决定转基因表达的启动子序列
丁香园
9725
1. 引言
传统的观点认为真核生物基因的表达是由位于同一分子（顺式元件）上的 DNA 序列与不同 DNA 分子（反式作用信号）编码的蛋白质或 RNA 之间的相互作用来调控的。这些顺式作用元件包括 CAAT 框和 TATA 框、启动子本身的响应元件、编码区远上游或下游端的增强子元件、5' 及 3' 非编码区（UTR ) 、内含子、多聚腺苷酸信号等。然而，这个相当简单的观点一直受到怀疑，特别是随着人们对哺乳动物生物学知识了解的逐步深入，该观点也得到更新和完善。（见示例 [1, 2 ] ) 。虽然人们对基因表达调控的信号系统尚不完全清楚，但利用较短的启动子序列（至 多 2 kb) ，以及启动子下游转录单元内增强基因表达的内含子序列，可以清楚地阐明重组基因的表达模式 [3~6 ] 。
在设计植物转化试验时，首个难题就是构建一个包含目的基因和合适的调控序列的表达框，以产生预期的基因表达模式。如果所选择的启动子序列以前并未在将要转化的物种上检测过，建议首先使用可量化的报告基因做检测，如 gfp 或者 uidA (GUS) 基因。本章综述了在禾谷类作物中已被证实为组成型、组织特异性或诱导型表达的各种启动子序列。表 11.1 列出了小麦、大麦或燕麦的所有已用于报告基因表达研究的启动子及代表性文献，以便读者可以进一步查询。我们亦可从“性状” （非报告基因）或选择性标记基因的表达，推断启动子功能的相关信息，但表 1 1.1 中并未列出这些研究。此外，所列出的启动子序列已应用于大面积种植的水稻和玉米的研究中。
2. 组成型基因的表达
若要获得稳定的转基因株系、进行瞬时超表达方法等转化研究工作，往往要求基因在所有组织和发育各阶段均能够高强度表达。为了使转入基因能产生普遍的组成型表达 ，在双子叶植物中通常使用附加或未附加不同上游激活区（ UAR ) 的花椰菜花叶病毒 35S ( CaMV35S) 启动子[49, 50 ]。该启动子也用于禾谷类作物以表达选择性标记基因和性状基因。在水稻中，通过检测报告基因，已掌握了该启动子的一些表达特征[51~53]。然而，关于 CaMV35S 在小麦、大麦或燕麦转化中的表现，学者们各持己见。一些作者认为，与单子叶植物启动子相比， CaMV35S 出现标记基因低水平表达和髙频率失活现象[12 , 54, 55]。在禾谷类作物中，已得到检测的还有其他一些病毒启动子序列，包括从水稻东格鲁杆状病毒（ RTBV ) 和夜香树黄曲叶病毒（ CmYLCV ) 获得的序列。启动子缺失试验表明， RTBV 启动子在转基因水稻的维管束或韧皮部特异表达 [56~61]。在转基因小麦中， RTBV启动子可驱动 GUS 在花粉和根组织以外的各种组织中强烈表达（作者未发表结果）。在转基因拟南芥、烟草、玉米及水稻中，CmYLCV 启动子可以激活 GUS 或 GFP 组成型表达[62]。
在禾谷类作物的转化试验中，通常优先使用两个单子叶植物启动子（Ubil和 Actl) ，而不是病毒序列。 Ubil 由玉米泛素启动子与其第一个外显子和第一个内含子构建而成 [10，11 ]， Atl 由水稻肌动蛋白启动子与其内含子构建而成 [63 ]。在水稻[64, 65]、玉米 [ 66 ] 和小麦 [ 3，14 ] 中，用玉米 Ubil 启动子驱动的 uWA ( GUS ) 报告基因，能够在多数组织中强烈表达。在幼嫩、代谢旺盛的组织和花粉中，通常 GUS 的表达水平较强；而在老叶、根部及花药的体细胞组织中，GUS 表达水平很低或者不表达 [ 图 11.1 (a ) 和图 11.1 (b ) ]。虽然玉米 Ubil 启动子具有很高水平的本底表达，但依然受热激和胁迫的诱导 [64]，而这种诱导性在大多数玉米 Ubil 启动子的转基因研究中并未被积极利用。玉米 Ubil 的热激元件已被鉴定，当采用豌豆凝集素启动子的一个碱性结构域/亮氨酸拉链因子结合位点替换 Ubil 的热激元件时，则会改变基因在种子内的表达分布，即表达由胚转向胚乳 [66]。其他一些泛素启动子在单子叶植物转化中也得到鉴定，包括从水稻中分离出的 RUBQ2 和 Rubi3 [ 67, 68 ]，以及从甘蔗中分离出的 ubi4 和 ubi9 [ 69 ] 启动子，所有这些启动子的表达活性与玉米 Ubil 或 CaMV35S 相当甚至更高。玉米 Ubil 驱动 GtAS 基因在转基因拟南芥、番茄、烟草和松树的根中也获得了很好的表达[70]。
Actin 基因的启动子是可以替代泛素的另一个理想启动子。水稻 Actin 启动子 ( Actl- D) 由 Act1 基因翻译起始密码子上游 2.1 kb 的一段序列组成。该序列包含了 GUS 高水平表达的所有 5' 调控因子，其为水稻和玉米瞬时转化[16, 63 ] 以及水稻稳定转化 [ 7 1 ] 所必需。除启动子区的序列外， Actl- D 还包括了 Act1 基因 5' 内含子和第一外显子的部分序列。据报道， Actl- D :: GUS 在转基因水稻株系的孢子体和配子体组织中均呈组成型表达[71 ]。定量分析表明，转化体中 GUS 蛋白量占总可溶性蛋白的 3%[71]。在小麦中， Actl- D 启动子能驱动基因在各发育阶段的所有主要组织中强烈表达 ( 作者未发表的数据）。在转基因燕麦中，水稻 Actl- D 和截短的 Act1 启动子（ActlF ) 均被用来驱动以必表达[12, 18]，但尚未有这些转基因株系详细分析的报道。
在谷类作物中得到鉴定和应用的其他类型的组成型表达的启动子还有：携带 Ubi 第一个内含子的玉米 H2B 启动子 [25]、玉米 Adh1 启动子[8 , 9 ] 、水稻细胞色素 c 启动子（OsCc1 ) [72]，以及来自地三叶草矮化病毒（SCSV) 的一系列调控序列[73]。
3. 组织特异性基因的表达
在自然状态下，依据基因产物的作用，大多数植物基因的表达具有器官或组织特异性。当用 1 kb 或 2 kb 的启动子构建异源表达框时，基因在新的表达框通常仍能保持其原有的空间特异性。例如，小麦高分子质量谷蛋白亚基基因的 1.2 kb 启动子序列，在转基因硬粒小麦中驱动报告基因以必在淀粉胚乳中特异性表达 [ 26 ] [ 图 11. 1(d ) ]。该启动子及其他的谷蛋白启动子，已被用于驱动外源基因在小麦胚乳中的表达( 综述 [ 74 ] ) 。其他已经证实的参与种子储藏物调控的启动子还包括：低分子质量谷蛋白亚基启动子[27]、 B- 和 D- 醇溶蛋白启动子[22, 28 , 30 ]。通过检测报告基因，Em、 asi、 aamyl、 aamy 2 ，以及嘌呤吲哚蛋白（puroindoline) a 和 b 等启动子均有在种子内特异表达的特点 [ 图11.1 ( e ) ] [ 31， 33 , 35, 36 ]。另外，作者未发表的资料表明，球蛋白启动子序列具有在糊粉层和胚胎表达的特征[图11 .1 ( f ) ]。
4 . 诱导型基因的表达
通过外界刺激调节候选基因表达的开启与关闭，对于许多研究的应用具有重要价值( 综述 [75~77 ] ) 。当仅对特定组织施以激活刺激时，诱导型启动子会产生所谓的组织特异性，包括 a1cR ( 乙醇诱导 [ 78 ~ 81 ] ) 、谷胱甘肽 S- 转移酶/I n 2-2 (安全剂诱导[82, 83 ] ) 、各种热激诱导多聚半乳糖醛酸酶抑制蛋白 [84~87] ( HSP，损伤诱导[88] ) 、 TetR 和 tRA ( 分别受四环素诱导和抑制 [ 89~93] ) 、EcR ( 蜕皮激素诱导 [ 94~97 ] ) 在内的多种诱导型启动子已在转基因植物中开展研究。然而，大多数的研究都是在双子叶植物中开展的，虽然其中的一些结果可类推到单子叶植物，但是我们确实需要验证转基因禾谷物作物的最佳诱导体系。
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传统的观点认为真核生物基因的表达是由位于同一分子（顺式元件）上的 DNA 序列与不同 DNA 分子（反式作用信号）编码的蛋白质或 RNA 之间的相互作用来调控的。这些顺式作用元件包括 CAAT 框和 TATA 框、启动子本身的响应元件、编码区远上游或下游端的增强子元件、5' 及 3' 非编码区（UTR ) 、内含子、多聚腺苷酸信号等。然而，这个相当简单的观点一直受到怀疑，特别是随着人们对哺乳动物生物学知识了解的逐步深入，该观点也得到更新和完善。（见示例 [1, 2 ] ) 。虽然人们对基因表达调控的信号系统尚不完全清楚，但利用较短的启动子序列（至 多 2 kb) ，以及启动子下游转录单元内增强基因表达的内含子序列，可以清楚地阐明重组基因的表达模式 [3~6 ] 。
在设计植物转化试验时，首个难题就是构建一个包含目的基因和合适的调控序列的表达框，以产生预期的基因表达模式。如果所选择的启动子序列以前并未在将要转化的物种上检测过，建议首先使用可量化的报告基因做检测，如 gfp 或者 uidA (GUS) 基因。本章综述了在禾谷类作物中已被证实为组成型、组织特异性或诱导型表达的各种启动子序列。表 11.1 列出了小麦、大麦或燕麦的所有已用于报告基因表达研究的启动子及代表性文献，以便读者可以进一步查询。我们亦可从“性状” （非报告基因）或选择性标记基因的表达，推断启动子功能的相关信息，但表 1 1.1 中并未列出这些研究。此外，所列出的启动子序列已应用于大面积种植的水稻和玉米的研究中。
2. 组成型基因的表达
若要获得稳定的转基因株系、进行瞬时超表达方法等转化研究工作，往往要求基因在所有组织和发育各阶段均能够高强度表达。为了使转入基因能产生普遍的组成型表达 ，在双子叶植物中通常使用附加或未附加不同上游激活区（ UAR ) 的花椰菜花叶病毒 35S ( CaMV35S) 启动子[49, 50 ]。该启动子也用于禾谷类作物以表达选择性标记基因和性状基因。在水稻中，通过检测报告基因，已掌握了该启动子的一些表达特征[51~53]。然而，关于 CaMV35S 在小麦、大麦或燕麦转化中的表现，学者们各持己见。一些作者认为，与单子叶植物启动子相比， CaMV35S 出现标记基因低水平表达和髙频率失活现象[12 , 54, 55]。在禾谷类作物中，已得到检测的还有其他一些病毒启动子序列，包括从水稻东格鲁杆状病毒（ RTBV ) 和夜香树黄曲叶病毒（ CmYLCV ) 获得的序列。启动子缺失试验表明， RTBV 启动子在转基因水稻的维管束或韧皮部特异表达 [56~61]。在转基因小麦中， RTBV启动子可驱动 GUS 在花粉和根组织以外的各种组织中强烈表达（作者未发表结果）。在转基因拟南芥、烟草、玉米及水稻中，CmYLCV 启动子可以激活 GUS 或 GFP 组成型表达[62]。
在禾谷类作物的转化试验中，通常优先使用两个单子叶植物启动子（Ubil和 Actl) ，而不是病毒序列。 Ubil 由玉米泛素启动子与其第一个外显子和第一个内含子构建而成 [10，11 ]， Atl 由水稻肌动蛋白启动子与其内含子构建而成 [63 ]。在水稻[64, 65]、玉米 [ 66 ] 和小麦 [ 3，14 ] 中，用玉米 Ubil 启动子驱动的 uWA ( GUS ) 报告基因，能够在多数组织中强烈表达。在幼嫩、代谢旺盛的组织和花粉中，通常 GUS 的表达水平较强；而在老叶、根部及花药的体细胞组织中，GUS 表达水平很低或者不表达 [ 图 11.1 (a ) 和图 11.1 (b ) ]。虽然玉米 Ubil 启动子具有很高水平的本底表达，但依然受热激和胁迫的诱导 [64]，而这种诱导性在大多数玉米 Ubil 启动子的转基因研究中并未被积极利用。玉米 Ubil 的热激元件已被鉴定，当采用豌豆凝集素启动子的一个碱性结构域/亮氨酸拉链因子结合位点替换 Ubil 的热激元件时，则会改变基因在种子内的表达分布，即表达由胚转向胚乳 [66]。其他一些泛素启动子在单子叶植物转化中也得到鉴定，包括从水稻中分离出的 RUBQ2 和 Rubi3 [ 67, 68 ]，以及从甘蔗中分离出的 ubi4 和 ubi9 [ 69 ] 启动子，所有这些启动子的表达活性与玉米 Ubil 或 CaMV35S 相当甚至更高。玉米 Ubil 驱动 GtAS 基因在转基因拟南芥、番茄、烟草和松树的根中也获得了很好的表达[70]。
Actin 基因的启动子是可以替代泛素的另一个理想启动子。水稻 Actin 启动子 ( Actl- D) 由 Act1 基因翻译起始密码子上游 2.1 kb 的一段序列组成。该序列包含了 GUS 高水平表达的所有 5' 调控因子，其为水稻和玉米瞬时转化[16, 63 ] 以及水稻稳定转化 [ 7 1 ] 所必需。除启动子区的序列外， Actl- D 还包括了 Act1 基因 5' 内含子和第一外显子的部分序列。据报道， Actl- D :: GUS 在转基因水稻株系的孢子体和配子体组织中均呈组成型表达[71 ]。定量分析表明，转化体中 GUS 蛋白量占总可溶性蛋白的 3%[71]。在小麦中， Actl- D 启动子能驱动基因在各发育阶段的所有主要组织中强烈表达 ( 作者未发表的数据）。在转基因燕麦中，水稻 Actl- D 和截短的 Act1 启动子（ActlF ) 均被用来驱动以必表达[12, 18]，但尚未有这些转基因株系详细分析的报道。
在谷类作物中得到鉴定和应用的其他类型的组成型表达的启动子还有：携带 Ubi 第一个内含子的玉米 H2B 启动子 [25]、玉米 Adh1 启动子[8 , 9 ] 、水稻细胞色素 c 启动子（OsCc1 ) [72]，以及来自地三叶草矮化病毒（SCSV) 的一系列调控序列[73]。
3. 组织特异性基因的表达
在自然状态下，依据基因产物的作用，大多数植物基因的表达具有器官或组织特异性。当用 1 kb 或 2 kb 的启动子构建异源表达框时，基因在新的表达框通常仍能保持其原有的空间特异性。例如，小麦高分子质量谷蛋白亚基基因的 1.2 kb 启动子序列，在转基因硬粒小麦中驱动报告基因以必在淀粉胚乳中特异性表达 [ 26 ] [ 图 11. 1(d ) ]。该启动子及其他的谷蛋白启动子，已被用于驱动外源基因在小麦胚乳中的表达( 综述 [ 74 ] ) 。其他已经证实的参与种子储藏物调控的启动子还包括：低分子质量谷蛋白亚基启动子[27]、 B- 和 D- 醇溶蛋白启动子[22, 28 , 30 ]。通过检测报告基因，Em、 asi、 aamyl、 aamy 2 ，以及嘌呤吲哚蛋白（puroindoline) a 和 b 等启动子均有在种子内特异表达的特点 [ 图11.1 ( e ) ] [ 31， 33 , 35, 36 ]。另外，作者未发表的资料表明，球蛋白启动子序列具有在糊粉层和胚胎表达的特征[图11 .1 ( f ) ]。
4 . 诱导型基因的表达
通过外界刺激调节候选基因表达的开启与关闭，对于许多研究的应用具有重要价值( 综述 [75~77 ] ) 。当仅对特定组织施以激活刺激时，诱导型启动子会产生所谓的组织特异性，包括 a1cR ( 乙醇诱导 [ 78 ~ 81 ] ) 、谷胱甘肽 S- 转移酶/I n 2-2 (安全剂诱导[82, 83 ] ) 、各种热激诱导多聚半乳糖醛酸酶抑制蛋白 [84~87] ( HSP，损伤诱导[88] ) 、 TetR 和 tRA ( 分别受四环素诱导和抑制 [ 89~93] ) 、EcR ( 蜕皮激素诱导 [ 94~97 ] ) 在内的多种诱导型启动子已在转基因植物中开展研究。然而，大多数的研究都是在双子叶植物中开展的，虽然其中的一些结果可类推到单子叶植物，但是我们确实需要验证转基因禾谷物作物的最佳诱导体系。
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