Buzeli Reginaldo A A, Cascardo Júlio C M, Rodrigues Leonardo A Z, Andrade Maxuel O, Almeida Raul S, Loureiro Marcelo E, Otoni Wagner C, Fontes Elizabeth P B
Departamento de Biologia Vegetal, BIOAGRO, UFV, Viçosa, MG, Brazil.
Plant Mol Biol. 2002 Nov;50(4-5):757-71. doi: 10.1023/a:1019994721545.
The binding protein BiP is an endoplasmic reticulum (ER)-resident member of the HSP70 stress-related protein family, which is essential for the constitutive function of the ER. In addition to responding to a variety of environmental stimuli, plant BiP exhibits a tissue-specific regulation. We have isolated two soybean BiP genomic clones, designated gsBiP6 and gsBiP9, and different extensions of their 5' flanking sequences were fused to beta-glucuronidase (GUS) reporter gene and introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Transgenic plants displayed prominent GUS activity in the vascular bundles of roots and shoots as well as in regions of intense cell division, such as procambial region and apical meristems. Promoter deletion analyses identified two cis-regulatory functional domains that are important for the spatially-regulated activation of BiP expression under normal plant development. While an AT-rich enhancer-like sequence, designated cis-acting regulatory domain 1, CRD1 (-358 to -211, on gsBiP6), activated expression of the BiP minimal promoter in all organs analyzed, BiP promoter activity in meristematic tissues and phloem cells required the presence of a second activating domain, CRD2 (-211 to -80). Apparently, the CRD2 sequence also harbors negative cis-acting elements, because removal of this region caused activation of gsBiP6 promoter in parenchymatic xylem rays. These results suggest that the tissue-specific control of BiP gene expression requires a complex integration of multiple cis-acting regulatory elements on the promoter.
结合蛋白BiP是热休克蛋白70（HSP70）应激相关蛋白家族的内质网（ER）驻留成员，对ER的组成功能至关重要。除了对多种环境刺激做出反应外，植物BiP还表现出组织特异性调控。我们分离出了两个大豆BiP基因组克隆，命名为gsBiP6和gsBiP9，并将其5'侧翼序列的不同延伸片段与β-葡萄糖醛酸酶（GUS）报告基因融合，通过根癌农杆菌介导的转化导入烟草。转基因植物在根和茎的维管束以及细胞分裂活跃的区域，如原形成层区域和顶端分生组织中表现出显著的GUS活性。启动子缺失分析确定了两个顺式调控功能域，它们对于正常植物发育过程中BiP表达的空间调控激活很重要。一个富含AT的增强子样序列，命名为顺式作用调控域1（CRD1，在gsBiP6上为-358至-211），在所有分析的器官中激活了BiP最小启动子的表达，而分生组织和韧皮部细胞中的BiP启动子活性需要第二个激活域CRD2（-211至-80）的存在。显然，CRD2序列也含有负性顺式作用元件，因为去除该区域会导致gsBiP6启动子在薄壁木质射线中被激活。这些结果表明，BiP基因表达的组织特异性控制需要启动子上多个顺式作用调控元件的复杂整合。
