Bassett Carole L, Callahan Ann M, Artlip Timothy S, Scorza Ralph, Srinivasan Chinnathambi
US Department of Agriculture, the Agricultural Research Service, Appalachian Fruit Research Station, 2217 Wiltshire Road, Kearneysville, West Virginia 25430, USA.
BMC Biotechnol. 2007 Aug 14;7:47. doi: 10.1186/1472-6750-7-47.
Promoters with tissue-specificity are desirable to drive expression of transgenes in crops to avoid accumulation of foreign proteins in edible tissues/organs. Several photosynthetic promoters have been shown to be strong regulators of expression of transgenes in light-responsive tissues and would be good candidates for leaf and immature fruit tissue-specificity, if expression in the mature fruit were minimized.
A minimal peach chlorophyll a/b-binding protein gene (Lhcb2*Pp1) promoter (Cab19) was isolated and fused to an uidA (beta-glucuronidase [GUS]) gene containing the PIV2 intron. A control vector carrying an enhanced mas35S CaMV promoter fused to uidA was also constructed. Two different orientations of the Cab19::GUS fusion relative to the left T-DNA border of the binary vector were transformed into tomato. Ten independent regenerants of each construct and an untransformed control line were assessed both qualitatively and quantitatively for GUS expression in leaves, fruit and flowers, and quantitatively in roots.
The minimal CAB19 promoter conferred GUS activity primarily in leaves and green fruit, as well as in response to light. GUS activity in the leaves of both Cab19 constructs averaged about 2/3 that observed with mas35S::GUS controls. Surprisingly, GUS activity in transgenic green fruit was considerably higher than leaves for all promoter constructs; however, in red, ripe fruit activities were much lower for the Cab19 promoter constructs than the mas35S::GUS. Although GUS activity was readily detectable in flowers and roots of mas35S::GUStransgenic plants, little activity was observed in plants carrying the Cab19 promoter constructs. In addition, the light-inducibility of the Cab19::GUS constructs indicated that all the requisite cis-elements for light responsiveness were contained on the Cab19 fragment. The minimal Cab19 promoter retains both tissue-specificity and light regulation and can be used to drive expression of foreign genes with minimal activity in mature, edible fruit.
具有组织特异性的启动子有利于驱动转基因在作物中的表达，以避免外源蛋白在可食用组织/器官中积累。几种光合启动子已被证明是光响应组织中转基因表达的强调节因子，如果在成熟果实中的表达降至最低，它们将是叶片和未成熟果实组织特异性的良好候选者。
分离出一个最小化的桃叶绿素a/b结合蛋白基因（Lhcb2*Pp1）启动子（Cab19），并将其与含有PIV2内含子的uidA（β-葡萄糖醛酸酶[GUS]）基因融合。还构建了一个携带与uidA融合的增强型mas35S CaMV启动子的对照载体。将Cab19::GUS融合相对于二元载体左T-DNA边界的两种不同方向转化到番茄中。对每个构建体的10个独立再生植株和一个未转化的对照系进行了定性和定量评估，以检测其在叶片、果实和花中的GUS表达，并对根进行了定量检测。
最小化的CAB19启动子主要在叶片和绿色果实中赋予GUS活性，并且对光有响应。两种Cab19构建体叶片中的GUS活性平均约为mas35S::GUS对照的2/3。令人惊讶的是，对于所有启动子构建体，转基因绿色果实中的GUS活性显著高于叶片；然而，对于Cab19启动子构建体，红色成熟果实中的活性远低于mas35S::GUS。虽然在mas35S::GUS转基因植物的花和根中很容易检测到GUS活性，但在携带Cab19启动子构建体的植物中几乎没有观察到活性。此外，Cab19::GUS构建体的光诱导性表明，Cab19片段上包含了光响应所需的所有顺式元件。最小化的Cab19启动子保留了组织特异性和光调节功能，可用于驱动外源基因的表达，且在成熟可食用果实中的活性最小。
