红小豆铁蛋白基因的克隆及在水稻中的转化
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摘要
本研究应用快速扩增cDNA末端技术(rapid amplification cDNA ends,简称RACE,下同),克隆得到红小豆铁蛋白基因的cDNA序列;对RACE技术进行了探讨和改进。应用基因枪转化法将该铁蛋白基因的编码区(CDS)转入水稻中,为培育含铁量高的转基因水稻,提高水稻的营养品质,解决人类的铁缺乏症提供一条方便、经济、有效的途径。主要研究结果如下:
1、根据现有的大豆、豌豆等十几种植物铁蛋白基因的同源序列,设计简并引物,应用3’RACE扩增出红小豆铁蛋白基因cDNA的3’端序列,通过三次5’RACE扩增5’端序列,得到了红小豆铁蛋白基因cDNA,长度为1030bp,得到了完整的编码序列为768bp,共编码255个氨基酸。此序列在国内外尚未见报道,现已录入Genbank,登录号:AY663197。
2、通过反转录后回收较大分子量DNA后再用连接酶环化的策略,减轻了利用TaKaRa公司5’RACE试剂盒扩增基因5’端时,常常出现小片段之间重组问题的影响,提高了扩增成功的机率,为利用RACE克隆新基因提供了技术支持。
3、以RACE扩增得到的红小豆铁蛋白基因的CDS作为目的片段,连接含有玉米泛素启动子的1301载体,构建成pCambia1301-UbiN-AFerCDS植物表达载体。
4、采用基因枪转化法,将含有红小豆铁蛋白基因的pCambia1301-UbiN-AFerCDS植物表达载体转入水稻中。对转基因水稻叶片进行PCR检测和铁含量测定,证明目的片段已经整合到水稻基因组中。
5、应用湿灰法对转基因水稻叶片中的铁含量进行测定,转基因植株叶片的铁含量均高于非转基因植株,最高达到240.0μg/gDW,是非转基因植株叶片铁含量的2.17倍。
Through technique of rapid amplification cDNA ends (RACE), cDNA of adzuki bean ferritin gene was cloned.To improve the iron content of rice, we transferred the entire coding sequence of the adzuki bean ferritin gene into rice by particle gun transformation, which not only helped to breed new variety of transgenic rice with ferritin, but also solve the problem of dietary iron deficiency. The main results and significances are as follows:1, According to homology sequence from soybean, pea and so on, by means of designing degenerate primer, we gained ferritin gene cDNA from adzuki bean with 3'-RACE and 5'-RACE techniques. The ferritin gene cDNA is 1030bp long with an open reading frame of 768bp encoding for a protein of 255 amino acids. The access number in Genbank is AY663197.2 By means of taking measures of reclaiming DNA with big molecular weight which come from reverse transcript before the circularity of ligase enzyme.problems of recombination among small segments were setteled. The probability of successful amplification was greatly improved.With this way, technical supports were supplied for RACE.3 In order to transfer rice and study ferritin gene expression in rice, we constructed the plant expression vector-the pCambial301-UbiN-AFerCDS, which is an expression vector that take the ubiquitin to carry.4 Through the approach of particle gun transformation, the expression vector with ferritin gene was transformed into upland rice by PCR analysis.5 The iron level in leaf of transgenic plants varies from 144.1 to 240.0 μ g/gDW. Given that the iron content in leaf of nontransgenic plants was 110.6μg/gDW, we estimated an increase of 2.17 times in iron content of leaf from the transgenic upland rice lines.
1、根据现有的大豆、豌豆等十几种植物铁蛋白基因的同源序列,设计简并引物,应用3’RACE扩增出红小豆铁蛋白基因cDNA的3’端序列,通过三次5’RACE扩增5’端序列,得到了红小豆铁蛋白基因cDNA,长度为1030bp,得到了完整的编码序列为768bp,共编码255个氨基酸。此序列在国内外尚未见报道,现已录入Genbank,登录号:AY663197。
2、通过反转录后回收较大分子量DNA后再用连接酶环化的策略,减轻了利用TaKaRa公司5’RACE试剂盒扩增基因5’端时,常常出现小片段之间重组问题的影响,提高了扩增成功的机率,为利用RACE克隆新基因提供了技术支持。
3、以RACE扩增得到的红小豆铁蛋白基因的CDS作为目的片段,连接含有玉米泛素启动子的1301载体,构建成pCambia1301-UbiN-AFerCDS植物表达载体。
4、采用基因枪转化法,将含有红小豆铁蛋白基因的pCambia1301-UbiN-AFerCDS植物表达载体转入水稻中。对转基因水稻叶片进行PCR检测和铁含量测定,证明目的片段已经整合到水稻基因组中。
5、应用湿灰法对转基因水稻叶片中的铁含量进行测定,转基因植株叶片的铁含量均高于非转基因植株,最高达到240.0μg/gDW,是非转基因植株叶片铁含量的2.17倍。
Through technique of rapid amplification cDNA ends (RACE), cDNA of adzuki bean ferritin gene was cloned.To improve the iron content of rice, we transferred the entire coding sequence of the adzuki bean ferritin gene into rice by particle gun transformation, which not only helped to breed new variety of transgenic rice with ferritin, but also solve the problem of dietary iron deficiency. The main results and significances are as follows:1, According to homology sequence from soybean, pea and so on, by means of designing degenerate primer, we gained ferritin gene cDNA from adzuki bean with 3'-RACE and 5'-RACE techniques. The ferritin gene cDNA is 1030bp long with an open reading frame of 768bp encoding for a protein of 255 amino acids. The access number in Genbank is AY663197.2 By means of taking measures of reclaiming DNA with big molecular weight which come from reverse transcript before the circularity of ligase enzyme.problems of recombination among small segments were setteled. The probability of successful amplification was greatly improved.With this way, technical supports were supplied for RACE.3 In order to transfer rice and study ferritin gene expression in rice, we constructed the plant expression vector-the pCambial301-UbiN-AFerCDS, which is an expression vector that take the ubiquitin to carry.4 Through the approach of particle gun transformation, the expression vector with ferritin gene was transformed into upland rice by PCR analysis.5 The iron level in leaf of transgenic plants varies from 144.1 to 240.0 μ g/gDW. Given that the iron content in leaf of nontransgenic plants was 110.6μg/gDW, we estimated an increase of 2.17 times in iron content of leaf from the transgenic upland rice lines.
引文
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