Isolation and expression analysis of a LEAFY/FLORICAULA homolog and its promoter from London plane (Platanus acerifolia Willd.)

The LEAFY/FLORICAULA (LFY/FLO) homologous genes are necessary for normal flower development in diverse angiosperm species. To understand the genetic and molecular mechanisms underlying floral initiation and development in Platanaceae, an early divergent eudicot family consisting of large monoecious trees, we isolated a homolog of LFY/FLO, PlacLFY, and its promoter from London plane (Platanus acerifolia). PlacLFY is 1,419?bp in length, with an ORF of 1,122?bp encoding a predicted polypeptide of 374 amino acids and 5'/3'-UTR of 54 and 213?bp, respectively. The putative PlacLFY protein showed a high degree of identity (56-84?%) with LFY/FLO homologs from other species, including two highly conserved regions, the N and C domains, and a less conserved amino-terminal proline-rich region. Real-time PCR analysis showed that PlacLFY was expressed mainly in male inflorescences from May of the first year to March of next year, with the highest expression level in December, and in female inflorescences from June to April of next year. PlacLFY mRNA was also detected strongly in subpetiolar buds of December from 4-year-old and adult trees, and slightly in stem of young seedling and young leaf of adult plant. Additionally, we cloned 1,138?bp promoter sequence of PlacLFY and we drove GUS expression in transgenic tobacco by the chimerical pPlacLFY::GUS construction. Histological GUS staining analysis indicated that PlacLFY promoter can drive GUS gene expression in shoot apex, stem, young leaf and petiole, flower stalk, petal tip, and young/semi-mature fruits of transgenic tobacco, which is almost identical to the expression pattern of PlacLFY in London plane. The results revealed that the PlacLFY gene isolated from London plane is expressed not only in reproductive organ but also in vegetative organs. Moreover, this expression pattern is consistent with the expression pattern in tobacco of a GUS reporter gene under the control of the potential promoter region of PlacLFY.     

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.     

Cloning and characterization of the WAX2 gene of Arabidopsis involved in cuticle membrane and wax production

Insertional mutagenesis of Arabidopsis ecotype C24 was used to identify a novel mutant, designated wax2, that had alterations in both cuticle membrane and cuticular waxes. Arabidopsis mutants with altered cuticle membrane have not been reported previously. Compared with the wild type, the cuticle membrane of wax2 stems weighed 20.2% less, and when viewed using electron microscopy, it was 36.4% thicker, less opaque, and structurally disorganized. The total wax amount on wax2 leaves and stems was reduced by >78% and showed proportional deficiencies in the aldehydes, alkanes, secondary alcohols, and ketones, with increased acids, primary alcohols, and esters. Besides altered cuticle membranes, wax2 displayed postgenital fusion between aerial organs (especially in flower buds), reduced fertility under low humidity, increased epidermal permeability, and a reduction in stomatal index on adaxial and abaxial leaf surfaces. Thus, wax2 reveals a potential role for the cuticle as a suppressor of postgenital fusion and epidermal diffusion and as a mediator of both fertility and the development of epidermal architecture (via effects on stomatal index). The cloned WAX2 gene (verified by three independent allelic insertion mutants with identical phenotypes) codes for a predicted 632-amino acid integral membrane protein with a molecular mass of 72.3 kD and a theoretical pI of 8.78. WAX2 has six transmembrane domains, a His-rich diiron binding region at the N-terminal region, and a large soluble C-terminal domain. The N-terminal portion of WAX2 is homologous with members of the sterol desaturase family, whereas the C terminus of WAX2 is most similar to members of the short-chain dehydrogenase/reductase family. WAX2 has 32% identity to CER1, a protein required for wax production but not for cuticle membrane production. Based on these analyses, we predict that WAX2 has a metabolic function associated with both cuticle membrane and wax synthesis. These studies provide new insight into the genetics and biochemistry of plant cuticle production and elucidate new associations between the cuticle and diverse aspects of plant development.     

Cloning and characterization of a cathepsin D inhibitor gene from Solanum tuberosum L.

A DNA clone encoding a cathepsin D inhibitor CathInh was isolated from a potato genomic library using a CathInh cDNA as hybridization probe. The amino acid sequence of the coding region is nearly identical with a CathInh cDNA and CathInh proteins previously isolated from a tuber-specific cDNA library and from tubers, respectively. Analysis of GUS activity resulting from expression of chimeric CathInh promoter-GUS genes in transgenic potato plants revealed expression exclusively confined to potato tubers. No GUS activity could be detected in any other organ of the transgenic plants either constitutively or after wounding or treatment with abscisic and jasmonic acid (JA). Interestingly, part of the promoter region of the CathInh gene, essential for GUS activity in tubers, shows striking similarity to promoter regions of tuber-specific class I patatin genes.     

AtSTP3启动子控制的外源基因在转基因水稻中的特异表达研究

利用PCR技术从哥伦比亚型拟南芥基因组DNA中分离了AtSTP3绿色组织特异表达的启动子,序列分析表明,扩增片段(1774bp)与已报道序列的相应区域同源性达99.9%。将其与GUS报告基因融合在一起,构建了植物表达载体,并由农杆菌介导法导入水稻品种‘中花11’中。对转基因水稻植株中的GUS活性进行定性与定量测定结果表明,AtSTP3启动子可驱动GUS报告基因在转基因水稻植株叶片中特异性表达,而在根和种子等器官中不表达或表达活性极弱,AtSTP3启动子表现出明显的组织特异性。     

Expression patterns of two genes for the delta-subunit of mitochondrial F1-ATP synthase from sweet potato in transgenic tobacco plants and cells.

Two nuclear genes, F1 delta-1 and F1 delta-2, coding for the delta-subunit of mitochondrial F1-ATP synthase, which corresponds to oligomycin-sensitivity conferring protein in animal and yeast mitochondria, were isolated from sweet potato. The gene for the delta-subunit was composed of 6 exons and these two genes shared high sequence similarities to each other not only in exons but also in introns and in the 5'-upstream regions. However, the 5'-upstream regions of F1 delta-1 and F1 delta-2 were distinguishable by the presence of novel sequences, designated Ins-1 and Ins-2, respectively. Ins-1 and Ins-2 contained a terminal direct repeat of 10 bp and 12 bp, respectively, and various forms of repeat sequences. The promoter fusion of both F1 delta-1 and F1 delta-2 with the GUS coding sequence gave expression of GUS activity in transformed tobacco BY-2 cells, although the levels of GUS activity and the patterns of expression during the growth of cells were different between the two. In transgenic tobacco plants, the two fusion genes showed similar levels of expression in leaves and stems, while F1 delta-2:GUS gave significantly higher levels of expression in roots than F1 delta-1:GUS. Deletion of Ins-1 from the 5'-upstream region of F1 delta-1:GUS did not affect the expression of the fusion gene in various organs of transgenic plants. However, it caused significant enhancement of expression in transformed tobacco BY-2 cells.     

西瓜果实特异启动子WSP功能区域的初步定位

西瓜AGPase的大亚基基因wml1的 5′端上游 15 73bp序列 ,是一个果实特异性启动子 (命名为WSP)。根据WSP内部酶切位点 ,获得了 3个不同 5′端缺失的启动子片段 (长分别为 12 0 1bp、898bp、795bp) ,并构建成植物瞬间表达载体 ,与含WSP的瞬间表达载体一起用基因枪的方法转入西瓜叶、茎、花及不同发育期果实中。瞬时表达结果表明 ,15 73bp、12 0 1bp、898bp的片段均能指导GUS基因在西瓜果实和花中特异性表达 ,但是表达强度和表达时期有所不同 ,795bp的片段不能指导GUS基因表达。推测在 180bp 5 5 1bp之间可能存在促进外源基因在果实发育后期表达的顺式作用元件 ,而果实特异调控区域可能位于 85 4bp 95 7bp之间。     

拟南芥2-甲基-6-叶绿基-1,4-苯醌甲基转移酶启动子的分离及表达特性分析

维生素E是一类人体必需的脂溶性抗氧化剂, 具有重要的生理功能。2-甲基-6-叶绿基-1,4-苯醌甲基转移酶(MPBQ MT)是天然维生素E合成途径中的关键酶之一, 催化MPBQ甲基化, 生成DMPBQ。从拟南芥分离了MPBQ MT基因1018bp的启动子序列, 构建了含该启动子和GUS报告基因的植物表达载体, 通过农杆菌介导转化拟南芥, 获得了转基因植株。GUS组织化学染色结果表明, 在MPBQ MT启动子驱动下, 报告基因GUS在拟南芥的茎、叶、花萼、雄蕊、种荚均有表达, 且在茎、叶、种荚中表达量较高, 而在根、花瓣和种子中则没有观察到GUS基因的表达, 表明MPBQ MT基因可能仅在拟南芥幼嫩茎、叶、种荚等绿色组织中特异性高表达。     

Expression and Promoter Activity of Genes for Isozymes of Horseradish Peroxidase

The expression and promoter activity of genes for isozymes ofhorseradish peroxidase, namely, prxCla, prxClb, prxC2 and prxC3,were studied. Organ-specific expression of these genes in horseradishplants was examined by Northern blot analysis. The group ofprxCl genes was expressed mostly in stems, while prxC2 and prxC3were expressed to a greater extent in roots. Hardly any expressionof any of the genes was detected in leaves. In transient-expressionassays with tobacco protoplasts, about 500 bp of the 5'-noncodingregions of each of the genes, ligated to the gene for ß-glucuronidase(GUS), exhibited significant promoter activity. In particular,the fragments extending from the initiation codon of the prxC2gene to –529 bp and –1 kbp supported high levelsof GUS activity, which were 4.4 and 11.4 times respectively,the activity observed under control of the 35S promoter fromcauliflower mosaic virus (CaMV). Conserved enhancer sequencesof human genes were found in the 5'-flanking region of prxC2,and deletion of the regions that contained the enhancer sequencesreduced the GUS activity. High levels of GUS activity were observedin transgenic tobacco plants that contained 1 kbp of the 5'flanking region of prxC2 fused to the GUS gene. GUS activitywas diminished when deletion from the 5' end extended as faras the CAAT box. No significant organ-specific expression ofGUS was observed with any such deletion. (Received April 15, 1992; Accepted September 11, 1992)     

Chalcone Synthase Promoters in Petunia Are Active in Pigmented and Unpigmented Cell Types

Chalcone synthase (CHS) catalyzes the first step in the biosynthesis of flavonoids that function in flower pigmentation, protection against stress, and induction of nodulation. The petunia genome contains eight complete chs genes, of which four are differentially expressed in floral tissues and UV-light-induced seedlings. The 5[prime]-flanking regions of these four chs genes were fused to the [beta]-glucuronidase (GUS) reporter gene and introduced into petunia plants by Agrobacterium-mediated transformation. We show that expression of each construct is identical to the expression of the authentic chs gene, implying that the differences in expression pattern between these chs genes are caused at least in part by their promoters. Histochemical analyses of GUS expression show that chs promoters are not only active in pigmented cell types (epidermal cells of the flower corolla and tube and [sub] epidermal cells of the flower stem) but also in a number of unpigmented cell types (mesophylic cells of the corolla, several cell types in the ovary and the seed coat). Comparison of chs-GUS expression and flavonoid accumulation patterns in anthers suggests that intercellular transport of flavonoids and enzymes occurs in this organ. Analysis of the flavonoids accumulated in tissues from mutant lines shows that only a subset of the genes that control flavonoid biosynthesis in the flower operates in the ovary and seed. This implies that (genetic) control of flavonoid biosynthesis is highly tissue specific.     

水稻OsWTF1基因启动子的克隆与表达分析

目的：分析水稻OsWTF1基因启动子的功能及核心序列。方法：利用PCR技术从水稻日本晴基因组中克隆了转录因子WTF1编码区5上游大小为2049bp的调控区域,命名为OsWTF1,将它和长度为1631、608、474、415bp的5端缺失体分别与GUS基因融合构建表达载体,并用农杆菌介导法转化水稻。结果：GUS组织化学分析表明,OsWTF1、Os1631能够驱动GUS基因在根、茎、叶、叶鞘、花药、颖壳上的表达,Os608,Os474,Os415能驱动GUS在根、茎、花药、颖壳中表达,在叶鞘中未表达,而且在叶中的表达也很微弱。结论：OsWTF1启动子核心序列可能位于-1bp--415bp之间,在-608bp--1631bp之间可能存在与基因叶肉特异表达相关的重要元件。