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.     

Functional characterization of the pollen-specific <Emphasis Type="Italic">SBgLR</Emphasis> promoter from potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

SBgLR (Solanum tuberosum genomic lysine-rich) gene was isolated from a potato genomic library using SB401 (S. berthaultii 401) cDNA as probe. RT-PCR analysis of SBgLR gene expression profile and microscopic analysis of green fluorescent protein (GFP) expression in tobacco plants transformed with SBgLR promoter-GFP reporters indicate that SBgLR is a pollen-specific gene. A series of 5′deletions of SBgLR promoter were fused to the β-glucuronidase (GUS) gene and stably introduced into tobacco plants. Histochemical and quantitative assays of GUS expression in transgenic plants allowed us to localize an enhancer of SBgLR promoter to the region −345 to −269 relative to the translation start site. This 76 bp (−345 to −269) fragment enhanced GUS expression in leaves, stems and roots when fused to −90/+6 CaMV 35S minimal promoter. Deletion analysis showed that a cis-element, which can repress gene expression in root hairs, was located in the region −345 to −311. Further study indicated that the −269 to −9 region was sufficient to confer pollen-specific expression of GFP when fused to CaMV 35S enhancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Authors Zhihong Lang and Peng Zhou contributed equally to this work.     

Characterization of the tissue-specific expression of phenylalanine ammonia-lyase gene promoter from loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis>) in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

We isolated the 5′ flanking region of a gene for phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) from Pinus taeda, PtaPAL. To investigate the tissue-specific expression of the PtaPAL promoter, histochemical assay of GUS activity was performed using the transgenic tobacco expressing the PtaPAL promoter-GUS. The region of −897 to −420 in PtaPAL promoter showed high activities in the secondary xylem and response to bending stress. To characterize the cis-regulatory functions of the promoters for enzymes in phenylpropanoid biosynthesis, we examined the activity of chimeric promoters of PtaPAL and a 4-coumarate CoA ligase, Pta4CLα. The chimeric promoter showed similar activity as the Pta4CLα promoter. Electrophoretic mobility shift assays implicated −897 to –674 of PtaPAL promoter containing cis-elements of the expression in xylem of Pinus taeda. The results suggested that AC elements of PtaPAL have multiple functions in the expression under the various developmental stages and stress conditions in the transgenic tobacco. The nucleotide sequence data reported will appear in the EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under the accession number AB449103 (PtaPAL promoter sequence).     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

Molecular cloning and characterization of the <Emphasis Type="Italic">Dicer-like</Emphasis> 2 gene from <Emphasis Type="Italic">Brassica rapa</Emphasis>

Dicer-like proteins (DCLs) are involved in small RNA-mediated development and viral defense in plants. In model plants, at least four DCLs have been found and a number of studies have helped to understand their function. However, the function of the Dicer or DCLs in other plants is still unclear. Here, we report the full-length cDNA sequence of Brassica rapa ssp. chinensis DCL2 (BrDCL2) gene, which contains a 4,179 bp open reading frame (ORF) encoding a protein of 1,392 amino acids. At the 3′ end of BrDCL2, clones with three different lengths of 3′ untranslated region were found. An alternative splice variant of BrDCL2, BrDCL2sv, in which one intron was retained between exon9 and exon10, was also cloned. Because of a change in the coding sequence resulting in a premature terminal codon, BrDCL2sv was expected to translate a short peptide containing the whole DEXHc domain.     

Molecular cloning,sequence analysis,expression and characterization of the endochitinase gene from <Emphasis Type="Italic">Trichoderma</Emphasis> sp. in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21

The endochitinase DNA and cDNA from Trichoderma sp. were cloned, sequenced and expressed. The cloned DNA and cDNA sequences were 1,476 and 1,275 bp in length, respectively. There were three introns in DNA sequence in comparison with the cDNA sequence. The endochitinase protein contained three regions: the signal peptide, the prepro-region and the mature protein region. The gene fragment encoding the mature endochitinase was ligated into the expression vector pET-28a+, yielding pET-1. The plasmid pET-1 was transformed into the Escherichia coli BL21 (DE3). The clone bearing pET-1 was picked and cultured at 30°C for the expression of endochitinase. SDS-PAGE analysis showed that the endochitinase was expressed in the periplasmic space and the purified protein showed a single band. The activity of 70.2 U/mg was obtained from the cellular extract of the recombinant strain. The activity of endochitinase was 2.5-fold higher at 24 h than at 16 h in the periplasmic space. The optimal pH and temperature of the recombinant endochitinase were determined to be 7.0 and 35°C, respectively. It was relatively stable within the pH range of 5–8. Significant activity stimulation by 1 mM Mg²⁺ and 5 mM Fe²⁺ and inhibition by 5 mM Co²⁺ and 5 mM Hg²⁺ were observed. The kinetic constants K_m, V_max and K_cat for the hydrolysis of the colloidal chitin were 1.5 mM, 1.37 μmol min⁻¹ and 6.23 min⁻¹, respectively.     

High-level expression of the xylanase from <Emphasis Type="Italic">Thermomyces lanuginosus</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

According to the amino acid sequence, a codon-optimized xylanase gene (xynA1) from Thermomyces lanuginosus DSM 5826 was synthesized to construct the expression vector pHsh-xynA1. After optimization of the mRNA secondary structure in the translational initiation region of pHsh-xynA1, free energy of the 70 nt was changed from −6.56 to −4.96 cal/mol, and the spacing between AUG and the Shine-Dalgarno sequence was decreased from 15 to 8 nt. The expression level was increased from 1.3 to 13% of total cell protein. A maximum xylanase activity of 47.1 U/mL was obtained from cellular extract. The recombinant enzyme was purified 21.5-fold from the cellular extract of Escherichia coli by heat treatment, DEAE-Sepharose FF column and t-Butyl-HIC column. The optimal temperature and pH were 65 °C and pH 6.0, respectively. The purified enzyme was stable for 30 min over the pH range of 5.0–8.0 at 60 °C, and had a half-life of 3 h at 65 °C.     

Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> with thaumatin II gene to control fungal diseases

Transgenic plants of hyacinth (Hyacinthus orientalis L.) cvs. Edisson and Chine Pink have been obtained by Agrobacterium-mediated transformation. Leaf explants of the both hyacinth cultivars regenerated shoots on MS medium containing 2.2 μM BAP and 0.3 μM NAA at a frequency of 95%. A. tumefaciens strain CBE21 carrying binary vector pBIThau35 was used for transformation. Plasmid pBIThau35 has been produced by cloning preprothaumatin II cDNA into pBI121 instead of uidA gene. Inoculated leaf explants formed calli and shoots at high frequency on selective medium with 100 mg l⁻¹ kanamycin. Four hyacinth transgenic lines of cv. Chine Pink and one line of cv. Edisson have been selected on medium containing 200 mg l⁻¹ kanamycin. The insertion of thaumatin II gene into hyacinth genome has been confirmed by PCR-analysis. All transgenic plants expressed substantial amounts of thaumatin II (between 0.06 and 0.28% of the total soluble protein). Hyacinth transgenic lines were assayed for resistance to the pathogenic fungi Fusarium culmorum and Botrytis cinerea. There were no significant differences between nontransformed control and transgenic leaves of both cultivars. At the same time the bulbs of the transgenic line Н7401 cv. Chine Pink showed the higher level of resistance to B. cinerea, the bulbs of the transgenic line Н7404 were more resistant to F. culmorum. In both cases the signs of the fungal disease were developed more slowly. The resistance of the bulbs cv. Edisson line to these fungi was not changed. All transgenic hyacinth plant were successfully transferred to soil for further evaluation.     

Analysis of two promoters that control the expression of the <Emphasis Type="Italic">GTP cyclohydrolase I</Emphasis> gene in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

GTP cyclohydrolase I (GTPCH) is a key enzyme in the de novo synthesis of tetrahydrobiopterin. Previously, the Drosophila melanogaster GTPCH gene has been shown to be expressed from two different promoters (P1 and P2). In our study, the 5′-flanking DNA regions required for P1 and P2 promoter activities were characterized using transient expression assay. The DNA regions between −98 and +31, and between −73 and +35 are required for efficient P1 and P2 promoter activities, respectively. The regions between −98 and −56 and between −73 and −41 may contain critical elements required for the expression of GTPCH in Drosophila. By aligning the nucleotide sequences in the P1 and P2 promoter regions of the Drosophila melanogaster and Drosophila virilis GTPCH genes, several conserved elements including palindromic sequences in the regions critical for P1 and P2 promoter activities were identified. Western blot analysis of transgenic flies transformed using P1 or P2 promoter-lacZ fusion plasmids further revealed that P1 promoter expression is restricted to the late pupae and adult developmental stages but that the P2 promoter driven expression of GTPCH is constitutive throughout fly development. In addition, X-gal staining of the embryos and imaginal discs of transgenic flies suggests that the P2 promoter is active from stage 13 of embryo and is generally active in most regions of the imaginal discs at the larval stages.     

Molecular cloning and characterization of the promoter for the multiple stress-inducible gene <Emphasis Type="Italic">BjCHI1</Emphasis> from <Emphasis Type="Italic">Brassica juncea</Emphasis>

We have previously isolated a Brassica juncea cDNA encoding a novel chitinase BjCHI1 with two chitin-binding domains (Zhao and Chye in Plant Mol Biol 40:1009–1018, 1999). The expression of BjCHI1 was highly inducible by methyl jasmonate (MeJA) treatment, wounding, caterpillar feeding, and pathogenic fungal infection. These observations suggest that the promoter of BjCHI1 gene might contain specific cis-acting elements for stress responses. Here, we report the cloning and characterization of the BjCHI1 promoter. A 1,098 bp BjCHI1 genomic DNA fragment upstream of the ATG start codon was isolated by PCR walking and various constructs were made by fusing the BjCHI1 promoter or its derivatives to β-glucuronidase reporter gene. The transgenic Arabidopsis plants showed that the BjCHI1 promoter responded to wounding and MeJA treatment, and to treatments with either NaCl or polyethyleneglycol (PEG 6000), indicating that the BjCHI1 promoter responses to both biotic and abiotic stresses. A transient gene expression system of Nicotiana benthamiana leaves was adopted for promoter deletion analysis, and the results showed that a 76 bp region from −695 to −620 in the BjCHI1 promoter was necessary for MeJA-responsive expression. Furthermore, removal of a conserved T/G-box (AACGTG) at −353 to −348 of the promoter greatly reduced the induction by MeJA. This is the first T/G-box element identified in a chitinase gene promoter. Gain-of-function analysis demonstrated that the cis-acting element present in the 76 bp region requires coupling with the T/G-box to confer full magnitude of BjCHI1 induction by MeJA.     

Transformation of <Emphasis Type="Italic">PttKN1</Emphasis> gene to cockscomb

We have established a shoot regeneration system and genetic transformation of cockscomb (Celosia cristata and Celosia plumosus). The best results in terms of frequency of shoot regeneration and number of shoot buds per explant are observed on media supplemented with 0.5 mg l⁻¹ 6-BA (for explants of apical meristems of C. cristata) or 2.0 mg l⁻¹ 6-BA, 0.5  mg l⁻¹ NAA and 0.5  mg l⁻¹ IAA (for hypocotyls explants of C. plumosus). We use apical meristems of C. cristata and hypocotyls of C. plumosus as the starting material for transformation. A novel KNOTTED1-like homeobox1 (KNOX), PttKN1 (Populus tremula × P. tremuoides knotted1) isolated from the vascular cambial region of hybrid aspen, is introduced into cockscomb by Agrobacterium. A series of novel phenotypes are obtained from the transgenic cockscomb plants, including lobed or rumpled leaves, partite leaves and two or three leaves developed on the same petiole, on the basis of their leaf phenotypes. Transformants are selected by different concentrations of kanamycin. Transformants are confirmed by PCR of the NptII gene and PCR or RT-PCR of PttKN1 gene. Furthermore, RT-PCR shows that 35S:: PttKN1 RNA levels do not correlate with phenotypic severity. It is discussed that our results bring elements on possible function of PttKN1 gene. To our knowledge, genetic transformation of cockscomb is first reported.     

High level expression of a recombinant phospholipase C from <Emphasis Type="Italic">Bacillus cereus</Emphasis> in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70°C) at acidic pH (pH 3.5–6) and were selected for cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed by an acetoin-controlled expression system. For PLC516, 13.7 U g⁻¹ wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted in pure PLC516 with a specific activity of 13,190 U mg⁻¹ protein.     

High-resolution mapping and gene prediction of <Emphasis Type="Italic">Xanthomonas Oryzae</Emphasis> pv. <Emphasis Type="Italic">Oryzae</Emphasis> resistance gene <Emphasis Type="Italic">Xa7</Emphasis>

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease in rice worldwide. The resistance gene Xa7, which provides dominant resistance against the pathogen with avirulence (Avr) gene AvrXa7, has proved to be durably resistant to BB. A set of SSR markers were selected from the “gramene” database based on the Xa7 gene initial mapping region on chromosome 6. These markers were used to construct a high-resolution genetic map of the chromosomal region surrounding the Xa7 gene. An F₂ mapping population with 721 highly susceptible individuals derived from a cross between the near isogenic lines (NILs) IRBB7 and IR24 were constructed to localize the Xa7 gene. In a primary analysis with eleven polymorphic SSR markers, Xa7 was located in approximately the 0.28-cM region. To walk closer to the target gene, recombinant F₂ individuals were tested using newly developed STMS (sequence tagged microsatellite) markers. Finally, the Xa7 gene was mapped to a 0.21-cM interval between the markers GDSSR02 and RM20593. The Xa7-linked markers were landed on the reference sequence of cv. Nipponbare through bioinformatics analysis. A contig map corresponding to the Xa7 gene was constructed. The target gene was assumed to span an interval of approximately 118.5-kb which contained a total of fourteen genes released by the TIGR Genome Annotation Version 5.0. Candidate-gene analysis of Xa7 revealed that the fourteen genes encode novel domains that have no amino acid sequence similar to other cloned Xa(xa) genes. Shen Chen and Zhanghui Huang are contributed equally to this work.     

Purification and Properties of an <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase from <Emphasis Type="Italic">Streptomyces cerradoensis</Emphasis>

An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The K_m and V_max values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg⁻¹ protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity was strongly inhibited by Cu²⁺ and Hg²⁺ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose.