农杆菌介导的橡胶树遗传转化体系的建立
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摘要
天然橡胶具有合成橡胶无法比拟的弹性、延展性及导热性,是重要的工业原料,主要来源于巴西橡胶树(Hevea brasiliensis Muell.Arg.)。巴西橡胶树为多年生异花授粉木本作物,通过常规杂交育种手段,培育一个新品种需要20-30年。转基因技术是缩短橡胶树育种周期、加快新品种培育十分有效的途径,也是橡胶树功能基因鉴定等研究的重要技术手段。尽管我国于2000年已开始橡胶树遗传转化研究,但依然没有建立起橡胶树遗传转化体系,这已成为制约橡胶树基因工程遗传改良的技术瓶颈。我国植胶区处于热带北缘,在大寒年份寒害给生产带来重大损失。因此,通过基因工程改良现有的优良无性系以提高其抗寒能力是一项非常重要的任务。CBF1转录因子能促进多个冷诱导基因表达,使得转基因植株的抗寒性比单一功能基因的转化强。
为建立橡胶树遗传转化体系,本研究首先筛选了适宜农杆菌侵染的橡胶树受体材料,然后以适宜侵染的花药再生体系为对象,筛选高频体胚发生的橡胶树无性系,再通过对抗生素种类和浓度、农杆菌菌株、共培养温度和共培养时间等影响遗传转化效率因素研究,最终建立以花药愈伤组织为受体的农杆菌介导的橡胶树遗传转化体系。并在此基础上,研究了拟南芥冷诱导基因的转录因子CBF1转化橡胶树优良无性系热研7-33-97。主要研究结果如下：
1.适宜农杆菌侵染的橡胶树外植体筛选
以花药和内珠被愈伤组织为受体研究了适宜农杆菌侵染的橡胶树遗传转化受体,结果表明内珠被愈伤组织在农杆菌侵染后极易褐化,很难恢复生长,因此不适宜作为转化受体。而花药愈伤组织耐侵染,容易抑菌,很快就能恢复生长,因此,花药愈伤组织更适宜作为根癌农杆菌的转化受体。
2.高频体胚发生的橡胶树无性系筛选
对5个生长、产胶等性状良好的橡胶树无性系体胚发生能力进行了研究,结果显示,热研7-33-97、热研6-62具有高的愈伤组织诱导率和体细胞胚胎发生能力且综合性状优良。
3.抑菌和筛选抗生素种类及浓度筛选
500 mg·L-1特泯丁抑制胚性愈伤组织生长但促进体胚发生,而羧苄青霉素、头孢霉素和特泯丁三种抑制农杆菌抗生素均为促进愈伤组织生长但抑制体胚发生。潮霉素、卡那霉素和巴龙霉素三种筛选抗生素均抑制愈伤组织生长和体胚发生,三者抑制能力顺序为潮霉素>卡那霉素>巴龙霉素。故特泯丁可以作为农杆菌介导巴西橡胶树花药愈伤组织遗传转化的抑菌抗生素,500 mg·L-1使用浓度效果较好,卡那霉素作为筛选抗生素,50 mg·L-1使用浓度效果较好。
4.影响遗传转化效率因素的研究
农杆菌菌株、共培养温度和共培养时间等对遗传转化效率具有显著影响,EHA105菌株侵染力最强,共培养温度为22℃和共培养时间为6 d时,GUS瞬时表达率最高。共培养基中添加As无助于橡胶树花药愈伤组织转化效率的提高。
5.转基因植株鉴定及转化体系的建立
2.2万个花药愈伤组织在EHA 105侵染后,通过抗性筛选、GUS染色、PCR、测序、Southern检测及Northern检测,鉴定出11株转基因植株,并检测到目的基因表达,随后通过嫁接和次生体胚发生,获得来自8个转基因株系681株转基因植株,移栽成活253株。
6.转基因植株移栽
以未经增殖直接大田移栽、嫁接到室外砧木和通过次生体胚发生增殖后大田移栽三种方式进行移栽,结果3株未经增殖直接移栽到大田,均未成活；3株通过籽苗芽接进行1轮室外嫁接繁殖,获得5株转基因单株,全部移栽成活；其余5个在胚状体阶段进行3次次生体胚发生增殖后诱导植株再生,最终获得676株转基因单株,移栽成活248株。
7.转基因体系验证
利用上述遗传转化及增殖、移栽体系将拟南芥转录因子CBF1导入橡胶树,获得2株GUS染色、PCR检测、PCR产物测序阳性植株,并通过籽苗芽接和次生体胚发生增殖为44株,移栽成活34株。
Natural rubber performes better at elasticity, ductibility, and heat transfer properties than synthetic rubber. Rubber tree (Hevea brasiliensis Muell. Arg) is the most important source of natural rubber. Rubber tree is a perennial woody plant, developping a new Hevea variety by crossing method will take 20-30 years, but genetic transformation provides an effective alternative for shortening breeding cycle. The rubber tree planting area in China is located in the north edge of tropical area, cold damage brought great loss to the rubber production. Therefore,improving the cold resistant ability of elite clones by genetic engineering is very important. CBF1 transcriptional factor could induce the expression of several cold-resistant genes, which improved cold resistant ability of transgenic plant much more than only one functional gene transformation.
Since the establishment of genetic transformation system of Hevea have been studied for 10 years in China, but failed. It limits genetic engineering breeding in Hevea. So, In order to establish Agrobacterium tumefaciens-mediated Hevea anther calli transformation system, we surveyed the factors influencing transformation frequency. Firstly, the optimal acceptors for Agrobacterium infection and clones with high somatic embryogenesis were screened, then the factors influencing transformation frequency, including strains of Agrobacterium, co-culture temperature, co-culture time and acetosyringone (AS), and the methods for transformed embryos regeneration, were surveyed, finally, the successful genetic transformation system of Hevea was established. And based on the system, Arabidopsis transcriptional factor CBF1 was transformed to Hevea elite clone CATAS7-33-97 and regenerated transgenic plantlets. The main results as follows:
1.Selection of the acceptors for transformation
Anther and inner integument calli were used as acceptors to study their reaction to Agrobacterium. The results showed that the inner integument calli browned easily after infection and was hard to resume their gowth; however, anther calli could endure infection, and was easy to eliminate bacteria, resume gowth fast. So, anther calli was more optimal for transformation.
2. Selection of the clones with high embryogenesis efficiency
Five elite clones with some important agronomic traits were selected for comparison of their regeneability. Three were good at yield and timber, one was elite in comprehensive tratits. The results showed that two genotypes, namely, CATAS6-62 and CATAS7-33-97 have predominant somatic embryogenesis ability. The identification of these two elite clons will afford good acceptor variety for genetic transformation in Hevea and accelerate the application of transgenic material in breeding.
3. Selection of antibiotics for transformation
The sensitivity of 30-days old anther calli of Hevea brasiliensis to antibiotics was measured. Two groups of antibiotics were tested:antibiotics commonly used to eliminate Agrobacterium from tissue culture (carbenicillin, cefotaxime and timentin), and antibiotics for the selection of transformed tissue (kanamycin, paromomycin and hygromycin).Results show that, except 500 mg·L-1 timentin inhibited callus growth but promoted embryogenesis, the first group significantly increased callus growth but inhibited embryogenesis; while the second group inhibited callus growth and embryogenesis, their inhibited ability ranked as follows:hygromycin> kanamycin> paromomycin; but hygromycin completely inhibited embryogenesis, while paromomycin had no obvious effect on embryogenesis.So in Agrobacterium-mediated transformation of Hevea brasiliensis anther callus, timentin could be used to eliminate Agrobacterium, optimal concentration was 500 mg·L-1; kanamycin to select transformant, optimal concentration was 50 mg·L-1.
4. Factors influencing transformation frequency
GUS transient expression frequency was significantly affected by Agrobacterium strains and co-culture temperature/time, which was 5.07 blue spots/callus for EHA105,higher than that for GV3101 and LBA4404. The highest GUS transient expression frequency was 8.43 and 19.10 blue spots/callus under 22℃co-cultured temperature and 6 days co-cultured time respectively. The AS was not helpful for improving the transformation efficiency.
5. Establishment of Agrobacterium tumefaciens-mediated Hevea genetic transformation system
Twenty two thousand anther embryogenic calli were transformed by EHA105 with pCAMBIA2301.In the results,11 transgenic plantlets were obtained through GUS staining, PCR, sequencing of PCR products and Southern detection. Subsequently,253 plants derived from 8 trangenic plantlets survived in the field.
6. Acclimation of transgenic plants
Three methods were used for acclimation of transgenic plants:directly acclimation without propagation, budding then acclimation, and acclimation after propagation by secondary embryogenesis of transgenic embryos. Three planted directly in the field died;three as scions were budded onto seedlings, producing five budded transgenic plants; five were multiplicated at embryo phase by secondary embryogenesis, producing 676 transgenic plantlets, among which 248 survived in the field.
7. CBF1 gene transformation into Hevea
Above genetic transformation system was used to transfer the Arabidopsis transcriptional factor CBF1 to Hevea. In the result, two transgenic plantlets were regenerated through GUS staining, PCR, sequencing of PCR products. These two plantlets were multiplicated by mini-seedling budding and transgenic embryo secondary embryogenesis, and 44 plantlets were obtained and acclimated,34 suvived in soil.
为建立橡胶树遗传转化体系,本研究首先筛选了适宜农杆菌侵染的橡胶树受体材料,然后以适宜侵染的花药再生体系为对象,筛选高频体胚发生的橡胶树无性系,再通过对抗生素种类和浓度、农杆菌菌株、共培养温度和共培养时间等影响遗传转化效率因素研究,最终建立以花药愈伤组织为受体的农杆菌介导的橡胶树遗传转化体系。并在此基础上,研究了拟南芥冷诱导基因的转录因子CBF1转化橡胶树优良无性系热研7-33-97。主要研究结果如下：
1.适宜农杆菌侵染的橡胶树外植体筛选
以花药和内珠被愈伤组织为受体研究了适宜农杆菌侵染的橡胶树遗传转化受体,结果表明内珠被愈伤组织在农杆菌侵染后极易褐化,很难恢复生长,因此不适宜作为转化受体。而花药愈伤组织耐侵染,容易抑菌,很快就能恢复生长,因此,花药愈伤组织更适宜作为根癌农杆菌的转化受体。
2.高频体胚发生的橡胶树无性系筛选
对5个生长、产胶等性状良好的橡胶树无性系体胚发生能力进行了研究,结果显示,热研7-33-97、热研6-62具有高的愈伤组织诱导率和体细胞胚胎发生能力且综合性状优良。
3.抑菌和筛选抗生素种类及浓度筛选
500 mg·L-1特泯丁抑制胚性愈伤组织生长但促进体胚发生,而羧苄青霉素、头孢霉素和特泯丁三种抑制农杆菌抗生素均为促进愈伤组织生长但抑制体胚发生。潮霉素、卡那霉素和巴龙霉素三种筛选抗生素均抑制愈伤组织生长和体胚发生,三者抑制能力顺序为潮霉素>卡那霉素>巴龙霉素。故特泯丁可以作为农杆菌介导巴西橡胶树花药愈伤组织遗传转化的抑菌抗生素,500 mg·L-1使用浓度效果较好,卡那霉素作为筛选抗生素,50 mg·L-1使用浓度效果较好。
4.影响遗传转化效率因素的研究
农杆菌菌株、共培养温度和共培养时间等对遗传转化效率具有显著影响,EHA105菌株侵染力最强,共培养温度为22℃和共培养时间为6 d时,GUS瞬时表达率最高。共培养基中添加As无助于橡胶树花药愈伤组织转化效率的提高。
5.转基因植株鉴定及转化体系的建立
2.2万个花药愈伤组织在EHA 105侵染后,通过抗性筛选、GUS染色、PCR、测序、Southern检测及Northern检测,鉴定出11株转基因植株,并检测到目的基因表达,随后通过嫁接和次生体胚发生,获得来自8个转基因株系681株转基因植株,移栽成活253株。
6.转基因植株移栽
以未经增殖直接大田移栽、嫁接到室外砧木和通过次生体胚发生增殖后大田移栽三种方式进行移栽,结果3株未经增殖直接移栽到大田,均未成活；3株通过籽苗芽接进行1轮室外嫁接繁殖,获得5株转基因单株,全部移栽成活；其余5个在胚状体阶段进行3次次生体胚发生增殖后诱导植株再生,最终获得676株转基因单株,移栽成活248株。
7.转基因体系验证
利用上述遗传转化及增殖、移栽体系将拟南芥转录因子CBF1导入橡胶树,获得2株GUS染色、PCR检测、PCR产物测序阳性植株,并通过籽苗芽接和次生体胚发生增殖为44株,移栽成活34株。
Natural rubber performes better at elasticity, ductibility, and heat transfer properties than synthetic rubber. Rubber tree (Hevea brasiliensis Muell. Arg) is the most important source of natural rubber. Rubber tree is a perennial woody plant, developping a new Hevea variety by crossing method will take 20-30 years, but genetic transformation provides an effective alternative for shortening breeding cycle. The rubber tree planting area in China is located in the north edge of tropical area, cold damage brought great loss to the rubber production. Therefore,improving the cold resistant ability of elite clones by genetic engineering is very important. CBF1 transcriptional factor could induce the expression of several cold-resistant genes, which improved cold resistant ability of transgenic plant much more than only one functional gene transformation.
Since the establishment of genetic transformation system of Hevea have been studied for 10 years in China, but failed. It limits genetic engineering breeding in Hevea. So, In order to establish Agrobacterium tumefaciens-mediated Hevea anther calli transformation system, we surveyed the factors influencing transformation frequency. Firstly, the optimal acceptors for Agrobacterium infection and clones with high somatic embryogenesis were screened, then the factors influencing transformation frequency, including strains of Agrobacterium, co-culture temperature, co-culture time and acetosyringone (AS), and the methods for transformed embryos regeneration, were surveyed, finally, the successful genetic transformation system of Hevea was established. And based on the system, Arabidopsis transcriptional factor CBF1 was transformed to Hevea elite clone CATAS7-33-97 and regenerated transgenic plantlets. The main results as follows:
1.Selection of the acceptors for transformation
Anther and inner integument calli were used as acceptors to study their reaction to Agrobacterium. The results showed that the inner integument calli browned easily after infection and was hard to resume their gowth; however, anther calli could endure infection, and was easy to eliminate bacteria, resume gowth fast. So, anther calli was more optimal for transformation.
2. Selection of the clones with high embryogenesis efficiency
Five elite clones with some important agronomic traits were selected for comparison of their regeneability. Three were good at yield and timber, one was elite in comprehensive tratits. The results showed that two genotypes, namely, CATAS6-62 and CATAS7-33-97 have predominant somatic embryogenesis ability. The identification of these two elite clons will afford good acceptor variety for genetic transformation in Hevea and accelerate the application of transgenic material in breeding.
3. Selection of antibiotics for transformation
The sensitivity of 30-days old anther calli of Hevea brasiliensis to antibiotics was measured. Two groups of antibiotics were tested:antibiotics commonly used to eliminate Agrobacterium from tissue culture (carbenicillin, cefotaxime and timentin), and antibiotics for the selection of transformed tissue (kanamycin, paromomycin and hygromycin).Results show that, except 500 mg·L-1 timentin inhibited callus growth but promoted embryogenesis, the first group significantly increased callus growth but inhibited embryogenesis; while the second group inhibited callus growth and embryogenesis, their inhibited ability ranked as follows:hygromycin> kanamycin> paromomycin; but hygromycin completely inhibited embryogenesis, while paromomycin had no obvious effect on embryogenesis.So in Agrobacterium-mediated transformation of Hevea brasiliensis anther callus, timentin could be used to eliminate Agrobacterium, optimal concentration was 500 mg·L-1; kanamycin to select transformant, optimal concentration was 50 mg·L-1.
4. Factors influencing transformation frequency
GUS transient expression frequency was significantly affected by Agrobacterium strains and co-culture temperature/time, which was 5.07 blue spots/callus for EHA105,higher than that for GV3101 and LBA4404. The highest GUS transient expression frequency was 8.43 and 19.10 blue spots/callus under 22℃co-cultured temperature and 6 days co-cultured time respectively. The AS was not helpful for improving the transformation efficiency.
5. Establishment of Agrobacterium tumefaciens-mediated Hevea genetic transformation system
Twenty two thousand anther embryogenic calli were transformed by EHA105 with pCAMBIA2301.In the results,11 transgenic plantlets were obtained through GUS staining, PCR, sequencing of PCR products and Southern detection. Subsequently,253 plants derived from 8 trangenic plantlets survived in the field.
6. Acclimation of transgenic plants
Three methods were used for acclimation of transgenic plants:directly acclimation without propagation, budding then acclimation, and acclimation after propagation by secondary embryogenesis of transgenic embryos. Three planted directly in the field died;three as scions were budded onto seedlings, producing five budded transgenic plants; five were multiplicated at embryo phase by secondary embryogenesis, producing 676 transgenic plantlets, among which 248 survived in the field.
7. CBF1 gene transformation into Hevea
Above genetic transformation system was used to transfer the Arabidopsis transcriptional factor CBF1 to Hevea. In the result, two transgenic plantlets were regenerated through GUS staining, PCR, sequencing of PCR products. These two plantlets were multiplicated by mini-seedling budding and transgenic embryo secondary embryogenesis, and 44 plantlets were obtained and acclimated,34 suvived in soil.
引文
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