Structural analysis of the promoter of tomato 1-aminocyclopropane-l-carboxylate synthase 6 gene （Le-ACS6）

Ethylene plays an important role in the regulation of many growth and developmental processes of higher plants. In tomato, Le-ACS6, a member of the ACC synthase multigene family involved in system 1 ethylene biosynthesis during fruit ripening, is subject to negative feedback regulation by ethylene. To identify the cis-elements that are responsible for the negative feedback control, we established an in vitro transient assay system employing particle bombardment on mature-green tomato fruit pericarp to examine the expression of a luciferase （LUC） reporter gene driven by a 5＇-serially deleted Le-ACS6 promoter. The results localized putative cis-elements required for negative ethylene-response between -347 and -266 upstream from the translational start site ATG Several lines of stable transformation of the Le-ACS6 promoter and GUS reporter fusion gene containing internal deletion from -347 to -266 were generated. The expression pattern of the GUS reporter showed that removal of the nucleotides from -347 to -266 completely eliminated the response of the Le-ACS6 promoter to exogenous ethylene.     

Effect of 5′-deletion ofArabidopsis profilin2 promoter on its vascular specific expression

Based on the published sequence of profilin2 promoter ofArabidopsis thaliana, a full-length promoter (1667 bp) was amplified by PCR. The 5′-end deletion fragments with length of 1380, 1153, 969 and 597 bp were then fused withgus (uidA) gene respectively. Constructed plant expression vectors were individually transferred intoKalanchoe laciniata and transgenic plants regenerated. GUS histochemical assay confirmed that the full-length promoter Pfn1.7 was vascular-specific. Deletion assays showed that profilin2 promoter could be divided into three parts. Deletion of fragment 1 (−1667—−1380 bp) resulted in constitutive expression, suggesting that element(s) responsible for vascular-specific expression might exist in this region. Fragment 2 located at −1153—−597 bp strongly inhibitedgus gene expression. Fragment 3 (−597—−1 bp) is considered as a basic domain of profilin2.     

Function of resveratrol derived from transgenic plant expressing resveratrol synthase gene

Two genes from grapevine coding for resveratrol synthase, named RS1 and RS2, were cloned by RT-PCR. AnEscherichia coli expression vector was constructed by insertion of RS1 into pBV221. A specific protein with the same molecular weight (42 ku) as the resveratrol synthase was expressed and used to prepare the rabbit antiserum. A plant expression vector was constructed by inserting the RS1 gene into pBin438 downstream of the doubled CaMV 35S promoter and TMV-Ω fragment. PCR-positive transgenic tobacco plants were obtained after transformation withAgrobacterium tumefaciens LBA4404 harboring the plant expression vector. Southern blot analysis demonstrated that the foreign gene was integrated into the tobacco genome. The results of RT-PCR and Western blot indicated that the RS1 gene was transcribed and expressed. Formation of resveratrol in transgenic tobacco was further determined by thin-layer chromatography of silica gel and HPLC. Increased accumulation of human breast adenocarcinoma cells in G₀ and G₁ phases of cell cycle was observed in cells treated with resveratrol purified from transgenic tobacco as compared to the untreated cells.     

Molecular characterization of a K+ channel blocker in the scorpionButhus martensii Karsch

K⁺ channel blockers of scorpion venoms are of important value in studying pharmacology and physiology of specific K⁺ channel of cells. Based on the amino acid sequences of BmP01 previously characterized as a small-conductance Ca²⁺-activated K⁺ channel blocker, two “back to back” degenarate primers have been designed and synthesized for inverse PCR strategy, its full-length cDNA has been cloned from the venom gland of the Chinese scorpionButhus martensii. The cDNA is composed of 3 parts: 5′ UTR, ORF and 3′ UTR. The flanking sequence of translation initiation codon ATG is AAAATGA, which is highly conserved in scorpion Na⁺ channel toxin and protozoan genes, suggesting that these genes may have followed a common mechanism for translation initiation. The 3′ UTR contains poly(A) signal AATAAA. The open reading frame encodes a precursor of 57 residues with a signal peptide of 28 residues and a mature peptide of 29 residues. The signal peptide is rich in hydrophobic amino acid residues and its length is significantly different from that of the determined scorpion Na⁺ channel toxin. The deduced amino acid sequence of mature peptide is completely consistent with BmP01 previously determined by primary structure analysis.     

Delayed resistance to Cucumber mosaic virus mediated by 3’UTR-derived hairpin RNA

RNA silencing has been shown to function in the plant antivirus defense response, leading to viral RNA degradation induced by vsiRNA-containing RISC cleavage activity. Cucumber mosaic virus （CMV） 3′UTR sequences share a high conservation of nucleotide sequence and secondary structures that are important for CMV replication. Here, in an attempt to simultaneously target the multiple genomic and subgenomic RNAs of CMV for degradation, CMV 3′UTR were used to design hairpin RNA （hpRNA） to transform tobacco （Xanthi. nc） so as to constitutively produce viral siRNAs. Most of the transgenic plants expressing CMV Q strain （Q-CMV, subgroup Ⅱ strain） RNA3 3′UTR-derived hpRNA showed delayed resistance to Q-CMV infection and exhibited recovery phenotypes. Compared with Q-CMV-inoculated leaves, the upper leaves showed weak or no disease symptoms and a reduced accumulation level of viral RNAs. Together with transient assays, our results indicate that the 3′UTR-derived siRNAs were biologically active in targeting viral RNA for degradation. Recovery resistance in transgenic plants was also observed against subgroup IB strain SD-CMV infection, indicating a broad-spectrum anti-CMV effect of the 3′UTR-based antiviral silencing. Northern blot assays indicated that there was no strong correlation between the degree of resistance and the accumulation level of 3′UTR-derived siRNAs, suggesting that to target a highly structured RNA, such as the CMV 3′UTR, the quantity of siRNAs may not be the only determinant of silencing efficiency. Target RNA secondary structures may also affect target accessibility, siRNA-containing RISC-target recognition and the consequent antiviral effect.     

Cloning and characterization of vernalization-related gene (ver203F) cDNA 3′ end

Based on the cDNA fragment sequence of vernalization-related geneverc203 cloned by differential screening in our lab, the 5′ primer has been designed. The cDNA 3′ end ofver203 gene (1 197 bp) has been cloned by the RACE method. And it is identified by Northern blotting that its expression is special in vernalization treatment. After comparing the sequence in the nucleotide sequence databases of Genbank, EMBL and DDBJ, the gene has homology withHordeum vulgare jesmonate-induced protein gene. It is suggested that this gene might be related to the signal transduction mediated by jamonate.     

Evaluation and characterization of an endosperm-specific sbeⅡa promoter in wheat

Starch,the main component of the wheat grain,is the product of a complex biochemical pathway. The sbeⅡα gene plays a key role in controlling the synthesis of starch, in particular, the biosynthesis of amylopectin,in maturing wheat grain.To investigate its regulatory mechanisms and endosperm-specific expression pattern, the sbeⅡα promoter (3094 bp in length) was cloned using APCR and sequenced.The effect of a series of deletions was studied using a GUS transient assay system. Results showed that the 3094 bp sequence (sbe.g construct) exhibited full stable promoting activity and that the activities of 5′ or 3′ deletions reduced levels of GUS expression. Some constructs with internal deletions showed only weak activity, however,sbe.e, with a deletion from -1579—--1210 bp resulted in higher levels of expression than the full-length promoter sequence, sbe.g. This indicates that motifs such as the -300 bp element, G-box and/or P-box act as positive elements and are necessary in determining the promoter‘s endosperm-specific pattern and that negative repressor elements or motifs may also be present within the -1579—-1210 bp sequence. The age of wheate ndosperm tissue used in the GUS-transient assay system is shown to be of significant importance.     

Enhanced hydrosen production by insertional inactivation of adhE gene in Klebsiella oxytoca HP1

Ethanol is the main byproduct of anaerobic H2-producing fermentation in Klebsiella oxytoca HP1. Two moles of NAD（P）H are consumed to yield one mole of ethanol that may decrease bacterial hydrogen production. In this article the adhE gene that codes for acetaldehyde dehydrogenase was disrupted for the first time. A homologous recombination vector pTA-Str was constructed in which the adhE gene was disrupted by inserting an aminoglycoside-3＇-adenyltransferase （aadA） gene. As expected, the vector includes the insertion 5＇-adhE-aadA-adhE-3＇. The amplified DNA fragment 5＇-adhE-aadA-adhE-3＇ from pTA-Str was transformed into K. oxytoca HP1 and one recombinant was obtained. PCR analysis of the resulting genomic DNA indicated the appropriate deletion and insertion. Compared with the H2-production of wild type K. oxytoca HP1, the hydrogen yield of the mutant increased by 16.07% and ethanol concentration decreased by 77.47%, suggesting that inactivation of the adhE gene in K. oxytoca HP1 is a potential method for enhancing bacterial H2-production.     

Transformation of plant young proembryos by electroporation

It is first reported that plant young proembryos expressed exogenous reporter genes by electroporation. Young proembryos with 8–32 cells and globular proembryos with 250–400 cells could be isolated by enzymatic maceration combined with microdissection. After electroporation withGUS orGFP genes, the proembryos were cultured for 1–2 d in KM8p medium. At the field strength of electroporation 500–1500 V/cm, blue reaction of GUS or green fluorescence of GFP could be observed in the proembryos. The highest transient expression frequency of young proembryos (2.2%) was obtained at the field strength of 750 V/cm, whereas the highest frequency of globular proembryos (5.9%) was obtained at the field strength of 1 250 V/cm. Taking the proportion of transformed cells in the whole cells of proembryos as efficient transformation frequency, the efficient transformation frequency of the young proembryos was 7 times that of the globular proembryos.     

Resistance to rice blast (Pyricularia oryzae) caused by the expression of trichosanthin gene in transgenic rice plants transferred through agrobacterium method

The gene of trichosanthin has been transferred into rice plants through agrobacterium method. The single copy insertion and the expression of foreign gene have been proved in regenerated plants. In antifungal assay the degrees of rice blast (Pyricularia oryzae) infection of the transgenic plants expressing trichosanthin and expressingGUS gene as control have been evaluated. The differences such as the time of disease symptom observed, the number of infected plants and damaged leaves, the growth of infected plants of the two transgenic plants after being inoculated by rice blast (Pyricularia oryzae) are significant. The transgenic plants with trichosanthin gene grew faster than the plants withGUS gene, even when humidity environment was removed. The results show that the transgenic plants that expressed trichosanthin are able to delay the infection of rice blast compared with the plants as control. In addition, no damage caused by the expression of trichosanthin gene in transgenic plants has been observed.     

Construction of chimeric inducible promoters by elicitors of rice fungal blast pathogen and their expression in transgenic rice

The promoter fragments of wheatGstA1 and potatoGst1 have been amplified by PCR, cloned and fused respectively to the minimal promoter sequence of rice actin gene (Act1)) and its 5′ untranslated leader sequence together withGUS. The constructs with 2 chimeric promoters (WGA and PGA) have been transferred into rice in order to analyze their inducibility patterns in transgenic rice plants. The results show that: WGA and PGA are both inducible by elicitors ofPyricularia oryzae in transgenic rice cells; the intron I of riceAct1 gene is important for the heterogenic expression of monocot and dicot promoter elements in rice; and theAct1 minimal promoter and its 5′ untranslated leader sequence produced low level background expression in rice.     

Expression of human hepatitis C virus core antigen in tobacco plants by tobacco mosaic virus-based vector system

Tobacco mosaic virus (TMV) has the potential to highly express foreign gene. A novel TMV-basedin trans expression system was constructed. A TMV mutant TSHc had its coat protein replaced with hepatitis C virus (HCV) core antigen gene. Anotherr TMV mutant TSBD was replicase-defective. Coinfection of the two mutants could cause systemic infection in tobacco plants byin trans complementation of their functions. TSHc could effectively replicate and assemble to viral particles, which were a little longer than that of wild-type TMV. HCV core antigen was expressed in whole tobacco plants. A similar expression level of HCV core antigen was detected on serial passages, which suggested that this viral expression system be stable.     

Discovery of C<Subscript>4</Subscript> species at high altitude in Qinghai-Tibetan Plateau

Plant specimens are collected from the areas between latitude 27°?2′N and 40°57′N, and longitude 88°93°E and 103°24′E, with an altitudinal range from 2210 to 5050 m above the sea level in Qinghai-Tibetan Plateau. The stable carbon isotope analysis indicates that two of Chenopodiaceae and six of Poaceae in the samples are C4 plants. Four of the C4 plants are found in 11 spots with altitudes above 3800 m, and Pennisetum centrasiaticum, Arundinella yunnanensis and Orinus thoroldii are present in six spots above 4000 m, even up to 4520 m. At low CO2 partial pressure, that sufficient energy of high light improving C4 plant's tolerance of low temperature and precipitations concentrating in growing season probably are favorable for C4 plants growing at high altitude in Qinghai-Tibetan Plateau.     

Molecular cloning, expression and biochemical property analysis of AtKP1, a kinesin gene from Arabidopsis thaliana

Kinesins are common in a variety of eukaryotic cells with diverse functions. A cDNA encoding a member of the Kinesin-14B subfamily is obtained using 3＇-RACE technology and named AtKP1 （for Arabidopsis kinesin protein 1）. This cDNA has a maximum open reading frame of 3.3 kb encoding a polypeptide of 1087 aa. Protein domain analysis shows that AtKP1 contains the motor domain and the calponin homology domain in the central and amino-terminal regions, respectively. The carboxyl-terminal region with 202 aa residues is diverse from other known kinesins. Northern blot analysis shows that AtKP1 is widely expressed at a higher level in seedlings than in mature plants. 2808 bp of the AtKP1 promoter region is cloned and fused to GUS. GUS expression driven by the AtKP1 promoter region shows that AtKP1 is mainly expressed in vasculature of young organs and young leaf trichomes, indicating that AtKP1 may participate in the differentiation or development of Arabidopsis thaliana vascular bundles and trichomes. A truncated AtKP1 protein containing the putative motor domain is expressed in E. coil and affinity-purified. In vitro characterizations indicate that the polypeptide has nucleotide-dependent microtubule-binding ability and microtubule-stimulated ATPase activity.     

Molecular cloning and nucleotide sequence of the attenuated hog cholera virus Lapinized Chinese strain

The genomic sequence of the attenuated hog cholera virus Lapinized Chinese strain (HCLV) was determined from overlapping cDNA clones. The viral RNA of HCLV stain comprised 12 310 nucleotide (nt) including 374 nt and 239 nt at the 5′ and 3′-noncoding region, respectively. The complete genome sequence contained one large open reading frame which encoded an amino acid sequence of 3 898 residues with a calculated molecular weight of 437×10³. Although there were mostly only small differences between the sequence of the HCLV strain and the published sequences of strains ALD, GPE⁻, Alfort and Brescia, there was one notable insertion of 12 nucleotides, TTTTCTTTTTTC in the 3′ non-coding region of HCLV strain. Supported by the National Pandeng Project, Genbank accession number AF091507 Wang Jiafu: born in 1972, Ph. D.     

On the thinness of non-tangential sets

Let Ω⊄R ^d and ξ∈∂Ω. LetE be any non-tangential subset of Ω. we prove that ifE is internal thin at ξ, then it is minimal thin at any minimal Martin boundary point of Ω. Supported by the National Natural Science Foundation of China Zhang Yiping: born in Dec. 1962. Professor     

Rice protoplasts isolated directly from mature-embryo-derived calli

Mature-embryo-derived calli of japonica rice (Oryza sativa L) Taipei 309 were used for replicated protoplast isolation experiments. Six out of nine callus lines produced protoplasts with satisfactory yield of 5.20×10⁶–8.96×10⁶ protoplasts/g FW (fresh weight). The remaining three callus lines initiated from seeds of cryopreserved-callus-derived plants had rooty calli, resulting in low yield of protoplasts and a large number of isolated banana-shape intact cells. Viability of protoplasts ranged 87.46%–94.15%. The average size of protoplasts was 207.49–379.04 μm² in different callus lines. Comparitive experiments were also carried out using both calli and suspension culture cells for protoplast isolation. The results demonstrated that protoplast isolation of calli was a substantially simplified and reliable method for preparing rice protoplasts. Jin Deming: born in Jan. 1959, Associated Professor