Plant & Soil Sciences, University of Kentucky, Lexington, KY 40546, USA.
BMC Biotechnol. 2009 Nov 18;9:94. doi: 10.1186/1472-6750-9-94.
In soybean somatic embryo transformation, the standard selection agent currently used is hygromycin. It may be preferable to avoid use of antibiotic resistance genes in foods. The objective of these experiments was to develop a selection system for producing transgenic soybean somatic embryos without the use of antibiotics such as hygromycin.
When tested against different alternate selection agents our studies show that 0.16 microg/mL glufosinate, 40 mg/L isopropylamine-glyphosate, 0.5 mg/mL (S-(2 aminoethyl)-L-cysteine) (AEC) and the acetolactate synthase (ALS) inhibitors Exceed and Synchrony both at 150 microg/mL inhibited soybean somatic embryo growth. Even at the concentration of 2 mg/mL, lysine+threonine (LT) were poor selection agents. The use of AEC may be preferable since it is a natural compound. Unlike the plant enzyme, dihydrodipicolinate synthase (DHPS) from E. coli is not feed-back inhibited by physiological concentrations of lysine. The dapA gene which codes for E. coli DHPS was expressed in soybean somatic embryos under the control of the CaMV 35S promoter. Following introduction of the construct into embryogenic tissue of soybean, transgenic events were recovered by incubating the tissue in liquid medium containing AEC at a concentration of 5 mM. Only transgenic soybeans were able to grow at this concentration of AEC; no escapes were observed.
Genetically engineered soybeans expressing a lysine insensitive DHPS gene can be selected with the non-antibiotic selection agent AEC. We also report here the inhibitory effects of glufosinate, (isopropylamine-glyphosate) (Roundup), AEC and the ALS inhibitors Exceed and Synchrony against different tissues of soybean.
在大豆体细胞胚胎转化中，目前使用的标准选择剂是潮霉素。在食品中避免使用抗生素抗性基因可能更为可取。本实验的目的是开发一种不使用抗生素（如潮霉素）生产转基因大豆体细胞胚胎的选择系统。
当针对不同的替代选择剂进行测试时，我们的研究表明，0.16μg/mL 草铵膦、40mg/L 异丙胺草甘膦、0.5mg/mL(S-(2 氨基乙基)-L-半胱氨酸)(AEC)和乙酰乳酸合酶(ALS)抑制剂 Exceed 和 Synchrony 均在 150μg/mL 时抑制大豆体细胞胚胎生长。即使在 2mg/mL 的浓度下，赖氨酸+苏氨酸（LT）也是较差的选择剂。由于 AEC 是一种天然化合物，因此它可能是更好的选择。与植物酶不同，来自大肠杆菌的二氢二吡啶羧酸合酶（DHPS）不受赖氨酸的生理浓度的反馈抑制。编码大肠杆菌 DHPS 的 dapA 基因在 CaMV 35S 启动子的控制下在大豆体细胞胚胎中表达。在将构建体引入到大豆的胚胎发生组织中后，通过在含有 5mM AEC 的液体培养基中孵育组织来回收转基因事件。只有转基因大豆能够在这个 AEC 浓度下生长；没有观察到逃逸现象。
表达赖氨酸不敏感 DHPS 基因的转基因大豆可以用非抗生素选择剂 AEC 进行选择。我们还报告了草铵膦、（异丙胺草甘膦）（Roundup）、AEC 和 ALS 抑制剂 Exceed 和 Synchrony 对大豆不同组织的抑制作用。
