CN114774463A - 一种基于手动加压的高效瞬时茄科作物转基因方法 - Google Patents
一种基于手动加压的高效瞬时茄科作物转基因方法 Download PDFInfo
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Abstract
本发明提供了一种基于手动加压的高效瞬时茄科作物转基因方法,首先配制携带目标基因的农杆菌侵染液,然后将注射器空筒的细口处封闭,将大小适宜的茄科作物叶片和农杆菌侵染液置于注射器空筒内,并使茄科作物叶片完全浸入农杆菌侵染液中,通过反复按压注射器推杆促使菌液全部浸染茄科作物叶片,最后将浸染的茄科作物叶片取出,再培养获得瞬时转基因茄科作物叶片。该方法通过反复抽拉注射器将农杆菌侵染液多次正压渗入茄科作物叶片,从而将外源基因高效瞬时导入植物叶片细胞中。相比传统方法,本发明方法具有耗时短、操作简单、成本低、效率高等诸多优势。
Description
技术领域
本发明属于基因工程技术领域,涉及一种农杆菌介导的在茄科作物叶片中实现外源基因瞬时表达的方法,具体涉及一种基于手动加压的高效瞬时茄科作物转基因方法。
背景技术
番茄是一种在世界范围内广泛种植的园艺、经济作物。番茄具有生长周期短、自花授粉等特点,逐渐成为园艺作物中研究基因功能及作用机制的模式作物。近年来,番茄的转基因体系越来越成熟,但是获得稳定的转基因植株需要严格的无菌环境以及无菌操作,而且存在转化效率不稳定、转化周期长等问题。因此瞬时表达技术得到广泛应用。
瞬时表达技术是指将目的基因导入受体细胞中以建立暂时性的高效表达体系,从而在相对较短时间内使得目的基因大量表达的技术。利用植物瞬时技术表达重组蛋白时,所转化的基因无需整合到植物基因组中,节省了遗传转化和筛选等冗长过程,可在短期内便实现核酸或蛋白质的表达、表型的检验、分子机理的研究,极大缩减了实验研究所需时间。
农杆菌渗入法或农杆菌注射法是介导外源基因瞬时表达最常用的手段。传统的农杆菌渗入法需将农杆菌菌液与植物组织震荡培养2-3天,番茄叶片容易褐化并腐烂;农杆菌注射法则使用医用注射器对番茄叶片施加创口并注射菌液,此方法处理面积小,且造成大面积创口和组织损伤,可能对实验结果产生干扰。目前还有通过使用机械装置使植物组织被农杆菌菌液负压渗入的方法,该方法虽然没有创口,但是成本较高。因此目前需要在番茄中建立一种经济、快速、稳定的瞬时表达技术,为验证启动子活性、基因功能等发面提供有力手段。
发明内容
本发明要解决的技术问题是为了克服现有技术的不足,提供一种操作简单、成本低、效果好的茄科作物叶片外源基因瞬时表达方法。
本发明解决上述技术问题所采用的技术方案为:
一种基于手动加压的高效瞬时茄科作物转基因方法,包括以下步骤:
(1)将携带含有目标基因重组质粒的农杆菌置于含有相应抗生素的YEB固体培养基中活化菌株;将活化后的菌株接种到小规模培养基中扩增;随后进行扩大培养;最后将扩培的菌液离心去除上清液,收集农杆菌菌体;利用悬浮液悬浮并培养得到农杆菌侵染液;
(2)将注射器空筒的细口处封闭,将大小适宜的茄科作物叶片和农杆菌侵染液置于注射器空筒内,并使茄科作物叶片完全浸入农杆菌侵染液中,通过反复按压注射器推杆促使菌液全部浸染茄科作物叶片;
(3)当全部侵染结束后,取出叶片并擦干表面残留菌液,培养2-3天,获得瞬时转基因茄科作物叶片。
进一步的:所述步骤(1)中活化菌种的培养条件为:含有利福平50-100mg/mL、链霉素100-150mg/mL和相应抗生素的固体YEB培养基中,在26-28℃,倒置培养36-48h。
进一步的:所述步骤(1)中接种培养条件为:含有利福平50-100mg/mL、链霉素100-150mg/mL和相应抗生素的5mL YEB液体培养基中,在26-28℃,180r/min条件下震荡培养16-24h。
进一步的:所述步骤(1)中接种扩大培养条件为:含有吗啉乙磺酸10mmol/L、乙酰丁香酮100μmol/L、MgSO4 2mmol/L以及相应抗生素的YEB液体培养基中进行扩培,28℃,180r/min条件下震荡培养过夜。
进一步的:所述步骤(1)中悬浮液中含有吗啉乙磺酸10mmol/L、乙酰丁香酮100μmol/L和MgCl2 10mmol/L。
进一步的:所述步骤(1)中侵染液中农杆菌的浓度为OD600=0.8-1.0。
进一步的:所述步骤(2)中注射器为医用一次性无菌注射器,体积大小为大于等于10mL。
进一步的:所述步骤(2)中空筒内农杆菌侵染液体积为注射器体积的40-60%。
进一步的:所述步骤(2)中在10s中内反复按压注射器10-15次,保证整个茄科作物叶片可以完全侵染。
进一步的:所述步骤(3)中将侵染结束后叶片先进行12h的暗培养后,光照培养2-3天获得瞬时转基因茄科作物叶片。
进一步的:所述步骤(3)中光照培养条件为:26-28℃,光照14h/黑暗10h。
进一步的:所述茄科作物为番茄或烟草。
本发明的有益效果是,本发明通过简单反复抽拉注射器即可将农杆菌侵染液通过正压渗入茄科作物叶片,从而将外源基因高效瞬时导入植物叶片细胞中,在茄科作物叶片中实现了外源基因瞬时表达。相比传统方法,本发明方法具有耗时短、操作简单、成本低、效率高等诸多优势。
附图说明
图1.采用加压渗入法的番茄叶片基因瞬时转化效果。
图2.采用加压渗入法的烟草叶片基因瞬时转化效果。
图3.叶片侵染的装置示意图。
具体实施方式
实施例1在番茄叶片通过瞬时侵染表达GUS
1.番茄植株的培养
番茄种子放置在衬有湿润滤纸的培养皿里催芽,培养温度为:23-27℃。4-5天后将发芽的种子播种在有机质土中,在23-27℃,光照14h/黑暗10h,相对湿度40-60%的环境下培养到五叶期。选择大小合适的叶片进行侵染。
2.农杆菌侵染液的制备
(1)将含有pBI121质粒的农杆菌GV3101进行活化,将菌液在含有硫酸卡那霉素的YEB固体培养基中进行划线,在28℃的培养箱中倒置培养36h。培养农杆菌的YEB固体培养基配方为:胰蛋白胨10g/L,酵母提取物1g/L,蔗糖5g/L,MgSO4·7H2O 0.5g/L,琼脂15g/L,pH=7.0。分装后121℃高压灭菌20min,待冷却后加入过滤灭菌的硫酸卡那霉素、利福平和链霉素混匀,得到含有利福平50mg/mL、链霉素100mg/mL和50mg/L硫酸卡那霉素的固体YEB培养基,然后倒平板。
(2)挑取单菌落接种于含有硫酸卡那霉素50mg/mL、利福平50mg/mL和链霉素100mg/mL的5mL YEB培养基中,在28℃,180r/min条件下震荡培养20h。
(3)将上述菌液按照1:50的比例在含有吗啉乙磺酸10mmol/L、乙酰丁香酮100μmol/L、MgSO4 2mmol/L以及含有50mg/mL硫酸卡那霉素的YEB培养基中进行扩培,28℃,180r/min条件下震荡培养过夜,得到扩培菌液。
(4)将扩培菌液在4℃,4000r/min离心10min收集菌体,重悬于含有吗啉乙磺酸10mmol/L、乙酰丁香酮100μmol/L、MgCl2 10mmol/L的MMA溶液中,调节OD600为1.0,在26-28℃,180r/min条件下震荡培养3h以上以活化农杆菌,做为农杆菌侵染液。
3.农杆菌对番茄叶片的侵染
如图3所示,打开10ml医用无菌注射器包装,将推杆从后方取下,用封口膜将注射器细口处封闭。挑选肥厚大小适中的番茄叶片从基部剪下,取一片放入注射器中,加入4mL农杆菌菌液,并将番茄叶片完全浸入农杆菌侵染液中,在10秒内快速按压推杆10-15次,完成对一片叶片的侵染,重复上述步骤,完成对第二片、第三片叶片的侵染。
4.侵染叶片培养
取出叶片并擦干表面残留菌液,培养2天,获得瞬时转基因番茄叶片。
5.农杆菌侵染后番茄叶片中GUS基因的检测
(1)配置GUS染色液:
X-Gluc母液:5-溴-4-氯-3-吲哚-β-葡萄糖苷酸酯(X-Gluc),用N-N-二甲基酰胺(DMF)配成20mM的贮存液,分装成每管100μL,保存于-20℃。
X-Gluc基液(50mM PBS,pH7.0)。配制方法:50mM NaH2PO4;50mM Na2HPO4;10mMNa2EDTA;0.1%Triton-100;0.5mM K3[Fe(CN)6];0.5mM K4[Fe(CN)6]
染色液:50μL X-Gluc母液+450μL X-Gluc基液
(2)将瞬时转基因番茄叶片浸泡在染色液中,在37℃中避光保存12-24h。
(3)染色后将叶片取出,转入70%乙醇中脱色3-4次,至阴性对照呈白色。
(4)观察蓝色部位即为GUS表达位点,拍照记录,如图1所示。
实施例2在烟草叶片通过瞬时侵染表达GUS
1.烟草植株的培养
将烟草种子均匀撒在有机质土中,培养温度为:23-27℃,光照14h/黑暗10h,相对湿度40%-60%的环境下培养。选择大小合适的叶片进行侵染。
2.农杆菌侵染液的制备
(1)将含有pBI121质粒的农杆菌GV3101进行活化,将菌液在含有硫酸卡那霉素的YEB固体培养基中进行划线,在28℃的培养箱中倒置培养36h。培养农杆菌的YEB固体培养基配方为:胰蛋白胨10g/L,酵母提取物1g/L,蔗糖5g/L,MgSO4·7H2O 0.5g/L,琼脂15g/L,pH=7.0。分装后121℃高压灭菌20min,待冷却后加入过滤灭菌的硫酸卡那霉素、利福平和链霉素混匀,得到含有利福平50mg/mL、链霉素100mg/mL和50mg/L硫酸卡那霉素的固体YEB培养基,然后倒平板。
(2)挑取单菌落接种于含有硫酸卡那霉素50mg/mL、利福平50mg/mL和链霉素100mg/mL的5mL YEB液体培养基中,在28℃,180r/min条件下震荡培养20h。
(3)将上述菌液按照1:50的比例在含有吗啉乙磺酸10mmol/L、乙酰丁香酮100μmol/L、MgSO4 2mmol/L以及含有50mg/mL硫酸卡那霉素的YEB液体培养基中进行扩培,28℃,180r/min条件下震荡培养过夜,得到扩培菌液。
(4)将扩培菌液在4℃,4000r/min离心10min收集菌体,重悬于含有吗啉乙磺酸10mmol/L、乙酰丁香酮100μmol/L和MgCl2 10mmol/L的MMA溶液中,调节OD600为1.0,在28℃,180r/min条件下震荡培养3h以上以活化农杆菌。
3.农杆菌对烟草叶片的侵染
如图3所示,打开10ml医用无菌注射器包装,将推杆从后方取下,用封口膜将注射器细口处封闭。挑选肥厚大小适中的烟草叶片从基部剪下,取一片放入注射器中,加入4mL农杆菌菌液,并将烟草叶片完全浸入农杆菌侵染液中,在10秒内快速按压推杆10-15次,完成对一片叶片的侵染。重复上述步骤,完成对第二片、第三片叶片的侵染。
4.侵染叶片培养
取出叶片并擦干表面残留菌液,培养2天,获得瞬时转基因烟草叶片。
5.农杆菌侵染后烟草叶片中GUS基因的检测
(1)配置GUS染色液:
X-Gluc母液:5-溴-4-氯-3-吲哚-β-葡萄糖苷酸酯(X-Gluc),用N-N-二甲基酰胺(DMF)配成20mM的贮存液,分装成每管100μL,保存于-20℃。
X-Gluc基液(50mM PBS,pH7.0)。配制方法:50mM NaH2PO4;50mM Na2HPO4;10mMNa2EDTA;0.1%Triton-100;0.5mM K3[Fe(CN)6];0.5mM K4[Fe(CN)6]
染色液:50μL X-Gluc母液+450μL X-Gluc基液
(2)将瞬时转基因烟草叶片浸泡在染色液中,在37℃中避光保存12-24h。
(3)染色后将叶片取出,转入70%乙醇中脱色3-4次,至阴性对照呈白色。
(4)观察蓝色部位即为GUS表达位点,拍照记录,如图2所示。
Claims (10)
1.一种基于手动加压的高效瞬时茄科作物转基因方法,其特征在于:包括以下步骤:
(1)将携带含有目标基因重组质粒的农杆菌置于含有相应抗生素的YEB固体培养基中活化菌株;将活化后的菌株接种到小规模培养基中扩增;随后进行扩大培养;最后将扩培的菌液离心去除上清液,收集农杆菌菌体;利用悬浮液悬浮并培养得到农杆菌侵染液;
(2)将注射器空筒的细口处封闭,将大小适宜的茄科作物叶片和农杆菌侵染液置于注射器空筒内,并使茄科作物叶片完全浸入农杆菌侵染液中,通过反复按压注射器推杆促使菌液全部浸染茄科作物叶片;
(3)当全部侵染结束后,取出叶片并擦干表面残留菌液,培养2-3天,获得瞬时转基因茄科作物叶片。
2.如权利要求1所述的一种基于手动加压的高效瞬时茄科作物转基因方法,其特征在于:所述步骤(1)中活化菌种的培养条件为:含有利福平50-100mg/mL、链霉素100-150mg/mL和相应抗生素的固体YEB培养基中,在26-28℃,倒置培养36-48h。
3.如权利要求1所述的一种基于手动加压的高效瞬时茄科作物转基因方法,其特征在于:所述步骤(1)中接种培养条件为:含有利福平50-100mg/mL、链霉素100-150mg/mL和相应抗生素的5mLYEB液体培养基中,在26-28℃,180r/min条件下震荡培养16-24h。
4.如权利要求1所述的一种基于手动加压的高效瞬时茄科作物转基因方法,其特征在于:所述步骤(1)中接种扩大培养条件为:含有吗啉乙磺酸10mmol/L、乙酰丁香酮100μmol/L、MgSO42 mmol/L以及相应抗生素的YEB液体培养基中,在28℃,180r/min条件下震荡培养过夜。
5.如权利要求1所述的一种基于手动加压的高效瞬时茄科作物转基因方法,其特征在于:所述步骤(1)中悬浮液中含有吗啉乙磺酸10mmol/L、乙酰丁香酮100μmol/L和MgCl210mmol/L。
6.如权利要求1所述的一种基于手动加压的高效瞬时茄科作物转基因方法,其特征在于:所述步骤(1)中侵染液中农杆菌的浓度为OD600=0.8-1.0。
7.如权利要求1所述的一种基于手动加压的高效瞬时茄科作物转基因方法,其特征在于:所述步骤(2)中注射器为医用一次性无菌注射器,体积大小为大于等于10mL。
8.如权利要求1或7所述的一种基于手动加压的高效瞬时茄科作物转基因方法,其特征在于:所述步骤(2)中空筒内农杆菌侵染液体积为注射器体积的40-60%。
9.如权利要求1所述的一种基于手动加压的高效瞬时茄科作物转基因方法,其特征在于:所述步骤(2)中在10s中内反复按压注射器10-15次,保证整个茄科作物叶片完全侵染。
10.如权利要求1所述的一种基于手动加压的高效瞬时茄科作物转基因方法,其特征在于:所述茄科作物为番茄或烟草。
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DE19958961A1 (de) * | 1999-12-07 | 2001-06-28 | Bioplant Biotechnologisches Fo | Nukleinsäuren zur Herstellung von transgenen Pflanzen mit erhöhter Phytopathogen-Resistenz |
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