棉花基因枪转化体系的建立
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摘要
本研究采用基因枪轰击转化技术，将外源双价抗虫基因Bt+CpTI导入棉花茎尖分生细织，获得了转基因植株；将GUS报告基因导入棉花胚性愈伤组织，获得了卡那霉素抗性的胚状体；并尝试了基因枪和农杆菌协同转化法在棉花转基因上的应用。
采用棉花茎尖分生组织为轰击受体，优化出了3日苗龄的茎尖进行基因枪轰击，并摸索出了一条适合茎尖再生的途径。对轰击参数进行综合比较，优选出最佳轰击组合。确定了适合基因枪茎尖转化体系的梯度筛选法。结果表明，基因枪轰击过程中，采用轰击压力1100psi，真空度28inch Hg，轰击距离9cm，轰击1次，转化效果最佳。卡那霉素抗性苗平均转化率4.23％，转化周期80天左右。后期对抗性苗进行嫁接，成活率90％以上。对获得的卡那霉素抗性植株进行PCR检测，证明外源基因已经整合到棉花基因组中。
采用棉花胚性愈伤组织为轰击受体，通过GUS瞬间表达，对胚性愈伤组织渗透处理的渗透剂种类、浓度、时间以及轰击过程中轰击距离、轰击次数、DNA用量、真空度等进行了优化。并确定了适合基因枪棉花胚性愈伤组织转化体系的筛选方法。结果表明，采用0.3mol／L的甘露醇在轰击前4小时，轰击后16小时进行渗透处理，轰击距离9cm，轰击次数2次，DNA用量0.5-1.0μg／枪，真空度28inch HgGUS表达效果最佳，恢复培养后采用150mg／L卡那霉素进行直接筛选，获得抗性胚状体。2个月后，对卡那霉素抗性的胚性愈伤组织进行GUS基因的稳定表达分析，初步证明GUS基因已转入棉花基因组中。
同时对基因枪和农杆菌协同转化棉花茎尖进行了初步探索。结果表明，以棉花茎尖为受体材料，植株很难再生，需要进行系统的研究和探讨。
研究结果为棉花现代分子育种增添了新的内容和新的种质材料，同时对棉花规模化转基因技术体系的建立和抗虫基因工程的开展具有重要的意义。
In this paper, many transgenic cotton plants which were introduced an insect-resistant gene via partical bombardment of apical meristems were obtained successfully. Simultaneously, a large amount of kanamycin-resistant somatic embryos of cotton in which a GUS gene was integrated via partical bombardment of embryogenic callus were also obtained. A new transformation system that combined Agrobacterium-mediated and particle bombardment transformation was attempted in cotton.
Shoot-tip culled from 3-day-old seedlings was opmtimal to the holistic transformation system. Thereafter, a more efficient regeneration system was established by comprehensive contrastes and the parameters combinations were as follows: Helium pressure at 1 lOOpsi, the vaccum pressure at 28 inches Hg, target distance at 9cm, 1 time bombardment. Subsequently, the meristems tissue bombarded were cultured in the selective media containing kanamycin, and then the concentration of kanamycin was steped to a higher amount in the series of subculture media. In the end, many regenerated resistant plants to kanamycin were obtained, with a transformation rate reaching 4.23%, and the cycles of transformation were approximate 80 days. The surviving rates of transplanting of the transgenic plants exceeded 90% by grafting, thereafter; the plants grafted were confirmed by PCR assay.
Embryogenic callus particle bombardment system was improved through GUS expression, and get a more efficient system: firstly, osmotic treated to embryogenic callus in solid medium with 0.3mol/L mannitol for 4hr before and 16hr after bombardments. Secondly, bombard the calli treated with the parameters showed as follow: vaccum pressure at 28 inches Hg, target distance at 9cm, 2 times bombardments, target DNA of 0.5-1.0 g per bombardment. After these, the calli was transferred to culture in selective medium containing kanamycin 150mg/L after recovery culture of 10-15days, and two months later, the somatic embryos resistant to kanamycin were obtained while a stable expression of GUS was detected in the embryogenic callus, which showed the GUS gene had been introduced the genome of cotton.
The apical merstems of cotton were very difficult to germinate after treatment of combination of Agrobocterium-med\a.ted transformation and particle bombardment in my exploration. So the new method need further research.
This research added new content and germplasm to modern cotton molecular breeding. Meanwhile, it was significant to the construction of a technical system of transformtion on a large-scale and insect-resistant gene engineering in cotton.
采用棉花茎尖分生组织为轰击受体，优化出了3日苗龄的茎尖进行基因枪轰击，并摸索出了一条适合茎尖再生的途径。对轰击参数进行综合比较，优选出最佳轰击组合。确定了适合基因枪茎尖转化体系的梯度筛选法。结果表明，基因枪轰击过程中，采用轰击压力1100psi，真空度28inch Hg，轰击距离9cm，轰击1次，转化效果最佳。卡那霉素抗性苗平均转化率4.23％，转化周期80天左右。后期对抗性苗进行嫁接，成活率90％以上。对获得的卡那霉素抗性植株进行PCR检测，证明外源基因已经整合到棉花基因组中。
采用棉花胚性愈伤组织为轰击受体，通过GUS瞬间表达，对胚性愈伤组织渗透处理的渗透剂种类、浓度、时间以及轰击过程中轰击距离、轰击次数、DNA用量、真空度等进行了优化。并确定了适合基因枪棉花胚性愈伤组织转化体系的筛选方法。结果表明，采用0.3mol／L的甘露醇在轰击前4小时，轰击后16小时进行渗透处理，轰击距离9cm，轰击次数2次，DNA用量0.5-1.0μg／枪，真空度28inch HgGUS表达效果最佳，恢复培养后采用150mg／L卡那霉素进行直接筛选，获得抗性胚状体。2个月后，对卡那霉素抗性的胚性愈伤组织进行GUS基因的稳定表达分析，初步证明GUS基因已转入棉花基因组中。
同时对基因枪和农杆菌协同转化棉花茎尖进行了初步探索。结果表明，以棉花茎尖为受体材料，植株很难再生，需要进行系统的研究和探讨。
研究结果为棉花现代分子育种增添了新的内容和新的种质材料，同时对棉花规模化转基因技术体系的建立和抗虫基因工程的开展具有重要的意义。
In this paper, many transgenic cotton plants which were introduced an insect-resistant gene via partical bombardment of apical meristems were obtained successfully. Simultaneously, a large amount of kanamycin-resistant somatic embryos of cotton in which a GUS gene was integrated via partical bombardment of embryogenic callus were also obtained. A new transformation system that combined Agrobacterium-mediated and particle bombardment transformation was attempted in cotton.
Shoot-tip culled from 3-day-old seedlings was opmtimal to the holistic transformation system. Thereafter, a more efficient regeneration system was established by comprehensive contrastes and the parameters combinations were as follows: Helium pressure at 1 lOOpsi, the vaccum pressure at 28 inches Hg, target distance at 9cm, 1 time bombardment. Subsequently, the meristems tissue bombarded were cultured in the selective media containing kanamycin, and then the concentration of kanamycin was steped to a higher amount in the series of subculture media. In the end, many regenerated resistant plants to kanamycin were obtained, with a transformation rate reaching 4.23%, and the cycles of transformation were approximate 80 days. The surviving rates of transplanting of the transgenic plants exceeded 90% by grafting, thereafter; the plants grafted were confirmed by PCR assay.
Embryogenic callus particle bombardment system was improved through GUS expression, and get a more efficient system: firstly, osmotic treated to embryogenic callus in solid medium with 0.3mol/L mannitol for 4hr before and 16hr after bombardments. Secondly, bombard the calli treated with the parameters showed as follow: vaccum pressure at 28 inches Hg, target distance at 9cm, 2 times bombardments, target DNA of 0.5-1.0 g per bombardment. After these, the calli was transferred to culture in selective medium containing kanamycin 150mg/L after recovery culture of 10-15days, and two months later, the somatic embryos resistant to kanamycin were obtained while a stable expression of GUS was detected in the embryogenic callus, which showed the GUS gene had been introduced the genome of cotton.
The apical merstems of cotton were very difficult to germinate after treatment of combination of Agrobocterium-med\a.ted transformation and particle bombardment in my exploration. So the new method need further research.
This research added new content and germplasm to modern cotton molecular breeding. Meanwhile, it was significant to the construction of a technical system of transformtion on a large-scale and insect-resistant gene engineering in cotton.
引文
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