Isolation and characterization of <Emphasis Type="Italic">Brittle2</Emphasis> promoter from <Emphasis Type="Italic">Zea Mays</Emphasis> and its comparison with <Emphasis Type="Italic">Ze19</Emphasis> promoter in transgenic tobacco plants

ADP-glucose pyrophosphorylase (AGPase) represents a key regulatory step in starch synthesis. A 0.9 kb of 5′ flanking region preceding Brittle2 gene, encoding the small subunit of maize endosperm AGPase, was cloned from maize genome and its expression pattern was studied via the expression of β-glucuronidase (GUS) gene in transgenic tobacco. Analysis of GUS activities showed that the 0.9 kb fragment flanking Brittle2 gene was sufficient for driving the seed-preferred expression of the reporter gene. The activity of the 0.9 kb 5′ flanking fragment was compared with that of the tandem promoter region from a zein gene (zE19, encoding a maize 19 kDa zein protein). The results indicated that both promoters were seed-preferred in a dicotyledonous system as tobacco and the activity of zE19 promoter was three to fourfold higher than that of the 0.9 kb fragment flanking Brittle2 gene in transgenic tobacco seeds. At the same time, zE19-driven GUS gene expressed earlier than Brittle2 promoter during seed development. Histochemical location of GUS activity indicated that both promoters showed high expression in embryos, which is different from similar promoters tested in maize.     

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.     

Authentic seed-specific activity of the <Emphasis Type="Italic">Perilla</Emphasis> oleosin 19 gene promoter in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Perilla (Perilla frutescens L. cv. Okdong) oleosin gene, PfOle19, produces a 19-kDa protein that is highly expressed only in seeds. The activity of the −2,015 bp 5′-upstream promoter region of this gene was investigated in transgenic Arabidopsis plants using the fusion reporter constructs of enhanced green fluorescent protein (EGFP) and β-glucuronidase (GUS). The PfOle19 promoter directs Egfp expression in developing siliques, but not in leaves, stems or roots. In the transgenic Arabidopsis, EGFP fluorescence and histochemical GUS staining were restricted to early seedlings, indehiscent siliques and mature seeds. Progressive 5′-deletions up to the −963 bp position of the PfOle19 promoter increases the spatial control of the gene expression in seeds, but reduces its quantitative levels of expression. Moreover, the activity of the PfOle19 promoter in mature seeds is 4- and 5-fold greater than that of the cauliflower mosaic virus 35S promoter in terms of both EGFP intensity and fluorometric GUS activity, respectively.     

pib基因启动子及其诱导启动性初探

将pib基因上游5.7 kb区段取代pCAMBIA1301中gus基因上游的35S启动子构建了pib拟启动区-GUS+ 35S-hpt 基因表达载体pNAR604。经农杆菌介导转化水稻成熟胚愈伤,获得了转基因抗潮霉素愈伤和36株转基因水稻植株。 转基因抗性愈伤和转基因植株根的组织化学GUS活性检测表明,光照培养下的抗性愈伤和转基因植株根不能使X-gluc显色,而暗处理24 h后的抗性愈伤和定植后转基因植株的根能使X-gluc显色。转基因植株GUS荧光定量分析结果表明,GUS表达具有器官特异性,黑暗处理前根的GUS活性最高、茎次之,分别是是叶片的7倍和3倍,叶片中仅有痕量本底。24 h黑暗处理后根、茎、叶中GUS活性都有增加,且叶片中的增加比例最大,其活性仅次于根。5 mmol/L水杨酸和0.3 mol/L NaCl叶面喷施转基因植株24 h后叶片中GUS活性分别为处理前的2.7和3.6倍。初步确定pib拟启动区是一个诱导型启动子。黑暗、水杨酸和NaCl能诱导该启动子启动活性。     

Isolation of the maize <Emphasis Type="Italic">Zpu1</Emphasis> gene promoter and its functional analysis in transgenic tobacco plants

By screening a genomic library of maize, a 2.2 kb 5′ flanking fragment of Zpu1 gene, encoding the pullulanase-type starch debranching enzyme, was isolated. Promoter fragments of various lengths, including the full 5′ flanking sequence (−2267 to −1) (Z1), a 3′ deletion (−2267 to −513) (Z5) and three 5′ deletions extending to −1943 (Z2), −1143 (Z3) and −516 (Z4) upstream of the translational initiation codon (ATG), were fused to the GUS reporter gene and introduced into tobacco. When these constructs were tested in transgenic tobacco plants, seed-preferred GUS activity was observed in pZ1-transgenic lines. In pZ2-transgenic lines, the GUS activity was not only restricted to seeds, but was also detected in calyxes, petals, stamens and mature leaves. At the same time, negligible GUS activity was detected in roots, stems, young leaves, stigmas and ovaries from the transgenic tobacco plants, which had integrated the full isolated sequence of Zpu1 promoter or its deletions. Deletion analysis indicated that the promoter contained a putative positive cis-regulatory element and the proximal region (−516 to −1) was essential for directing the expression of gus reporter gene. Analysis of GUS activity during the fruit development and seed germination suggested that Zpu1 promoter is active both in starch anabolism and in starch catabolism, which is consistent with the function of the endogenous gene in maize. GUS activity in leaves under light and darkness confirmed that Zpu1 promoter functions in the starch degradation of photosynthetic tissues in the dark phase of the diurnal cycle.     

Functional characterization of a cotton late embryogenesis-abundant D113 gene promoter in transgenic tobacco

Previous studies have shown that mRNA and protein encoded by late embryogenesis-abundant (LEA) gene D113 from Gossypium hirsutum L. accumulate at high levels in mature seeds and also in response to abscisic acid (ABA) in young embryo. In this study, we studied the expression of four promoter 5′ deletion constructs (−1383, −974, −578 and −158) of the LEA D113 gene fused to beta-glucuronidase (GUS). GUS activity analysis revealed that the −578 promoter fragment was necessary to direct seed-specific GUS expression in transgenic tobacco plants (Nicotiana tabacum L.). To further investigate the expression pattern of LEA D113 promoter under environmental stresses, 2-week-old transgenic tobacco seedlings were exposed to ABA, dehydration, high salinity and cold treatments. GUS activity in the seedlings was quantified fluorimetrically, and expression was also observed by histochemical staining. An apparent increase in GUS activity was found in plants harboring constructs −1383, −974 and −578 after 24 h of ABA or high-salinity treatments, as well as after 10 days of dehydration. By contrast, only a slight increase was observed in all the three lines after cold treatment. Virtually no change in expression was found in construct −158 in response to dehydration, salinity and cold, but there was a moderate response to ABA, suggesting that the region between −574 and −158 was necessary for dehydration- and salinity-dependent expression, whereas ABA-responsive cis-acting elements might be located in the −158 region of the promoter.     

香蕉果实成熟相关基因ACO1启动子区的克隆及其功能初探

根据已报道的香蕉课实表达ACC氧化酶基因（ACO1）的序列,用改进的接头连接PCR法从香蕉基因组中扩增并克隆了此基因5′旁侧区1526bp的片段,其中包含一个推测的TATA盒序列;与已公布的两个香蕉ACC氧化酶基因启动子序列（分别为934bp和1451bp）的相似性各为97．3％（Lopez-Gomez等）和88．8％（May和Kipp)。将4个含有不同大小启动子区的克隆片段与GUS基因编码区连接构建成嵌合成基因,通过基因枪轰击转入香蕉叶、根和果实的细胞后,瞬时表达结果表明不同大小的ACO1启动子区段都只在果实细胞中指导GUS基因表达,证明该启动子具有指导基因在果实中表达的功能,并推测负责果实特异性的顺式元件可能位于启动子近端0．7kb区段之内,在468至822的355bp区段内可能在与正控制有关的顺式元件。     

Developmental regulation of peach ACC oxidase promoter--GUS fusions in transgenic tomato fruits

A genomic DNA sequence (PpACO1) encoding 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) from peach (Prunus persica L. Batsch cv. Loring) was isolated. It has four exons interrupted by three introns and 2.9 kb of flanking region 5' of the translational start codon. Previous work with the cDNA demonstrated that accumulation of the peach ACO message correlated with increasing amounts of ethylene synthesized by the fruit as they ripened. To identify regulatory elements in the peach ACC oxidase gene, chimeric fusions between 403, 610, 901, 1319, 2141, and 2919 bp of the 5' flanking region of the PpACO1 sequence and the beta-glucuronidase (GUS) coding sequence were constructed and used to transform tomato (Lycopersicon esculentum [Mill] cv. Pixie). Fruits from the various promoter lines were analysed for GUS expression by histochemical GUS staining, GUS quantitative enzyme activity determination, and measuring the relative amounts of GUS mRNA. Constructs with the smallest promoter of 403 bp had significant GUS expression in fruit, but not in other tissues, indicating the presence of a region that affects tissue-specific expression. An increase in GUS expression was observed with promoters longer than 901 bp, indicating an enhancer region between -1319 and -901. The full-length promoter of 2919 bp directed GUS expression in the green stage of fruit development, and increased GUS expression as fruit matured, indicating a regulatory region between -2919 and -2141 that controls the temporal expression of the gene in fruit. Only the full-length promoter sequence demonstrated responsiveness to ethylene.     

A quantitative and histological comparison of gus expression with different promoter constructs used in microprojectile bombardment of peanut leaf tissue

Summary β-glucuronidase (GUS) expression driven with different promoter constructs was quantitatively and histologically compared in peanut leaf tissue following microprojectile bombardment. X-Gluc staining patterns varied with the construct used. Tissues bombarded with the pAC2GUS construct had larger foci and a greater percentage of area staining blue. pEmuGN exhibited the greatest numbers of blue spots compared to pAC2GUS and pTRA140. Histological evaluations of blue staining foci showed a diffusion gradient of blue precipitate from a central, prominently-staining cell outward to as many as seven cell layers. The intensity of X-gluc product in centrally-staining cells varied. Gold microprojectile particles were usually located within the three surface cell layers. Depending on the plasmid construct, 72–90% of the centrally-staining cells had at least one gold particle. However, the presence of GUS expression did not appear to require a microprojectile within the nucleus, which was observed in 37% or fewer of the centrally-staining cells. With the pAC2GUS construct, staining patterns varied with location within leaflets and had an “edge effect,” i.e., blue spots were frequently larger at the margin versus central regions. This enhanced activity could be anticipated with an actin promoter in the more mitotically active marginal leaf cells. Total GUS activity as determined by fluorometric analyses was correlated with the percentage of X-gluc stained area. The pAC2GUS construct exhibited the highest total GUS activity among the three constructs.     

A promoter directing high level expression in pistils of transgenic plants

The promoter of the potato (Solanum tuberosum L.) SK2 gene, encoding a pistil-specific basic endochitinase, was cloned. Various fragments of the SK2-promoter, from 1 kb down to 0.23 kb in length, were fused to the GUS reporter gene. Chimaeric SK2 promoter-GUS fusion constructs were transformed into potato by Agrobacterium tumefaciens-mediated transformation. The SK2-GUS transgenic potato plants exhibited a highly specific GUS activity in the pistil. Expression in the pistil was shown to be developmentally regulated. In addition to the GUS activity in pistils, transgenic plants also showed a much weaker ectopic expression in anthers. In other tissues no systematic expression was detectable. All SK2 promoter fragments analysed conferred pistil-specific expression without significant qualitative or quantitative differences, demonstrating that the regulatory elements mediating this expression pattern are located within a 230 bp SK2 promoter fragment. The SK2 promoter may be used to engineer high levels of expression in pistils of transgenic plants.