CN117802156A - 硼酸转运蛋白基因在转基因植株筛选中的应用及筛选方法 - Google Patents
硼酸转运蛋白基因在转基因植株筛选中的应用及筛选方法 Download PDFInfo
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Abstract
本发明公开了一种硼酸转运蛋白基因在转基因植株筛选中的应用及筛选方法。所述硼酸转运蛋白基因为新型筛选标记基因,其属于植物内源性基因,在自然界众多植物中都存在相同功能的高度相似序列,不会造成生物安全风险,是一种绿色、安全的标记基因。所述用硼酸筛选方法对植物细胞影响较小,毒害作用较轻,可以有效缩短筛选转基因植株的周期,能够降低遗传转化过程中培养基污染的风险,并且可以通过叶片形态差异进行转基因植株的快速初步筛选,筛选过程绿色环保。
Description
技术领域
本发明属于分子生物学领域,特别涉及一种硼酸转运蛋白基因通过农杆菌转化法转化植株,然后以硼酸作为筛选剂筛选得到转基因植株。由此构建的硼酸筛选体系在植物转化筛选过程中有良好表现,且具有污染风险低、生物安全性高等特点。
背景技术
植物遗传转化技术是指利用重组DNA技术、细胞组织培养技术或种质***转化技术,有计划地将外源基因或DNA片段***到目标植物的基因组中,然后通过细胞***的方式获得新植株。在植物遗传转化的过程中,只有一小部分细胞能够保持稳定的遗传特性,为了筛选出这些具有稳定遗传特性的细胞,通常需要使用高效的选择标记。
目前,抗生素抗性基因和除草剂抗性基因是植物遗传转化中最常使用的传统选择标记基因。然而,随着转基因植物应用领域的不断发展,此类基因的缺陷也愈发凸显。这些标记基因存在的问题在于:一旦植物转化成功,它们仍然存在于植物体内并稳定遗传,但不再起到作用,这可能会带来潜在的安全隐患。因此,研究具有生物安全性的选择标记基因变得尤为重要。
发明内容
鉴于现有技术的不足,本发明提供一种可实现快速筛选且绿色环保、安全的硼酸转运蛋白基因在转基因植株筛选中的应用及筛选方法。
本发明的第一方面,提供一种硼酸转运蛋白基因作为筛选标记基因在转基因植株筛选中的应用。
根据本发明的具体实施方式,硼酸转运蛋白基因为植物来源,优选为拟南芥AtBOR4基因。
根据本发明的具体实施方式,植株为任何种类的植物,优选为烟草。
某些硼酸转运蛋白能够将细胞中多余的硼酸外转运出细胞,从而使得过表达它们的植株对于硼酸的耐受性提高。硼是植物正常生长发育所必需的微量元素,主要以硼酸分子的形式被植物根系吸收。研究发现,低浓度硼能够促进植物生长发育,而高浓度硼则会引发植物硼中毒。同时由于许多植物(例如水稻)对硼毒性十分敏感,由此导致的作物产量下降也成为一个世界性问题。在拟南芥中,拟南芥AtBOR4基因已被报道能够赋予植物对高浓度硼的耐受性,其过表达的硼酸转运蛋白通过将根细胞中过量的硼外运出细胞,从而使得细胞内的硼维持在相对适宜的浓度。与源自细菌的基因不同,拟南芥AtBOR4基因在大多数植物中均有同源序列,在高效筛选的同时兼具极高的生物安全性。本发明所用拟南芥AtBOR4基因参照NCBI中NM_101415.4(mRNA),NP_172999.1(蛋白质),在核苷酸序列上有个别碱基差异,但翻译出来的蛋白并无差异。
本发明的第二方面,提供一种应用前述的硼酸转运蛋白基因作为筛选标记基因筛选转基因植株的方法,包括:S1、构建过表达筛选标记基因的植物载体;S2、将植物表达载体电转化到农杆菌感受态细胞中,得到含有筛选标记基因的农杆菌;S3、用含有筛选标记基因的农杆菌感染植物细胞,经共培养以及含有硼酸的培养基选择培养,得到再生植株;S4、根据再生植株的叶片形态差异情况进行筛选,得到初步筛选的转基因植株。
根据本发明的具体实施方式,含有硼酸的培养基中,硼酸的终浓度小于5 mM,优选为3~4 mM。
根据本发明的具体实施方式,根据再生植株的叶片形态差异情况进行筛选的判定标准为:转基因植株的叶片为正常形态,例如圆形或椭圆形;非转基因植株的叶片为异形,例如长条形。
根据本发明的具体实施方式,植物表达载体还含有荧光标记基因,此时可进行荧光可视化筛选。具体地,在紫外灯下根据再生植株的荧光效应情况进行筛选,判定标准为:转基因植株的根部具有荧光,非转基因植株的根部无荧光。
根据本发明的具体实施方式,还包括对再生植株进行PCR鉴定以验证是否为转基因植株。
本发明的有益效果为:
1、本发明提供了一种新型筛选标记基因,其属于植物内源性基因,在自然界众多植物中都存在相同功能的高度相似序列,不会造成生物安全风险,是一种绿色、安全的标记基因。
2、本发明提供的以硼酸转运蛋白基因作为筛选标记基因,通过农杆菌转化法转化植株,然后以硼酸作为筛选剂筛选转基因植株的方法,构建了一套全新的筛选标记***。与传统采用卡那霉素抗性基因作为筛选标记基因、以卡那霉素作为筛选剂的筛选方法相比,具有以下优点:1)硼酸筛选相对卡那霉素等抗生素筛选对植物细胞影响较小,毒害作用较轻,可以有效缩短筛选转基因植株的周期(卡那霉素筛选需85~90天,硼酸筛选需65~75天;2)卡那霉素筛选转基因烟草植株的过程中,植株叶片均正常生长,为圆形或椭圆形,因此无法从叶片形态上鉴定是否为转基因植株。硼酸筛选时,愈伤芽长出新植株时可见转基因植株的叶片为正常形态,即圆形或椭圆形;而非转基因植株叶片形态则为长条状,叶片形态怪异,这种形态差异可以进行阳性植株的快速初步筛选;3)硼酸作为一种广谱杀菌剂,在一定程度上可以抑制多种细菌和真菌的生长和繁殖,降低遗传转化过程中培养基污染的风险;4)硼作为一种植物所需的微量元素,在土壤中天然存在,高浓度会引发植物硼毒害,但不会影响到由硼酸筛选出来的转基因植株。因此,在浓度合适的条件下,硼酸也可作为一种除草剂使用,且开发基于硼酸的除草剂不会对土壤造成污染。
附图说明
图1为植物表达载体pBI(aBYe)质粒图谱;
图2为卡那霉素筛选转基因烟草阳性鉴定(泳道M为Marker;泳道C为质粒阳性对照Control;其余泳道为转基因植株鉴定;a、b、c分别为三对引物35sP-Sq/AtBOR4-3c、AtBOR4-5F/NosDw-nR以及AtBOR4-5F/AtBOR4-3c的PCR鉴定);
图3为3 mM硼酸筛选转基因烟草阳性鉴定(泳道M为Marker;泳道C为质粒阳性对照;其余泳道为转基因植株鉴定;a、b、c分别为三轮鉴定,同图2);
图4为3 mM硼酸筛选共培养(A)、选择培养(B)、继代培养(C)及生根培养(D)生长阶段;
图5为3 mM硼酸筛选共培养(A)、选择培养(B)、继代培养(C)及生根培养(D)的荧光可视化;
图6为未被农杆菌侵染以及被农杆菌侵染后45天的烟草叶片经不同筛选剂筛选得到的愈伤再生小植株情况对比,其中:A:3 mM硼酸筛选(未侵染);B:80 μg/mL Kan筛选(侵染);C:3 mM硼酸筛选(侵染);D:5 mM硼酸筛选(侵染);E:7 mM硼酸筛选(侵染)。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地说明。显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1 拟南芥植物表达载体pBI(aBYe)的构建
该实施例中所用到的引物如下:
拟南芥AtBOR4基因为自合成,命名为BORfv,编码区核苷酸序列如SEQ ID NO:1所示。
质粒载体pBI(aTYe)为本实验室构建并储存。
1、将BORfv质粒用Xba I和BamH I双酶切,跑胶回收约2.1 kb的基因片段,取名为BORfv(XB):取一PCR管,加入约1 μg BORfv质粒、5 μL 10×Buffer O、13 μL ddH2O,再加XbaⅠ和BamHⅠ各1 μL。用枪吸打混匀,于PCR仪中,37℃酶切6 h。取1 μL电泳检测酶切完全后,将酶切产物用胶回收试剂盒进行胶回收。
2、将pBI(aTYe)质粒用XbaI和BamHI双酶切,跑胶回收约14.5 kb的大片段,取名为pBI(aTYe)XB,步骤同上。
3、用T4 DNA连接酶将基因片段BORfv(XB)与载体片段pBI(aTYe)XB连接:于一PCR管中加2 μL 10×T4 DNA Ligase Buffer、4 μL BORfv(XB) (25 ng/ μL)、3 μL pBI(aTYe)XB(50 ng/μL)、2 μL T4 DNA ligase、9 μL ddH2O,加好体系后吸打混匀,于PCR仪上22 ℃孵育4 h。
取10 μL连接产物转化DH5α感受态细胞,涂LB板(Kan抗性):100 μL感受态DH5α于冰上解冻后,加入10 μL连接产物(上述产物),吸打混匀,冰浴30 min。42 ℃热激90 s,冰浴2 min。在超净工作台加入200 μL新鲜LB培养基,37 ℃,200 rpm培养1 h。取适量菌液涂布于LB固体培养基(Kan抗性),倒置过夜培养。
4、挑单菌落进行PCR鉴定阳性克隆:分别用35sP-Sq/AtBOR4-3c、AtBOR4-5F/NosDw-nR以及AtBOR4-5F/AtBOR4-3c三对引物进行菌落PCR鉴定,条带大小分别为2180 bp、3648 bp以及2096 bp。阳性质粒取名为pBI(aBYe),质粒图谱如图1所示,大小约16.6 kb。
5、摇菌提质粒、保菌并测序。
实施例2 烟草农杆菌侵染转化与培养
本实施例中农杆菌(EHA105)感受态为市购获得。
1、阳性质粒转农杆菌及阳性菌落鉴定
取100 μL EHA105感受态,加入1 μg实施例1阳性质粒,吸打混匀后移入预冷的电击杯中。1800 V电压电击后迅速取出电击杯,加入500 μL LB(无抗生素)液体培养基,迅速吸打混匀(电击杯必须擦干水分,以防爆杯)。吸出电击杯中的培养基,移入到无菌的1.5 mL无菌离心管中,28 ℃振荡培养4 h。取转化后的菌液200 μL于超净台中涂布于LB固体培养基(50 ng/mL Kan+200 ng/mL Rif),28 ℃倒置培养36~48 h。挑取单菌落进行PCR鉴定,鉴定过程同上述鉴定。
2、烟草转化及培养
该步骤中配制的激素、抗生素、筛选剂的母液浓度如下表所示:
该步骤中使用到的培养基配方如下表所示:
各时期MS培养基(100 mL)所需激素、抗生素、筛选剂配置如下:
挑取已鉴定的农杆菌阳性克隆,经烟草叶盘转化法将目的基因导入烟草,经图4所示共培养、选择培养、继代培养及生根培养得到转基因烟草植株:将野生型烟草无菌苗的叶片在超净台中剪切为约1 cm×1 cm的方形(尽量去除叶脉),置于含有植物表达载体pBI(aBYe)的EHA105农杆菌侵染液中,不断摇晃混匀侵染15 min。侵染完成后,用灭菌ddH2O洗去表面多余菌液(3~5遍),用滤纸吸干表面水分。叶片背面朝上,置于共培养培养基上(表面覆盖高压灭菌过的滤纸),28 ℃培养箱暗培养3 天。
随后将叶片转移至选择培养基以诱导愈伤生成和愈伤芽的再生。当观察到愈伤再生芽长至1 cm左右高小植株时,将其切下,转移并***到生根培养基中继续诱导生根直至长成新植株。
图4为3 mM硼酸筛选共培养、选择培养、继代培养及生根培养阶段图片。
图5为3 mM硼酸筛选共培养、选择培养、继代培养及生根培养阶段的荧光可视化。由于植物载体pBI(aBYe)中含有YGFPuv荧光标签,因此可以在紫外灯下通过荧光观察来判断是否为转基因植株。
图6为未被农杆菌侵染以及被农杆菌侵染后45天的烟草叶片经不同筛选剂筛选得到的愈伤再生小植株情况对比,可以看出,在3 mM硼酸浓度下,未侵染的野生型烟草再生植株生长被严重抑制,且叶片形态不正常,卡那霉素筛选的烟草再生植株叶片形态正常,为圆形或椭圆形,侵染后转拟南芥AtBOR4基因的烟草再生植株则生长较快,叶片形态正常。而高浓度时即使有转基因再生植株也会被抑制甚至不能生长。因此在合适的硼酸浓度下,拟南芥AtBOR4基因确实能够快速筛选出阳性植株,且可以通过叶片形态差异完成初筛。
对比例
与实施例2的区别在于:(1)将卡那霉素(Kan)抗性基因nptⅡ替代拟南芥AtBOR4基因作为选择标记基因,用Kan筛选;(2)烟草转化及培养中各时期MS培养基(100 mL)所需的激素和抗生素配置如下:
实验例 转基因烟草植株的鉴定
使用植物DNA提取试剂盒提取烟草基因组,以烟草基因组DNA为模板,进行PCR阳性鉴定。鉴定所需的三对引物同上。分别对实施例2及对比例方法筛选长出的转基因植株进行PCR鉴定,如图2~3所示,结果如下表:
可以看出,本发明通过采用拟南芥AtBOR4基因代替常用的卡那霉素抗性筛选基因,通过3 mM硼酸抑制生长代替卡那霉素杀死细胞的方式,转化所用时间由85~90 天缩短至65~75 天,从而明显提高了筛选效率。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
Claims (9)
1.硼酸转运蛋白基因作为筛选标记基因在转基因植株筛选中的应用,所述硼酸转运蛋白基因为拟南芥AtBOR4基因,其编码区核苷酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的应用,其特征在于,所述植株为烟草。
3.一种应用权利要求1~2之一所述硼酸转运蛋白基因作为筛选标记基因筛选转基因植株的方法,其特征在于,所述的方法包括:
S1、构建过表达筛选标记基因的植物载体;
S2、将植物表达载体电转化到农杆菌感受态细胞中,得到含有筛选标记基因的农杆菌;
S3、用含有筛选标记基因的农杆菌侵染植物细胞,经共培养以及含有硼酸的培养基选择培养,得到再生植株;
S4、根据再生植株的叶片形态差异情况进行筛选,得到初步筛选的转基因植株。
4.根据权利要求3所述的方法,其特征在于,所述含有硼酸的培养基中,硼酸的终浓度小于5 mM。
5.根据权利要求4所述的方法,其特征在于,所述硼酸的终浓度为3~4 mM。
6.根据权利要求3所述的方法,其特征在于,所述根据再生植株的叶片形态差异情况进行筛选的判定标准为:转基因植株的叶片为正常形态,非转基因植株的叶片为异形。
7.根据权利要求3所述的方法,其特征在于,所述植物表达载体还含有荧光标记基因。
8.根据权利要求7所述的方法,其特征在于,在紫外灯下根据再生植株的荧光效应情况进行筛选,判定标准为:转基因植株的根部具有荧光,非转基因植株的根部无荧光。
9.根据权利要求3所述的方法,其特征在于,还包括S5——对再生植株进行PCR鉴定以验证是否为转基因植株。
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CN118048396A (zh) * | 2024-04-15 | 2024-05-17 | 云南师范大学 | 植物遗传转化筛选体系的建构方法及应用 |
Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5242945A (en) * | 1991-04-12 | 1993-09-07 | American Home Products Corporation | Tetronic and thiotetronic acid derivatives as phospholipase a2 inhibitors |
CN1699579A (zh) * | 2005-05-17 | 2005-11-23 | 中国科学院武汉病毒研究所 | 耐盐基因作为植物遗传转化筛选标记基因的方法与应用 |
US20070079394A1 (en) * | 2004-03-15 | 2007-04-05 | Toru Fujiwara | Proteins imparting boron-tolerance and genes thereof |
CN101096703A (zh) * | 2006-06-28 | 2008-01-02 | 华中农业大学 | 一种非抗生素筛选小麦转基因植物的方法 |
WO2008069318A1 (ja) * | 2006-12-07 | 2008-06-12 | Japan Science And Technology Agency | 植物に過剰ホウ素耐性を付与したホウ素耐性植物、前記植物の作出方法及び前記作出に利用する遺伝子 |
CN101353696A (zh) * | 2007-07-24 | 2009-01-28 | 财团法人工业技术研究院 | 包括肝纤维化和/或肝硬化的肝纤维损伤的生物标志及其检测方法 |
WO2013005211A2 (en) * | 2011-07-05 | 2013-01-10 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Boron complexing plant materials and uses thereof cross-reference to related applications |
CN104711277A (zh) * | 2015-03-19 | 2015-06-17 | 安徽农业大学 | 玉米丝氨酸消旋酶基因作为筛选标记在南抗杨转化体系中的应用 |
CN107012166A (zh) * | 2017-04-05 | 2017-08-04 | 华南农业大学 | 水稻OsAT1蛋白在改善植物硼含量中的应用 |
WO2018112435A1 (en) * | 2016-12-16 | 2018-06-21 | The Regents Of The University Of California | Compounds for the inhibition of a prolyl isomerase and methods of use thereof |
CN108588115A (zh) * | 2018-05-09 | 2018-09-28 | 湖北文理学院 | 异戊烯转移酶基因作为筛选标记基因在培育转基因拟南芥植株中的应用 |
CN112375763A (zh) * | 2020-11-03 | 2021-02-19 | 浙江农林大学 | Ebs1基因在增强拟南芥抗碳酸氢盐胁迫能力中的应用 |
-
2024
- 2024-02-29 CN CN202410224626.4A patent/CN117802156A/zh active Pending
Patent Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5242945A (en) * | 1991-04-12 | 1993-09-07 | American Home Products Corporation | Tetronic and thiotetronic acid derivatives as phospholipase a2 inhibitors |
US20070079394A1 (en) * | 2004-03-15 | 2007-04-05 | Toru Fujiwara | Proteins imparting boron-tolerance and genes thereof |
CN1699579A (zh) * | 2005-05-17 | 2005-11-23 | 中国科学院武汉病毒研究所 | 耐盐基因作为植物遗传转化筛选标记基因的方法与应用 |
CN101096703A (zh) * | 2006-06-28 | 2008-01-02 | 华中农业大学 | 一种非抗生素筛选小麦转基因植物的方法 |
WO2008069318A1 (ja) * | 2006-12-07 | 2008-06-12 | Japan Science And Technology Agency | 植物に過剰ホウ素耐性を付与したホウ素耐性植物、前記植物の作出方法及び前記作出に利用する遺伝子 |
CN101353696A (zh) * | 2007-07-24 | 2009-01-28 | 财团法人工业技术研究院 | 包括肝纤维化和/或肝硬化的肝纤维损伤的生物标志及其检测方法 |
WO2013005211A2 (en) * | 2011-07-05 | 2013-01-10 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Boron complexing plant materials and uses thereof cross-reference to related applications |
CN104711277A (zh) * | 2015-03-19 | 2015-06-17 | 安徽农业大学 | 玉米丝氨酸消旋酶基因作为筛选标记在南抗杨转化体系中的应用 |
WO2018112435A1 (en) * | 2016-12-16 | 2018-06-21 | The Regents Of The University Of California | Compounds for the inhibition of a prolyl isomerase and methods of use thereof |
CN107012166A (zh) * | 2017-04-05 | 2017-08-04 | 华南农业大学 | 水稻OsAT1蛋白在改善植物硼含量中的应用 |
CN108588115A (zh) * | 2018-05-09 | 2018-09-28 | 湖北文理学院 | 异戊烯转移酶基因作为筛选标记基因在培育转基因拟南芥植株中的应用 |
CN112375763A (zh) * | 2020-11-03 | 2021-02-19 | 浙江农林大学 | Ebs1基因在增强拟南芥抗碳酸氢盐胁迫能力中的应用 |
Non-Patent Citations (8)
Title |
---|
LING LIU等: "Identification and function characterization of BnaBOR4 genes reveal their potential for Brassica napus cultivation under high boron stress", ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY, 23 January 2024 (2024-01-23), pages 1 - 12 * |
MASATAKA KA JIKAWA等: "Expression of the Arabidopsis Borate Efflux Transporter Gene, AtBOR4, in Rice Affects the Xylem Loading of Boron and Tolerance to Excess Boron", BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY, 7 December 2011 (2011-12-07), pages 2421 * |
THEOLOGIS, A. 等: "HCO3- transporter family [Arabidopsis thaliana]", GENBANK DATABASE, 20 October 2022 (2022-10-20), pages 172999 * |
THEOLOGIS, A.等: "Arabidopsis thaliana HCO3- transporter family (BOR4), mRNA", GENBANK DATABASE, 20 October 2022 (2022-10-20), pages 101415 * |
刘凯;游艾青;: "硼在植物中的吸收和转运机制研究进展", 湖北农业科学, no. 24, 25 December 2016 (2016-12-25), pages 6331 - 6336 * |
刘春光;何小娇;: "过量硼对植物的毒害及高硼土壤植物修复研究进展", 农业环境科学学报 * |
刘春光等: "过量硼对植物的毒害及高硼土壤植物修复研究进展", 农业环境科学学报, vol. 31, no. 2, 20 February 2012 (2012-02-20), pages 230 * |
夏金婵;何奕;: "植物对硼元素的吸收转运机制", 中国生物化学与分子生物学报, no. 08, 20 August 2009 (2009-08-20), pages 702 - 702 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN118048396A (zh) * | 2024-04-15 | 2024-05-17 | 云南师范大学 | 植物遗传转化筛选体系的建构方法及应用 |
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