摘要
为了使来自原核生物的苏云金芽孢杆菌（ＢａｃｉｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓＢｅｒｌｉｎｅｒ，Ｂｔ）的杀虫晶体蛋白（ＩＣＰ）基因能够在高等植物中得到很好的表达，对编码这种杀虫晶体蛋白基因的核苷酸顺序进行了必要而又合理的修饰和改造：不改变杀虫晶体蛋白基因的氨基酸编码顺序，将杀虫晶体蛋白基因的密码子更换成植物基因组中常用的优化密码子，同时更换掉Ｂｔ杀虫晶体蛋白结构基因中的不稳定元件，人工全合成了长度为１８２４ｂｐ的杀虫晶体蛋白基因———ＧＦＭＣｒｙＩＡ基因。为了检测合成的ＧＦＭＣｒｙＩＡ基因的生物杀虫活性，将ＧＦＭＣｒｙＩＡ基因导入了烟草（ＮｉｃｏｔｉａｎａｔａｂａｃｕｍＬ．），经Ｓｏｕｔｈｅｒｎ杂交证实了ＧＦＭＣｒｙＩＡ基因已整合到转基因烟草植株的基因组中。用再生的转ＧＦＭＣｒｙＩＡ基因烟草植株的叶片喂养二龄棉铃虫（ＨｅｌｉｏｔｈｉｓａｒｍｉｇｅｒａＨüｂｎｅｒ），进行杀虫活性检测，结果表明，转ＧＦＭＣｒｙＩＡ基因烟草植株能显著地抑制棉铃虫的生长发育，表现出有效的杀虫活性。与此相反，对照植物即未转ＧＦＭＣｒｙＩＡ基因烟草植株受到棉铃虫的严重危害。这一实验结果证明：人工合成的ＧＦＭＣｒｙＩＡ基因在烟草植株中获得?
GFM CryIA gene is a fully modified synthetic gene derived from insecticidal crystal protein gene of Bacillus thuringiensis Berliner (Bt). It was synthesized based on the codon usage of plant genes instead of changing the primary sequences of amino acids of insecticidal crystal protein (ICP) gene of Bacillus thuringiensis Hübner. To test the function of the synthetic GFM CryIA gene, we introduced the GFM CryIA gene into tobacco plant cells via an Agrobacterium tumefaciens (Smith et Townsedn) Conn binary vector system. As expected, the GFM CryIA gene is expressed under control of the cauliflower mosaic virus (CaMV) 35S promoter and allows efficient production of lepidopteran insectspecific toxic proteins in the transformed tobacco plants. Bioassays using transgenic tobacco plants with tobacco bollworm showed that the transgenic tobacco plants expressing proteins of GFM CryIA gene had effective control to tobacco bollworm. In this paper the authors firstly report the complete synthesis of GFM CryIA gene and the construction of plant expression vector pGBI4AB. The authors performed introduction of the synthetic GFM CryIA gene into the tobacco plants, and the integration of GFM CryIA gene into tobacco genome was confirmed by Southern blot analysis of the tobacco genomic DNA. The gene was efficiently expressed in the transgenic tobacco plants and effective tobacco bollworm control was verified by the insectbioassays.
