CN116411021B - 一种番茄草甘膦筛选体系的转化方法 - Google Patents
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Abstract
本发明属于基因工程和转基因技术领域,涉及一种番茄草甘膦筛选体系的转化方法。所述的转化方法包括如下步骤:(1)将番茄子叶在预培养培养基上进行预培养;(2)将预培养后的番茄子叶用转化有抗草甘膦基因的农杆菌菌液进行侵染后吹干;(3)将侵染后的番茄子叶在共培养培养基上进行共培养;(4)将共培养后的番茄子叶在诱导培养基上进行诱导愈伤培养;(5)将诱导愈伤培养后的番茄子叶在筛选培养基上进行筛选培养,获得抗性芽;(6)将抗性芽在再生培养基上进行再生培养,获得再生芽;(7)将再生芽在生根培养基上进行生根培养,获得生根壮苗。利用本发明的番茄草甘膦筛选体系的转化方法,能够实现番茄草甘膦筛选体系的高效遗传转化。
Description
技术领域
本发明属于基因工程和转基因技术领域,涉及一种番茄草甘膦筛选体系的转化方法。
背景技术
番茄(Solanum lycopersicum L.)属于茄科茄属植物,是世界范围内广泛种植的蔬菜和经济作物,具有基因组较小、生长周期短、自花授粉等特点,是生产转基因植物和植物生物制药产品的模式植物。
番茄目前较为常见的遗传转化方法有基因枪法、超声波法及农杆菌介导的遗传转化法等。其中农杆菌介导的遗传转化法因操作方便,成本较低,在植物转基因中广泛应用。农杆菌介导的遗传转化法有许多优点,比如高效稳定的外源基因插入、插入外源DNA的片段较长、外源基因的拷贝数低并且符合孟德尔遗传规律等。
目前番茄遗传转化常用的筛选体系有:潮霉素筛选体系、Kan筛选体系、Bar筛选体系等。
发明内容
本发明的目的是提供一种番茄草甘膦筛选体系的转化方法,以能够通过对预培养方式、侵染方法、各个阶段培养基成分及筛选培养基中的草甘膦筛选剂浓度等进行优化,从而实现番茄草甘膦筛选体系的高效遗传转化。
为实现此目的,在基础的实施方案中,本发明提供一种番茄草甘膦筛选体系的转化方法,所述的转化方法包括如下步骤:
(1)将番茄子叶在预培养培养基上进行预培养;
(2)将预培养后的番茄子叶用转化有抗草甘膦基因的农杆菌菌液进行侵染后吹干;
(3)将侵染后的番茄子叶在共培养培养基上进行共培养;
(4)将共培养后的番茄子叶在诱导培养基上进行诱导愈伤培养;
(5)将诱导愈伤培养后的番茄子叶在筛选培养基上进行筛选培养,获得抗性芽;
(6)将抗性芽在再生培养基上进行再生培养,获得再生芽;
(7)将再生芽在生根培养基上进行生根培养,获得生根壮苗,
所述的筛选培养基与所述的再生培养基中分别含有40-100mg/L的草甘膦。
在一种优选的实施方案中,本发明提供一种番茄草甘膦筛选体系的转化方法,其中所述的预培养培养基为4.3g/L MS盐+0.5ml/L MS维生素+30g/L蔗糖 +0.5-3mg/L 玉米素(Zeatin)+0.02-0.3mg/L IAA+8g/L琼脂,pH=5.8,预培养为48h的暗培养。
在一种优选的实施方案中,本发明提供一种番茄草甘膦筛选体系的转化方法,其中步骤(2)中,所述的转化有抗草甘膦基因的农杆菌为转化CP4 EPSPS抗草甘膦基因的农杆菌EHA105。
在一种优选的实施方案中,本发明提供一种番茄草甘膦筛选体系的转化方法,其中步骤(2)中,所述的侵染的时间为10-15min。
在一种优选的实施方案中,本发明提供一种番茄草甘膦筛选体系的转化方法,其中步骤(3)中,所述的共培养培养基为4.3g/L MS盐+0.5ml/L MS维生素+20g/L蔗糖+10g/L葡萄糖+1g/L MES +2mg/L 玉米素 +100mg/L 半胱氨酸 +0.1mg/L IAA+100µM AS+8g/L琼脂,pH=5.8,共培养为22℃下暗培养48h。
在一种优选的实施方案中,本发明提供一种番茄草甘膦筛选体系的转化方法,其中步骤(4)中,所述的诱导培养基为4.3g/L MS盐+0.5ml/L MS维生素+30g/L蔗糖 +1g/LMES +2mg/L 玉米素 +0.02-0.3mg/L IAA+200-500mg/L 特美汀 + 8g/L 琼脂,pH=5.8,诱导培养为25℃,16h光/8h暗条件下进行愈伤组织的诱导培养。
在一种优选的实施方案中,本发明提供一种番茄草甘膦筛选体系的转化方法,其中步骤(5)中,所述的筛选培养基为4.3g/L MS盐+0.5ml/L MS维生素 + 30g/L蔗糖 +1g/LMES +0.5-3mg/L 玉米素 +0.02-0.3mg/L IAA+ 40-100mg/L 草甘膦 +200-500mg/L 特美汀+ 8g/L琼脂,pH=5.8,筛选培养为25℃,16h光/8h暗条件下培养21天。
在一种优选的实施方案中,本发明提供一种番茄草甘膦筛选体系的转化方法,其中步骤(6)中,所述的再生培养基为4.3g/L MS盐+0.5ml/L MS维生素 + 30g/L蔗糖 +1g/LMES+0.5-3mg/L 玉米素 +0.02-0.3mg/L IAA+ 40-100mg/L 草甘膦 +200-500mg/L 特美汀+ 8g/L琼脂,pH=5.8。
在一种优选的实施方案中,本发明提供一种番茄草甘膦筛选体系的转化方法,其中步骤(7)中,所述的生根培养基为2.15g/L MS盐+0.25ml/L MS维生素+ 30 g/L 蔗糖 +0.1-1 mg/L IBA+ 8g/L琼脂,pH=5.8。
在一种优选的实施方案中,本发明提供一种番茄草甘膦筛选体系的转化方法,其中所述的转化方法还在步骤(1)前进行番茄种子的消毒与萌发,以产生所述的番茄子叶。
本发明的有益效果在于,利用本发明的番茄草甘膦筛选体系的转化方法,能够通过对预培养方式、侵染方法、各个阶段培养基成分及筛选培养基中的草甘膦筛选剂浓度等进行优化,从而实现番茄草甘膦筛选体系的高效遗传转化。
附图说明
图1为实施例3得到的诱导愈伤培养基中维生素对诱导愈伤率的影响图,左、中、右的培养皿分别为加入MS维生素、B5维生素、N&N维生素的情况。
图2为实施例5得到的各个阶段的生长情况图,其中上左图为萌发阶段,上中图为诱导愈伤阶段,上右图为筛选阶段,下左图为再生阶段,下右图为生根阶段。
具体实施方式
以下结合实施例和附图对本发明的具体实施方式作出进一步的说明。
实施例1:草甘膦筛选剂浓度对转化效率影响检测
筛选培养基、再生培养基中不同的筛选剂浓度是影响转化效率检测结果的关键因素。本发明使用草甘膦作为筛选剂,在筛选和再生阶段添加筛选剂进行处理,筛选剂设置了10mg/L、20mg/L、40mg/L、 60mg/L、80mg/L、100mg/L和120mg/L共7种不同的浓度,各培养阶段培养基组成、转基因操作和培养条件同实施例5,测试最佳的筛选效果,结果如下表1所示。
表1 筛选剂浓度对转化效率及阳性率的影响
序号 | 处理方式 | 侵染外植体数 | 出苗数 | 阳性苗数 | 阳性率(阳性苗平均数/出苗平均数) | 转化效率(阳性苗平均数/侵染外植体数) |
1 | 10mg/L | 100 | 40±3.74 | 0 | 0% | 0% |
2 | 20mg/L | 100 | 36±3.27 | 0.33±0.47 | 0.9% | 0.33% |
3 | 40mg/L | 100 | 33±2.94 | 4±0.82 | 12% | 4% |
4 | 60mg/L | 100 | 28±4.08 | 10±2.16 | 35.7% | 10% |
5 | 80mg/L | 100 | 18±2.45 | 14±2.94 | 77.8% | 14% |
6 | 100mg/L | 100 | 3±0.82 | 3±0.82 | 100% | 3% |
7 | 120 mg/L | 100 | 0 | 0 | 0 | 0 |
注:每种处理方式处理三次,取三次结果的平均值。
表1的结果表明:草甘膦筛选剂浓度为20mg/L时,仅1株阳性苗再生,阳性率1%,转化效率0.33%;草甘膦筛选剂浓度在40-100mg/L时,均可获得较多阳性植株,其中草甘膦筛选剂浓度在80mg/L时,转化效率最高。
实施例2:预培养及浸泡的影响
方法1:不进行预培养直接将番茄子叶切成0.5cm左右的小块进行侵染,侵染后将番茄子叶吹干放入共培养培养基中培养2d,共培养培养基为4.3g/L MS盐+0.5ml/L MS维生素+20g/L蔗糖+10g/L葡萄糖+1g/L MES +2mg/L 玉米素 +100mg/L 半胱氨酸 +0.1mg/LIAA+100µM AS+8g/L琼脂,pH=5.8,共培养条件为22℃下暗培养48h。将共培养结束后的番茄子叶放入诱导愈伤培养基中培养21d左右,诱导愈伤培养为25℃,16h光/8h暗。诱导培养结束后统计诱导愈伤率(诱导愈伤率=产生愈伤组织数/外植体总数(污染除外)×100%)。诱导愈伤培养基为4.3g/L MS盐+0.5ml/L MS维生素+30g/L蔗糖 +1g/L MES +2mg/L 玉米素 +0.02-0.3mg/L IAA+200-500mg/L 特美汀 + 8g/L 琼脂,pH=5.8。
方法2:将切成0.5cm左右的小块的番茄子叶放入侵染液中浸泡1h后,将子叶放到预培养培养基上进行预培养。预培养2d后将经过预培养的子叶放入配制好的农杆菌EHA105菌液中进行侵染,后续愈伤诱导培养基及培养条件同方法1。
方法3:将切成0.5cm左右的小块的番茄子叶直接放到预培养培养基上进行预培养。预培养2d后将经过预培养的子叶放入配制好的农杆菌EHA105菌液中进行侵染,后续愈伤诱导培养基及培养条件同方法1。
上述方法中,侵染液成分为4.3g/L MS盐+0.5ml/L MS维生素+20g/L蔗糖+10g/L葡萄糖+1g/L MES +2mg/L 玉米素,pH=5.6,农杆菌EHA105菌液OD600为0.5左右,侵染时间为10-15min。
预培养培养基为4.3g/L MS盐+0.5ml/L MS维生素+30g/L蔗糖 +1mg/L 玉米素(Zeatin) +0.02-0.3mg/L IAA+8g/L琼脂,pH=5.8,预培养条件为25℃下暗培养48h。
三种方法的诱导愈伤率检测结果如下表2所示。
表2三种方法的诱导愈伤率检测结果
序号 | 处理方式 | 平均诱导愈伤率 |
1 | 方法1 | 43.67%±1.53% |
2 | 方法2 | 91.67%±4.16% |
3 | 方法3 | 90.67%±2.08% |
注:每种方法处理三次,取三次诱导愈伤率的平均值。
表2的结果表明:
(1)经过2d预培养后再进行侵染(方法2和方法3),后期诱导愈伤率明显提高,说明在侵染之前对番茄子叶进行预培养,可以明显提高诱导愈伤率;
(2)在预培养前是否对番茄子叶在侵染液中进行浸泡,对后期诱导愈伤率无明显影响。
实施例3:诱导愈伤培养基中维生素对诱导愈伤率的影响
诱导愈伤培养基中的维生素是影响诱导愈伤率的关键因素,本发明对诱导愈伤培养基中的不同维生素种类进行了测试(在按实施例2的处理方式2处理后),诱导培养基为4.3g/L MS盐+0.5ml/L MS维生素+30g/L蔗糖 +1g/L MES +2mg/L 玉米素 +0.02-0.3mg/LIAA+200-500mg/L 特美汀 + 8g/L 琼脂,pH=5.8,设置三种维生素添加处理,分别为:0.5ml/L MS维生素(采购于上海麦克林生化科技股份有限公司,货号:M909604 )、0.5ml /L B5维生素(采购于上海麦克林生化科技股份有限公司,货号:B909599)0.5ml/L N&N维生素(采购于上海麦克林生化科技股份有限公司,货号:N909607 )。
结果如下表3及图1所示。
表3 维生素对诱导愈伤率影响的检测结果
序号 | 处理方式 | 维生素浓度 | 平均诱导愈伤率 |
1 | MS维生素 | 0.5ml /L | 92.33%±1.15% |
2 | B5维生素 | 0.5ml /L | 90.33%±1.53% |
3 | N&N维生素 | 0.5ml /L | 83.33%±5.69% |
注:每种处理方式处理三次,取三次诱导愈伤率的平均值。
表3、图1的结果表明:使用MS维生素与B5维生素,诱导愈伤率较高,其中MS维生素愈伤生长速度最快且分化较好有利于后期抗性芽的分化;B5维生素愈伤生长速度较慢且愈伤组织较硬不利于后期抗性芽的分化。
实施例4:侵染方法的影响
通常在侵染阶段对受体材料(番茄子叶)进行热击、超声波或者真空渗透等处理可以明显提高转化效率。本发明尝试使用了超声波的方法对番茄子叶(按实施例2的方法3进行预处理后)进行处理,分别设置为无超声波、超声波30s和2min三种处理,超声波功率为40Hz。超声波处理结束后侵染10-15min(侵染液成分为4.3g/L MS盐+0.5ml/L MS维生素+20g/L蔗糖+10g/L葡萄糖+1g/L MES +2mg/L 玉米素,pH=5.6,农杆菌EHA105菌液OD600为0.5左右)。后期的步骤按照实施例2的方法3进行,待出苗后进行阳性苗的检测,转化效率=阳性苗数/起始侵染的子叶数,结果如下表4所示。
表4 超声处理对转化效率的影响结果
序号 | 处理方式 | 平均转化效率 |
1 | 无超声波 | 12%±1.73% |
2 | 超声波30s | 10.33%±2.08% |
3 | 超声波2min | 4.67%±1.52% |
注:每种处理方式处理三次,取三次转化效率的平均值。
表4的结果表明:无超声波与超声波30s转化效率无明显差异,超声波2min子叶受损影响转化效率。
实施例5:按实施例1-4的参数优化后的转化试验
1、番茄种子消毒与萌发
将番茄Ailsa Craig种子用75%(v/v)的酒精浸泡2-3 min,再用5-10%(m/v)的次氯酸钠溶液浸泡10-15 min(灭菌过程中需要轻摇),然后再用无菌水清洗3-5次。
将灭菌后的番茄种子在超净工作台中接种在萌发培养基(2.15g/L MS盐+0.25ml/L MS维生素+30g/L 蔗糖+8g/L琼脂,pH=5.8),尽量让种子散开,以获得最大的生长空间。将番茄种子放置于25℃,16h光/8h暗的光照培养室中进行培养,大约7d-10d幼苗子叶展开即可(子叶展开但不露心叶)。
2、子叶预培养
当番茄幼苗子叶展平时,在无菌环境下将子叶用剪刀或手术刀切成长宽为0.5 cm左右的小块,将切好的子叶放置于预培养培养基(4.3g/L MS盐+0.5ml/L MS维生素+30g/L蔗糖 +1mg/L 玉米素 +0.02-0.3mg/L IAA+8g/L琼脂,pH=5.8),预培养为25℃下暗培养48h。
3、农杆菌悬浮液的制备
将转化CP4 EPSPS抗草甘膦基因的农杆菌EHA105接种于YP培养基(含有相应抗生素)中,将划好的菌板放在28℃恒温培养箱中,培养2天。挑取菌板上单菌落重新活化到新的YP菌板上。重新活化的菌板在28℃培养箱培养1天后用于实验。
在侵染液(4.3g/L MS盐+0.5ml/L MS维生素+20g/L蔗糖+10g/L葡萄糖+1g/L MES+2mg/L 玉米素,pH=5.6)中加入乙酰丁香酮(AS),4.3g/L MS盐+0.5ml/L MS维生素使AS的终浓度为200µmol/L。用接菌环取农杆菌并充分悬浮于已加入AS的侵染液中,农杆菌的OD660值控制在0.5左右。
4、浸染转化
将预培养48 h的番茄子叶充分浸泡于已加入AS的侵染液中,侵染10-15min(侵染过程中需要轻摇)。完成侵染后,将子叶置于无菌滤纸上吸取残留的农杆菌菌液,并放在超净工作台上吹干(吹干子叶表面的菌液即可,不要使子叶吹干脱水)。
5、共培养
在共培养培养基(4.3g/L MS盐+0.5ml/L MS维生素+20g/L蔗糖+10g/L葡萄糖+1g/L MES +2mg/L 玉米素 +100mg/L 半胱氨酸 +0.1mg/L IAA+100µM AS+8g/L琼脂,pH=5.8)上放一层滤纸,将吹干菌液的子叶叶背面朝上放置在共培养培养基上22℃下暗培养48h。
6、诱导愈伤
共培养2-3d后,将共培养结束后的子叶置于诱导培养基(4.3g/L MS盐+0.5ml/LMS维生素+30g/L蔗糖 +1g/L MES +2mg/L 玉米素 +0.02-0.3mg/L IAA+200-500mg/L 特美汀 + 8g/L 琼脂,pH=5.8)上并于25℃,16h光/8h暗条件下进行愈伤组织的诱导。诱导3-4周直至长出愈伤组织即可,每7-10d左右继代一次。
7、筛选
将愈伤组织放在筛选培养基(4.3g/L MS盐+0.5ml/L MS维生素 + 30g/L蔗糖 +1g/L MES +0.5-3mg/L 玉米素 +0.02-0.3mg/L IAA+ 40-100mg/L 草甘膦 +200-500mg/L特美汀+ 8g/L琼脂,pH=5.8)上进行筛选,在25℃,16h光/8h暗条件下培养21天即可获得抗性芽。
8、再生
将抗性芽放置于再生培养基(4.3g/L MS盐+0.5ml/L MS维生素+ 30g/L蔗糖 +1g/L MES+0.5-3mg/L 玉米素 +0.02-0.3mg/L IAA+ 40-100mg/L 草甘膦 +200-500mg/L 特美汀+ 8g/L琼脂,pH=5.8)上,进行再生,在25℃,16h光/8h暗条件下培养30天左右即可获得再生芽。
9、生根
待再生芽长到3-5cm时,将再生的植株放入生根培养基(2.15g/L MS盐+0.25ml/LMS维生素 + 30 g/L 蔗糖 + 0.5 mg/L IBA+ 8g/L琼脂,pH=5.8)中生根壮苗,在25℃,16h光/8h暗条件下培养30天左右获得生根壮苗,即可进行移栽。
上述各个阶段的生长情况如图2所示。对按上述方法得到的生根壮苗的转化效率进行检测,结果如下表5所示。
表5 生根壮苗的转化效率检测结果
序号 | 起始植株数量 | 阳性植株数量 | 转化效率 |
1 | 96 | 9 | 9.38% |
2 | 112 | 20 | 17.86% |
3 | 80 | 10 | 12.50% |
4 | 145 | 23 | 15.86% |
5 | 250 | 33 | 13.20% |
平均值 | 13.76% |
注:序号1-5为分别进行的5次实验
表5的结果表明:成功建立了番茄草甘膦筛选体系的转化方法,并对各个转化阶段的激素配比及草甘膦筛选剂浓度进行了确认。通过对各个环节的优化,最终番茄草甘膦转化体系的平均转化效率达到13.76%。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若对本发明的这些修改和变型属于本发明权利要求及其同等技术的范围之内,则本发明也意图包含这些改动和变型在内。上述实施例或实施方式只是对本发明的举例说明,本发明也可以以其它的特定方式或其它的特定形式实施,而不偏离本发明的要旨或本质特征。因此,描述的实施方式从任何方面来看均应视为说明性而非限定性的。本发明的范围应由附加的权利要求说明,任何与权利要求的意图和范围等效的变化也应包含在本发明的范围内。
Claims (2)
1.一种番茄草甘膦筛选体系的转化方法,其特征在于,所述的转化方法包括如下步骤:
(1)将番茄子叶在预培养培养基上进行预培养;
(2)将预培养后的番茄子叶用转化有抗草甘膦基因的农杆菌菌液进行侵染后吹干;
(3)将侵染后的番茄子叶在共培养培养基上进行共培养;
(4)将共培养后的番茄子叶在诱导培养基上进行诱导愈伤培养;
(5)将诱导愈伤培养后的番茄子叶在筛选培养基上进行筛选培养,获得抗性芽;
(6)将抗性芽在再生培养基上进行再生培养,获得再生芽;
(7)将再生芽在生根培养基上进行生根培养,获得生根壮苗,
所述的筛选培养基与所述的再生培养基中分别含有40-100mg/L的草甘膦,
其中:
步骤(1)中,所述的预培养培养基为4.3g/L MS盐+0.5ml/L MS维生素+30g/L蔗糖 +0.5-3mg/L 玉米素+0.02-0.3mg/L IAA+8g/L琼脂,pH=5.8,预培养为48h的暗培养;
步骤(2)中,所述的转化有抗草甘膦基因的农杆菌为转化CP4 EPSPS抗草甘膦基因的农杆菌EHA105;
步骤(2)中,所述的侵染的时间为10-15min;
步骤(3)中,所述的共培养培养基为4.3g/L MS盐+0.5ml/L MS维生素+20g/L蔗糖+10g/L葡萄糖+1g/L MES +2mg/L 玉米素 +100mg/L 半胱氨酸 +0.1mg/L IAA+100µM AS+8g/L琼脂,pH=5.8,共培养为22℃下暗培养48h;
步骤(4)中,所述的诱导培养基为4.3g/L MS盐+0.5ml/L MS维生素+30g/L蔗糖 +1g/LMES +2mg/L 玉米素 +0.02-0.3mg/L IAA+200-500mg/L 特美汀 + 8g/L 琼脂,pH=5.8,诱导培养为25℃,16h光/8h暗条件下进行愈伤组织的诱导培养;
步骤(5)中,所述的筛选培养基为4.3g/L MS盐+0.5ml/L MS维生素 + 30g/L蔗糖 +1g/L MES +0.5-3mg/L 玉米素 +0.02-0.3mg/L IAA+ 40-100mg/L 草甘膦 +200-500mg/L 特美汀+ 8g/L琼脂,pH=5.8,筛选培养为25℃,16h光/8h暗条件下培养21天;
步骤(6)中,所述的再生培养基为4.3g/L MS盐+0.5ml/L MS维生素 + 30g/L蔗糖 +1g/L MES+0.5-3mg/L 玉米素 +0.02-0.3mg/L IAA+ 40-100mg/L 草甘膦 +200-500mg/L 特美汀+ 8g/L琼脂,pH=5.8;
步骤(7)中,所述的生根培养基为2.15g/L MS盐+0.25ml/L MS维生素 + 30 g/L 蔗糖+ 0.1-1 mg/L IBA+ 8g/L琼脂,pH=5.8。
2.根据权利要求1所述的转化方法,其特征在于:所述的转化方法还在步骤(1)前进行番茄种子的消毒与萌发,以产生所述的番茄子叶。
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