农杆菌介导的百合ACO反义基因转化东方百合‘索邦’的研究
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摘要
百合是世界著名的球根花卉和鲜切花花卉,在世界花卉市场中占有极其重要的位置。百合作为一种呼吸跃变型花卉,在开花过程中伴随着乙烯的大量释放而迅速衰老,如能有效控制百合花期乙烯生成量即可延长其观赏寿命,增加百合的观赏价值。
本研究以东方百合‘索邦’为材料,利用根癌农杆菌介导法将百合ACO反义基因导入组培苗的鳞茎及叶柄组织。通过GUS基因组织化学染色、PCR、基于梯状回收的PCR法及Southern杂交等检测方法对外源基因导入情况进行鉴定。并对组培苗进行移栽试验。获得以下研究成果:
1通过根癌农杆菌介导法将ACO反义基因导入东方百合‘索邦’。获得潮霉素抗性植株165株。
2优化了本实验室建立的根癌农杆菌介导法的转化百合的遗传转化体系。外植体不经预培养,侵染时抽真空1 min,侵染30 min,菌液浓度OD=0.8,共培养7天可获得15.76%的转化率。
3应用GUS基因组织化学法检测、PCR检测、基于梯状胶回收的PCR和Southern杂交等转基因检测方法对转化植株进行检测。获得PCR检测阳性植株26株,对其中4个植株进行了Southern杂交检测和1个植株的PCR-Southern杂交检测获得PCR-Southern阳性植株1株。
4研究了潮霉素筛选时间对农杆菌介导法转化的百合植株体内外源基因的影响,确定持续抗生素筛选对转化植株的作用。
5对东方百合‘索邦’组培苗的进行炼苗移栽,移栽苗成活率达92.4%。筛选出较适宜‘索邦’组培苗移栽的基质为珍珠岩/泥炭1/1。初步研究不同外源激素对移栽苗生长的影响,喷施的较适宜浓度分别为IBA 50mg/L;NAA 50mg/L;GA3 6mg/L。
Lily is a world famous bulbous-flower and cut-flower, take very important part in world flower market. As a kind of ethylene lead decay flowers, flower decay rapidly while blooming with ethylene’s release. Liliaceous view and admire life will be prolonged, and the value of lily be raised if we can effectively control the release of ethylene. We did the genetic transformation of ACO antisense gene into lily’s bulb and leafstalk by Agrobacterium tumfaciens-mediated method. And exogenous genes were identified by GUS gene coloration, PCR, ladderlike purification-based PCR method and southern blot analysis. Besides, we did some research on transplant of tissue culture seeding.
1. 165 hygromycin-resistant plantlets were obtained through Agrobacterium tumefaciens-mediated transformation of ACO antisense gene into lilium oriental‘sorbonne’
2. Ameliorated the protocol of Agrobacterium tumefaciens-mediated transformation established by our lab. 15.76% genetic transformation rate was obtained according to the procedures: the explants without pre-cultured, immersed into bacterial suspension (OD600=0.8) vacuum degassed for 1min, slowly waggled for 30 min, and then co-cultivated for 7d.
3. Transformation plants were identified by histochemical GUS gene detection, PCR, ladderlike purification-based PCR method,southern blot and PCR-southern blot analysis. 26 positive plants were got out of 85 plants by PCR analysis, 1 positive plant was obtained by PCR-Southern.
4. According to studies on the effect of the selection time on hygB added medium on lilium exogenous genes transformed by Agrobacterium tumefaciens-mediated method, the effects of persist selection to transformation plants was ascertained.
5. A Transplant system was initiated from lilium oriental‘sorbonne’. The medium Perlite/peat 1/1 was identified as the best transplant medium. And we did some elementary research on different exogenous phytohormone’s effect on the transplant seeding. The suitable consistency is IBA 50mg/L; NAA 50mg/L; GA3 6mg/L.
本研究以东方百合‘索邦’为材料,利用根癌农杆菌介导法将百合ACO反义基因导入组培苗的鳞茎及叶柄组织。通过GUS基因组织化学染色、PCR、基于梯状回收的PCR法及Southern杂交等检测方法对外源基因导入情况进行鉴定。并对组培苗进行移栽试验。获得以下研究成果:
1通过根癌农杆菌介导法将ACO反义基因导入东方百合‘索邦’。获得潮霉素抗性植株165株。
2优化了本实验室建立的根癌农杆菌介导法的转化百合的遗传转化体系。外植体不经预培养,侵染时抽真空1 min,侵染30 min,菌液浓度OD=0.8,共培养7天可获得15.76%的转化率。
3应用GUS基因组织化学法检测、PCR检测、基于梯状胶回收的PCR和Southern杂交等转基因检测方法对转化植株进行检测。获得PCR检测阳性植株26株,对其中4个植株进行了Southern杂交检测和1个植株的PCR-Southern杂交检测获得PCR-Southern阳性植株1株。
4研究了潮霉素筛选时间对农杆菌介导法转化的百合植株体内外源基因的影响,确定持续抗生素筛选对转化植株的作用。
5对东方百合‘索邦’组培苗的进行炼苗移栽,移栽苗成活率达92.4%。筛选出较适宜‘索邦’组培苗移栽的基质为珍珠岩/泥炭1/1。初步研究不同外源激素对移栽苗生长的影响,喷施的较适宜浓度分别为IBA 50mg/L;NAA 50mg/L;GA3 6mg/L。
Lily is a world famous bulbous-flower and cut-flower, take very important part in world flower market. As a kind of ethylene lead decay flowers, flower decay rapidly while blooming with ethylene’s release. Liliaceous view and admire life will be prolonged, and the value of lily be raised if we can effectively control the release of ethylene. We did the genetic transformation of ACO antisense gene into lily’s bulb and leafstalk by Agrobacterium tumfaciens-mediated method. And exogenous genes were identified by GUS gene coloration, PCR, ladderlike purification-based PCR method and southern blot analysis. Besides, we did some research on transplant of tissue culture seeding.
1. 165 hygromycin-resistant plantlets were obtained through Agrobacterium tumefaciens-mediated transformation of ACO antisense gene into lilium oriental‘sorbonne’
2. Ameliorated the protocol of Agrobacterium tumefaciens-mediated transformation established by our lab. 15.76% genetic transformation rate was obtained according to the procedures: the explants without pre-cultured, immersed into bacterial suspension (OD600=0.8) vacuum degassed for 1min, slowly waggled for 30 min, and then co-cultivated for 7d.
3. Transformation plants were identified by histochemical GUS gene detection, PCR, ladderlike purification-based PCR method,southern blot and PCR-southern blot analysis. 26 positive plants were got out of 85 plants by PCR analysis, 1 positive plant was obtained by PCR-Southern.
4. According to studies on the effect of the selection time on hygB added medium on lilium exogenous genes transformed by Agrobacterium tumefaciens-mediated method, the effects of persist selection to transformation plants was ascertained.
5. A Transplant system was initiated from lilium oriental‘sorbonne’. The medium Perlite/peat 1/1 was identified as the best transplant medium. And we did some elementary research on different exogenous phytohormone’s effect on the transplant seeding. The suitable consistency is IBA 50mg/L; NAA 50mg/L; GA3 6mg/L.
引文
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