Improvement Fe Content of Wheat(Triticum aestivum) Grain by Soybean Ferritin Expression Cassette without Vector Backbone Sequence | 无载体框架结构的大豆铁蛋白线性表达盒提高小麦籽粒铁含量的研究

Chino. 培育和推广铁强化型小麦品种对于经济有效地解决贫困人口的铁营养不良问题具有重要意义。本研究通过RT-PCR得到了大豆(Glycine max)铁蛋白基因，构建了无载体框架的大豆铁蛋白线性表达盒(即小麦籽粒特异的高分子量谷蛋白亚基1Dx5的启动子-大豆铁蛋白基因-NOS终止子酶切片段)；用基因枪法转化“京花1号”小麦(Triticum aestivum)幼胚愈伤组织，经草胺膦筛选和分化后，得到276株转基因T0植株，通过PCR筛选出阳性植株65株；RT-PCR检测发现29个株系的大豆铁蛋白基因在灌浆期籽粒中表达；与对照相比，对8031个T1代株系所结的T2代籽粒进行了铁含量测定，有265个株系成熟籽粒的铁含量均比对照有不同幅度提高，增幅在4.93%~64.03%之间，其中22个株系的籽粒铁含量显著或极显著提高。结果提示，利用无载体框架的大豆铁蛋白线性表达盒可以有效地培育出籽粒铁含量显著提高的转基因小麦。

Inglés. Iron (Fe) is important and indispensable element for the normal physiological processes in the human body, and iron deficiency is a serious nutritional problem in the world, so it is economical and effective to breed and grow wheat cultivars with high grain iron content, especially for those population in poor-developed areas. In this paper, the soybean (Glycine max) ferritin gene was obtained by RT-PCR, and cloned into the pBAC47P vector. The soybean ferritin expression cassette without vector backbone sequence but including wheat endosperm-specific HMW-GS 1Dx5 promoter and NOS terminator was constructed by digestion and purification, and has been transformed through particle bombardment of PDS1000/He system into winter wheat(Triticum aestivum) cultivars, Jinghua No.1. After phosphinothricin (PPT) selection, differentiation and regeneration, 276 plants were regenerated from immature embryos callus. By PCR screening and PCR-Southern blot using specific primers for soybean ferritin gene 65 transgenic plants of T0 generation were selected, Among them, kernels of 29 plants of T1 generation were positive by RT-PCR on the RNA levels. The grain iron content of 8031 T1 transgenic lines have been determined. Compared to the control, there were 265 lines of them have increased iron content, ranging from 4.93 to 64.03 percent, and 22 lines of them increased significantly, which suggests that Fe content of wheat grain can be improved by the soybean ferritin expression cassette without vector backbone sequence.