Inheritance of tissue-specific expression of barley hordein promoter-uidA fusions in transgenic barley plants

 Barley (Hordeum vulgare L.) hordeins are alcohol-soluble redundant storage proteins that accumulate in protein bodies of the starchy endosperm during seed development. Strong endosperm-specific β-glucuronidase gene-(uidA; gus) expression driven by B₁- and D-hordein promoters was observed in stably transformed barley plants co-transformed with the selectable herbicide resistance gene, bar. PCR analysis using DNA from calli of 22 different lines transformed with B₁- or D-hordein promoter-uidA fusions showed the expected 1.8-kb uidA fragment after PCR amplification. DNA-blot analysis of genomic DNA from T₀ leaf tissue of 13 lines showed that 12 (11 independent) lines produced uidA fragments and that one line was uidA-negative. T₁ progeny from 6 out of 12 independent regenerable transgenic lines tested for uidA expression showed a 3 : 1 segregation pattern. Of the remaining six transgenic lines, one showed a segregation ratio of 15 : 1 for GUS, one expressed bar alone, one lacked transmission of either gene to T₁ progeny, and three were sterile. Stable GUS expression driven by the hordein promoters was observed in T₅ progeny in one line, T₄ progeny in one line, T₃ progeny in three lines and T₂ or T₁ progeny in the remaining two fertile lines tested; homozygous transgenic plants were obtained from three lines. In the homozygous lines the expression of the GUS protein, driven by either the B₁- or D-hordein promoters, was highly expressed in endosperm at early to mid-maturation stages. Expression of bar driven by the maize ubiquitin promoter was also stably transmitted to T₁ progeny in seven out of eight lines tested. However, in most lines PAT expression driven by the maize ubiquitin promoter was gradually lost in T₂ or later generations; one homozygous line was obtained. In contrast, six out of seven lines stably expressed GUS driven by the hordein promoters in T₂ or later generations. We conclude that the B₁- and D-hordein promoters can be used to engineer, and subsequently study, stable endosperm-specific gene expression in barley and potentially to modify barley seeds through genetic engineering. Received: 28 May 1998 / Accepted: 19 December 1998     

Functional analysis of six drought-inducible promoters in transgenic rice plants throughout all stages of plant growth

There are few efficient promoters for use with stress-inducible gene expression in plants, and in particular for monocotyledonous crops. Here, we report the identification of six genes, Rab21, Wsi18, Lea3, Uge1, Dip1, and R1G1B that were induced by drought stress in rice microarray experiments. Gene promoters were linked to the gfp reporter and their activities were analyzed in transgenic rice plants throughout all stages of plant growth, from dry seeds to vegetative tissues to flowers, both before and after drought treatments. In fold induction levels, Rab21 and Wsi18 promoters ranged from 65- and 36-fold in leaves to 1,355- and 492-fold in flowers, respectively, whereas Lea3 and Uge1 were higher in leaves, but lower in roots and flowers, as compared with Rab21 and Wsi18. Dip1 and R1G1B promoters had higher basal levels of activity under normal growth conditions in all tissues, resulting in smaller fold-induction levels than those of the others. In drought treatment time course, activities of Dip1 and R1G1B promoters rapidly increased, peaked at 2 h, and remained constant until 8 h, while that of Lea3 slowly yet steadily increased until 8 h. Interestingly, Rab21 activity increased rapidly and steadily in response to drought stress until expression peaked at 8 h. Thus, we have isolated and characterized six rice promoters that are all distinct in fold induction, tissue specificity, and induction kinetics under drought conditions, providing a variety of drought-inducible promoters for crop biotechnology.     

Analysis of Inheritability and Expression Profile of Single and Multi-copy Transgene(s) in Rice Over Generations

Transformation of Oryza sativa subsp indica variety Pusa Basmati 1 with Agrobacterium tumefaciens strain LBA4404(pTOK233) carrying genes coding for neomycin phosphotransferase (nptII), β-glucuronidase (gus) and hygromycin phosphotransferase (hph) under the control of plant-specific promoters (pnos and pCaMV35S) within its T-DNA region produced transgenics with single and multiple copies of T-DNA. Simple Mendelian as well as complex patterns of the inheritance for hygromycin resistance trait were observed in R1 and R2 generations. Non-segregating lines selected in R2 generation did not show further segregation of the resistance trait in R3 and R4 generations accompanied by stabilization of integrated transgenes. One of these lines showed the presence of truncated T-DNA in R1 generation. The single copy transgenics showed high stability of expression of gus gene, whereas multi-copy transgenics were prone to silencing up to R3 generation after which no further reduction in gene expression was observed.     

Characterization of the root-predominant gene promoter HPX1 in transgenic rice plants

Gene promoter(s) specialized in root tissues is an important component for crop biotechnology. In our current study, we report results of promoter analysis of the HPX1, a gene expressed predominantly in roots. The HPX1 promoter regions were predicted, linked to the gfp reporter gene, and transformed into rice. Promoter activities were analyzed in various organs and tissues of six independent transgenic HPX1:gfp plants using the fluorescent microscopy and q-RT-PCR methods. GFP fluorescence levels were high in root elongation regions but not in root apex and cap of the HPX1:gfp plants. Very low levels of GFP fluorescence were observed in anthers and leaves. Levels of promoter activities were 16- to 190-fold higher in roots than in leaves of the HPX1:gfp plants. The HPX1 promoter directs high levels of gene expression in root tissues producing GFP levels up to 0.39 % of the total soluble protein. Thus, the HPX1 promoter is predominantly active in the root elongation region during the vegetative stage of growth.     

对增强辣椒疫霉菌抗性的研究

病原物诱导型启动子能精确控制抗病基因在侵染位点的表达,是抗病基因工程的有效工具。prp1-1是来自马铃薯谷胱甘肽巯基转移酶基因启动子的一个273bp的片段,能够快速准确地启动被侵染位点抗病基因的表达;Rs-AFP2是具有对致病性丝状真菌的广谱抗性。该研究构建prp1-1调控Rs-AFP2基因表达的载体,经农杆菌介导转化法导入辣椒。逆转录PCR检测发现,转基因辣椒只在受到疫霉菌孢子侵染时,才由prp1-1启动Rs-AFP2基因的转录。用疫霉菌孢子灌根接种转基因辣椒T1代植株,35株T1代辣椒中有29株表现出明显的疫霉菌抗性。另将23株T1代辣椒种于人工气候箱,发现其形态和发育特征与相同条件下的非转基因植株无明显区别。研究表明,prp1-1调控Rs-AFP2的诱导表达达到了增强辣椒疫霉菌抗性的目的,而且避免了负面效应的发生。     

Isolation of four rice seed-specific promoters and evaluation of endosperm activity

Cereal grains are major targets for genetically improving the nutritional value of food and for producing recombinant proteins. Strong and tissue-specific promoters are highly desired for effectively controlling expression in the seed or endosperm. In this study, we isolated four rice promoters from the 5′ upstream region of putative seed-specifically expressed genes: PROLAM26, RAL2, RAL4 and CAPIP. By generating transgenic rice plants carrying promoter-reporter constructs, we found these four promoters to be specifically expressed in seeds, with three having endosperm-specific or -preferential activity. The strength of each promoter in the endosperm was determined and compared to a constitutively expressed OsACTIN promoter and an endosperm-specifically expressed Glu4-B promoter in single-copy transgenic plants. The promoter of RAL2 exhibited relatively high activity, and the promoters of RAL4 and CAPIP exhibited activities comparable with those of OsACTIN and Glu4-B. In addition, monitoring activities in high-generation (T₃–T₄) homozygous progeny of single-copy plants revealed maintenance of expression for all four promoters, with no evidence of silencing. Taken together, our findings offer four stable rice seed-specific promoters of different strengths for endosperm expression.     

Gene interactions and genetics of blast resistance and yield attributes in rice (Oryza sativa L.)

Blast disease caused by the pathogen Pyricularia oryzae is a serious threat to rice production. Six generations viz., P₁, P₂, F₁, F₂, B₁ and B₂ of a cross between blast susceptible high-yielding rice cultivar ADT 43 and resistant near isogenic line (NIL) CT13432-3R, carrying four blast resistance genes Pi1, Pi2, Pi33 and Pi54 in combination were used to study the nature and magnitude of gene action for disease resistance and yield attributes. The epistatic interaction model was found adequate to explain the gene action in most of the traits. The interaction was complementary for number of productive tillers, economic yield, lesion number, infected leaf area and potential disease incidence but duplicate epistasis was observed for the remaining traits. Among the genotypes tested under epiphytotic conditions, gene pyramided lines were highly resistant to blast compared to individuals with single genes indicating that the nonallelic genes have a complementary effect when present together. The information on genetics of various contributing traits of resistance will further aid plant breeders in choosing appropriate breeding strategy for blast resistance and yield enhancement in rice.     

Structural and functional analysis of new plant promoter pro-SmAMP1 from <Emphasis Type="Italic">Stellaria media</Emphasis>

The nucleotide sequence of a fragment of the promoter region of pro-SmAMP1 gene, having a length of 1257 bp and encoding antifungal peptides, was determined in chickweed (Stellaria media (L.) Vill.). Computer analysis of the nucleotide sequence revealed a number of cis-elements that are typical strong plant promoters. Five 5′-deletion variants were created taking into account the distribution of cis-elements:–1235,–771,–714,–603, and–481 bp of pro-SmAMP1 gene promoter, which were fused to the coding region of the uidA reporter gene in pCambia1381Z plant expression vector. The efficacy of pro-SmAMP1 promoter deletion variants was determined by transient expression in plants of Nicotiana benthamiana and using sequential generations of transgenic Nicotiana tabacum plants. It was found that the levels of GUS reporter protein activity in the extracts from transgenic and agroinfiltrated plants using all deletion variants of pro-SmAMP1 gene promoter were 3–5 times higher than those of 35S CaMV viral promoter. The highest activity of GUS protein was observed in the leaves of transgenic tobacco plants and closely correlated with the mRNA level of encoding gene. The levels of GUS activity did not differ significantly among 11 independent homozygous lines of T₂ generation of N. tabacum plants with different deletion variants of pro-SmAMP1 promoter. The results give reason to assume that all deletion variants of pro-SmAMP1 promoter provide stable and high level of expression of controlled genes. The shortest deletion variant–481 bp of pro-SmAMP1 promoter should be viewed as a potentially strong plant promoter for the genetic engineering of plants.     

Direct BRLF1 binding is required for cooperative BZLF1/BRLF1 activation of the Epstein – Barr virus early promoter, BMRF1

Disruption of Epstein – Barr virus (EBV) latency is mediated through the activation of the viral immediate-early proteins, BZLF1 (Z) and BRLF1 (R).i.; (Chevallier-Greco, A., et al., (1986) EMBO J., 5, 3243 – 9; Countryman, and Miller, G. (1985) Proc. Natl. Acad. Sci. USA, 82, 4085 – 4089). We have previously demonstrated that these proteins cooperatively activate the EBV early promoter BMRF1 in lymphoid cells but not in epithelial cells. Although cooperative transactivation by these proteins has been demonstrated with a number of EBV promoters, the mechanism of this interaction is not well understood. We now show that the cooperative activation of the BMRF1 promoter by Z-plus-R requires an intact R binding site and at least one functional Z response element (ZRE). Despite the presence of an R binding site, the BMRF1 promoter is only moderately responsive to R alone in either HeLa or Jurkat cells. Efficient activation of the BMRF1 promoter by Z alone in HeLa cells requires two ZREs (located at − 59 and − 106), whereas two additional Z binding sites (located at − 42 and − 170) contribute very little to Z-induced activation. In the absence of ZREs, Z acted as a repressor of R-induced transactivation. These observations, along with observations made by other investigators (Giot, J.F. et al., (1991) Nucleic Acids Res., 19, 1251 – 8), suggest that Z-plus-R cooperative activation is dependent upon 1) direct binding by R and Z to responsive promoter elements and 2) contributions by cell-specific factors.     

Lithium nuclear magnetic resonance measurements in halotolerant bacterium Ba1

Measurements of relaxation times T₁ and T₂, were carried out on a high-salt- and low-salt-grown bacterial pellets of halotolerant bacterium B_a1. In our measurements, T₁ ? T₂ and both were frequency-independent. In the high-salt-grown pellet the relaxation time values were much shorter than in the case of low-salt growth medium. Intensity measurements show that only 55% of the lithium in the high-salt pellet is detected; for the low-salt pellet almost all the lithium is detected. Growth measurements were carried out on the B_a1. It is suggested that there is some form of adaptation of the bacteria to the growth medium. The adaptation is reflected in the lithium NMR results.     

DNA methylation at promoter regions of interleukin 1B, interleukin 6, and interleukin 8 in non-small cell lung cancer

Epidemiologic and experimental evidences support the concept that inflammation promotes the development and progression of cancers. Interleukins (ILs) regulate the expression of several molecules and signaling pathways involved in inflammation. High expression of some ILs in the tumor microenvironment has been associated with a more virulent tumor phenotype. To examine the role of IL-1β, IL-6, and IL-8 in non-small cell lung cancer, we measured mRNA levels and promoter DNA methylation in a panel of cultured human lung cells (n = 23) and in matched pair lung tumor versus adjacent non-tumorous tissues (n = 24). We found that lung cancer cells or tissues had significantly different DNA methylation and mRNA levels than normal human bronchial epithelial cells or adjacent non-tumorous tissues, respectively. High DNA methylation of ILs promoters in lung cancer cells or tissues was associated with low mRNA levels. We found an inverse correlation between DNA methylation of IL1B, IL6, and IL8 gene promoters and their corresponding mRNA levels, such inverse correlation was more significant for IL1B (i.e., all cancer cell lines used in this study had a hypermethylated IL1B promoter which was associated with silencing of the gene). Our results underline for the first time the role of epigenetic modifications in the regulation of the expression of key cytokines involved in the inflammatory response during lung cancer development.     

The production of aflatoxin B1 or G1 by Aspergillus parasiticus at various combinations of temperature and water activity is related to the ratio of aflS to aflR expression

The influence of varying combinations of water activity (a_w) and temperature on growth, aflatoxin biosynthesis and aflR/aflS expression of Aspergillus parasiticus was analysed in the ranges 17–42°C and 0.90–0.99 a_w. Optimum growth was at 35°C. At each temperature studied, growth increased from 0.90 to 0.99 a_w. Temperatures of 17 and 42°C only supported marginal growth. The external conditions had a differential effect on aflatoxin B₁ or G₁ biosynthesis. The temperature optima of aflatoxin B₁ and G₁ were not at the temperature which supported optimal growth (35°C) but either below (aflatoxin G₁, 20–30°C) or above (aflatoxin B₁, 37°C). Interestingly, the expression of the two regulatory genes aflR and aflS showed an expression profile which corresponded to the biosynthesis profile of either B₁ (aflR) or G₁ (aflS). The ratios of the expression data between aflS:aflR were calculated. High ratios at a range between 17 and 30°C corresponded with the production profile of aflatoxin G₁ biosynthesis. A low ratio was observed at >30°C, which was related to aflatoxin B₁ biosynthesis. The results revealed that the temperature was the key parameter for aflatoxin B₁, whereas it was water activity for G₁ biosynthesis. These differences in regulation may be attributed to variable conditions of the ecological niche in which these species occur.     

<Emphasis Type="Italic">Hymenobacter sedentarius</Emphasis> sp. nov., isolated from a soil

A novel Gram-negative and red-pinkish bacterium designated DG5B^T was isolated from a dry soil. Cells were rods that were catalase- and oxidase-positive, and non-motile. The strain was found to grow at temperatures from 10 to 30°C (optimum 25°C) and pH 6.0–8.0, (optimum pH 7) on R2A broth. 16S rRNA gene sequence (1,452 bp) analysis of this strain identified it as a member of the genus Hymenobacter that belongs to the class Cytophagia. The highest gene sequence similarities were with Hymenobacter arizonensis OR362-8^T (98.3%), Hymenobacter humi DG31A^T (97.6%), and Hymenobacter glaciei VUG-A130^T (96.6%). Strain DG5B^T exhibited <70% DNA-DNA relatedness with H. arizonensis (34.7 ± 7.0%; reciprocally, 29.7 ± 1.2%) and H. humi (39.4 ± 4.3%; reciprocally, 39.5 ± 3.3%) as a different genomic species, and its genomic DNA G+C content was 59.8%. Strain DG5B^T had the following chemotaxonomic characteristics: the major fatty acids are iso-C_15:0, anteiso-C_15:0, C_16:1ω5c, and summed feature 3 (C_16:1ω7c / C_16:1ω6c); polar lipid profile contained phosphatidylethanolamine (PE), unknown aminophospholipid (APL), unknown glycolipids (GL), unknown phospholipids (PL), and unknown polar lipids (L); the major quinone is MK-7. The absorbance peak of pigment is at 481.0 nm. Strain DG5B^T showed low-level resistance to gamma-ray irradiation. Phenotypic, chemotaxonomic, and genotypic properties indicated that isolate DG5B^T represents a novel species within the genus Hymenobacter for which the name Hymenobacter sedentarius sp. nov. is proposed. The type strain is DG5B^T (=KCTC 32524^T =JCM 19636^T).