拟南芥悬浮细胞生物学及其遗传转化模式
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摘要
本文从拟南芥悬浮细胞及其愈伤组织生物学特性研究入手，以期制备适合农杆菌高效转化的受体材料，并制定科学、合理、有效的转化方案；利用所构成的多个质粒载体，进行拟南芥悬浮细胞基因转化，以检验多种转化方案的可行性，并优化转化条件；检测转化细胞报告基因的瞬时表达和稳定表达，以期建立有效的转化模式，并获得可利用的转化材料。本论文所做的主要工作如下：
1、拟南芥悬浮细胞及其愈伤组织生长特性与培养条件
试验研究了拟南芥悬浮细胞及其愈伤组织的生长特性及其继代培养条件，旨在摸索维持拟南芥悬浮细胞正常稳定生长的培养技术，并为拟南芥悬浮细胞进一步的遗传操作提供良好的受体材料和科学依据。结果表明，在每个7d一次的继代周期中，拟南芥悬浮细胞的生长表现明显的S型生长曲线。正常培养条件下，继代后2-4 d出现快速生长时期，此后生长量迅速下降，但接种量不同，其继代后悬浮细胞生物量及快速增长发生时期不同。维持7d继代周期的理想接种量为起始悬浮液按10ml／50ml稀释。接种量小，生长迟缓，甚至不能增殖。培养基中不同激素及不同蔗糖含量均会对其生长产生极明显的影响，在B5+0.2mg／L NAA条件下反复继代后的细胞，再加入外源NAA时，反而会抑制其生长。蔗糖含量以30g／L最好；蔗糖含量低，悬浮生物量及细胞活性明显下降。
拟南芥愈伤组织生长也呈现一定的规律性，正常条件下，需经历约各10d的诱导时期、快速生长时期和生长停滞及老化时期。愈伤组织诱导及其生长对外界条件也特别敏感，一定的表面湿度及较低的培养温度是必须的。
2、拟南芥悬浮细胞及其愈伤组织对抗生素的反应
试验研究了拟南芥悬浮及其愈伤组织对潮霉素、卡那霉素、头孢霉素等3种抗生素的反应，为拟南芥悬浮细胞转化子抗生素选择浓度和选择方案的制定提供依据。结果表明：潮霉素对拟南芥悬浮细胞生长、愈伤组织诱导及其生长均有极明显的抑制作用，但作用效果和特性均有所不同。拟南芥悬浮细胞继代培养阶段、愈伤组织诱导阶段和愈伤组织增殖阶段的临界致死浓度分别为25，2.5，1.5mg／L，液体与固体培养条件下差异极大。当采用潮霉素抗性标记筛选转化子时，应当分别采用相应阶段的临界致死浓度作为转化子选择浓度。与此同时，潮霉素需要一定处理时间才能充分表现其抑制细胞生长或杀死细胞的效果，一般液体培养条件下3d以后、固体培养条件下5d以后效果趋于稳定，但不同的处理浓度有较大差异，建议选择时间在液体培养条件下不少于3d，在固体培养条件下不少于5d。否则，假阳性率会很高，从而大大加重后续转化子检测的工作量。
卡那霉素与潮霉素有类似的影响，在愈伤组织诱导阶段，适宜的选择浓度为50mg／L，根据潮霉素作用特性推测，其在液体培养阶段和愈伤组织生长阶段的适宜选择浓度分别在100mg／L、25mg／L左右。后续的转化试验中，若利用卡那霉素选择，我们将采用这一选择浓度。
试验中意外发现头孢霉素能促进绿色悬浮细胞的获得，并具有逆转悬浮细胞
Transformation of Arabidopsis thaliana suspension cells was performed. Different protocols and parameters affecting the transformation procedure were tested, and an optimized protocol was determined. Transformants was selected with antibiotic resistant gene screening and checked with histochemical GUS and PCR assay. Prior to transformation experiments, some relational biology characteristics of Arabidopsis thaliana suspension cells and Agrobacteria haboring certein plasmid were observed. The main works and results are shown as follows.
Subculture Conditions and Growth Characteristics of Arabidopsis Suspension Culture
The experiments of Arabidopsis thaliana suspension culture were performed. Some subculture conditions were determined, and the growth characteristics of suspension culture was observed. The typical S-like growth curve in a subculture cycle was obtained. In normal condition, a logarithmic phase of growth of suspension biomass was occurred in 2-4 days after subculture. Than the growth of biomass slowed down quickly. However, its growth and time of logarithmic phase were changed with inoculation amount. 10ml/50ml of diluting ratio is advisable to subculture. The growth was slowed down even halted with 10ml/50ml of diluting ratio. Different hormones in media affected growth of suspension cells, but the result was unexpected. The biomass of suspension cells subcultured in B5 medium without any hormones was higher than with 0.1mg/L of BAP and 0.2mg/L NAA (as normal medium). Concentration of sucrose greatly affects growth of suspension cells. Changing it from normal 30g/L to 10,20,40 g/L, more or less inhibition of growth and viability were observed.
The pattern of callus growth was observed. In general, each a 10 d of period is presented in callus induction phase, logarithmic growth phase and growth-stopped phase. Incubation temperature and humidity had a remarkable impact on both inducement and growth of callus. A certain surface humidity on plate and lower incubation temperature were indispensable.
1、拟南芥悬浮细胞及其愈伤组织生长特性与培养条件
试验研究了拟南芥悬浮细胞及其愈伤组织的生长特性及其继代培养条件，旨在摸索维持拟南芥悬浮细胞正常稳定生长的培养技术，并为拟南芥悬浮细胞进一步的遗传操作提供良好的受体材料和科学依据。结果表明，在每个7d一次的继代周期中，拟南芥悬浮细胞的生长表现明显的S型生长曲线。正常培养条件下，继代后2-4 d出现快速生长时期，此后生长量迅速下降，但接种量不同，其继代后悬浮细胞生物量及快速增长发生时期不同。维持7d继代周期的理想接种量为起始悬浮液按10ml／50ml稀释。接种量小，生长迟缓，甚至不能增殖。培养基中不同激素及不同蔗糖含量均会对其生长产生极明显的影响，在B5+0.2mg／L NAA条件下反复继代后的细胞，再加入外源NAA时，反而会抑制其生长。蔗糖含量以30g／L最好；蔗糖含量低，悬浮生物量及细胞活性明显下降。
拟南芥愈伤组织生长也呈现一定的规律性，正常条件下，需经历约各10d的诱导时期、快速生长时期和生长停滞及老化时期。愈伤组织诱导及其生长对外界条件也特别敏感，一定的表面湿度及较低的培养温度是必须的。
2、拟南芥悬浮细胞及其愈伤组织对抗生素的反应
试验研究了拟南芥悬浮及其愈伤组织对潮霉素、卡那霉素、头孢霉素等3种抗生素的反应，为拟南芥悬浮细胞转化子抗生素选择浓度和选择方案的制定提供依据。结果表明：潮霉素对拟南芥悬浮细胞生长、愈伤组织诱导及其生长均有极明显的抑制作用，但作用效果和特性均有所不同。拟南芥悬浮细胞继代培养阶段、愈伤组织诱导阶段和愈伤组织增殖阶段的临界致死浓度分别为25，2.5，1.5mg／L，液体与固体培养条件下差异极大。当采用潮霉素抗性标记筛选转化子时，应当分别采用相应阶段的临界致死浓度作为转化子选择浓度。与此同时，潮霉素需要一定处理时间才能充分表现其抑制细胞生长或杀死细胞的效果，一般液体培养条件下3d以后、固体培养条件下5d以后效果趋于稳定，但不同的处理浓度有较大差异，建议选择时间在液体培养条件下不少于3d，在固体培养条件下不少于5d。否则，假阳性率会很高，从而大大加重后续转化子检测的工作量。
卡那霉素与潮霉素有类似的影响，在愈伤组织诱导阶段，适宜的选择浓度为50mg／L，根据潮霉素作用特性推测，其在液体培养阶段和愈伤组织生长阶段的适宜选择浓度分别在100mg／L、25mg／L左右。后续的转化试验中，若利用卡那霉素选择，我们将采用这一选择浓度。
试验中意外发现头孢霉素能促进绿色悬浮细胞的获得，并具有逆转悬浮细胞
Transformation of Arabidopsis thaliana suspension cells was performed. Different protocols and parameters affecting the transformation procedure were tested, and an optimized protocol was determined. Transformants was selected with antibiotic resistant gene screening and checked with histochemical GUS and PCR assay. Prior to transformation experiments, some relational biology characteristics of Arabidopsis thaliana suspension cells and Agrobacteria haboring certein plasmid were observed. The main works and results are shown as follows.
Subculture Conditions and Growth Characteristics of Arabidopsis Suspension Culture
The experiments of Arabidopsis thaliana suspension culture were performed. Some subculture conditions were determined, and the growth characteristics of suspension culture was observed. The typical S-like growth curve in a subculture cycle was obtained. In normal condition, a logarithmic phase of growth of suspension biomass was occurred in 2-4 days after subculture. Than the growth of biomass slowed down quickly. However, its growth and time of logarithmic phase were changed with inoculation amount. 10ml/50ml of diluting ratio is advisable to subculture. The growth was slowed down even halted with 10ml/50ml of diluting ratio. Different hormones in media affected growth of suspension cells, but the result was unexpected. The biomass of suspension cells subcultured in B5 medium without any hormones was higher than with 0.1mg/L of BAP and 0.2mg/L NAA (as normal medium). Concentration of sucrose greatly affects growth of suspension cells. Changing it from normal 30g/L to 10,20,40 g/L, more or less inhibition of growth and viability were observed.
The pattern of callus growth was observed. In general, each a 10 d of period is presented in callus induction phase, logarithmic growth phase and growth-stopped phase. Incubation temperature and humidity had a remarkable impact on both inducement and growth of callus. A certain surface humidity on plate and lower incubation temperature were indispensable.
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