利用人工microRNA技术构建CHSJ基因沉默载体及适于矮牵牛转化株系的筛选
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摘要
矮牵牛具有易于组培、植株再生容易、生长周期较长、易于管理、花期长、对环境要求不严格等优点。因此,矮牵牛不但是重要的观赏花卉,同时在基因研究中发挥重要的作用。在植物进行遗传转化过程中,基因型是影响遗传转化效果的重要因素。本实验构建矮牵牛CHSJ(X14599基因)基因沉默植物表达载体,并在对其农杆菌转化过程中,对矮牵牛株系进行筛选,选出适于进行遗传转化的株系。且对筛选出的株系,进行农杆菌转化条件优化。结果如下：
1.利用人工miRNA技术,构建矮牵牛CHSJ(X14599基因)基因沉默植物表达载体pCMF604。
2.种子在氯气中消毒4h,获得种子发芽率最高且污染率最低；在获得无菌苗的过程中,把氯气消毒种子与升汞消毒的外植体和种子相比较发现：氯气消毒种子发芽所需时间较短,且操作简单。对无菌苗进行再生率的优化之前对无菌苗进行农艺性状的观察筛选出26个株系。对26个株系再生率同对照A01相比较,得到再生率较高于对照A01的株系：C01、C02、C03、C04、C05和C06。
3.对C01、C02、C03、C04、C05和C06六个株系,农杆菌侵染后进行染色率和染色点数统计,于对照A01比较得出了染色率较高的株系C01、C04和C06。
4.对C01、C04和C06三个株系进行转化条件的优化,结果表明：共培养2-3d、菌液浓度OD600=0.3-0.4、侵染时间5-10min和在侵染液中加入AS时会达到最佳的侵染效果,并对pCMF604侵染获得抗性愈伤组织。
Petunia have many advantages, including easy to regeneration, easy to manage, the longer of growth cycle, the longer of flowering period, not strictly for the environ-mental requirements and so on. So, petunia is not only the important ornamental flow ers, but also the important model plant in the research of gene. Genotype is the impor-tant factors In the Agro bacterium-mediated transformation. In this study, we selected the suitable petunia lines for the Agro bacterium-mediated transformation. We carried out optimization for the selected petunia lines. The results are as following:
1. We successfully constructed a petunia pMADS9 miRNA gene silencing vector for plant expression vector by overlaping PCR method, Using Tools carrier RS300, the intermediate vector pMF604 and plant expression vector pC2301,
2. After the Chlorine disinfection in 4h, the seed has the highest seed germination rate and the lowest pollution; to compared with the seed or the explants by the mercuric disinfection, the seed by the Chlorine disinfection have many advantages, as short to germination time and easy to operate.In agronomic traits, we seleced 26 petunia lines. Compared with the petunia lines A01, we selected 6 petunia lines (C01, C02, C03, C04, C05 and C06).
2. After the infection by agrobacterium, we gathered statistics of the selected 6 petunia lines (C01, C02, C03, C04, C05 and C06) in GUS transient expression. Compared with the petunia lines A01, we selected the petunia lines C01, C04 and C06.
3. For the three petunia lines C01, C04 and C06, we optimized the transformation conditions, the results show that:In a certain ranges of the concentration of Agro bacterium (OD600=0.3-0.4), time of inoculation(5-10 min) and time of co-culture(2-3 d); we would have higher conversion and lower contamination rate.
1.利用人工miRNA技术,构建矮牵牛CHSJ(X14599基因)基因沉默植物表达载体pCMF604。
2.种子在氯气中消毒4h,获得种子发芽率最高且污染率最低；在获得无菌苗的过程中,把氯气消毒种子与升汞消毒的外植体和种子相比较发现：氯气消毒种子发芽所需时间较短,且操作简单。对无菌苗进行再生率的优化之前对无菌苗进行农艺性状的观察筛选出26个株系。对26个株系再生率同对照A01相比较,得到再生率较高于对照A01的株系：C01、C02、C03、C04、C05和C06。
3.对C01、C02、C03、C04、C05和C06六个株系,农杆菌侵染后进行染色率和染色点数统计,于对照A01比较得出了染色率较高的株系C01、C04和C06。
4.对C01、C04和C06三个株系进行转化条件的优化,结果表明：共培养2-3d、菌液浓度OD600=0.3-0.4、侵染时间5-10min和在侵染液中加入AS时会达到最佳的侵染效果,并对pCMF604侵染获得抗性愈伤组织。
Petunia have many advantages, including easy to regeneration, easy to manage, the longer of growth cycle, the longer of flowering period, not strictly for the environ-mental requirements and so on. So, petunia is not only the important ornamental flow ers, but also the important model plant in the research of gene. Genotype is the impor-tant factors In the Agro bacterium-mediated transformation. In this study, we selected the suitable petunia lines for the Agro bacterium-mediated transformation. We carried out optimization for the selected petunia lines. The results are as following:
1. We successfully constructed a petunia pMADS9 miRNA gene silencing vector for plant expression vector by overlaping PCR method, Using Tools carrier RS300, the intermediate vector pMF604 and plant expression vector pC2301,
2. After the Chlorine disinfection in 4h, the seed has the highest seed germination rate and the lowest pollution; to compared with the seed or the explants by the mercuric disinfection, the seed by the Chlorine disinfection have many advantages, as short to germination time and easy to operate.In agronomic traits, we seleced 26 petunia lines. Compared with the petunia lines A01, we selected 6 petunia lines (C01, C02, C03, C04, C05 and C06).
2. After the infection by agrobacterium, we gathered statistics of the selected 6 petunia lines (C01, C02, C03, C04, C05 and C06) in GUS transient expression. Compared with the petunia lines A01, we selected the petunia lines C01, C04 and C06.
3. For the three petunia lines C01, C04 and C06, we optimized the transformation conditions, the results show that:In a certain ranges of the concentration of Agro bacterium (OD600=0.3-0.4), time of inoculation(5-10 min) and time of co-culture(2-3 d); we would have higher conversion and lower contamination rate.
引文
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