Molecular cloning of a fulllength cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1–25 amino acid sequence is a predicted signal peptide and the other 26–216 amino acid sequence is a mature peptide. The 26–45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca²⁺-dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.     

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.     

Identification of a novel DNA molecule associated with Tobacco leaf curl virus

A circular DNA molecule, designated as DNAβ, was identified in tobacco plants infected with Tobacco leaf curl virus (TLCV) isolates Y5 and Y8 by PCR using primers based on the conserved region of the two reported DNAβ sequences of whitefly-transmitted geminiviruses (WTGs). The complete nucleotide sequences of DNAβ of Y5 and Y8 (TLCV DNAβ) were determined. Y5 DNAβ comprises 1333 nucleotides encoding 8 predicted ORFs with 4 ORFs in virion-sense DNA and 4 ORFs in complementary-sense DNA; Y8 DNAβ consists of 1338 nucleotides encoding 7 predicted ORFs with 4 ORFs in virion-sense DNA and 3 ORFs in complementary-sense DNA. TLCV DNAβ has little sequence homology to DNA-A of TLCV., except that it shares conserved TAATATTAC loop sequence with TLCV DNA-A. Sequence comparison showed that Y5 DNAβ shared 85% sequence homology with Y8 DNAβ, and both Y5 DNAβ and Y8 DNAβ had relatively low sequence identity (51%–65%) with the reported DNAβ molecules associated with Ageratum yellow vein virus and Cotton leaf curl virus. The immunotrapping PCR and whitefly transmission tests showed that DNAβ molecule could be encapsidated in virus particle and transmitted by Bemisia tabaci. This is the first report of DNAβ associated with WTGs in China.     

红色赤潮藻对球形棕囊藻的化感作用研究

红色赤潮藻(Akashiwo sanguinea)和球形棕囊藻(Phaeocystis globosa)都是近年来在我国近岸海域频繁引发赤潮的藻类。为了探索红色赤潮藻和球形棕囊藻之间是否存在化感作用及作用方式,本研究采用不同起始密度培养红色赤潮藻与球形棕囊藻,并将前者的完整细胞培养液、无细胞滤液、藻液超声破碎液分别与球形棕囊藻进行混合培养,分析红色赤潮藻对球形棕囊藻细胞生长的影响。结果显示,在红色赤潮藻细胞起始密度保持5×10⁵ cells/L的混合培养条件下,起始密度分别为5×10⁵、1×10⁶、2×10⁶、1×10⁷ cells/L的球形棕囊藻生长均受到明显的抑制;无论是含有红色赤潮藻完整细胞的培养液,还是其无细胞滤液或藻液超声破碎液,均对球形棕囊藻的生长具有显著抑制作用(P<0.01),抑制强度由强至弱依次为完整细胞培养液、无细胞滤液和藻液超声破碎液;相反,球形棕囊藻的存在对红色赤潮藻细胞的生长无显著影响(P>0.05)。因此,红色赤潮藻具有抑制球形棕囊藻生长...     

Improving DNA damage repair ability ofE. coli to UV-radiosensitivity by DNA-mediated gene transfer of low energy ion beam

Using calcium chloride method of transfer gene as control, a new technique of transferring gene by low energy ion beam has been applied to the study of improving DNA damage repair ability ofE. coli to UV-radiosensitivity. The genome DNA pieces ofDeinococcus radiodurans, as “foreign” genetic materials, were introduced into the UV-radiosensitive strains ofE. coli by implantation of 20 keV Ar⁺ at doses ranging from 1 × 10¹⁵ to 2 × 10¹⁵ ions/cm². Results show that the transfected strains present higher UV-radioresistance than that of un-transfected ones and start ones. The survival rate of transfected strains and their unscheduled DNA synthesis (UDS) ability is increased, indicating that the transfer gene is a success.     

Tobacco curly shoot virus isolated in Yunnan is a distinct species of Begomovirus

Virus isolate Y1 was obtained from tobacco showing curly shoot symptoms in Baoshan, Yunnan Province. Whitefly transmission test and virion morphology observation showed that it is a begomovirus. In reactions with 14 monoclonal antibodies raised against begomoviruses, Y1 was readily differentiated from begomoviruses reported in China, Pakistan and India. The complete nucleotide sequence of DNA-A was determined, it contains 2746 nucleotides, with two ORFs in virion-sense DNA and four ORFs in complementary-sense DNA. Comparisons with total DNA-A, intergenic region and deduced amino acid sequences of individual ORFs showed that Yl is a distinct Begomovirus species, for which the name Tobacco curly shoot virus (TCSV) is proposed. The total DNA-A of TCSV is most closely related to that of Tomato leaf curl virus from India (85% sequence identity). In contrast, the deduced coat protein of TCSV is most like that of Cotton leaf curl virus 72b isolate from Pakistan (98% amino acid sequence identity).     

Experimental proof for the regulation ofSalmonella typhimurium purB bypurR

Salmonella typhimurium purB encodes adenylosuccinate lyase (ASL), the enzyme that catalyzes step 8 in the pathway forde novo synthesis of inosine 5′-monophosphate (IMP) and also the final reaction in the two-step sequence from IMP to adenosine monophosphate (AMP). The nucleotide sequence ofpurB was obtained by the genetic map and sequence homologous analysis. The conservedpur operator inpurB was identified to be located 185 bp downstream of the initiation codon and overlaps codons 62 – 67 in the protein-coding region. The binding of PurR to this operator was demonstrated by gel retardation experiment and site-directed mutagenesis, indicating that thepurB is under the control ofpurR. We also answered why previous study had conflicting report concerning the regulation ofpurB bypurR by identifying the junction site ofpurB tolacZ in apurB- MudJ (lacZ,Kan ^r) fusion strain. This result strongly supports that thepurB is the second gene in theycfC-purB operon.     

秀丽线虫对根际益生菌P13和S3-1传播作用及对植物生长的影响

通过缓慢杀线实验、偏好性实验和共培养毒性实验分析了秀丽线虫(Caenorhabditis elegans)对荧光假单胞菌P13(Pseudomonas fluorescens P13)和解淀粉芽孢杆菌S3-1(Bacillus amyloliquefaciens S3-1)传播的可能性.通过显微观察与平板稀释涂布对秀丽线虫细菌携带作用进行了定性、定量分析,并对细菌—线虫—植物三者交互作用进行初步探究.结果发现,P13和S3-1对秀丽线虫的慢性致死率分别为12.12%和3.00%,每10 s身体弯曲次数分别为4.68和4.33.相对于尿嘧啶缺陷型大肠杆菌(OP50),线虫对P13和S3-1选择系数分别为0.13和0.52,P13、S3-1携带菌量分别为(4.02×103±47)和(9.67×102±22)CFU/条.携细菌线虫将细菌定向传播至植物根际,细菌定殖菌量为105CFU,有效促进植物生长发育.     

Root and xylem ABA changes in response to soil drying in two woody plants

Two woody plants, Platycladus orientalise (tolerant to drought) and Acacia auriculi-formis (sensitive to drought), have been subjected to rapid and slow soil drying. ABA levels in their roots and xylem sap have been determined using radioimmunoassay (RIA, sensitivity is 0.4 pmol per assay vial) with a monoclonal antibody against ( + )-ABA. ABA contents of P. orientalise and A. auriculiformis growing in well watered soil are 0.3 and 2.5 nmol·gDW-1 in roots and 1.6 and 0.4 μmol in xylem saps, respectively. A rapid soil drying has been applied to these two plants with soil water content (SWC) being reduced to 0.02 and 0.06 g·gDW-1 respectively. Under such treatment, ABA was increased by 22 times and 2 times in roots and by 7 times and 34 times in xylem saps respectively for P. orientalise and A. auriculiformis. After rewatering for 6 d, ABA in roots and xylem sap of both species returned to control levels. When a slow soil drying was applied, SWC was reduced to 0.1 and 0.13 g·gDW-1 respectively for P. orientalise and A. auriculiformis. ABA was increased by 5 times and 1.6 times in roots and by 6 times and 19 times in xylem saps respectively for these two plants. ABA in roots and xylem saps decreased to near control levels 8 d after watering. Plant leaf water potentials of both plants hardly changed at times when root and xylem ABA showed substantial increase in response to soil drying. It is concluded that ABA levels in the roots and xylem saps of P. orientalise and A. auriculiformis are more sensitively regulated than leaf water potential in response to soil drying and can act as a chemical signal in root-shoot communications of the drought stress.     

海绵Pseudoceratina sp.共栖细菌多样性及其抑制甘蔗鞭黑粉菌活性研究

【目的】探讨广西斜阳岛附近海域海绵共附生细菌的多样性,并筛选对甘蔗鞭黑粉菌有抑制作用的活性物质,为发现潜在的细菌新物种及开发农用生防菌肥奠定基础。【方法】采用稀释涂布法,通过6种复合营养分离培养基,从海绵Pseudoceratina sp.中分离可培养的共附生细菌。采取细菌形态学观察去除冗余,通过PCR扩增菌株的16SrRNA基因序列,并通过构建系统发育树分析物种多样性。采用牛津杯法进行抑制甘蔗鞭黑粉菌活性筛选实验。【结果】从海绵Pseudoceratina sp.中共分离到可培养细菌54株,经16SrRNA基因序列对比后,获得24株细菌,隶属于13科14属。其中,菌株BGMRC 2118与已发表有效菌株的16SrRNA基因序列的最高相似率为96.46%,可能为潜在新种。抑菌筛选实验结果显示,有8株细菌的发酵粗提物对甘蔗鞭黑粉菌具有很强的抑菌活性。【结论】广西斜阳岛附近海域中海绵共附生细菌具有较高的物种多样性,蕴藏着丰富的新物种资源,富含生物活性菌株。     

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.     

固定化污泥与固定化小球藻共培养去除污水氮磷研究

为减少污水处理装置排出水中氮元素(N)、磷元素(P)的含量,减轻收纳土地和水体的污染,降低水华暴发的频率,本研究采用海藻酸钠分别固定小球藻和活性污泥,并以不同比例组合成共培养体系,处理经Up-flow Anaerobic Sludge Bed/Blanket(USAB)工艺处理后的真实畜禽养殖废水,同时对比传统悬浮细胞方法对N、P的去除效果。结果表明,在72 h内,较低固定化污泥/固定化小球藻初始比例(R=1/3)下的共培养体系对N、P有较好的去除效率;48 h时NH_~+4-N去除率为83.2%,PO_4~(3-)-P去除率为95.1%;3个半连续批次处理中,NH_~+4-N和PO_4~(3-)-P的去除率保持相对稳定。以上结果说明藻类能促进N、P元素的去除,固定化工艺可提升去除效率,具有应用于畜禽养殖废水处理的潜力。     

Expression and anti-apoptotic mechanism of Spodoptera littoralis Nucleopolyhedrovirus P49 protein

A novel cloned Spodoptera littoralis Nucleopolyhedrovirus (SlNPV) p49 gene is able to suppress apoptosis of insect cells Sf9 triggered by virus. The amino acid sequence of P49 expressed in baculovirus expression system is the same as predicted, indicating that the expression of P49 is correct. Metabolic labeling revealed that p49 was able to be expressed both in the early and late phases after the viral infection, and only in the late phase was the expression driven by polyhedra promoter, but the amount of expression was higher than that of wtSlNPV. In summary, the early gene of SlNPV p49 as well as p35 of AcMNPV is able to be expressed in the late phase, but its promoter is weaker compared with polyhedra promoter. In vitro, P49 can be cut by Bm caspase and human caspase-3, yielding 10 and 40 ku fragments. Purified P49 blocks the substrate cleavage by Bm caspase and human caspase-3, showing that P49 inhibits downstream caspases in the apoptotic pathway.     

Site-directed mutagenesis reveals new and essential elements for iron-coordination of the sulfur oxygenase reductase from the acidothermophilic Acidianus tengchongensis

Previous study on refolding of sulfur oxygenase reductase (SOR) inclusion bodies from recombinant Escherichia coli showed that iron was critical to the activity of the SOR from Acidianus ambivalens. In this study, enzymatic assays showed that 2,2′-Dipyridyl, Tiron and 8-hydroxyquinoline, which are specific for chelating ferrous or ferric ions, strongly inhibited the activity of SOR from A. tengchongensis, suggesting that iron atom is essential for SOR activity. Alignment of several functionally identified SORs and SOR-like sequences from genome database revealed a conserved, putative iron binding motif, H⁸⁶-X₃-H⁹⁰-X _n -E¹¹⁴-X _n -E¹²⁹ (numbering according to the Acidianus tengchongensis SOR sequence). Three mutants of SOR were generated by site-directed mutagenesis of H₈₆, H₉₀ and E₁₂₉ into phenylalanine or alanine residue in this study. Circular dichroism spectrum determination indicated that there was no change of the secondary structures of mutant SORs, H⁸⁶F, H⁹⁰F and E¹²⁹A, but all mutants were completely inactive. Through determination of iron contents we found that SOR mutants of H⁸⁶F, H⁹⁰F and E¹²⁹A completely or partially lost iron, while mutants of C³¹S, C¹⁰¹S, and C¹⁰⁴S (generated in a previous study) did not. This result indicated that H⁸⁶, H⁹⁰ and E¹²⁹ but not C³¹, C¹⁰¹, and C¹⁰⁴ were involved in binding to iron atom. Based on this and previous studies, it is proposed that the conserved motifs, C³¹-X _n -C¹⁰¹-X₂-C¹⁰⁴ and H⁸⁶-X₃-H⁹⁰-X₂₃-E¹¹⁴-X₁₄-(E/D)¹²⁹, are respectively for sulfur and molecular oxygen binding and activation. These two conserved motifs are essential elements for the SOR activity. Supported by National Natural Science Foundation of China (Grant Nos. 30670018, 30621005) and State Key Basic Research and Development Program of China (Grant No. 2004CB719602)     

特色壮药黄花倒水莲高效栽培技术研究

本研究旨在探索适用于黄花倒水莲(Polygala fallax Hemsl.)的栽培措施，为黄花倒水莲的资源开发利用提供参考。以黄花倒水莲组培苗为材料，研究种植模式、立地条件、种植密度、肥料种类、施肥次数、施肥时间对黄花倒水莲产量的影响。结果表明：采用山地、林下种植模式，黄花倒水莲的产量和成活率明显提高。选择在4月移栽幼苗，成活率可高达(97±2)%,高于其他月份。种植密度为50 cm×50 cm时，平均产量最高。施用有机肥时，产量较施用复合肥、不施肥明显增加；当施肥量为750 kg/666.7 m²时，产量达到最大；每年于6月施肥效果最好，其次是4月。综合考量，为提高黄花倒水莲的产量，种植地应选在山地，采用林下种植模式；4月移栽；种植密度为50 cm×50 cm; 4-6月施用有机肥约750 kg/666.7 m²(每株约0.2 kg),分两次施肥。     

Chromosomalin situ hybridization ofCyprinus carpio genomic DNA repetitive sequence CR1

Common carpCyprinus carpio genomic DNA repetitive sequence CR1 has been DIG-labeled and hybridizedin situ against chromosomes of red common carp (Cyprinus carpio L. Xingguo red var. ). It is found that the repetitive sequence CR1 is mainly localized at the centromeric regions of chromosomes of the red common carp. The application of the chromosomalin situ hybridization technique on fish and the relationship between CR1 repetitive sequence distribution and its function have been discussed.     

人WDR79基因启动子表达载体的构建及鉴定

人WDR79是一种重要的支架蛋白,在端粒酶组装、卡哈尔体(Cahar body)形成和DNA损伤修复等过程中发挥着重要作用。采用PCR扩增技术获得人WDR79基因启动子上游DNA序列,构建人WDR79基因启动子荧光素酶报告基因载体pGL3-WDR79-promoter;通过双酶切、琼脂糖凝胶电泳、DNA测序和荧光素酶活性测定等实验手段,验证质粒pGL3-WDR79-promoter构建的正确性及其表达活性。本研究结果为进一步探讨人WDR79基因表达的调控机制奠定实验基础。