摘要
铁蛋白是广泛存在于生物体中的一种长期贮藏铁的蛋白,调节铁的吸收和释放,是植物贮藏铁的主要形式。大豆是含铁量高的植物,其内大约90%的铁以铁蛋白的形式存在。研究发现大豆铁蛋白中铁的吸收利用率和FeSO4基本相当。本研究使用农杆菌介导的转化方法将大豆铁蛋白Ferritin基因转入玉米,期望在玉米胚乳特异表达基因15 kDβ-Zein启动子的驱动下,特异表达,以提高籽粒中微量元素铁的含量,为强化玉米营养品质,解决以玉米为主食人群的缺铁问题提供帮助。主要结果如下:
1.从玉米自交系鲁2548分离获得胚乳特异表达基因15 kDβ-Zein的启动子序列,并构建了表达载体pBI121-Zein-GUS。
2.从大豆品种95-5883克隆并获得大豆铁蛋白Ferritin基因的cDNA序列,全长753 bp,包含251个氨基酸残基,构建植物表达载体pBI121-35s-Ferritin,pCUbi1390-Ferritin,pBI121-Zein-Ferritin,pCUbi1390-Ferritin-Epsps。
3.诱导玉米自交系R18-599和齐319的胚性愈伤组织,为遗传转化提供受体材料。利用三亲交配法将重组载体pCUbi1390-Ferritin,pBI121-Zein-Ferritin导入农杆菌LBA4404,通过农杆菌介导法将表达载体转入玉米自交系R18-599和齐319的胚性愈伤组织,得到转基因植株。提取转基因植株的DNA,经PCR鉴定,初步证明Ferritin基因整合到转基因玉米基因组中。
Ferritin is a long-term iron binding and storage protein widely in the organism. It regulates iron absorption and release.It is a major form of iron storage in plant.Soybeans in particular have a high iron,and about 90% of Total iron in seeds stored inside ferritin.It has been demonstrated that iron from soybean ferritin is equal in bioavailability to FeSO4. In this study, soybean ferritin gene was transferred into maize by Agrobacterium-mediated transformation, in order to realize the specific expression of soybean ferritin gene in maize endosperm driven by the promoter of endosperm-specific expression gene 15 kDβ-Zein.With this study, we expect to obtain maize with higher iron content. And this would lay the foundation for the enhancement nutritional quality of maize and iron deficiency of human corn-based food. The main results were summarized as follows:
1. The promoter of endosperm-specific expression gene 15 kDβ-Zein was cloned from maize inbred lines Lu 2548 genomic. Sequence analysis and promoter function prediction indicated that the cloned sequence would function as a promoter and make the endosperm-specific expression. In order to confirm its function, we construct the plant expressing vector pBI121-Zein-GUS.
2. According to the reported sequence of soybean ferritin, ferritin gene cDNA sequence was cloned from soybean varieties Zhongpin 95-5883. The ferritin gene cDNA sequence is 753 bp including all encoding sequence,and 251 amino acid residues.In order to study soybean ferritin gene expression in maize, we constructed the plant expression vector pBI121-35s-Ferritin, pCUbi1390-Ferritin, pBI121-Zein- Ferritin, pCUbi1390-Ferritin-Epsps.
3. In order to provide receptor material for the Agrobacterium-mediated transformation of maize, we cultivated embryogenic callus from maize inbred lines R18-599 and Qi319. Through the approach of Triparental mating, the plant expression vector pCUbi1390-Ferritin, pBI121-Zein-Ferritin were introduced into an Agrobacterium strain LBA 4404, then were transformed embryogenic callus of maize by Agrobacterium-mediated transformation. And transgenic plants had been obtained. The genome DNA was extracted from transformed maize leaves. And it was briefly proved that Ferritin gene cDNA had been integrated into the transgenic maize genome by PCR analysis.
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