered vector and the fragment were connected in a molar ratio of more than 1:10. The direction of the inserted gene was identified by PCR, and the clone of the hygromycin resistance gene on the promoter side was found for further identification, and a new marker gene plant expression vector pHygfpbar controlled by the CaMV35S promoter was constructed.
实施例3：水稻和拟南芥转化Example 3: Rice and Arabidopsis transformation
所用菌株为根癌农杆菌。质粒经电击法导入农杆菌中。挑取单菌到25 ml YEB培养基（50mg/l 利福平）培养过夜，取5 ml菌液转接到100 ml YEB培养基（50mg/l 利福平），培养至OD600 = 0.7-0.8，菌液冰上放置10分钟，5000 rpm离心10 min ，4℃，收集菌体，加入100ml 无菌双蒸水清洗两次。加入2 ml 10%甘油悬浮菌体，转到50 ml离心管。5500 rpm离心10min ，4℃。收集菌体，加入500 µl 10%甘油悬浮菌体，转到1.5 ml离心管。取70µl感受态细胞，分别加入1µl重组质粒pHygfpbar。用去头的200µl枪头混匀，转到0.1cm 电击杯中。电击参数：200Ω，1.7 KV， 2.5F，电击后立即加入800µl SOC 培养液。培养1小时后，取100µl 涂抗性板筛选转化子，28℃培养。The strain used was Agrobacterium tumefaciens. Plasmids were introduced into Agrobacterium by electroporation. Pick a single bacteria into 25 ml of YEB medium (50mg/l rifampicin) and culture overnight, transfer 5 ml of bacterial liquid to 100 ml of YEB medium (50mg/l rifampicin), and cultivate to OD600 = 0.7- 0.8, place the bacterial liquid on ice for 10 minutes, centrifuge at 5000 rpm for 10 minutes, 4 °C, collect the bacterial cells, and add 100 ml of sterile double-distilled water to wash twice. Add 2 ml of 10% glycerol suspended bacteria and transfer to a 50 ml centrifuge tube. Centrifuge at 5500 rpm for 10 min at 4°C. Collect the bacteria, add 500 µl of 10% glycerol to suspend the bacteria, and transfer to a 1.5 ml centrifuge tube. Take 70µl of competent cells and add 1µl of recombinant plasmid pHygfpbar to each. Mix well with a decapitated 200µl pipette tip and transfer to a 0.1cm shock cup. Electric shock parameters: 200Ω, 1.7 KV, 2.5F, add 800µl SOC medium immediately after electric shock. After culturing for 1 hour, take 100 µl of the resistant plate to screen transformants and culture at 28°C.
水稻转化以N6培养基为基本培养基。去壳的种子，授粉后12-15天的幼胚，经表面消毒后接种到N6D2培养基中诱导愈伤组织（N6培养基，水解乳蛋白500mg/L，蔗糖30g/L，2,4-D 2mg/L，植物凝胶2.5g/L，pH5.8）；培养4-7天后取愈伤组织进行转化。农杆菌培养OD600=0.8-1.0后离心5000 g离心 8分钟，去离子H2O清洗一次，等体积MS培养液悬浮侵染8分钟后，吸干放置在MS+NAA 1 mg/L +BA 2 mg/L的培养基中，22度共培养3天。然后转入筛选培养基（加入头孢Cb(500 µg/ml) 和潮霉素HYG(50 µg/ml)，转化后的愈伤组织在含有抗性培养基上培养3-4代，转入分化培养基中（2 mg/L KT）；幼芽长至2 mm转移到生根培养基（1/2MS+0.5mg/L IBA）。以上培养基中分别加入500 mg/L酶水解乳蛋白(CH)，0-700 mg/L谷氨酰胺或精氨酸，蔗糖30-80 g/L，琼脂6 g，pH 5.8。继代周期为25 d。将淡黄色的胚性愈伤组织转入分化培养基中，30 d左右分化出芽。光照强度1 500-2 000lx，12-14 h/d。For rice transformation, N6 medium was used as the basic medium. Shelled seeds, young embryos 12-15 days after pollination, inoculated into N6D2 medium after surface disinfection to induce callus (N6 medium, hydrolyzed milk protein 500mg/L, sucrose 30g/L, 2,4- D 2mg/L, phytogel 2.5g/L, pH 5.8); after culturing for 4-7 days, take callus for transformation. Agrobacterium cultured at OD600 =0.8-1.0, centrifuged at 5000 g for 8 minutes, washed once with deionized H2 O, suspended and infected with an equal volume of MS medium for 8 minutes, blotted dry and placed in MS+NAA 1 mg/L +BA In 2 mg/L medium, the cells were co-cultured at 22 degrees for 3 days. Then transferred to screening medium (adding cephalosporin Cb (500 µg/ml) and hygromycin HYG (50 µg/ml), the transformed callus was cultured on medium containing resistance for 3-4 passages, transferred to differentiation Medium (2 mg/L KT); shoots grow to 2 mm and transfer to rooting medium (1/2MS+0.5mg/L IBA). Add 500 mg/L enzymatically hydrolyzed milk protein (CH ), 0-700 mg/L glutamine or arginine, 30-80 g/L sucrose, 6 g agar, pH 5.8. The subculture cycle is 25 d. The pale yellow embryogenic callus was transferred into differentiation In the medium, about 30 days of differentiation and budding. The light intensity is 1 500-2 000lx, 12-14 h/d.
拟南芥转化采用蘸花法。含目的质粒的农杆菌菌株单菌落接菌在5毫升含对应抗生素的LB培养基中28℃培养2天。将5毫升菌液转到500毫升的液体LB培养基中28℃培养16-24小时（OD=1.5-2.0）。室温下离心收集菌体，4000g离心10分钟。用等体积5%的新鲜蔗糖溶液悬浮。加入0.02%的Silwet-77混匀后转移到烧杯中。每个菌株用300毫升转化，转2-3钵。隔7天后再转化1次。将拟南芥倒置后浸入菌液中10秒钟。莲座和花序都要侵染。侵染后将转化植株菌液空干3-5秒。用保鲜膜将转化植株圈好，平放16-24小时。转化后不要放置在高温和强光下。揭开保鲜膜，保持一定湿度，再生长1个月后收种子。Arabidopsis transformation was performed using the dip flower method. A single colony of the Agrobacterium strain containing the target plasmid was inoculated in 5 ml of LB medium containing the corresponding antibiotics and cultured at 28°C for 2 days. Transfer 5 ml of bacterial broth to 500 ml of liquid LB medium and cultivate at 28°C for 16-24 hours (OD=1.5-2.0). The cells were collected by centrifugation at room temperature and centrifuged at 4000g for 10 minutes. Suspend with an equal volume of 5% fresh sucrose solution. Add 0.02% Silwet-77, mix and transfer to a beaker. 300 ml of each strain were used to transform into 2-3 bowls. Convert again every 7 days. Invert the Arabidopsis thaliana and immerse them in the bacterial solution for 10 seconds. Both rosettes and inflorescences are infested. After infection, the transformed plants were air-dried for 3-5 seconds. Wrap the transformed plants with plastic wrap and lay flat for 16-24 hours. Do not place in high temperature and strong light after conversion. Remove the plastic wrap, maintain a certain humidity, and harvest the seeds after another month of growth.
实施例4：转基因植物的验证Example 4: Validation of transgenic plants
HYG抗性的表达：Expression of HYG resistance:
作为新型筛选标记基因，Hygfpbar具有潮霉素抗性，潮霉素抗性基因HPT是一种较理想的检测转基因植株及其后代的报告基因。水稻在抗性愈伤筛选过程中采用50mg/L潮霉素B作为筛选压，可以区别转基因和非转基因愈伤组织。拟南芥种子可以通过潮霉素筛选获得转基因植株。As a new selection marker gene, Hygfpbar has hygromycin resistance, and the hygromycin resistance gene HPT is an ideal reporter gene for detecting transgenic plants and their progeny. Rice used 50 mg/L hygromycin B as the screening pressure in the selection process of resistant callus, which could distinguish transgenic and non-transgenic callus. Arabidopsis seeds can be screened by hygromycin to obtain transgenic plants.
另外利用水稻的潮霉素抗性检测叶片法，也可以检测转基因植株对潮霉素的抗性。剪取约2cm长的叶片放入含有检测溶液（1.0mg/L的6-苄氨基嘌呤（6-BA）和30mg/L的潮霉素B+1%Silwet-L77）的平皿中，Silwet-L77是一种高效有机硅表面活性剂，能够极大的降低水的表面张力(水的表面张力为72.4mN/m，0.1%的Silwet-L77系列有机硅溶液的表面张力约为21mN/m)，这使Silwet-L77有机硅溶液可轻易湿润水稻的叶面，相对于传统助剂，显著提高了在靶标生物的覆盖面。在16 h光照，8 h黑暗，25℃条件下培养，定期观察叶片变色情况，以判断其抗性水平。到第5天时，在含潮霉素B的检测液中，对照组水稻叶片段均产生褐斑甚至枯死，而转基因水稻在含潮霉素的检测溶液中的叶片仍保持鲜绿色，形成鲜明对比。In addition, the resistance of transgenic plants to hygromycin can also be detected by using the hygromycin resistance detection leaf method of rice. Cut leaves about 2cm long and put them in a plate containing detection solution (1.0mg/L 6-benzylaminopurine (6-BA) and 30mg/L hygromycin B+1% Silwet-L77), Silwet- L77 is a high-efficiency silicone surfactant, which can greatly reduce the surface tension of water (the surface tension of water is 72.4mN/m, and the surface tension of 0.1% Silwet-L77 series silicone solution is about 21mN/m) , which makes the Silwet-L77 organosilicon solution can easily wet the leaves of rice, which significantly improves the coverage of target organisms compared to traditional adjuvants. Cultivated under the conditions of 16 h light, 8 h dark, and 25 °C, and regularly observed the discoloration of leaves to judge the resistance level. On the 5th day, in the test solution containing hygromycin B, the leaves of the control group had brown spots or even withered, while the leaves of the transgenic rice in the test solution containing hygromycin remained bright green, forming a sharp contrast. .
Bar除草剂抗性基因的表达：Expression of Bar herbicide resistance genes:
转基因水稻种子（未脱壳），浸于蒸馏水中，置于25℃，16h 光照、8h 黑暗。七天后将萌发一致的水稻苗移栽到土里。22℃，16h 光照、8h 黑暗，待到水稻苗长出四个叶左右时，分别喷洒100-500mmol/L等不同浓度的草铵膦除草剂，五天后，观察发现，喷洒草铵膦浓度为100mmol/L、200mmol/L、300mmol/L 的水稻苗没有发生变黄或者死亡的现象，有个别出现褐色斑点，但是对照组和转基因水稻对比不是很明显。喷洒浓度为500mmol/L的草铵膦的对照组水稻和转基因水稻植株均出现死亡现象，但是对照组死亡现象更严重。喷洒浓度为400mmol/L的草铵膦除草剂的对照组水稻发黄现象严重甚至死亡，而三种转基因水稻植株只有叶片出现个别发黄，大体生长状态较好。Transgenic rice seeds (not hulled) were immersed in distilled water and placed at 25°C under 16h light and 8h dark. After seven days, the rice seedlings with uniform germination were transplanted into the soil. 22°C, 16h light, 8h darkness, when the rice seedlings grow four leaves, spray 100-500mmol/L of different concentrations of glufosinate-ammonium herbicide respectively. The rice seedlings of 100 mmol/L, 200 mmol/L and 300 mmol/L did not turn yellow or die, and some brown spots appeared, but the comparison between the control group and the transgenic rice was not very obvious. Both the control and transgenic rice plants that were sprayed with glufosinate-ammonium at a concentration of 500 mmol/L died, but the death of the control group was more serious. In the control group sprayed with glufosinate-ammonium herbicide at a concentration of 400 mmol/L, the yellowing phenomenon was serious or even death, while the three transgenic rice plants only had individual yellowing on the leaves, and the general growth state was good.
转基因拟南芥草铵膦浓度为100mmol/L。The concentration of glufosinate-ammonium in transgenic Arabidopsis was 100 mmol/L.
转基因GFP基因的表达:Expression of the transgenic GFP gene:
用荧光显微镜分别观察1,3两个含有GFP标记基因的转基因水稻和拟南芥根尖的绿色荧光蛋白的表达。结果显示转基因植物表达了绿色荧光蛋白。The expression of green fluorescent protein in the root tips of 1 and 3 transgenic rice and Arabidopsis thaliana containing the GFP marker gene was observed by fluorescence microscope, respectively. The results showed that the transgenic plants expressed green fluorescent protein.
序列表sequence listing
<110> 上海市农业科学院<110> Shanghai Academy of Agricultural Sciences
<120> 一种新型转基因植物标记基因构建及应用<120> Construction and application of a novel transgenic plant marker gene
<130> 2019<130> 2019
<160> 8<160> 8
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
<211> 2412<211> 2412
<212> DNA<212> DNA
<213> Artificial sequence<213> Artificial sequence
<400> 1<400> 1
atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac 60atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac 60
agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat 120agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat 120
gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat 180gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat 180
cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt 240cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt 240
ggggagttta gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg 300ggggagttta gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg 300
caagacctgc ctgaaaccga actgcccgct gttctacaac cggtcgcgga ggctatggat 360caagacctgc ctgaaaccga actgcccgct gttctacaac cggtcgcgga ggctatggat 360
gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga 420gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga 420
atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat 480atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat 480
cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag 540cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag 540
ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc 600ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc 600
tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg 660tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg 660
atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct 720atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct 720
tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgcca 780tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgcca 780
cgactccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac 840cgactccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac 840
ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga 900ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga 900
gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc 960gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc 960
tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag 1020tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag 1020
aaatagctgt tgaatttcga tcttcttaag cttgctggtg atgttgaatc caacccaggt 1080aaatagctgt tgaatttcga tcttcttaag cttgctggtg atgttgaatc caacccaggt 1080
ccaatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 1140ccaatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 1140
gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 1200gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 1200
tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc 1260tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc 1260
accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg 1320accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg 1320
aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 1380aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 1380
ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 1440ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 1440
ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 1500ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 1500
cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag 1560cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag 1560
aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc 1620aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc 1620
gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 1680gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 1680
cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg 1740cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg 1740
gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag 1800gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag 1800
taactgttga atttcgatct tcttaagctt gctggtgatg ttgaatccaa cccaggtcca 1860taactgttga atttcgatct tcttaagctt gctggtgatg ttgaatccaa cccaggtcca 1860
atgagcccag aacgacgccc ggccgacatc cgccgtgcca ccgaggcgga catgccggcg 1920atgagcccag aacgacgccc ggccgacatc cgccgtgcca ccgaggcgga catgccggcg 1920
gtctgcacca tcgtcaacca ctacatcgag acaagcacgg tcaacttccg taccgagccg 1980gtctgcacca tcgtcaacca ctacatcgag acaagcacgg tcaacttccg taccgagccg 1980
caggaaccgc aggagtggac ggacgacctc gtccgtctgc gggagcgcta tccctggctc 2040caggaaccgc aggagtggac ggacgacctc gtccgtctgc gggagcgcta tccctggctc 2040
gtcgccgagg tggacggcga ggtcgccggc atcgcctacg cgggcccctg gaaggcacgc 2100gtcgccgagg tggacggcga ggtcgccggc atcgcctacg cgggcccctg gaaggcacgc 2100
aacgcctacg actggacggc cgagtcgacc gtgtacgtct ccccccgcca ccagcggacg 2160aacgcctacg actggacggc cgagtcgacc gtgtacgtct ccccccgcca ccagcggacg 2160
ggactgggct ccacgctcta cacccacctg ctgaagtccc tggaggcaca gggcttcaag 2220ggactgggct ccacgctcta cacccacctg ctgaagtccc tggaggcaca gggcttcaag 2220
agcgtggtcg ctgtcatcgg gctgcccaac gacccgagcg tgcgcatgca cgaggcgctc 2280agcgtggtcg ctgtcatcgg gctgcccaac gacccgagcg tgcgcatgca cgaggcgctc 2280
ggatatgccc cccgcggcat gctgcgggcg gccggcttca agcacgggaa ctggcatgac 2340ggatatgccc cccgcggcat gctgcgggcg gccggcttca agcacgggaa ctggcatgac 2340
gtgggtttct ggcagctgga cttcagcctg ccggtaccgc cccgtccggt cctgcccgtc 2400gtgggtttct ggcagctgga cttcagcctg ccggtaccgc cccgtccggt cctgcccgtc 2400
accgagatct ga 2412accgagatct ga 2412
<210> 2<210> 2
<211> 57<211> 57
<212> DNA<212> DNA
<213> Artificial sequence<213> Artificial sequence
<400> 2<400> 2
ctgttgaatt tcgatcttct taagcttgct ggtgatgttg aatccaaccc aggtcca 57ctgttgaatt tcgatcttct taagcttgct ggtgatgttg aatccaaccc aggtcca 57
<210> 3<210> 3
<211> 31<211> 31
<212> DNA<212> DNA
<213> Artificial sequence<213> Artificial sequence
<400> 3<400> 3
ccgctcgaga tgaaaaagcc tgaactcacc g 31ccgctcgaga tgaaaaagcc tgaactcacc g 31
<210> 4<210> 4
<211> 59<211> 59
<212> DNA<212> DNA
<213> Artificial sequence<213> Artificial sequence
<400> 4<400> 4
acatcaccag caagcttaag aagatcgaaa ttcaacagct atttctttgc cctcggacg 59acatcaccag caagcttaag aagatcgaaa ttcaacagct atttctttgc cctcggacg 59
<210> 5<210> 5
<211> 60<211> 60
<212> DNA<212> DNA
<213> Artificial sequence<213> Artificial sequence
<400> 5<400> 5
cttaagcttg ctggtgatgt tgaatccaac ccaggtccaa tggtgagcaa gggcgaggag 60cttaagcttg ctggtgatgt tgaatccaac ccaggtccaa tggtgagcaa gggcgaggag 60
<210> 6<210> 6
<211> 59<211> 59
<212> DNA<212> DNA
<213> Artificial sequence<213> Artificial sequence
<400> 6<400> 6
acatcaccag caagcttaag aagatcgaaa ttcaacagtc agatctcggt gacgggcag 59acatcaccag caagcttaag aagatcgaaa ttcaacagtc agatctcggt gacgggcag 59
<210> 7<210> 7
<211> 60<211> 60
<212> DNA<212> DNA
<213> Artificial sequence<213> Artificial sequence
<400> 7<400> 7
cttaagcttg ctggtgatgt tgaatccaac ccaggtccaa tgagcccaga acgacgcccg 60cttaagcttg ctggtgatgt tgaatccaac ccaggtccaa tgagcccaga acgacgcccg 60
<210> 8<210> 8
<211> 30<211> 30
<212> DNA<212> DNA
<213> Artificial sequence<213> Artificial sequence
<400> 8<400> 8
ccgctcgagt cagatctcgg tgacgggcag 30ccgctcgagt cagatctcgg tgacgggcag 30
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910705015.0ACN110468149A (en) | 2019-08-01 | 2019-08-01 | A kind of novel Study of Marker Genes in Transgenic Plants building and application |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910705015.0ACN110468149A (en) | 2019-08-01 | 2019-08-01 | A kind of novel Study of Marker Genes in Transgenic Plants building and application |
| Publication Number | Publication Date |
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| CN110468149Atrue CN110468149A (en) | 2019-11-19 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910705015.0APendingCN110468149A (en) | 2019-08-01 | 2019-08-01 | A kind of novel Study of Marker Genes in Transgenic Plants building and application |
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| CN (1) | CN110468149A (en) |
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