农杆菌介导调节基因Lc转化菊花的研究
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摘要
菊花(Chrysanthemum×morifolium Ramat.)是重要的盆花和切花,具有巨大的经济价值和观赏价值。目前菊花花色育种仍存在一定的缺陷:1.由于缺乏类黄酮-3′,5′-羟基化酶基因(F3`5`H),不能产生蓝色的飞燕草色素而缺少蓝色系品种;2.红色和紫色品种中色素亮度低,花色暗淡,缺少色彩鲜艳的红色品种。由于菊花花色基因库的有限性,用传统育种方法至今仍无法解决这两大缺陷。根据菊花的花色形成机理和花色分子育种的研究进展,利用转基因技术将花色相关调节基因转入菊花中,研究转录因子对菊花花色结构基因的调控,研究其花色形成的分子机理,对于通过分子手段改良菊花花色具有重要的理论和实践意义。
本研究主要取得了如下成果:
1.对pBI121载体Gus基因后的终止子进行2次PCR扩增,在原SacⅠ酶切位点后添加了新的SalⅠ酶切位点,利用组织化学染色法检测,结果表明改造后的载体上的Gus基因能正常表达,终止子功能正常,载体改造成功。用改造的pBI121N构建了含有Lc基因的植物表达载体pBI121N-Lc。
2.利用农杆菌介导菊花节间薄层(tTCLs)和叶盘进行遗传转化。共对7个品种1751个外植体进行了侵染。通过分化培养,除‘Repulse'外的6个品种均获得了抗性芽,通过抗性芽生根筛选,‘日切桃红'、‘Regan Elite Sunny'、‘Regan Elite Improved'3个品种获得了生根的抗性苗,‘墨菊'、‘粉荷'和‘粉牡丹'未获得生根抗性苗。说明菊花抗性芽再生和生根具有基因型差异。
3.提取48株生根‘日切桃红'抗性苗的基因组DNA,以Lc基因和CaMv35S启动子序列为引物,分别对Lc基因和35S启动子进行PCR检测,获得了7个Lc基因和35S启动子的阳性株系;PCR结果表明Lc基因已经转入菊花中,转化率1.4%。在‘日切桃红'节间薄层(tTCLs)遗传转化过程中,产生的抗性愈伤组织中有红色愈伤组织出现,在已获得的‘日切桃红'转基因植株中发现部分转基因株系的根系有变红的现象。
Chrysanthemum(Chrysanthemum×morifolium Ramat.) is one of the most famous pot flower and cut flower in the world.But there are two faults in chrysanthemum flower color breeding:1. Chrysanthemum have no blue-flowering cultivars.A main reason for the lack of blue flowers is the absence of 3′,5′-hydroxylated anthocyanins,which are synthesized by a key enzyme,flavonoid 3′, 5′-hydroxylase(F3′5′H);2.Few colorful red cultivars are found in Chrysanthemum.The application of genetic engineering including introducing transcription factor gene and enhancing coordinated expression of a number of genes can achieve a better plant transformation result.
The results were as follows:
1.The one new restriction enzyme SalI site was added to terminator behind Gus gene of pBI121 vector by PCR.Gus histochemical assay indicated that Gus gene can express as same as original pBI121 vector and the function of terminator was normal.The expression vector pBI121N-Lc was constructed by Lc gene using reconstruct vector pBI121N.
2.The vector pBI121N-Lc was transferred into Chrysanthemum seven cultivars by Agrobacterium-mediated using leaf disk and tTCLs as the explants.Kanamycin-resistant shoot have been obtained in the six cultivars;only in three of the six cultivars kanamycin-resistant root were obtained.Results indicated that different Chrysanthemum cultivars have genotypic differences in the transformed and regenerated.
3.The 48 Chrysanthemum 'riqietaohong' generation resistance plants were detected by PCR assay, 7 of them have the same positive band as Lc gene and 35S.The results indicated that Lc gene had been successfully introduced and integrated into the genome.And some transgenic lines roots become red.
本研究主要取得了如下成果:
1.对pBI121载体Gus基因后的终止子进行2次PCR扩增,在原SacⅠ酶切位点后添加了新的SalⅠ酶切位点,利用组织化学染色法检测,结果表明改造后的载体上的Gus基因能正常表达,终止子功能正常,载体改造成功。用改造的pBI121N构建了含有Lc基因的植物表达载体pBI121N-Lc。
2.利用农杆菌介导菊花节间薄层(tTCLs)和叶盘进行遗传转化。共对7个品种1751个外植体进行了侵染。通过分化培养,除‘Repulse'外的6个品种均获得了抗性芽,通过抗性芽生根筛选,‘日切桃红'、‘Regan Elite Sunny'、‘Regan Elite Improved'3个品种获得了生根的抗性苗,‘墨菊'、‘粉荷'和‘粉牡丹'未获得生根抗性苗。说明菊花抗性芽再生和生根具有基因型差异。
3.提取48株生根‘日切桃红'抗性苗的基因组DNA,以Lc基因和CaMv35S启动子序列为引物,分别对Lc基因和35S启动子进行PCR检测,获得了7个Lc基因和35S启动子的阳性株系;PCR结果表明Lc基因已经转入菊花中,转化率1.4%。在‘日切桃红'节间薄层(tTCLs)遗传转化过程中,产生的抗性愈伤组织中有红色愈伤组织出现,在已获得的‘日切桃红'转基因植株中发现部分转基因株系的根系有变红的现象。
Chrysanthemum(Chrysanthemum×morifolium Ramat.) is one of the most famous pot flower and cut flower in the world.But there are two faults in chrysanthemum flower color breeding:1. Chrysanthemum have no blue-flowering cultivars.A main reason for the lack of blue flowers is the absence of 3′,5′-hydroxylated anthocyanins,which are synthesized by a key enzyme,flavonoid 3′, 5′-hydroxylase(F3′5′H);2.Few colorful red cultivars are found in Chrysanthemum.The application of genetic engineering including introducing transcription factor gene and enhancing coordinated expression of a number of genes can achieve a better plant transformation result.
The results were as follows:
1.The one new restriction enzyme SalI site was added to terminator behind Gus gene of pBI121 vector by PCR.Gus histochemical assay indicated that Gus gene can express as same as original pBI121 vector and the function of terminator was normal.The expression vector pBI121N-Lc was constructed by Lc gene using reconstruct vector pBI121N.
2.The vector pBI121N-Lc was transferred into Chrysanthemum seven cultivars by Agrobacterium-mediated using leaf disk and tTCLs as the explants.Kanamycin-resistant shoot have been obtained in the six cultivars;only in three of the six cultivars kanamycin-resistant root were obtained.Results indicated that different Chrysanthemum cultivars have genotypic differences in the transformed and regenerated.
3.The 48 Chrysanthemum 'riqietaohong' generation resistance plants were detected by PCR assay, 7 of them have the same positive band as Lc gene and 35S.The results indicated that Lc gene had been successfully introduced and integrated into the genome.And some transgenic lines roots become red.
引文
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