香蕉与龙眼转化受体系统建立及转化PEAS基因初步研究
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摘要
本研究分别以香蕉(Musa spp．)品种天宝蕉(AAA类型)试管苗茎尖的横切薄片和龙眼(Dimocarpus longan Lour．)品种“红核子”幼胚诱导并长期继代保持的胚性愈伤组织(Embryogenie calli，简称EC)作为试验材料，建立了香蕉与龙眼基因转化的受体系统，并采用根癌农杆菌介导法进行香蕉和龙眼转化的初步研究。主要试验结果如下：
1 建立了香蕉基因转化的适宜受体系统。采用横切薄层培养方法建立了香蕉的高效再生系统。结果发现：在30℃、黑暗培养15d，而后转入24℃、光照培养5d的香蕉横切薄层的出芽率较高，植株生长较健壮，适宜用作转基因的受体；羧苄青霉素对香蕉薄片的生长没有明显的抑制作用，可以作为转化的抑菌剂：香蕉对潮霉素比较敏感，能够显著抑制香蕉横切薄层的出芽率，确定用40mg／L的潮霉素作为筛选的工作浓度。
2 建立了龙眼基因转化的适宜受体系统。采用生长量测定的方法确定了继代培养10—15d的龙眼EC生长速度最快，生活力最强，适宜用作转基因的受体；同时采用生长量测定结合定性观察发现高浓度的潮霉素严重抑制龙眼EC的生长，确定50mg／L为有效的筛选浓度。
3 进行了对香蕉和龙眼基因转化的初步研究。采用根癌农杆菌(带hpt基因)介导的方法进行了目的基因PEAS导入香蕉横切薄片和龙眼胚性愈伤组织的研究。采用附加潮霉素的筛选培养基对感染后的材料进行筛选，共得到10个系列的香蕉抗性芽和5个龙眼抗性细胞系。由于时间的关系，对这些抗性芽和抗性EC的分子检测只进行了hpt抗性基因的PCR初步检测。随机抽取的15株香蕉抗性植株中发现有2株整合进了hpt抗性基因；龙眼5个抗性细胞系中各随机抽取1份进行检测，发现只有1个细胞系整合进了hpt抗性基因。关于后续的PEAS基因的PCR检测、Southern杂交、Northern杂交以及转基因植株田间的表达等问题在以后的试验中将继续进行研究。
本研究为香蕉和龙眼转基因的研究提供了技术平台，可用于香蕉、龙眼的生物技术育种。
In this experiment, a high efficient receptor system was established using the slices induced from the thin cell layer of Musa spp. cv. Tianbaojiao(AAA group) and the long-term maintained embryogenic call! (EC) induced from immature embryos of Dimocarpus longan Lour. cv. Honghezi, and then the preliminary study on the introduction of PEAS gene mediated by Agrobacterium tumefaciens was performed. The main results were described as follows:
1) An appropriate receptor system for banana genetic transformation was established. The high regeneration system was established by thin-cell-layer culture.It was found that the high differentiation of banana shoots was obtained from the slices cultured at 24C in light for 5d after they had been cultured at 30*C in darkness for 15d and, which grew well and was suitable for the receptor system; the sensitivity of banana thin cell layer to carbenicillin was limited, and it did not restrain the growth of banana, which could be used as the bacteriostasis for banana transformation; and banana was sensitive to hygromycin B, and it remarkably restrained the growth of banana., which could be used as the screening agent, and the concentration of 50mg/L for hygromycin B was the effective concentration for banana transgenic selection.
2) An appropriate receptor system for longan genetic transformation was established. Calli pre-cultured for 10-15 d were proved at their rapid-growth stage and their strong vitality stage through the measurement of EC fresh weight increase and suistable for transgenics. The sensitivity of longan EC to hygromycin B was evaluated through the method of the measurement of fresh weight increase combined with qualitative obserbation, which showed that high concentration of hygromycin B obviously depressed the growth of longan EC. The concentration of 50mg/L for hygromycin B was the effective concentration for longan EC transgenic selection.
3) The preliminary study was performed on the transformation of banana and longan. The target gene PEAS was introduced to the thin cell layer of banana and EC of longan mediated by Agrobacterium tumefaciens with hpt gene. After infection the materials were selected on MS medium supplemented with hygromycin B, finally 10
lines of banana and 5 cell lines of longan were obtained. PCR assays indicated that hpt gene had been integrated into both the genomes of the 2 lines from 15 tested banana plantlets and one line from 5 tested longan lines. Something about the assays of PEAS gene> Southern blotting, Northern blotting and the expression of the transplants in field were to be further studied.
The establishment and the preliminary study on transformation of thin cell layer of banana and EC of longan would be the technique plat of genetic transformation of banana and longan, which could be used for genetic improvement of banana and longan.
1 建立了香蕉基因转化的适宜受体系统。采用横切薄层培养方法建立了香蕉的高效再生系统。结果发现：在30℃、黑暗培养15d，而后转入24℃、光照培养5d的香蕉横切薄层的出芽率较高，植株生长较健壮，适宜用作转基因的受体；羧苄青霉素对香蕉薄片的生长没有明显的抑制作用，可以作为转化的抑菌剂：香蕉对潮霉素比较敏感，能够显著抑制香蕉横切薄层的出芽率，确定用40mg／L的潮霉素作为筛选的工作浓度。
2 建立了龙眼基因转化的适宜受体系统。采用生长量测定的方法确定了继代培养10—15d的龙眼EC生长速度最快，生活力最强，适宜用作转基因的受体；同时采用生长量测定结合定性观察发现高浓度的潮霉素严重抑制龙眼EC的生长，确定50mg／L为有效的筛选浓度。
3 进行了对香蕉和龙眼基因转化的初步研究。采用根癌农杆菌(带hpt基因)介导的方法进行了目的基因PEAS导入香蕉横切薄片和龙眼胚性愈伤组织的研究。采用附加潮霉素的筛选培养基对感染后的材料进行筛选，共得到10个系列的香蕉抗性芽和5个龙眼抗性细胞系。由于时间的关系，对这些抗性芽和抗性EC的分子检测只进行了hpt抗性基因的PCR初步检测。随机抽取的15株香蕉抗性植株中发现有2株整合进了hpt抗性基因；龙眼5个抗性细胞系中各随机抽取1份进行检测，发现只有1个细胞系整合进了hpt抗性基因。关于后续的PEAS基因的PCR检测、Southern杂交、Northern杂交以及转基因植株田间的表达等问题在以后的试验中将继续进行研究。
本研究为香蕉和龙眼转基因的研究提供了技术平台，可用于香蕉、龙眼的生物技术育种。
In this experiment, a high efficient receptor system was established using the slices induced from the thin cell layer of Musa spp. cv. Tianbaojiao(AAA group) and the long-term maintained embryogenic call! (EC) induced from immature embryos of Dimocarpus longan Lour. cv. Honghezi, and then the preliminary study on the introduction of PEAS gene mediated by Agrobacterium tumefaciens was performed. The main results were described as follows:
1) An appropriate receptor system for banana genetic transformation was established. The high regeneration system was established by thin-cell-layer culture.It was found that the high differentiation of banana shoots was obtained from the slices cultured at 24C in light for 5d after they had been cultured at 30*C in darkness for 15d and, which grew well and was suitable for the receptor system; the sensitivity of banana thin cell layer to carbenicillin was limited, and it did not restrain the growth of banana, which could be used as the bacteriostasis for banana transformation; and banana was sensitive to hygromycin B, and it remarkably restrained the growth of banana., which could be used as the screening agent, and the concentration of 50mg/L for hygromycin B was the effective concentration for banana transgenic selection.
2) An appropriate receptor system for longan genetic transformation was established. Calli pre-cultured for 10-15 d were proved at their rapid-growth stage and their strong vitality stage through the measurement of EC fresh weight increase and suistable for transgenics. The sensitivity of longan EC to hygromycin B was evaluated through the method of the measurement of fresh weight increase combined with qualitative obserbation, which showed that high concentration of hygromycin B obviously depressed the growth of longan EC. The concentration of 50mg/L for hygromycin B was the effective concentration for longan EC transgenic selection.
3) The preliminary study was performed on the transformation of banana and longan. The target gene PEAS was introduced to the thin cell layer of banana and EC of longan mediated by Agrobacterium tumefaciens with hpt gene. After infection the materials were selected on MS medium supplemented with hygromycin B, finally 10
lines of banana and 5 cell lines of longan were obtained. PCR assays indicated that hpt gene had been integrated into both the genomes of the 2 lines from 15 tested banana plantlets and one line from 5 tested longan lines. Something about the assays of PEAS gene> Southern blotting, Northern blotting and the expression of the transplants in field were to be further studied.
The establishment and the preliminary study on transformation of thin cell layer of banana and EC of longan would be the technique plat of genetic transformation of banana and longan, which could be used for genetic improvement of banana and longan.
引文
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