Cloning of the promoter region of plasma membrane aquaporin BnPIP1 from Brassica napus and its functional analysis

Aquaporins make water-selective channels in plants, facilitating the permeation of water through membranes and adjusting water fast transport during seed germination, cell elongation, stoma movement, fertilization and responses to environmental stresses. They belong to the MIP (major intrinsic protein) family with molecular weight of 2629 kD and are characterized by six membrane-spanning a-helixes connected by five loops and short N-terminal and C-terminal domains in the cytoplasm[13]. The p…     

从水稻细胞质型果糖-1,6-二磷酸酶启动子上游中去除93 bp可以彻底改变其表达模式

细胞质型果糖-1,6-二磷酸基因ATG上游1 195bp侧翼序列可调控GUS基因在水稻(Oryza sativa L.)中特异性表达,因此该片段包含有使报告基因在叶肉细胞中特异性表达的所有顺式元件.为了研究其调控特异表达的顺式元件,对启动子5′端进行了一系列的缺失,得到4种与GUS基因融合的植物表达载体,通过基因枪法转入水稻.结果表明,自启动子5′端-1 195 bp缺失至-1 102 bp时,GUS基因由叶肉细胞特异性表达变为组成型表达,且表达活性有所提高,推测在该区段中存在调控叶肉细胞特异性表达的顺式元件.进一步缺失仍然保持组成性表达的模式,即在转化株的根、茎和叶中的所有细胞中均有表达,同时启动子活性有所提高.这一结果暗示该启动子具有很大的应用潜力.     

Cloning of the promoter region of plasma membrane aquaporin<Emphasis Type="Italic">BnPIP1</Emphasis> from<Emphasis Type="Italic">Brassica napus</Emphasis> and its functional analysis

A 1.6 kb upstream regulatory sequence (GenBank accession no. AF472487) of plasma membrane aquaporinBnPIP1 gene fromBrassica napus was obtained by genomic walking based on ligation-mediated PCR method. Sequence analysis indicated that this fragment contained seed germination specific and vascular specific sequences. The 1.6 kb upstream sequence and various 5′ end deleted sequences were fused withuidA gene and constructed into plant expression vectors which were used for tobacco transformation. GUS histochemical assay showed that the 1.6 kb fragment had high levels of promoter activity and the GUS staining was mainly distributed in vascular systems and tissues with rapid expanding and proliferating cells. Promoter deletion analysis showed that the deletion of -1610 — -1030 bp resulted in a dramatic reduction in GUS activity. It was assumed that there might be cis-acting element(s) existing in this region. Whereas, the region located at -1030 — -902 bp strongly inhibited the expression ofgus and probably contained negative regulatory element(s). The fragment of -902 — -19 bp could also directgus expression at high level.     

A GATA box in the GATA-1 gene hematopoietic enhancer is a critical element in the network of GATA factors and sites that regulate this gene

A region located at kbp -3.9 to -2.6 5' to the first hematopoietic exon of the GATA-1 gene is necessary to recapitulate gene expression in both the primitive and definitive erythroid lineages. In transfection analyses, this region activated reporter gene expression from an artificial promoter in a position- and orientation-independent manner, indicating that the region functions as the GATA-1 gene hematopoietic enhancer (G1HE). However, when analyzed in transgenic embryos in vivo, G1HE activity was orientation dependent and also required the presence of the endogenous GATA-1 gene hematopoietic promoter. To define the boundaries of G1HE, a series of deletion constructs were prepared and tested in transfection and transgenic mice analyses. We show that G1HE contains a 149-bp core region which is critical for GATA-1 gene expression in both primitive and definitive erythroid cells but that expression in megakaryocytes requires the core plus additional sequences from G1HE. This core region contains one GATA, one GAT, and two E boxes. Mutational analyses revealed that only the GATA box is critical for gene-regulatory activity. Importantly, G1HE was active in SCL(-/-) embryos. These results thus demonstrate the presence of a critical network of GATA factors and GATA binding sites that controls the expression of this gene.     

Nuclease sensitivity and functional analysis of a maize histone H3 gene promoter

A 1 kb region of a maize H3 histone gene promoter has been analysed at a structural and functional level. Micrococcal nuclease digestion of isolated nuclei showed that the promoter region is organized into nucleosomes but a zone extending over approximately one nucleosome (20 to 230 bp upstream of the TATA box) displays remarkable accessibility to digestion. Three DNase I-hypersensitive sites were found within this zone at the vicinity of consensus sequences, some of which are already known to act ascis elements. This promoter region is able to direct faithful expression of the GUS reporter gene in meristematic tissues of transgenic tobacco plants.     

水稻OsGSTL1启动子的分离和表达分析

从水稻基因组文库中筛选得到一个水稻GST基因,命名为OsGSTL1.半定量RT-PCR分析表明OsGSTL1基因的表达不受绿磺隆、乙烯利、脱落酸、水杨酸和茉莉酸甲酯的诱导,因此该基因可能与植物抗逆性无关.为了研究OsGSTL1启动子在植物体内的表达特性,将OsGSTL1起始位点5＇端上游不同长度的调控序列与报告基因GUS融合,并在洋葱表皮瞬间表达和拟南芥中稳定表达.研究表明：在洋葱表皮细胞中,160bp及更长的上游调控序列均能启动GUS基因的表达;而在转基因拟南芥中,含有2155 bp的上游序列的PGZ2.1：：GUS具有时空表达的特性,在转基因的早期幼苗中GUS基因在子叶中特异性表达,但在根中没有表达;而在幼苗生长的后期,根、茎、叶中都有少量的表达.但包含1 224 bp的上游序列的PGZ1.2：：GUS却表现为组成型表达的特性.由此推测,OsGSTL1启动子启动的基因表达可能与幼苗的营养代谢相关;而OsGSTL1启动子的时空表达相关元件可能位于OsGSTL1翻译起始位点5＇端上游-2155 bp至-1224 bp范围内.     

高羊茅FaChit1基因启动子的功能分析

用含有不同长度FaChitl基因启动子区域与GUS基因融合构建植物表达载体pFaChitlP—I、pFaChitlP-Ⅱ以及pFaChitlP-Ⅲ并分别对烟草进行转化,经真菌激发子、干旱、机械损伤以及乙烯等多种胁迫处理后测定GUS活性。启动子缺失分析实验结果显示,真菌激发子对FaChitl基因启动子所介导的GUS诱导表达效果最强,而机械损伤只能微弱地诱导GL靥基因表达;FaChitl基因启动子-651bp以内的序列均能介导GUS基因的诱导表达,同时-935bp与-233bp之间的区域是该启动子响应真菌激发子、乙烯以及机械损伤胁迫所必需的。表明FaChitl启动子是一个多胁迫诱导型启动子。