刺槐高频再生体系的建立及转Bt Cry1A基因的初步研究
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摘要
刺槐(Robinia pseudoacacia L.),为豆科Leguminosae刺槐属(Robinia L.)落叶乔木,原产北美。中国于1877年引入栽培,主要包括刺槐R.pseudoacaciaL.和毛刺槐R.hispida L.两种,常见于中部和东部,由于其生长迅速、木材坚韧、纹理细致、有弹性、耐水湿、抗腐朽、根系发达,具根瘤,有一定的抗旱、抗烟、耐盐碱能力(国家林业局国有林场和林木种苗工作总站,2001),现已成为华北、西北等地区优良的保持水土、防风固沙、改良土壤的优质树种,深受群众欢迎(荀守华等,2009)。刺槐还具有花香浓郁、抗逆性强、病虫害少、价格低等优点,且易于栽培、成活率高、绿化效果好,适宜于景观绿化与经济用途(张亚玲等,2009)。
然而,近年来,生态环境等各方面的影响使刺槐也面临一些严重的病虫害:病害——紫纹羽病和烂皮病等;虫害——白蚁、叶蝉、蚧、小皱蝽(叶)、豆荚螟虫、麦蛾、小蜂(种子)等。而因为以下种种原因,刺槐的转基因工程研究进展较缓慢。其一,一直以来,木本植物的遗传转化成功几率都比较低,而刺槐为大型木本植物,基因组庞大,外源基因很难插入并表达,即使有少数转化成功的株系,在以后的继代培养中也容易发生基因丢失或基因沉默等现象,这是木本植物转基因研究中迄待解决的问题(荀守华等,2009);其二,刺槐的遗传转化工作缺乏再生频率高、重复性好的受体系统;其三,缺乏优良的目的基因的克隆和启动子的构建;其四,转化频率低、实验重复性差,没有形成第一代工程品种(郑进等,2005)。针对上述刺槐的广泛用途及其病虫害特点,更兼于豆科植物是被子植物第二大科,本实验展开了刺槐离体转化受体系统的建立及基因转化的研究,拟建立一个高效的刺槐转化受体系统,并进一步开展抗病虫害的转基因研究,旨在为刺槐及其他豆科植物的抗病虫害方面提供借鉴及良好的应用价值。
本实验首先以生长良好的刺槐植株的带腋芽茎段为外植体,研究了在刺槐高频再生体系的建立过程中不同基本培养基、不同激素浓度及其配比对愈伤组织的诱导、芽的分化及生根的影响;然后在得到高频再生体系的基础上,通过农杆菌介导法转化Bt Cry1A抗虫基因,以GUS染色组织分析为依据探讨了影响转化效率的各种因素,建立了高效、可重复的基因转化体系,为刺槐目的基因的导入打下了基础。试验取得如下研究结果:
1.建立了刺槐高频再生基因转化受体系统。在刺槐离体茎段为外植体诱导愈伤组织的过程中,芽的分化以MS+6-BA 0.3mg/L+NAA 0.04mg/L+蔗糖30g/L+琼脂9g/L条件下分化率最高,每个外植体的平均分化芽数可达到7.3个;诱导生根以MS+NAA0.04mg/L+KT 0.05mg/L+蔗糖30g/L,效果最佳,生根率最高可达93.3,平均生根率达86.7%;在移栽生根苗时,先在温室中驯化炼苗15～20天后移入大田,成活率可达83%以上。
2.以GUS染色检测为依据初步建立了刺槐高效遗传转化体系。以农杆菌菌株LBA4404介导Bt Cry1A基因的转化,首先确定了转化培养中最佳的抑菌素及其浓度和选择压:头孢霉素(Cef)400mg/L即可有效地抑制农杆菌菌株LBA4404的生长;卡那霉素(Km)50mg/L为选择压较适宜。并进一步优化了转化体系:以离体茎段为外植体,预培养1-2天,OD值为0.5～0.7的农杆菌LBA4404菌液浸染外植体以8～10分钟为宜;共培养时间3～4天较适合;共培养培养基中添加AS 30mg/L,GUS+阳性发生率可达最高值。共培养后转入附加Cef 400mg/L的培养基中培养,15天后转入附加Km 50mg/L的培养基中筛选抗性植株。
Robinia pseudoacacia L,a member of Robinia in Leguminosae was originated from North America.It possesses many important features,such as rapid growth,strong.and elastic woody fibers,anti-adversity,well-nodulated root system and so on,so that it becomes widespread in north and northwest of China for soil and water Conservation. Robinia pseudoacacia is also an indispensable species for urban avenue virescence due to its rich fragrance,easy planting,and valuable appearance for public attention.
Robinia pseudoacacia ever featured in strong resistance to infection of pathogenic organisms.However,in recent years,it has been found to suffer some serious diseases, such as violet root rot and skin ulceration caused by pathogenic microorganisms,and invasion from bugs small wrinkle(leaf),pod borer,Angoumois grain moth,small bees (seeds).
Though transgenic engineering has been successfully used to introduce advantageous genes into some important plant species to improve resistance to attacking from pests and diseases,it has been not available in Robinia pseudoacacia mainly due to lacking a well-established receptor system.
In present study,we used the growing-well stems of Robinia pseudoacacia with axillary's buds as explants.Firstly,we studied the influence of several factors on differentiation of buds and roots,including different media,different hormones and different concentration ratio of some hormones mixtures.And then,based on availability of a high frequency regeneration system,we tested the effects of various factors on transformation efficiency of Bt CrylA gene mediated by Agrobacterium tumefaciens with histochemical assays of GUS.The transformation procedure established here was proved to be reproducible and effective,and opened up a possibility of more beneficial genes to be introduced into Robinia pseudoacacia.Detailed experiments and data were listed as the following
(1) Establishment of a high frequency regeneration system,of Robinia pseudoacacia:we found that the most effective medium to induce callus is MS basic medium containing 0.3mg/L of 6-BA(6-benzyladenine),0.04mg/L of NAA (naphthalene acetic acid) and 30g/L of sucrose and 9g/L of agar,and that the mean bud differentiation rate is 80%,the mean number of buds differentiated from an explant reaches up to 7.3,and MS medium supplemented with 0.04mg/L ofNAA,0.05mg/L of KT(kinetin) and 30g/L of sucrose is very efficient to improve rooting.The highest rooting frequency is 93.3%and the mean rooting frequency is 86.7%.Our data showed that the plants cultivated in tubes should be acclimated in green house for 15～20 days before planting into outdoor soils,making the surviving rate reach higher than 83%.
(2) Establishment of a high efficient transformation system of Robinia pseudoacacia:in the transformation of Bt CrylA gene mediated by Agrobacterium tumefaciens strain LBA4404,the most suitable bacteriostatic agent is 400mg/L Cef(cephalosporin) and the selecting concentration is 50mg/L of Km(kalamycin ). Based on further optimization of necessary factors to improve the transformation frequency by GUS histochemical assays,we established an efficient transformation system of Robinia pseudoacacia:including:(1) stems should be pre-cultured in-vitro for 1～2 days;(2),Agrobacterium tumefaciens was inoculated till OD600 reached 0.5～0.7;(3) they were co-cultured for 3～4 days.Our data also suggested adding 30mg/L of Acetosyringone(AS) into the co-cultured medium could dramatically improve transformation frequency;(4) the co-cultured explants were transferred to MS medium containing 400mg/L of Cef;(5) After about 15 days,they were transferred to the same medium with 50mg/L of Km to select Km-resistant shoots.
然而,近年来,生态环境等各方面的影响使刺槐也面临一些严重的病虫害:病害——紫纹羽病和烂皮病等;虫害——白蚁、叶蝉、蚧、小皱蝽(叶)、豆荚螟虫、麦蛾、小蜂(种子)等。而因为以下种种原因,刺槐的转基因工程研究进展较缓慢。其一,一直以来,木本植物的遗传转化成功几率都比较低,而刺槐为大型木本植物,基因组庞大,外源基因很难插入并表达,即使有少数转化成功的株系,在以后的继代培养中也容易发生基因丢失或基因沉默等现象,这是木本植物转基因研究中迄待解决的问题(荀守华等,2009);其二,刺槐的遗传转化工作缺乏再生频率高、重复性好的受体系统;其三,缺乏优良的目的基因的克隆和启动子的构建;其四,转化频率低、实验重复性差,没有形成第一代工程品种(郑进等,2005)。针对上述刺槐的广泛用途及其病虫害特点,更兼于豆科植物是被子植物第二大科,本实验展开了刺槐离体转化受体系统的建立及基因转化的研究,拟建立一个高效的刺槐转化受体系统,并进一步开展抗病虫害的转基因研究,旨在为刺槐及其他豆科植物的抗病虫害方面提供借鉴及良好的应用价值。
本实验首先以生长良好的刺槐植株的带腋芽茎段为外植体,研究了在刺槐高频再生体系的建立过程中不同基本培养基、不同激素浓度及其配比对愈伤组织的诱导、芽的分化及生根的影响;然后在得到高频再生体系的基础上,通过农杆菌介导法转化Bt Cry1A抗虫基因,以GUS染色组织分析为依据探讨了影响转化效率的各种因素,建立了高效、可重复的基因转化体系,为刺槐目的基因的导入打下了基础。试验取得如下研究结果:
1.建立了刺槐高频再生基因转化受体系统。在刺槐离体茎段为外植体诱导愈伤组织的过程中,芽的分化以MS+6-BA 0.3mg/L+NAA 0.04mg/L+蔗糖30g/L+琼脂9g/L条件下分化率最高,每个外植体的平均分化芽数可达到7.3个;诱导生根以MS+NAA0.04mg/L+KT 0.05mg/L+蔗糖30g/L,效果最佳,生根率最高可达93.3,平均生根率达86.7%;在移栽生根苗时,先在温室中驯化炼苗15～20天后移入大田,成活率可达83%以上。
2.以GUS染色检测为依据初步建立了刺槐高效遗传转化体系。以农杆菌菌株LBA4404介导Bt Cry1A基因的转化,首先确定了转化培养中最佳的抑菌素及其浓度和选择压:头孢霉素(Cef)400mg/L即可有效地抑制农杆菌菌株LBA4404的生长;卡那霉素(Km)50mg/L为选择压较适宜。并进一步优化了转化体系:以离体茎段为外植体,预培养1-2天,OD值为0.5～0.7的农杆菌LBA4404菌液浸染外植体以8～10分钟为宜;共培养时间3～4天较适合;共培养培养基中添加AS 30mg/L,GUS+阳性发生率可达最高值。共培养后转入附加Cef 400mg/L的培养基中培养,15天后转入附加Km 50mg/L的培养基中筛选抗性植株。
Robinia pseudoacacia L,a member of Robinia in Leguminosae was originated from North America.It possesses many important features,such as rapid growth,strong.and elastic woody fibers,anti-adversity,well-nodulated root system and so on,so that it becomes widespread in north and northwest of China for soil and water Conservation. Robinia pseudoacacia is also an indispensable species for urban avenue virescence due to its rich fragrance,easy planting,and valuable appearance for public attention.
Robinia pseudoacacia ever featured in strong resistance to infection of pathogenic organisms.However,in recent years,it has been found to suffer some serious diseases, such as violet root rot and skin ulceration caused by pathogenic microorganisms,and invasion from bugs small wrinkle(leaf),pod borer,Angoumois grain moth,small bees (seeds).
Though transgenic engineering has been successfully used to introduce advantageous genes into some important plant species to improve resistance to attacking from pests and diseases,it has been not available in Robinia pseudoacacia mainly due to lacking a well-established receptor system.
In present study,we used the growing-well stems of Robinia pseudoacacia with axillary's buds as explants.Firstly,we studied the influence of several factors on differentiation of buds and roots,including different media,different hormones and different concentration ratio of some hormones mixtures.And then,based on availability of a high frequency regeneration system,we tested the effects of various factors on transformation efficiency of Bt CrylA gene mediated by Agrobacterium tumefaciens with histochemical assays of GUS.The transformation procedure established here was proved to be reproducible and effective,and opened up a possibility of more beneficial genes to be introduced into Robinia pseudoacacia.Detailed experiments and data were listed as the following
(1) Establishment of a high frequency regeneration system,of Robinia pseudoacacia:we found that the most effective medium to induce callus is MS basic medium containing 0.3mg/L of 6-BA(6-benzyladenine),0.04mg/L of NAA (naphthalene acetic acid) and 30g/L of sucrose and 9g/L of agar,and that the mean bud differentiation rate is 80%,the mean number of buds differentiated from an explant reaches up to 7.3,and MS medium supplemented with 0.04mg/L ofNAA,0.05mg/L of KT(kinetin) and 30g/L of sucrose is very efficient to improve rooting.The highest rooting frequency is 93.3%and the mean rooting frequency is 86.7%.Our data showed that the plants cultivated in tubes should be acclimated in green house for 15～20 days before planting into outdoor soils,making the surviving rate reach higher than 83%.
(2) Establishment of a high efficient transformation system of Robinia pseudoacacia:in the transformation of Bt CrylA gene mediated by Agrobacterium tumefaciens strain LBA4404,the most suitable bacteriostatic agent is 400mg/L Cef(cephalosporin) and the selecting concentration is 50mg/L of Km(kalamycin ). Based on further optimization of necessary factors to improve the transformation frequency by GUS histochemical assays,we established an efficient transformation system of Robinia pseudoacacia:including:(1) stems should be pre-cultured in-vitro for 1～2 days;(2),Agrobacterium tumefaciens was inoculated till OD600 reached 0.5～0.7;(3) they were co-cultured for 3～4 days.Our data also suggested adding 30mg/L of Acetosyringone(AS) into the co-cultured medium could dramatically improve transformation frequency;(4) the co-cultured explants were transferred to MS medium containing 400mg/L of Cef;(5) After about 15 days,they were transferred to the same medium with 50mg/L of Km to select Km-resistant shoots.
引文
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