Nitric oxide suppresses stomatal opening by inhibiting inward-rectifying K<Stack><Subscript>in</Subscript><Superscript>+</Superscript></Stack> channels in <Emphasis Type="Italic">Arabidopsis</Emphasis> guard cells

We explore nitric oxide （NO） effect on K^＋in, channels in Arabidopsis guard cells. We observed NO inhibited K^＋in, currents when Ca^2＋ chelator EGTA （Ethylene glycol-bis（2-aminoethylether）-N,N,N′,N;tetraacetic acid） was not added in the pipette solution; K^＋in currents were not sensitive to NO when cytosolic Ca^2＋ was chelated by EGTA. NO inhibited the Arabidopsis stomatal opening, but when EGTA was added in the bath solution, inhibition effect of NO on stomatal opening vanished. Thus, it implies that NO elevates cytosolic Ca^2＋ by activating plasma membrane Ca^2＋ channels firstly, then inactivates K^＋in, chartnels, resulting in stomatal opening suppressed subsequently.     

Heterologous Expression of Mycobacterium tuberculosis AgSSB in Pichia pastoris

The DNA fragment encoding matureMycobacterium tuberculosis major secretory protein Ag85B was inserted into thePichia pastoris secretory expression vector pHBM905A, under the control of theAOX1 promoter. The recombinant plasmid pHBM905A-85B linearized bySal I was introduced intoPichia pastoris strain GS115 by PEG1000 transformation method. After phenotype screening and PCR identification, the resulting GS115-pHBM905A-85B strain was cultivated and induced with methanol. The recombinant Ag85B protein in secreted form was attained with molecular weight of 35×10³ approximately detected by SDS-PAGE and Western blot. ELISA experiment proved that the protein had good antigen specificity. Secretory expression of recombinantM. tuberculosis Ag85B inP. pastoris will open a door to mass production of the protein in heterologous host and allow ready evaluation of its immunological function. Foundation item: Supported by the Key Scientific and Technological Project of Wuhan(301121028) Biography: LIU Yan(1971-), female, Ph. D candidate, research direction: vaccine against tuberculosis.     

Construction and characterization of partiallyntrC-deleted mutants inAlcaligenes faecalis

To study the effect of ntrC gene product on the expression and regulation of other important nitrogen-fixing genes in Alcaligenes faecalis, partially ntrC-deleted mutants of A. faecalis have been generated. To start with, the ntrC gene of A. faecalis was cloned into a suicide plasmid pSUP202 to create a recombinant plasmid pSUM1. The ntrC gene in pSUM1 was then replaced by a lacZ-Km^r fragment resulted in the generation of a plasmid pSUM2. The lacZ fragment in pSUM2 was further removed and a plasmid pSUM3 produced. As a second step, the plasmid pSUM2 or pSUM3 was introduced into the wild type of A. faecalis A1501 by conjugation and two partially ntrC-deleted mutants A15CM1 (ntrC∷lacZ) and A15CM2 (ntrC ^- ) were obtained. To understand the regulatory effect of the NtrC on the expression of nifH and nifA, a nifH-lacZ gene or a nifH-lacZ gene was introduced into the ntrC^- mutant by conjugation. The results indicated that: (ⅰ) although the ntrC^-mutant was nif ⁺ , its nitrogen fixation activity was only 20% that of the wild type; (ⅱ) the ntrC^- mutant failed to grow on the medium containing nitrate as a sole nitrogen source; (ⅲ) the regulation of ntrC gene expression did not require its own product; (ⅳ) the expression of nifH in A . faecalis was positively regulated by the ntrC. Deletion of the ntrC resulted in the reduction of nifH expression or even totally inactivated nitrogen fixation; (ⅴ) there was no obvious influence on the expression of nifA in A. faecalis if the ntrC gene was deleted.     

A tumor suppressor genefhit prokaryotic expression and identification

The recombinant expression vector pGEMD-fhit which contains full encoding region offhit gene was constructed. The recombinant was introduced into the BL21 (DE3) strain ofE. coli and induced by 1 mmol/L IPTG to express a 29×10³ polypeptide offhit fusion protein. And the 29×10³ protein was sensitive and specific in reaction with anti-fhit antibody in Western blot. Foundation item: Supported by the National Natural Science Foundation of China (39770373) Biography: SUN Yan (1975−), female, Master of science, Research direction: gene engineering     

Construction of cDNA library from iron-deficiency induced maize roots and screening and identification of iron-stress geneFdr3

To isolate Fe-deficient related (Fdr) genes, an expression cDNA library of 4.5×10⁵ pfu/μg has been constructed from maize roots in iron-stress. 6 clones have been screened from the cDNA library by differential hybridization screening. It is proved that anFdr3 cDNA clone expressed stronger under iron-deficient condition than under iron-sufficient one by Northern blot and Western blot.     

基于生物信息数据库分析TEFM基因在DLBCL中表达研究

为了探讨线粒体延伸因子TEFM在弥漫性大B细胞淋巴瘤DLBCL中的表达,初步研究其表达与DLBCL患者的临床特征及预后的关系,从数据库中下载关于DLBCL患者TEFM mRNA二代测序数据及临床病理资料,分析TEFM转录水平与临床病理参数的相关性及其预后价值.结果显示TEFM在DLBCL组织中均较正常淋巴组织显著高表达...     

The expression of vasa gene during zebrafish ( Danio rerio ) oogenesis

vasa gene expression pattern during oogenesis of zebrafish was examined usingin situ hybridization and fluorescent quantitative RT-PCR. During zebrafish oogensis,vasa mRNA is expressed strongly and uniformly distributed in the cytoplasm in stage II oocytes, followed by a distribution among vacuome in stage III. Later in stage IV and V,vasa mRNA is enriched at the cortex and finally localized at the cortex. The fluorescent quantitative RT-PCR shows that the quantity ofvasa mRNA decreases from stage II to stage III, but remains relatively invariable from stage III to stage V. The observed differences invasa mRNA expression in the different stages of zebrafish oogenesis suggest thatvasa gene plays an important role during oogenesis. Foundation item: Supported by the National Natural Science Foundation of China (30370744, 30150005) Biography: XIANG Fang (1979-), male, Master candidate, research direction: molecular development of animals.     

Rapid induction of mRNAs forPC3 by partial hepatectomy and epidermal growth factor

The expression of immediate early gene plays a pivotal role in rat hepatocyte proliferation from G₀ to G₁ phases and the progression through G₁ phase of the cell cycle within several hours after 2/3 hepatectomy. We investigated the different gene expressions within 1 h after 2/3 hepatectomy by representational difference analysis. Sequence analysis indicated thatPC3 induced by NGF was a kind of immediate early gene and might be correlated with liver regeneration. Moreover, we found that 2/3 hepatectomy could induce the expressing ofPC3 mRNA by Northern blot with a peak 1–2 h after surgery. In primary cultures of rat hepatocytes, addition of EGF resulted in rapid and transient induction ofPC3 mRNA. It was first reported thatPC3 gene belongs to immediate early gene associated with liver regeneration.     

Localization of calmodulin and calmodulin-like protein and their functions in biomineralization in P. fucata

Calmodulin (CaM) and calmodulin-like protein (CaLP) are two proteins involved in biomineralization. Their localizations in Pinctada fucata mantle epithelia were studied by Western blot (WB) analysis of the nuclear/cytosol fraction of primary cultured Pinctada fucata mantle cells and immunogold electron microscopy. The results showed a completely different distribution of these two proteins at the subcellular level. CaM was distributed throughout both the nucleus and cytoplasm of the mantle epithelium but CaLP was distributed only in the cytoplasm. The functions of these two proteins in biomineralization were investigated by shell regeneration. During this process, the expressions of CaM and CaLP were greatly enhanced in different organelles of the mantle epithelium. Overexpression of these two proteins and a mutant of calmodulin-like protein (M-CaLP) that lacks an extra C-terminal tail in MC3T3-E1 promoted the mRNA expression of osteopontin, a biomineralization marker for osteoblasts. All of the results indicated that CaM and CaLP have completely different distributions in the mantle epithelium and affect the biomineralization process at different levels. The extra C-terminal tail of CaLP is important for its functions in biomineralization in Pinctada fucata.     

The foxtail millet Si69 gene is a Wali7 (wheat aluminum-induced protein 7) homologue and may function in aluminum tolerance

We isolated a clone, named Si69, from a foxtail millet immature seed cDNA library. The protein encoded by Si69 contains a conserved Wali7 (wheat aluminum induced protein 7) domain and shares high-level homology with aluminum-induced proteins from other species including rice and Arabidopsis. The Si69 gene presents as a single locus in foxtail millet genome and is globally expressed in all tissues examined. Its expression is up-regulated by aluminum. The sequence feature and expression pattern suggest that the Si69 gene is involved in aluminum tolerance or detoxification. To confirm its biological functions, Si69 controlled by the CaMV35S promoter was introduced into Arabidopsis. Transgenic plants did not show any visible morphological changes compared to wild-type plants under normal growth conditions. However, when treated with 20 or 50 μmol/L Aluminum (Al), the root apices of wild-type plants were heavily stained by hematoxylin, whereas those of Si69 transgenic plants were not stained when treated with 20 μmol/L Al and slightly stained when treated with 50 μmol/L Al. Scanning electron microscopy (SEM) results further demonstrated that the damage of the root apices was severer in wild-type plants than in transgenic plants. Inhibition of root growth and accumulation of malondialdehyde (MDA), an indicator of lipid peroxidation, were lower in transgenic plants than in wild-type plants. The results show that overexpression of Si69 may increase Al tolerance in transgenic plants, indicating that a series of Wali7-containing genes may play similar roles in Al tolerance/detoxification.     

Expression ofbcl-2 gene increases in apoptosis of vascular endothelial cells induced by rattlesnake venom

Apoptosis of vascular endothelial cells (VEC) has been induced by deprivation of survival factors (aFGF and serum) and by rattlesnake venom. The expression ofbcl-2 gene has been examined by Northern blotting in the two apoptosis inducing systems. Our results show that the expression ofbcl-2 has not been detected in normal culture cells and in apoptotic cells induced by deprivation of survival factors. But in apoptotic cells induced by rattlesnake venom (10 ng/mL), the expression ofbcl-2 increases, and its mRNA exhibits two bands. The data first suggest that increasing expression and splitting ofbcl-2 mRNA may play an important role in apoptosis of VEC induced by rattlesnake venom, and this finding is helpful to understanding the role ofbcl-2 in regulation of apoptosis.     

Characterization of the flagellar biosynthesis regulatory geneflbD inAzospirillum brasilense

A flagellar gene cluster fragment includingflbD ofAzospirillum brasilense was cloned and sequenced. TheflbD mutant strain was found to be nonmotile—losing both polar and lateral flagella (Fla⁻Laf⁻). Motility and flagella were regained by complementation with plasmid-borne multicopyflbD, but altered with larger swarming circle and fewer lateral flagella on the semisolid plate. This result indicated that FlbD plays an important role in the regulation of both polar and lateral flagellar biosynthesis inA. brasilense.     

Cloning and identification of a novel human glioma differentiation related geneGDR1

In order to identify the genes associated with glioblastoma differentiation, some ESTs, expressed differentially in the control cell and the differentiated human glioblastoma cell line BT-325 induced by the all-trans retinoid acid, have been isolated by the method of DDRT-PCR. Of the 46 ESTs sequenced, 19 are from new genes. A full-length 1 535-bp cDNA, termed geneGDR1, has been isolated from the human cDNA library using the probe designed according to one of the novel ESTs, HGBB098. The open reading frame ofGDR1 gene encodes a putative protein containing 334 amino acid residues. Blast against the current GenBank DNA and protein sequence database did not reveal significant homology with any known proteins. RT-PCR shows thatGDR1 mRNA level increased in the differentiated BT-325 cells after being treated with RA. The different expression patterns ofGDR1 mRNA in human tissues have been detected through the multiple tissue Northern blot hybridization.     

An interspecies conserved antigen ofPlasmodium falciparum containing clusters of asparagine residues

An interspecies conservedPlasmodium asparagine rich antigen, designated as ARK26, was isolated by immunoscreeningP. falciparum genomic DNA expression library with mouse convalescent anti-P. yeolii serum. Partial DNA sequence analysis reveals that ARK26 contains clusters of asparagines and no randomly repeated amino acid sequence motifs are observed. A 65×10³ GST fusion protein is expressed by recombinant plasmid PGEX-5X-1 (ARK26) inE. coli C strain ABLE-K. Computer programs predict that two asparagine rich regions are among the possible antigenic epitopes of p37 encoded by ARK26. Interestingly, the sequence of ARK26 displays significant similarity to yeast and several other species’ mitochondrial genes, and its possible function is discussed. Supported by a fellowship offered by International Center for Genetic Engineering & Biotechnology(ICGEB) Ma Donghui: born in 1969, Graduate student     

Expression and characterization of HGV E2 cDNA inPichia pastoris

A 559 base pair fragment of cDNA locating at the putative E2 region of GBV-C/HGV was inserted intoPichia pastoris expression vector pPIC9K in the reading frame of α-factor secreting signal peptide. The recombinant expression plasmid pPIC9K-E2 was introduced intoP. pastoris GS 115 with electroporation and recombined with the host genome by homological recombination. The His⁺Mut⁺ recombinant yeasts were selected and cultivated in the BMMY medium. After 3 days induction with 0.5% methanol, the target protein (E2) accumulated up to 30% of total proteins in the supernatant. The expressed E2 protein was proved possessing antigenicity and high specificity with Western blot and ELISA probed with sera from the immunized rabbits and the patients infected by GBV-C/HGV. Biography: Wang Zhuo-hua (1977-), female, Master, research direction: genetic engineering pharmaceuticals.     

Expression ofChlamydomonas actin-gfp fusion gene in tobacco suspension cell and polymerization of the actin-gfp proteinin vitro

The fusion gene of actin (cDNA ofChlamydomonas reinhardtii) and green fluorescence protein (gfp) had been constructed into two expression vectors which could be expressed inE. coli and tobacco suspension cells BY2. The correct expression was observed inE. coli and BY2 with a fluorescence microscopy. The fusion protein, which took part in the membrane skeleton, was mainly located peripherally along the membrane, specially the fusion protein was distributed around nucleus and cell plate, while the fusion protein also forms F-actin in the cell. The fusion protein was purified from Bl21plus by ammonium sulfate fractionation, ion exchange chromatography and hydrophobic interaction chromatography. The purified production could polymerize into F-actin when the actin polymerizing buffer was added. It was demonstrated that the characteristics and function of actin inChlamydomonas was similar with those of animals and higher plants.