抗盐草地早熟禾高效转基因体系的研究
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摘要
试验以草地早熟禾(P.pratensis L.)7个品种的成熟种子为外植体,对其再生体系和农杆菌介导的遗传转化体系进行了研究。试验表明:MSM培养基比较适宜于草地早熟禾的组织培养;愈伤组织的间接诱导率要显著高于愈伤组织的直接诱导率,各品种均可达93%以上;草地早熟禾品种‘优异’较适合本次试验所建立的组培再生体系,其愈伤组织分化率最高,为77.65%,且16周即可完成植株再生全程。采用农杆菌介导法将抗盐相关基因——rstB和mrstB转入草地早熟禾的愈伤组织,经筛选获得抗性愈伤,分化获取再生植株,通过PCR、Southern-blot和Northern-blot证明,基因rstB和mrstB已经分别整合到草地早熟禾的基因组中,7个品种的转化效率在0%～6.71%之间,遗传转化周期为24周。目前,已获得草地早熟禾品种‘优异’、‘百蒂雅’、‘自由’3个品种的转基因植株共计92株。通过耐盐性试验,失水处理后的叶片剩余重量百分比测定,和盐处理后植物叶片内的脯氨酸、丙二醛、叶绿素含量的测定,证明转基因植株较非转基因植株有较强的抗盐能力。
Mature bluegrass (P. pratensis L.) seeds were used as explant for callus initiation. The basic medium was medium MSM. Under the instructed plant regeneration system, the indirect callus induction frequency was significantly higher (93-99%) than that of direct induction way. The cultivar Merit performed the best of the seven cultivars tested, with a regeneration frequency of 77.65% and a regeneration period of 16 weeks. Embryogenic calli were infected with the A. tumefaciens strain EHA105 harboring the Ti plasmid pZGl or pZG2. The transformation frequencies of the seven cultivars ranged from 0 to 6.71%. In total, 92 transgenic plants were generated. PCR, Southern-blot and Northern-blot were used for molecular test, the results showed that the rstB or mrstB was integrated into the bluegrass genome. The cultivar Merit consumed the shortest time for genetic transformationm, it was about 24 weeks. Some of the transgenic plants could live longer than non-transgenic plant, under stress of different concentration of NaCl. Physiological tests also indicated that the water-retaining capacity and the chlorophyll and proline concentrations of some salt-treated transgenic lines were higher than those of non-transgenic plants, whereas the malondialdehyde contents were lower in some transgenic plants than in non-transgenic plants.
Mature bluegrass (P. pratensis L.) seeds were used as explant for callus initiation. The basic medium was medium MSM. Under the instructed plant regeneration system, the indirect callus induction frequency was significantly higher (93-99%) than that of direct induction way. The cultivar Merit performed the best of the seven cultivars tested, with a regeneration frequency of 77.65% and a regeneration period of 16 weeks. Embryogenic calli were infected with the A. tumefaciens strain EHA105 harboring the Ti plasmid pZGl or pZG2. The transformation frequencies of the seven cultivars ranged from 0 to 6.71%. In total, 92 transgenic plants were generated. PCR, Southern-blot and Northern-blot were used for molecular test, the results showed that the rstB or mrstB was integrated into the bluegrass genome. The cultivar Merit consumed the shortest time for genetic transformationm, it was about 24 weeks. Some of the transgenic plants could live longer than non-transgenic plant, under stress of different concentration of NaCl. Physiological tests also indicated that the water-retaining capacity and the chlorophyll and proline concentrations of some salt-treated transgenic lines were higher than those of non-transgenic plants, whereas the malondialdehyde contents were lower in some transgenic plants than in non-transgenic plants.
引文
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樊军锋,李嘉瑞,韩一凡,李玲,彭学贤.mtlD/gutD双价耐盐基因转化秦美猕猴桃的研究.西北农林科技大学学报(自然科学版),2002,6(3):53～58。
侯丙凯,夏光敏,陈正华.植物基因工程表达载体的改进和优化策略.遗传,2001,23(5):492-497.
黄复瑞、刘祖祺主编.现代草坪建植与管理技术.北京:中国农业出版社,1998
贾海燕,王洪刚.小麦愈伤组织诱导及其再生能力影响因素的研究.山东农业大学学报(自然科学版),2003,34(1):9～14.
李卫,郭光沁,郑国昌.根癌农杆菌介导遗传转化研究的若干新进展.科学通报,2000,45(8):798-807.
林拥军,陈浩,曹应龙,等.农杆菌介导的牡丹江8号高效转基因体系的建立.作物学报,2002,28(3):294-300.
刘俊君,彭学贤,王海云,黄绍兴.大肠杆菌mtlD基因和gutD基因的克隆、全序列测定和高效表达.生物工程学报,1995,11(2):157～161.
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施利利,王松文,蔡宝立,孙宗修,张欣,刘霞.影响农杆菌介导的水稻遗传转化效率的因素.天津农学院学报,2003,10(4):53-57
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