外源基因转化南荻和籼稻的研究
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摘要
本文对南荻基因枪转化系统的建立及其外源基因的转化进行了较为系统的
研究。同时，还对籼稻基因枪转化系统和农杆菌转化系统的优化，以及抗虫、抗
病目的基因对多个籼稻栽培品种的转化进行了一系列的研究。
1．南荻基因枪转化系统的建立及外源基因转化的研究
南荻是一种重要的经济植物，它既可提供优质的造纸、造板原材料，又具
有良好的生态效应。然而，它的虫害却十分严重。因此，开展南荻遗传转化的
研究，获得转基因抗虫南荻新品种，有着十分重要的意义。本研究在这方面取
得了以下结果：
建立了南荻胚性愈伤组织高频诱导与再生系统。研究结果表明，南荻幼穗
是诱导愈伤组织最理想的外植体，其愈伤组织诱导率高达100％，其愈伤组织分
化率可达87.5％；南荻愈伤组织最适合的基本培养基是CC培养基。
建立了南荻愈伤组织超低温冷冻保存方法。采用高渗预培养和冷冻保护剂
（10％DMSO＋0.5mol/L山梨醇）处理方法进行南荻愈伤组织液氮超低温冷冻保
存可获得满意的效果，冷冻6个月后的愈伤组织其存活率可保持68.4％，存活愈
伤的分化率仍高达78.3％。
建立了南荻抗性愈伤组织的筛选方案。对南荻愈伤组织的筛选，选用G418、
Hyg和Basta作筛选剂效果较好，其适宜的筛选浓度分别为150-200mg/L、20-
30mg/L和10-20mg/L，其筛选时间均以40-60天为宜。
建立了南荻愈伤组织基因枪转化的技术体系。其技术要点为，选用继代培
养60天以内的胚性愈伤组织作为转化受体，采用1300psi的压力、每枪250μg金
粉和0.5μg质粒DNA的用量、6cm的轰击距离和每皿材料轰击二次的转化参数，
可获得较好的转化效果。另外，使用45 g／L浓度的甘露醇对受体愈伤组织作轰
击前12小时至轰击后72小时的高渗处理可以明显提高转化效率。
获得了转基因南获植株。应用本研究所建立的基因枪转化系统，将马铃薯
蛋白酶抑制剂oinll）基因和花粉特异性mA酶（bamase）基因导入南获愈伤组
智 织，经筛选剂筛选获得52株抗性植株。通过对部分植株进行PCR点杂交和
Southern杂交等分子检测的结果表明，外源目的基因已整合进了南获转化植株
基因组。对转基因植株的抗虫性鉴定和育性分析正在进行中。
到目前为止，有关南获的遗传转化研究，在国内外还未见有任何报道，本
研究结果无疑为这一研究的进一步深入奠定了基础。
2．和稻基因枪转化系统和农杆苗转化系统的优化及其抗虫、抗病基因转化
的研究
本文进行了将苏云金芽抱杆菌毒蛋白（Bt）基因、马铃薯蛋白酶抑制剂（Pin
11）基因和雪花莲凝集素（GNA）基因等抗虫基因以及几丁质酶Khi）和葡聚糖
酶（Gin）等抗病基因导入 14种重要釉稻栽培品种或品系（其中包括不育系培矮645
＠及其恢复系E32、特青、288和93等重要材料）的研究。主要结果概括如下：
优化了和稻胚性愈伤组织诱导与分化的培养条件。研究结果表明，CC培养
基是绝大多数制稻材料愈伤组织的最适诱导与继代培养基，它可以明显减轻釉
稻愈伤组织的褐化程度；添加2．56 mg／L ABA可以有效地改善和稻愈伤组织的
生长状态，促进胚性愈伤组织的形成；植物凝胶作为培养基固化剂所培养的愈
伤组织质量明显优于用琼脂所培养的愈伤质量。
优化了釉稻抗性愈伤组织的筛选条件，并建立了狲稻苗期叶片筛选方案。
t稻抗性愈伤组织的最适筛选条件为：使用30＊0 mg／L浓度的Hyg筛选40天左
右，或用150E00 mg／L浓度的G418筛选50天左右，或用10上0 mg／L浓度的Basta
筛选50七O天：舢稻苗期叶片筛选鉴定的最佳方案为：用100二00 m叭浓度的 陀
筛选4－6天，或用 10－15 mwh浓度的Basta筛选46天。
．
优化了制稻愈伤组织基因枪转化技术体系。其最佳优化参数为：每枪 100 pg
金粉吸附0二 pg DNA的用量、900psi的压力、6cm的轰击距离和每个样品轰击两
2
枪，并且以60 g／L甘露醇浓度作轰击前12毛4小时至轰击后24－48小时的高渗处
理。
优化了釉稻愈伤组织农杆菌转化方法。实验结果表明，根癌农杆菌EHA105
菌株对和稻的转化效果优于LBA 4404和AGL；菌株的转化效果；头抱霉素对农
．杆菌的抑制效果优于Wt青霉素的抑制效果，使用300－400 mg／L头抱霉素浓度
可得到满意的结果；在共培养过程完成后进行适当的干燥处理既可增强抑菌效
果，又可提高转化频率。
获得了抗虫、抗病的转基因釉稻植株。应用本研究优化的基因枪和农杆菌
转化方法，将＊t、＊＂ill、＊N和＊IU等基因导入到了
In the first part of this thesis the biolistic bombardment transformation system for
Miscanthus saccharjflorus was established and introduction of some agronomically
useful genes was performed. In the second part of thesis the factors influencing the
transfonnation efficiency of several indica rice varieties for both Agrobacterium and
biolistic bombardment were investigated, and the insect and fungal resistant genes
were introduced into several indica rice varieties.
Establishment of biolistic bombardment transformation system for
Miscanthus Saccha lorus and its transformation with exogenous
genes
Miscanthus saccharzflorus is one of the important commercial plants, it is not
only an excellent raw material for papermaking and artificial board, also very
important for sustaining ecological system. But it is extremely vulnerable to the insect
attack. Therefore, any attempt that creates the insect resistant plants is encouraged. In
this thesis we are attempting to improve the plant resistance to insect via
biotechnology. As the first step the initiation and regeneration system for
embryogenic callus of Miscanthus saccharjflorus were established at very high
frequency. The result showed that the young inflorescence was the best explant for
callus induction and easier differentiation into entire plantlets compared to other
explants such as mature seeds, leaf, root etc. in Miscanthus saccharflorus. CC
medium was the best basic medium for subculture of the callus amongst all the media
tested.As the second step the selection scheme for tolerant callus of Miscanthus
saccharflorus was established. The suitable selection scheme was culturing the callus
on the medium containing 150-200 mg/L G4 18 or 20-30 mg/L Hyg or 10-20 mg(L
Basta for 40-60 days. By using the particle bombardment transformation system
potato proteinase inhibitor II (pinll) and pollen specific ribonuclease (barnase) genes
were introduced into the callus of Miscanthus saccharflorus and 52 transgenic plants
were obtained. PCR, Southern, and dot blot analysis showed that target genes were
integrated into the genome of these transgenic plants, the further bioassays on
resistance to insect and fertility for these transgenic plants are underway.
Since the differentiation capacity of the embryogenic callus degenerated with
period of subculture. The cryopreservation for embryogenic callus of Miscanthus
saccharjflorus was also investigated. With treatment of desiccation and addition of
protective agent (10 % DM50 + 0.5 M sorbitol) the callus cryopreserved in liquid
nitrogen for 6 months still kept at high survival rate (68.4%) and regeneration
frequency (78.3%), which makes all-year-around transformation possible.
The optimization of biolistic bombardment and Agrobacleriwn
transformation system for indica rice and their transformation with
insect and fungal resistant genes
Since the condition for initiation and regeneration of embryonic callus varied, the
14 different indica rice varieties were used in this thesis for investigation on these
aspects in detail. The results showed that for most indica rice CC medium was the
best for both callus initiation and subculture in which the browning of calli could be
mitigated significantly. With supplement of 2.5-Smg/L absisic acid (ABA) the quality
of calli can be improved and the rate of embryogenic calli were increased, the gelling
agents also significantly influence the quality of calli which phytagel was the best
among all the agars used.
The selective efficiency and selective peri
研究。同时，还对籼稻基因枪转化系统和农杆菌转化系统的优化，以及抗虫、抗
病目的基因对多个籼稻栽培品种的转化进行了一系列的研究。
1．南荻基因枪转化系统的建立及外源基因转化的研究
南荻是一种重要的经济植物，它既可提供优质的造纸、造板原材料，又具
有良好的生态效应。然而，它的虫害却十分严重。因此，开展南荻遗传转化的
研究，获得转基因抗虫南荻新品种，有着十分重要的意义。本研究在这方面取
得了以下结果：
建立了南荻胚性愈伤组织高频诱导与再生系统。研究结果表明，南荻幼穗
是诱导愈伤组织最理想的外植体，其愈伤组织诱导率高达100％，其愈伤组织分
化率可达87.5％；南荻愈伤组织最适合的基本培养基是CC培养基。
建立了南荻愈伤组织超低温冷冻保存方法。采用高渗预培养和冷冻保护剂
（10％DMSO＋0.5mol/L山梨醇）处理方法进行南荻愈伤组织液氮超低温冷冻保
存可获得满意的效果，冷冻6个月后的愈伤组织其存活率可保持68.4％，存活愈
伤的分化率仍高达78.3％。
建立了南荻抗性愈伤组织的筛选方案。对南荻愈伤组织的筛选，选用G418、
Hyg和Basta作筛选剂效果较好，其适宜的筛选浓度分别为150-200mg/L、20-
30mg/L和10-20mg/L，其筛选时间均以40-60天为宜。
建立了南荻愈伤组织基因枪转化的技术体系。其技术要点为，选用继代培
养60天以内的胚性愈伤组织作为转化受体，采用1300psi的压力、每枪250μg金
粉和0.5μg质粒DNA的用量、6cm的轰击距离和每皿材料轰击二次的转化参数，
可获得较好的转化效果。另外，使用45 g／L浓度的甘露醇对受体愈伤组织作轰
击前12小时至轰击后72小时的高渗处理可以明显提高转化效率。
获得了转基因南获植株。应用本研究所建立的基因枪转化系统，将马铃薯
蛋白酶抑制剂oinll）基因和花粉特异性mA酶（bamase）基因导入南获愈伤组
智 织，经筛选剂筛选获得52株抗性植株。通过对部分植株进行PCR点杂交和
Southern杂交等分子检测的结果表明，外源目的基因已整合进了南获转化植株
基因组。对转基因植株的抗虫性鉴定和育性分析正在进行中。
到目前为止，有关南获的遗传转化研究，在国内外还未见有任何报道，本
研究结果无疑为这一研究的进一步深入奠定了基础。
2．和稻基因枪转化系统和农杆苗转化系统的优化及其抗虫、抗病基因转化
的研究
本文进行了将苏云金芽抱杆菌毒蛋白（Bt）基因、马铃薯蛋白酶抑制剂（Pin
11）基因和雪花莲凝集素（GNA）基因等抗虫基因以及几丁质酶Khi）和葡聚糖
酶（Gin）等抗病基因导入 14种重要釉稻栽培品种或品系（其中包括不育系培矮645
＠及其恢复系E32、特青、288和93等重要材料）的研究。主要结果概括如下：
优化了和稻胚性愈伤组织诱导与分化的培养条件。研究结果表明，CC培养
基是绝大多数制稻材料愈伤组织的最适诱导与继代培养基，它可以明显减轻釉
稻愈伤组织的褐化程度；添加2．56 mg／L ABA可以有效地改善和稻愈伤组织的
生长状态，促进胚性愈伤组织的形成；植物凝胶作为培养基固化剂所培养的愈
伤组织质量明显优于用琼脂所培养的愈伤质量。
优化了釉稻抗性愈伤组织的筛选条件，并建立了狲稻苗期叶片筛选方案。
t稻抗性愈伤组织的最适筛选条件为：使用30＊0 mg／L浓度的Hyg筛选40天左
右，或用150E00 mg／L浓度的G418筛选50天左右，或用10上0 mg／L浓度的Basta
筛选50七O天：舢稻苗期叶片筛选鉴定的最佳方案为：用100二00 m叭浓度的 陀
筛选4－6天，或用 10－15 mwh浓度的Basta筛选46天。
．
优化了制稻愈伤组织基因枪转化技术体系。其最佳优化参数为：每枪 100 pg
金粉吸附0二 pg DNA的用量、900psi的压力、6cm的轰击距离和每个样品轰击两
2
枪，并且以60 g／L甘露醇浓度作轰击前12毛4小时至轰击后24－48小时的高渗处
理。
优化了釉稻愈伤组织农杆菌转化方法。实验结果表明，根癌农杆菌EHA105
菌株对和稻的转化效果优于LBA 4404和AGL；菌株的转化效果；头抱霉素对农
．杆菌的抑制效果优于Wt青霉素的抑制效果，使用300－400 mg／L头抱霉素浓度
可得到满意的结果；在共培养过程完成后进行适当的干燥处理既可增强抑菌效
果，又可提高转化频率。
获得了抗虫、抗病的转基因釉稻植株。应用本研究优化的基因枪和农杆菌
转化方法，将＊t、＊＂ill、＊N和＊IU等基因导入到了
In the first part of this thesis the biolistic bombardment transformation system for
Miscanthus saccharjflorus was established and introduction of some agronomically
useful genes was performed. In the second part of thesis the factors influencing the
transfonnation efficiency of several indica rice varieties for both Agrobacterium and
biolistic bombardment were investigated, and the insect and fungal resistant genes
were introduced into several indica rice varieties.
Establishment of biolistic bombardment transformation system for
Miscanthus Saccha lorus and its transformation with exogenous
genes
Miscanthus saccharzflorus is one of the important commercial plants, it is not
only an excellent raw material for papermaking and artificial board, also very
important for sustaining ecological system. But it is extremely vulnerable to the insect
attack. Therefore, any attempt that creates the insect resistant plants is encouraged. In
this thesis we are attempting to improve the plant resistance to insect via
biotechnology. As the first step the initiation and regeneration system for
embryogenic callus of Miscanthus saccharjflorus were established at very high
frequency. The result showed that the young inflorescence was the best explant for
callus induction and easier differentiation into entire plantlets compared to other
explants such as mature seeds, leaf, root etc. in Miscanthus saccharflorus. CC
medium was the best basic medium for subculture of the callus amongst all the media
tested.As the second step the selection scheme for tolerant callus of Miscanthus
saccharflorus was established. The suitable selection scheme was culturing the callus
on the medium containing 150-200 mg/L G4 18 or 20-30 mg/L Hyg or 10-20 mg(L
Basta for 40-60 days. By using the particle bombardment transformation system
potato proteinase inhibitor II (pinll) and pollen specific ribonuclease (barnase) genes
were introduced into the callus of Miscanthus saccharflorus and 52 transgenic plants
were obtained. PCR, Southern, and dot blot analysis showed that target genes were
integrated into the genome of these transgenic plants, the further bioassays on
resistance to insect and fertility for these transgenic plants are underway.
Since the differentiation capacity of the embryogenic callus degenerated with
period of subculture. The cryopreservation for embryogenic callus of Miscanthus
saccharjflorus was also investigated. With treatment of desiccation and addition of
protective agent (10 % DM50 + 0.5 M sorbitol) the callus cryopreserved in liquid
nitrogen for 6 months still kept at high survival rate (68.4%) and regeneration
frequency (78.3%), which makes all-year-around transformation possible.
The optimization of biolistic bombardment and Agrobacleriwn
transformation system for indica rice and their transformation with
insect and fungal resistant genes
Since the condition for initiation and regeneration of embryonic callus varied, the
14 different indica rice varieties were used in this thesis for investigation on these
aspects in detail. The results showed that for most indica rice CC medium was the
best for both callus initiation and subculture in which the browning of calli could be
mitigated significantly. With supplement of 2.5-Smg/L absisic acid (ABA) the quality
of calli can be improved and the rate of embryogenic calli were increased, the gelling
agents also significantly influence the quality of calli which phytagel was the best
among all the agars used.
The selective efficiency and selective peri
引文
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