Long-term stability of transgene expression driven by barley endosperm-specific hordein promoters in transgenic barley
Long-term stability of transgene expression driven by barley endosperm-specific hordein promoters in transgenic barley
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DOI：
10.1007/s00299-003-0630-9
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发表时间：
2003-07-01
影响因子：
6.2
通讯作者：
Cho, MJ
中科院分区：
文献类型：
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作者：
Choi, HW;Lemaux, PG;Cho, MJ
为了评价B-1和D-醇溶蛋白启动子在转基因大麦中表达的长期稳定性，我们分析了15个转基因大麦品系的植株，其中6个是uidA，9个是sgfp(S65T)，其中4个是二倍体，11个是四倍体。通过功能性转基因表达分析、聚合酶链式反应和荧光原位杂交(FISH)检测转基因的表达和遗传。由B-1或D-醇溶蛋白启动子驱动的转基因表达能力在T-4及其后代中遗传：T-4(2株)、T-5(8株)、T-6(3株)、T-8(1株)和T-9(1株)。从12个品系中获得了纯合的转基因植株[5个uidA和7个sgfp(S65T)]；其余的品系目前正在分析中。应用FISH技术对染色体进行物理定位，有助于通过检测转基因的存在来早期筛选纯合子植物。例如，一个表达uidA的品系，在一对同源染色体上有双重荧光信号，通过其分离比率证实为纯合品系；此外，该品系对T-9后代表现出稳定的转基因遗传。在由醇溶蛋白启动子驱动的大多数品系中，有14个品系(14个品系)的转基因表达稳定地传递到T-4或更高的世代，而其余品系从T-1世代开始有1：1的偏斜分离模式。相反，在15个品系中只有6个品系的后代在玉米泛素启动子控制下的转基因沉默或转基因缺失。
In order to evaluate the long-term stability of transgene expression driven by the B-1- and D-hordein promoters in transgenic barley (Hordeum vulgare L., 2n=2x=14), we analyzed plants from 15 independent transgenic barley lines [6 for uidA and 9 for sgfp(S65T)] produced via microprojectile bombardment of immature embryos; 4 were diploid and 11 were tetraploid. The expression and inheritance of transgenes were determined by analysis of functional transgene expression, polymerase chain reaction and fluorescence in situ hybridization (FISH). Ability to express transgenes driven by either B-1- or D-hordein promoter was inherited in T-4 and later generations: T-4 (2 lines), T-5 (8 lines), T-6 (3 lines), T-8 (1 line) and T-9 (1 line). Homozygous transgenic plants were obtained from 12 lines [5 for uidA and 7 for sgfp(S65T)]; the remaining lines are currently being analyzed. The application of the FISH technique for physical mapping of chromosomes was useful for early screening of homozygous plants by examining for presence of the transgene. For example, one line expressing uidA, and shown to have doublet fluorescence signals on a pair of homologous chromosomes was confirmed as a homozygous line by its segregation ratio; additionally this line showed stable inheritance of the transgene to T-9 progeny. The expression of transgenes in most lines (14 out of 15 lines) driven by hordein promoters was stably transmitted to T-4 or later generations, although there was a skewed segregation pattern (1:1) from the T-1 generation onward in the remaining line. In contrast, transgene silencing or transgene loss under the control of the maize ubiquitin promoter was observed in progeny of only 6 out of 15 lines.