CN102899282A - Sugarcane callus protoplast separation and purification method - Google Patents
- ️Wed Jan 30 2013
CN102899282A - Sugarcane callus protoplast separation and purification method - Google Patents
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- CN102899282A CN102899282A CN2012103918779A CN201210391877A CN102899282A CN 102899282 A CN102899282 A CN 102899282A CN 2012103918779 A CN2012103918779 A CN 2012103918779A CN 201210391877 A CN201210391877 A CN 201210391877A CN 102899282 A CN102899282 A CN 102899282A Authority
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Abstract
本发明涉及一种甘蔗愈伤组织原生质体的分离与纯化方法，包括甘蔗愈伤组织培养、甘蔗悬浮细胞培养、甘蔗原生质体的分离和甘蔗原生质体的纯化。利用本发明的方法可以快速从甘蔗胚性愈伤组织培养获得悬浮细胞系，并通过酶消解后分离纯化获得质量高、数量足、均一性好的完整原生质体，为甘蔗外源基因表达、启动子分析、基因沉默等转基因瞬时表达研究提供理想的实验材料。
The invention relates to a method for separating and purifying sugarcane callus protoplasts, comprising sugarcane callus culture, sugarcane suspension cell culture, separation of sugarcane protoplasts and purification of sugarcane protoplasts. The method of the present invention can be used to quickly obtain suspension cell lines from sugarcane embryogenic callus culture, and obtain high-quality, sufficient quantity, and good uniformity of complete protoplasts through separation and purification after enzymatic digestion, which can be used for the expression and activation of exogenous genes in sugarcane. It provides ideal experimental materials for transient expression studies of transgenes such as subanalysis and gene silencing.
Description
技术领域 technical field
本发明涉及一种植物原生质体的分离与纯化方法，具体涉及一种甘蔗愈伤组织原生质体的分离与纯化方法，属生物技术领域。 The invention relates to a method for separating and purifying plant protoplasts, in particular to a method for separating and purifying protoplasts from sugarcane callus, and belongs to the field of biotechnology.
背景技术 Background technique
植物原生质体克服了细胞壁这一屏障，是植物生理学、细胞生物学、体细胞遗传学等理论研究的理想材料，而且在基因工程及作物改良新品种培育方面有着重要应用前景。近年来，随着植物分子生物学发展，原生质体作为一个单细胞系统广泛地应用在基因定位、基因表达调控、RNA沉默机理、细胞钙信号转导及其调节机制等方面的研究。 Plant protoplasts have overcome the barrier of the cell wall, and are ideal materials for theoretical research on plant physiology, cell biology, and somatic cell genetics, and have important application prospects in genetic engineering and the cultivation of new varieties of crop improvement. In recent years, with the development of plant molecular biology, protoplasts, as a single-cell system, have been widely used in the research of gene localization, gene expression regulation, RNA silencing mechanism, cellular calcium signal transduction and its regulation mechanism, etc.
甘蔗愈伤组织原生质体的研究始于上世纪70年代，原生质体分离、培养和植株再生也获得成功。但是，与模式植物烟草或拟南芥叶肉细胞原生质体研究相比，甘蔗愈伤组织原生质体分离纯化效果不尽理想，植株再生频率很低，直至今日没有长足发展。以甘蔗愈伤组织原生质体为受体材料，开展甘蔗转基因瞬时表达未见报道。本发明建立高效、快速的甘蔗愈伤组织原生质体的分离和纯化方法，获得高质量、高产量的原生质体可作为外源基因表达、启动子分析、基因沉默等转基因瞬时表达研究的受体材料。 The research on sugarcane callus protoplasts began in the 1970s, and the isolation, cultivation and plant regeneration of protoplasts were also successful. However, compared with the model plant tobacco or Arabidopsis mesophyll cell protoplasts, the separation and purification of sugarcane callus protoplasts is not ideal, and the plant regeneration frequency is very low, so it has not made great progress until today. There is no report on the transient expression of sugarcane transgenes using sugarcane callus protoplasts as recipient materials. The present invention establishes an efficient and rapid separation and purification method of sugarcane callus protoplasts, and obtains high-quality, high-yield protoplasts, which can be used as receptor materials for transgene transient expression studies such as exogenous gene expression, promoter analysis, and gene silencing. .
发明内容 Contents of the invention
本发明的目的是提供一种快速、高效、简便的甘蔗愈伤组织原生质体的分离与纯化方法，克服现有甘蔗愈伤组织原生质体分离和纯化方法存在质量和数量上的不足的问题，为甘蔗外源基因表达、启动子分析、基因沉默等转基因瞬时表达研究提供高质量、高产量的甘蔗愈伤组织原生质体。 The purpose of the present invention is to provide a kind of fast, efficient, easy separation and purification method of sugarcane callus protoplast, to overcome the existing quality and quantitative problems of sugarcane callus protoplast separation and purification method, for Sugarcane exogenous gene expression, promoter analysis, gene silencing and other transgene transient expression studies provide high-quality, high-yield sugarcane callus protoplasts.
本发明的目的是通过以下方法实现的。 The object of the present invention is achieved by the following methods.
本发明的甘蔗愈伤组织原生质体的分离与纯化方法，包括甘蔗愈伤组织培养、甘蔗悬浮细胞培养、甘蔗原生质体的分离和甘蔗原生质体的纯化；其特征在于操作步骤如下： The method for separating and purifying sugarcane callus protoplasts of the present invention comprises sugarcane callus culture, sugarcane suspension cell culture, separation of sugarcane protoplasts and purification of sugarcane protoplasts; it is characterized in that the operation steps are as follows:
1、甘蔗愈伤组织培养 1. Sugarcane callus culture
取消毒后的甘蔗心叶组织，无菌条件下切成1-2 cm厚的薄片，在MS3固体培养基上，28 ℃暗培养1-2个月，每隔2周继代1次；所述MS3固体培养基为：MS+0.5 g/L水解酪蛋白+3 mg/L 2,4-D+30 g/L蔗糖+6.0 g/L琼脂粉；MS培养基为1962年，Murashige和Skoog公开的MS培养基（下同）； The detoxified sugarcane heart leaf tissue was cut into 1-2 cm thick slices under aseptic conditions, cultured in the dark at 28°C for 1-2 months on MS3 solid medium, and subcultured once every 2 weeks; the MS3 The solid medium is: MS+0.5 g/L hydrolyzed casein+3 mg/L 2,4-D+30 g/L sucrose+6.0 g/L agar powder; MS medium was published by Murashige and Skoog in 1962 MS medium (the same below);
2、甘蔗悬浮细胞培养 2. Sugarcane suspension cell culture
将甘蔗胚性愈伤组织捣碎后，在MS3液体培养基中，28 ℃暗培养2周，纱布过滤，滤液静置10-15 min，吸取底部胚性愈伤组织培养液，于恒温摇床震荡培养，转速120转/秒，28 ℃暗培养1-2月，每隔1周继代1次，直至悬浮细胞系出现；所述MS3液体培养基：MS+0.5 g/L水解酪蛋白+3 mg/L 2,4-D+30 g/L蔗糖，去离子水补足1升； After mashing the sugarcane embryogenic callus, culture it in MS3 liquid medium at 28°C for 2 weeks in the dark, filter it through gauze, let the filtrate stand for 10-15 min, absorb the embryogenic callus culture medium at the bottom, and put it on a constant temperature shaker. Shaking culture, rotation speed 120 rpm, 28 ℃ dark culture for 1-2 months, subculture once every 1 week, until the suspension cell line appears; the MS3 liquid medium: MS+0.5 g/L hydrolyzed casein+3 mg /L 2,4-D+30 g/L sucrose, make up 1 liter of deionized water;
3、甘蔗原生质体的分离 3. Isolation of sugarcane protoplasts
取步骤2的新鲜悬浮细胞分装到50 mL离心管，100 g离心2 min；弃上清液，用酶解缓冲液清洗悬浮细胞后，100 g离心2 min；弃上清液，取悬浮细胞2-3克加入6孔的细胞培养板内，用3 mL酶解反应液酶解过夜，分离出甘蔗愈伤组织原生质体；所述酶解缓冲液：0.02 mol/L MES+0.4 mol/L甘露醇+0.02 mol/L氯化钾，pH 5.7；所述酶解反应液：2 %纤维素酶+0.1 %果胶酶+0.01 mol/L氯化钙+0.1 %牛血清蛋白，溶解于酶解缓冲液；在酶解反应液中，各原料所占的百分比为各原料与酶解缓冲液的重量百分比； Take the fresh suspended cells from step 2 and put them into 50 mL centrifuge tubes, centrifuge at 100 g for 2 min; discard the supernatant, wash the suspended cells with enzymatic hydrolysis buffer, and centrifuge at 100 g for 2 min; discard the supernatant, and take the suspended cells Add 2-3 grams into a 6-well cell culture plate, and use 3 mL of enzymatic hydrolysis reaction solution to enzymolyze overnight to separate sugarcane callus protoplasts; the enzymatic hydrolysis buffer: 0.02 mol/L MES+0.4 mol/L Mannitol + 0.02 mol/L potassium chloride, pH 5.7; the enzymatic hydrolysis reaction solution: 2% cellulase + 0.1% pectinase + 0.01 mol/L calcium chloride + 0.1% bovine serum albumin, dissolved in enzyme solution buffer; in the enzymolysis reaction solution, the percentage of each raw material is the weight percentage of each raw material and the enzymolysis buffer;
4、甘蔗原生质体的纯化 4. Purification of sugarcane protoplasts
用无菌水清洗70 μm 孔径的尼龙网，然后用W5溶液润洗，取步骤3获得的原生质体转移至10 mL离心管中，100 g离心2 min；弃上清液，冰浴的W5溶液悬浮原生质体，100 g离心1 min；弃上清液后即为纯化的甘蔗愈伤组织原生质体，在电子显微镜下观察，形态正常的原生质体量达106个/mL；所述W5溶液：2 mmol/L MES+154 mmol/L 氯化纳+125 mmol/L 氯化钙+5 mmol/L 氯化钾，pH 5.7。 Wash the nylon mesh with a pore size of 70 μm with sterile water, then rinse with W5 solution, transfer the protoplasts obtained in step 3 to a 10 mL centrifuge tube, and centrifuge at 100 g for 2 min; discard the supernatant, and put the W5 solution on ice Suspend the protoplasts and centrifuge at 100 g for 1 min; discard the supernatant to obtain purified sugarcane callus protoplasts. Observed under an electron microscope, the amount of protoplasts with normal morphology reaches 10 6 /mL; the W5 solution: 2 mmol/L MES+154 mmol/L sodium chloride+125 mmol/L calcium chloride+5 mmol/L potassium chloride, pH 5.7.
本发明还保护利用上述方法分离与纯化的甘蔗愈伤组织原生质体在甘蔗转基因瞬时表达中的应用。 The invention also protects the application of the sugarcane callus protoplast isolated and purified by the above method in the transient expression of sugarcane transgene.
本发明使用的所有化学试剂为分析纯。 All chemical reagents used in the present invention are analytically pure.
本发明的优点是，快速从甘蔗胚性愈伤组织培养获得悬浮细胞系，并通过酶消解后分离纯化获得质量高、数量足、均一性好的完整原生质体，为甘蔗外源基因表达、启动子分析、基因沉默等转基因瞬时表达研究提供理想的实验材料。 The advantage of the present invention is that the suspension cell line can be quickly obtained from sugarcane embryogenic callus culture, and the complete protoplasts with high quality, sufficient quantity and good uniformity can be obtained by separating and purifying after enzymatic digestion, which can be used for the expression and activation of exogenous genes in sugarcane. It provides ideal experimental materials for transient expression studies of transgenes such as subanalysis and gene silencing.
附图说明 Description of drawings
图1是分离纯化后的甘蔗原生质体，标尺为0.2 μm。 Figure 1 is the isolated and purified sugarcane protoplasts, the scale bar is 0.2 μm.
图2是pUbi-EYFP-Nos和pUbi-P19-Nos（P19是番茄丛矮病毒编码的RNA沉默抑制子）质粒转化后，EYFP基因在原生质体上的瞬时表达，标尺为0.2 mm。 Figure 2 shows the transient expression of EYFP gene in protoplasts after pUbi-EYFP-Nos and pUbi-P19-Nos (P19 is an RNA silencing suppressor encoded by tomato bushy dwarf virus) plasmid transformation, the bar is 0.2 mm.
图3是pUbi-EYFP-Nos和pUbi-γb-Nos（γb是大麦条纹花叶病毒编码的RNA沉默抑制子）质粒转化后，EYFP基因在原生质体上的瞬时表达，标尺为0.2 mm。 Figure 3 shows the transient expression of EYFP gene in protoplasts after pUbi-EYFP-Nos and pUbi-γb-Nos (γb is the RNA silencing suppressor encoded by barley stripe mosaic virus) plasmid transformation, the scale bar is 0.2 mm.
图4是pUbi-EYFP-Nos和pUbi-Nos（不含任何沉默抑制子的空载体）质粒转化后，EYFP基因在原生质体上的瞬时表达，标尺为0.2 mm。 Figure 4 shows the transient expression of EYFP gene in protoplasts after pUbi-EYFP-Nos and pUbi-Nos (empty vector without any silencing suppressor) plasmid transformation, the scale bar is 0.2 mm.
图5是不同启动子质粒转化原生质体后GUS的瞬时表达活性，其中，A为pPr4-GUS-Nos，B为pUbi-GUS-Nos，C为p35S-GUS-Nos，D为pUbi-Nos（空载体）质粒；左侧的标尺表示GUS活性值，单位为pmol 4-MU/μg protein·min，图中误差线为标准误。 Figure 5 is the transient expression activity of GUS after different promoter plasmids transform protoplasts, wherein, A is pPr4-GUS-Nos, B is pUbi-GUS-Nos, C is p35S-GUS-Nos, D is pUbi-Nos (empty Vector) plasmid; the scale on the left indicates the GUS activity value, the unit is pmol 4-MU/μg protein min, and the error bars in the figure are standard errors.
具体实施方式 Detailed ways
为了进一步阐明本发明而不是限制本发明，以下结合实施例加以说明。 In order to further illustrate the present invention rather than limit the present invention, the following examples will be used for illustration.
实施例1：一种甘蔗愈伤组织原生质体的分离与纯化方法，包括以下步骤： Embodiment 1: a kind of separation and purification method of sugarcane callus protoplast comprises the following steps:
1、甘蔗愈伤组织培养：选取生长正常且无病虫害的甘蔗梢部，去除外围的老叶直至最高可见肥厚带叶和顶部长出的多余叶片，保留25 cm长的心叶组织；用体积比为75 %的酒精溶液进行表面擦洗消毒后，在无菌超净工作台里，将甘蔗心叶组织切成1.5 cm厚的薄片，在MS3固体培养基上，28 ℃暗培养1.5个月，每隔2周继代1次； 1. Sugarcane callus culture: select sugarcane tips that grow normally and are free from diseases and insect pests, remove peripheral old leaves until the highest visible hypertrophic leaves and excess leaves grow from the top, and retain 25 cm long heart leaf tissue; use volume ratio After scrubbing and disinfecting the surface with 75% alcohol solution, the sugarcane heart leaf tissue was cut into 1.5 cm thick slices in a sterile ultra-clean workbench, and cultured on MS3 solid medium at 28°C for 1.5 months in the dark, every Subculture once every 2 weeks;
2、甘蔗悬浮细胞培养：挑选致密、金黄色、颗粒状的胚性愈伤组织5克，捣碎后，移入100 mL MS3液体培养基，28℃暗培养2周后，将胚性愈伤组织培养液纱布过滤，滤液静置12 min，用移液管吸取底部10 mL细小胚性愈伤组织培养液，用100 mL MS3液体培养基在恒温摇床里震荡培养，转速120 转/秒，28℃暗培养1-2月，每隔1周继代1次，直至细小、均一的悬浮细胞系出现。 2. Suspension cell culture of sugarcane: Select 5 grams of dense, golden yellow, granular embryogenic callus, mash it, transfer it into 100 mL MS3 liquid medium, and culture it in the dark at 28°C for 2 weeks. The culture solution was filtered with gauze, the filtrate was allowed to stand for 12 min, and the bottom 10 mL of small embryogenic callus culture solution was sucked with a pipette, and 100 mL of MS3 liquid medium was used for shaking culture in a constant temperature shaker at a speed of 120 rpm, 28 Cultivate in dark at ℃ for 1-2 months, and subculture once every 1 week until fine and uniform suspension cell lines appear.
3、取步骤2的新鲜悬浮细胞分装到50 mL离心管，或者将步骤2的悬浮细胞继代培养3天后，分装到50 mL离心管；100 g离心2 min；弃上清液，加入40 mL 酶解缓冲液清洗悬浮细胞，100 g离心2 min；弃上清液，取悬浮细胞2-3 克加入6孔的细胞培养板内，用3 mL酶解反应液酶解过夜，分离出甘蔗愈伤组织原生质体； 3. Take the fresh suspension cells in step 2 and aliquot them into 50 mL centrifuge tubes, or subculture the suspension cells in step 2 for 3 days, then aliquot them into 50 mL centrifuge tubes; centrifuge at 100 g for 2 min; discard the supernatant, add Wash the suspended cells with 40 mL of enzymatic hydrolysis buffer, centrifuge at 100 g for 2 min; discard the supernatant, take 2-3 grams of suspended cells and add them to a 6-well cell culture plate, and use 3 mL of enzymatic hydrolysis reaction solution to hydrolyze overnight, and separate Sugarcane callus protoplasts;
4、用无菌水清洗70μm 孔径的尼龙网，然后用W5溶液润洗，将步骤3酶解过夜的甘蔗悬浮细胞通过尼龙网过滤，取5 mL滤液转移至10 mL离心管中，100 g离心2 min；弃上清液，1 mL冰浴的W5溶液悬浮原生质体，100 g离心1 min；弃上清液后即为纯化的甘蔗愈伤组织原生质体。 4. Wash the nylon mesh with a pore size of 70 μm with sterile water, and then rinse it with W5 solution. Filter the sugarcane suspension cells that were enzymatically hydrolyzed overnight in step 3 through the nylon mesh, transfer 5 mL of the filtrate to a 10 mL centrifuge tube, and centrifuge at 100 g. 2 min; discard the supernatant, suspend the protoplasts in 1 mL ice-bathed W5 solution, and centrifuge at 100 g for 1 min; discard the supernatant to obtain the purified sugarcane callus protoplasts.
取1 mL冰浴的W5溶液再次悬浮原生质体；用血球计数板在100倍显微镜下计算原生质体数量，完整的原生质体如图1所示，低温贮藏备用。 Take 1 mL of ice-bathed W5 solution to suspend protoplasts again; use a hemocytometer to count the number of protoplasts under a 100-fold microscope. The complete protoplasts are shown in Figure 1, and they are stored at low temperature for later use.
实施例2：利用从甘蔗品种CP72-1210心叶愈伤组织分离、纯化的原生质体，研究不同植物病毒编码的沉默抑制子的活性。 Example 2: Using protoplasts isolated and purified from heart leaf callus of sugarcane variety CP72-1210, the activities of silencing suppressors encoded by different plant viruses were studied.
1、PEG遗传转化 1. PEG genetic transformation
1.1 将制备好的甘蔗CP72-1210愈伤组织原生质体冰浴30 min，去除W5溶液，用1-2 mL MMG溶液悬浮原生质体浓度至每毫升2×105个。 1.1 Place the prepared sugarcane CP72-1210 callus protoplasts in an ice bath for 30 min, remove the W5 solution, and suspend the protoplasts with 1-2 mL MMG solution to a concentration of 2×10 5 per ml.
1.2 分别取5 μL的pUbi-EYFP-Nos和pUbi-P19-Nos质粒DNA（1 μg/μl），5 μL的pUbi-EYFP-Nos和pUbi-γb-Nos质粒DNA（1μg/μl），5 μL 的pUbi-EYFP –Nos和pUbi-Nos质粒DNA（1 μg/μl）到2 mL离心管。 1.2 Take 5 μL of pUbi-EYFP-Nos and pUbi-P19-Nos plasmid DNA (1 μg/μl), 5 μL of pUbi-EYFP-Nos and pUbi-γb-Nos plasmid DNA (1 μg/μl), 5 μL pUbi-EYFP-Nos and pUbi-Nos plasmid DNA (1 μg/μl) into 2 mL centrifuge tubes.
1.3 加入100 μL甘蔗CP72-1210原生质体，混合后，加入110 μL 40%聚乙二醇溶液（PEG-4000），混匀，室温温浴5-15 min。 1.3 Add 100 μL sugarcane CP72-1210 protoplasts, after mixing, add 110 μL 40% polyethylene glycol solution (PEG-4000), mix well, and incubate at room temperature for 5-15 minutes.
1.4 加入440 uL W5溶液，混匀，终止遗传转化，100 g离心2 min。弃上清液，1 mL WI溶液悬浮原生质体，然后移入6孔的细胞培养板，室温放置24 h。 1.4 Add 440 uL W5 solution, mix well, stop genetic transformation, and centrifuge at 100 g for 2 min. Discard the supernatant, suspend the protoplasts with 1 mL WI solution, then transfer to a 6-well cell culture plate, and place at room temperature for 24 h.
MMG溶液：4 mmol/L MES+0.4 mol/L 甘露醇+15 mmol/L 氯化镁，pH 5.7。 MMG solution: 4 mmol/L MES+0.4 mol/L mannitol+15 mmol/L magnesium chloride, pH 5.7.
WI溶液：4 mmol/L MES+0.5 mol/L甘露醇+20 mmol/L氯化钾，pH 5.7。 WI solution: 4 mmol/L MES+0.5 mol/L mannitol+20 mmol/L potassium chloride, pH 5.7.
聚乙二醇转化液：40% (W/V) 聚乙二醇（PEG4000）+0.2 mol/L 甘露醇+100 mmol/L氯化钙。 Polyethylene glycol conversion solution: 40% (W/V) polyethylene glycol (PEG4000) + 0.2 mol/L mannitol + 100 mmol/L calcium chloride.
2、在荧光显微镜下（YFP荧光滤片）观察表达EYFP原生质体数目，如图2、图3和图4所示，不同植物病毒编码的沉默抑制子的活性，表现为P19和γb沉默抑制子可以提高外源基因EYFP表达水平，平均增幅分别达63%和56%。 2. Observe the number of protoplasts expressing EYFP under a fluorescent microscope (YFP fluorescent filter). As shown in Figure 2, Figure 3 and Figure 4, the activities of silencing suppressors encoded by different plant viruses are expressed as P19 and γb silencing suppressors It can increase the expression level of exogenous gene EYFP, with an average increase of 63% and 56%, respectively.
实例3：利用从甘蔗品种CP72-1210心叶愈伤组织分离、纯化的原生质体，研究不同启动子的活性。 Example 3: Using the protoplasts isolated and purified from the heart leaf callus of sugarcane variety CP72-1210, the activities of different promoters were studied.
1、转基因瞬时表达 1. Transient expression of transgene
1.1 将制备好的甘蔗CP72-1210愈伤组织原生质体冰浴30 min，去除W5溶液，用1-2 mL MMG溶液悬浮原生质体浓度至每毫升2×105个。 1.1 Place the prepared sugarcane CP72-1210 callus protoplasts in an ice bath for 30 min, remove the W5 solution, and suspend the protoplasts with 1-2 mL MMG solution to a concentration of 2×10 5 per ml.
1.2 分别取10 μL质粒DNA（pPr4-GUS-NOS、pUbi-GUS-NOS、p35S-GUS-NOS，10 μg）到2 mL离心管中，设空载体（pUbi-NOS）、水为空白对照。 1.2 Take 10 μL of plasmid DNA (pPr4-GUS-NOS, pUbi-GUS-NOS, p35S-GUS-NOS, 10 μg) into 2 mL centrifuge tubes, set empty vector (pUbi-NOS) and water as blank control.
1.3 加入100 μL原生质体，混合后，加入110 μL 40 %聚乙二醇转化液，混匀，室温温浴5-15 min。 1.3 Add 100 μL of protoplasts, after mixing, add 110 μL of 40% polyethylene glycol conversion solution, mix well, and incubate at room temperature for 5-15 minutes.
1.4 加入440 μL W5溶液轻轻混匀，终止遗传转化，100 g离心2 min。弃上清液，1 mL WI溶液悬浮原生质体，然后移入6孔的细胞培养板，室温放置24 h。 1.4 Add 440 μL W5 solution and mix gently to terminate genetic transformation, and centrifuge at 100 g for 2 min. Discard the supernatant, suspend the protoplasts with 1 mL WI solution, then transfer to a 6-well cell culture plate, and place at room temperature for 24 h.
2、GUS活性测定 2. Determination of GUS activity
2.1 将甘蔗CP72-1210愈伤组织原生质体装入1.5 mL离心管，100 g离心2 min，弃上清液，取25 μL等量原生质体分装到1.5 mL离心管。 2.1 Put the sugarcane CP72-1210 callus protoplasts into a 1.5 mL centrifuge tube, centrifuge at 100 g for 2 min, discard the supernatant, and take 25 μL equal amount of protoplasts and aliquot them into 1.5 mL centrifuge tubes.
2.2 加入25 μL MUG底物反应液，37 ℃反应60 min。 2.2 Add 25 μL of MUG substrate reaction solution and react at 37 °C for 60 min.
2.3 加入950 μL 碳酸钠溶液（0.2 mol/L）终止反应。 2.3 Add 950 μL sodium carbonate solution (0.2 mol/L) to stop the reaction.
2.4 用荧光分光光度计测定荧光强度。 2.4 Measure the fluorescence intensity with a fluorescence spectrophotometer.
2.5 原生质质体总蛋白含量采用考马斯亮蓝法( Bradford法)。 2.5 Coomassie brilliant blue method (Bradford method) was used for the total protein content of protoplasts.
2.6 4-MU标准曲线的绘制 2.6 Drawing of 4-MU standard curve
在0.2 mol/L 碳酸钠溶液的终止液中加入4-MU, 配成不同4-MU 浓度的溶液, 然后利用荧光分光光度计测其产生的荧光(激发光 365 nm, 发射光 455 nm), 绘制标准曲线。 Add 4-MU to the stop solution of 0.2 mol/L sodium carbonate solution to make solutions with different 4-MU concentrations, and then use a fluorescence spectrophotometer to measure the fluorescence generated (excitation light 365 nm, emission light 455 nm), Draw a standard curve.
2.7 GUS活性计算 2.7 Calculation of GUS activity
GUS活性用每微克蛋白每分钟形成的4-MU的pmol表示,计算公式为 GUS activity is expressed in pmol of 4-MU formed per microgram of protein per minute, and the calculation formula is
GUS活性=MUG分解形成的4-MU（pmol）÷[可溶解性蛋白（μg）×反应时间（min）]。实验重复3 次。 GUS activity = 4-MU formed by MUG decomposition (pmol) ÷ [soluble protein (μg) × reaction time (min)]. The experiment was repeated three times.
GUS缓冲液：50 mmol/L 磷酸缓冲液（pH 7.0），10 mmol/L β-巯基乙醇，10 mmol/L乙二胺四乙酸（pH 8.0），0.1% (v/v) Triton X-100。 GUS buffer: 50 mmol/L phosphate buffer (pH 7.0), 10 mmol/L β-mercaptoethanol, 10 mmol/L ethylenediaminetetraacetic acid (pH 8.0), 0.1% (v/v) Triton X-100 .
MUG底物反应液：2 mmol/L MUG溶解于GUS缓冲液。 MUG substrate reaction solution: 2 mmol/L MUG dissolved in GUS buffer.
2.8 不同启动子活性差异：如图5所示，不同启动子活性有所差异，启动子活性依次为Pr4、Ubi、35S，其驱使GUS活性分别为40.86、15.71、4.99 pmol 4-MU/μg protein·min。 2.8 Differences in the activities of different promoters: As shown in Figure 5, the activities of different promoters are different. The promoter activities are Pr4, Ubi, and 35S in sequence, and the GUS activities driven by them are 40.86, 15.71, and 4.99 pmol 4-MU/μg protein respectively. min. the
Claims (2)
1.一种甘蔗愈伤组织原生质体的分离与纯化方法，包括甘蔗愈伤组织培养、甘蔗悬浮细胞培养、甘蔗原生质体的分离和甘蔗原生质体的纯化；其特征在于操作步骤如下： 1. a method for separating and purifying sugarcane callus protoplasts, comprising sugarcane callus culture, sugarcane suspension cell culture, the separation of sugarcane protoplasts and the purification of sugarcane protoplasts; it is characterized in that the operating steps are as follows: (1) 甘蔗愈伤组织培养 (1) Sugarcane callus culture 取消毒后的甘蔗心叶组织，无菌条件下切成1-2 cm厚的薄片，在MS3固体培养基上，28 ℃暗培养1-2个月，每隔2周继代1次；所述MS3固体培养基为：MS+0.5 g/L水解酪蛋白+3 mg/L 2,4-D+30 g/L蔗糖+6.0 g/L琼脂粉；MS培养基为1962年，Murashige和Skoog公开的MS培养基； The detoxified sugarcane heart leaf tissue was cut into 1-2 cm thick slices under aseptic conditions, cultured in the dark at 28°C for 1-2 months on MS3 solid medium, and subcultured once every 2 weeks; the MS3 The solid medium is: MS+0.5 g/L hydrolyzed casein+3 mg/L 2,4-D+30 g/L sucrose+6.0 g/L agar powder; MS medium was published by Murashige and Skoog in 1962 MS medium; (2) 甘蔗悬浮细胞培养 (2) Sugarcane suspension cell culture 将甘蔗胚性愈伤组织捣碎后，在MS3液体培养基中，28℃暗培养2周，纱布过滤，滤液静置10-15 min，吸取底部胚性愈伤组织培养液，于恒温摇床震荡培养，转速120转/秒，28 ℃暗培养1-2月，每隔1周继代1次，直至悬浮细胞系出现；所述MS3液体培养基：MS+0.5 g/L水解酪蛋白+3 mg/L 2,4-D+30 g/L蔗糖，去离子水补足1升； After mashing the sugarcane embryogenic callus, culture it in MS3 liquid medium at 28°C for 2 weeks in the dark, filter it through gauze, let the filtrate stand for 10-15 min, absorb the embryogenic callus culture medium at the bottom, and put it on a constant temperature shaker. Shaking culture, rotation speed 120 rpm, 28 ℃ dark culture for 1-2 months, subculture once every 1 week, until the suspension cell line appears; the MS3 liquid medium: MS+0.5 g/L hydrolyzed casein+3 mg /L 2,4-D+30 g/L sucrose, make up 1 liter of deionized water; (3) 甘蔗原生质体的分离 (3) Isolation of sugarcane protoplasts 取步骤（2）的新鲜悬浮细胞分装到50 mL离心管，100 g离心2 min；弃上清液，用酶解缓冲液清洗悬浮细胞后，100 g离心2 min；弃上清液，取悬浮细胞2-3 g加入6孔的细胞培养板内，用3 mL酶解反应液酶解过夜，分离出甘蔗愈伤组织原生质体；所述酶解缓冲液：0.02 mol/L MES+0.4 mol/L甘露醇+0.02 mol/L氯化钾，pH 5.7；所述酶解反应液：2 %纤维素酶+0.1 %果胶酶+0.01 mol/L氯化钙+0.1 %牛血清蛋白，溶解于酶解缓冲液；在酶解反应液中，各原料所占的百分比为各原料与酶解缓冲液的重量百分比； Take the fresh suspended cells from step (2) and put them into 50 mL centrifuge tubes, centrifuge at 100 g for 2 min; discard the supernatant, wash the suspended cells with enzymatic hydrolysis buffer, and centrifuge at 100 g for 2 min; discard the supernatant, take Add 2-3 g of suspended cells into a 6-well cell culture plate, and use 3 mL of enzymatic hydrolysis reaction solution to enzymolyze overnight to separate sugarcane callus protoplasts; the enzymatic hydrolysis buffer: 0.02 mol/L MES+0.4 mol /L mannitol+0.02 mol/L potassium chloride, pH 5.7; the enzymolysis reaction solution: 2% cellulase+0.1% pectinase+0.01 mol/L calcium chloride+0.1% bovine serum albumin, dissolved in the enzymolysis buffer; in the enzymolysis reaction solution, the percentage of each raw material is the weight percentage of each raw material and the enzymolysis buffer; (4) 甘蔗原生质体的纯化 (4) Purification of sugarcane protoplasts 用无菌水清洗70μm孔径的尼龙网，然后用W5溶液润洗，取步骤（3）获得的原生质体转移至10 mL离心管中，100 g离心2 min；弃上清液，冰浴的W5溶液悬浮原生质体，100 g离心1 min；弃上清液后即为纯化的甘蔗愈伤组织原生质体；所述W5溶液：2 mmol/L MES+154 mmol/L 氯化纳+125 mmol/L 氯化钙+5 mmol/L 氯化钾，pH 5.7。 Wash the nylon mesh with a pore size of 70 μm with sterile water, and then rinse it with the W5 solution. Transfer the protoplasts obtained in step (3) to a 10 mL centrifuge tube, centrifuge at 100 g for 2 min; discard the supernatant, and put the W5 on ice. Suspend the protoplasts in the solution, centrifuge at 100 g for 1 min; discard the supernatant to obtain the purified sugarcane callus protoplasts; the W5 solution: 2 mmol/L MES+154 mmol/L sodium chloride+125 mmol/L Calcium chloride + 5 mmol/L potassium chloride, pH 5.7. 2.由权利要求1的方法分离与纯化的甘蔗愈伤组织原生质体在甘蔗转基因瞬时表达中的应用。 2. the application of the sugarcane callus protoplast isolated and purified by the method of claim 1 in the transient expression of sugarcane transgene.
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| CN110669718A (en) * | 2019-09-30 | 2020-01-10 | 江苏省中国科学院植物研究所 | A method for isolating, purifying, and instantaneously efficient transformation of protoplasts from the roots, stems and leaves of Chinese fir |
| CN111202005A (en) * | 2020-03-24 | 2020-05-29 | 广西壮族自治区农业科学院 | Sugarcane variety embryonic callus induction method |
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| CN115287248A (en) * | 2022-07-06 | 2022-11-04 | 贵州大学 | Preparation method of eucommia ulmoides protoplast |
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| CN116463276B (en) * | 2023-04-28 | 2024-05-14 | 广西大学 | Method for isolating and culturing sugarcane suspended single cells and method for regenerating them into plants |
| CN116536242A (en) * | 2023-05-19 | 2023-08-04 | 云南省农业科学院甘蔗研究所 | Rapid and efficient sugarcane protoplast preparation method |
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