自1995年首个复合性状转化体获得商业化批准以来，通过杂交或载体聚合而开发的多基因复合性状转化体逐渐成为转基因作物开发的重要趋势。基因的载体聚合需要多种类型的调控元件，双向启动子具有降低载体大小、避免同源片段发生沉默的优势，因此是多基因聚合的优选元件。最近，陶氏益农公司就在油菜中发现了一个新的双向启动子EPSPS-A。
EPSPS（5-烯醇丙酮酰-3-磷酸转移酶）是生物体内芳香族氨基酸（苯丙氨酸、酪氨酸、色氨酸）生物合成途径中的关键酶。这些芳香族氨基酸是构建细胞蛋白最基本的氨基酸，在这一生物合成途径中，EPSPS酶催化莽草酸-3-磷酸转变为3-烯醇式丙酮酸莽草酸-5-磷酸。植物中的EPSPS是除草剂草甘膦的作用位点，抗除草剂作物就是因转入对草甘膦不敏感的II型EPSPS，从而保证了芳香族氨基酸的顺利合成。陶氏益农的研究人员发现，油菜中的EPSPS-A基因就被一个双向启动子驱动。通过两个报告基因gfp和gusA分别构建在启动子的上下游两端并转化到大豆中进行测试，结果表明EPSPS-A启动子两端都具有驱动效应，但是对下游基因的驱动力更强。此外，在启动子上游的基因在叶脉中表达极高，而在下游的基因在整个叶片中表达都很高。EPSPS-A基因启动子在构建多基因聚合载体上表现出极大的应用前景。
Transgenic Res. 2017 Sep 15.
Expression of a novel bi-directional Brassica napus promoter in soybean.
Author
Chennareddy S, Cicak T, Clark L, Russell S, Skokut M, Beringer J, Yang X, Jia Y, Gupta M*.
*: Dow AgroSciences, USA.
Abstract
The expression profile of a natural bi-directional promoter, derived from the Brassica napus EPSPS-A gene, was studied in transgenic soybean (Glycine max C.V. Maverick) lines. Two constructs, pDAB100331 and pDAB100333, were assembled to test the bi-directionality of the promoter. Two reporter genes, gfp and gusA, were employed and they were interchangeably placed in both constructs, one on each end of the promoter such that both proteins expressed divergently in each construct. In the T0 generation, GUS expression was more uniform throughout the leaf of pDAB100333 transgenic plants, where the gusA gene was expressed from the downstream or EPSPS-A end of the bi-directional promoter. Comparatively, GUS expression was more localized in the midrib and veins of the leaf of pDAB100331 transgenic plants, where the gusA gene was expressed from the upstream end of the bi-directional promoter. These observations indicated a unique expression pattern from each end of the promoter and consistently higher expression in genes expressed from the downstream end (e.g., EPSPS-A end) of the promoter in the tissues examined. The GFP expression pattern followed that of GUS when placed in the same position relative to the promoter. In the T1 generation, transcript analysis also showed higher expression of both gusA and gfp when those genes were located at the downstream end of the promoter. Accordingly, the pDAB100331 events exhibited a higher gfp/gusA transcript ratio, while pDAB100333 events produced a higher gusA/gfp transcript ratio consistent with the observations in T0 plants. These results demonstrated that the EPSPS-A gene bidirectional promoter can be effectively utilized to drive expression of two transgenes for the desired traits.
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