TURNIP MOSAIC VIRUS COAT PROTEIN GENE: CLONING AND CONSTRUCTION OF THE PLANT VECTOR

Complementary DNAs to turnip mosaic virus (a radish Raphanus stativus isolate) genomicRNA were synthesized using oligo (dT) as primer and cloned into the vector λ-ZAP Ⅱ. Afterhybridization with a single-stranded cDNA probe and the sequencing of inserted DNA,positive clones with poly--A tails were obtained. One clone containing 1429-base pair insertwas sequenced. The coat protein gene was identified based on the molecular weight of theTuMV coat protein and the consensus sequences of the polyprotein processing sites ofpotyviruses. The 5' end of the coat protein gene was modified by PCR to introduce aninitiation codon, ATG, and two restriction enzyme sites. The gene was then manipulatedinto a binary vector pBIN437 which was derived from pBI121, and the plant expressionvector is being used to transform Brassica napus.     

GENETIC ENGINEERING OF TOBACCO PLANTS WITH CMV RESISTANCE CONFERRED BY MONOMER cDNA OF SATELLITE RNA

The synthesized full-length cDNA to CMV satellite RNA-1 was integrated into plant expression vector RoKII with a CaMV 35S promoter. Infected by Agrobacterium tumefaciens harbouring the recombinant plasmid. The tobacco leaf discs (the G-140 variety which is widely cultivated in China) were regenerated into plants. After being inoculated with virulent strain CMV, most of the transgenic plants expressed satellite RNA at high levels and developed a much milder symptom than the untransformed ones.Basically in accordance with Mendel's law of segregation, the novel tobacco pure line engineered with viral resistance was screened out. The secondary generation transgenic plants still maintained high level satellite RNA expression and the resistance to CMV.     

TRANSFER OF THE ATRAZINE-RESISTANT GENE OF BLACK NIGHTSHADE TO SOYBEAN CHLOROPLAST GENOME AND ITS EXPRESSION IN TRANSGENIC PLANTS

The atrazine-resistant psbA gene of black nightshade was transferred to the chloroplast genome of atrazine-susceptible soybean by means of ovary microinjection during the stage of zygote. The identification was carried out by using the methods of spraying the leaves directly with atrazine solution, examining the change of leaf fluorescence kinetics under a brighter light induction, molecular hybridization, etc. The experimental results show that the transgenic soybean plants do have been obtained for the first time.     

RNA—PROTEIN CROSSLINK AND RNA CHAIN BREAK FORMATION ON UV-IRRADIATION OF TOBACCO MOSAIC VIRUS

Abstract— The ability of UV-irradiation (254 nm) to induce formation of RNA-protein crosslinks in tobacco mosaic virus (TMV) particles have been studied by Cs₂SO₄ density gradient centrifugation, analytical centrifugation, nitrocellulose filter binding and two-dimensional peptide mapping. RNA-protein crosslinks were found to be formed on UV-irradiation of TMV, but the parallel process of UV-induced RNA chain breakage complicated their quantitation. Using speciall devised equations, the quantum yield of RNA-protein crosslink formation was found to be 0.65 × 10⁻⁵ and that of RNA chain break formation 0.95 × 10⁻⁵.     

CLEAVAGES OF TRANSCRIPTS OF TOBACCO MOSAIC VIRUS BY SYNTHETIC RIBOZYMES in vitro

Based upon computer-assisted predictions on the secondary structures of tobacco mosaicvirus (TMV) genomic RNA (both polarities), hammerhead type ribozymes were synthesizedin vitro, which all shared a conserved domain adapted from satellite tobacco ringspot virus(sTobRV)RNA. Ribozymes RZ1, RZ2 and RZ3 were designed to cleave the phosphodiester bondsimmediate to the 3'--end of GUC between the residues 5384-5385 and 6312--6313 on the plusstrand and 1214-1215 on the minus strand, respectively. The in vitro data indicated that RZ1 wasable to cleave completely its substrates BT1(+ ) and BT2(+ ), representing partial sequencesof the plus strand of the TMV MP region at 50, 37 and 30℃ with a molar ratio of ribozymeto the target as low as 1:1. Its two iso-ribozymes RZ1A and RZ1B which were respectivelymodified to contain a CUUCGG sequence in the conserved region and in an additional 3'-ter-minal stem-loop of UUUUUCUUCGGAAAAA were able to cleave BT1(+) and BT2(+) asefficiently as RZ1. Ribozyme RZ3 cleaved, with l     

温敏梳状嵌段共聚物对PS微球阻抗蛋白吸附作用的研究

采用可逆加成断裂链转移聚合(RAFT)方法和大分子单体技术,制备了温敏性聚N-异丙基丙烯酰胺(PNIPAM)-聚乙烯基吡咯烷酮(PVP)与PNIPAM-聚氧化乙烯(PEO)梳状嵌段共聚物,这些共聚物具有PVP或PEO支链.以溶菌酶为蛋白模型研究了所得共聚物对聚苯乙烯(PS)微球表面蛋白吸附的抑制作用.通过絮凝实验、激光散射法表观粒径测定、电泳迁移率测定及蛋白吸附量的定量数据比较了不同梳状结构的抗蛋白吸附效果.结果表明,预吸附梳状嵌段共聚物可有效阻抗蛋白吸附,亲水支链增加阻抗性能提高,即使环境温度高于PNIPAM的相转变温度也能阻抗蛋白吸附.透射电镜和共聚物胶体粒径测试表明,梳状嵌段共聚物阻抗蛋白吸附的机制是预吸附后PVP或PEO亲水支链在微球表面形成了阻隔层.通过PS微球的变温絮凝实验可评价预吸附聚合物的抗蛋白吸附性能,快速获得定性结果.     

Expression of Human Hepatitis B Virus Surface Antigen Gene in Transgenic Tobacco

Expression of Human hepatitis B virus surface antigen (HBsAg) gene in plant was reported for the first time. The recombinant plasmid pRoKⅡ-HBsAg was constructed by inserting HBsAg gene into the downstream of CaMV 35S promoter of binary vector pRoKⅡ and then introduced into Agrobacterium tumefaciens LBA4404. The kanamycin-resistant plants were obtained by Agrobacterium-mediated transformation system. It was shown that HBsAg gene was expressed in transgenic tobacco plants and their progenies by ELISA. The spherical particles of ψ 22 nm in the leaf extract of trangenic tobacco were observed by immunosorbent electron microscopy.     

Electrochemical studies of 1,5-dihydroxynaphthalene as substrate for voltammetric enzyme immunoassay

Immunoassay is one of the biochemical analytical techniques using the specific antigen antibody com-plexation for analytical purposes. It has extensive ap-plication in clinical diagnostics, prevention and cure of diseases, and virus diagnostics. The presentation and progress of immunoassay methodology are one of the greatest achievements of bioanalytical chemistry. It is estimated that several-hundred millions of immuno-analytical determinations are carried out every year all over the world. E…     

DISSOCIATION AND RECONSTITUTION OF IMMOBILIZED RIBULOSE-1, 5-BISPHOS PHATE CARBOXYLASE//OXYGENASE FROM TOBACCO

Ribulose-1,5-bisphosphate carboxylase/oxygenase from tobacco covalently coupled to CNBr-activated Sepharose 4B was treated with urea. Analysis by electrophoresis showed that the small subunit was dissociated at 2—2.5 mol/L urea, while the large subunit was still bound to matrix. The large subunit core, L_8, was further dissociated into monomer at 3 mol/L urea. It is suggested that RuBPCase is coupled to Sepharose by virtue of ε-NH_2 on a large subunit. The activity of the immobilized enzyme was inversely proportional to the amount of small subunit dissociated by urea. The dissociated small subunits were almost completely bound back to the S-depleted immobilized RuBPCase, if the urea concentration was diluted to 0.5 mol/L. The enzyme activity could be recovered nearly to 100％. The activity of the S-depleted enzyme was linearly correlated on the concentration of small subunits in solution. These results indicate that the small subunit plays an important role in the maintenance of RuBPCase activity.     

DEVELOPMENT OF COMMON WHEAT GERM-PLASM RESISTANT TO BARLEY YELLOW DWARF VIRUS BY BIOTECHNOLOGY

Barley yellow dwarf virus (BYDV) is one of the most serious wheat diseases in China. So far no resistance has been described in common wheat. A certain level of BYDV resistance was found in thirteen Triticeae species. Thinopyrum intermedium, two octoploids derived from TH. intermedium/wheat, Zhong 4 awnless and TAF46, and one disomic addition line, L1 derived from TAF46, showed good resistance to BYDV by enzyme linked immunosorbent assay (ELISA). Two wheat/TA. intermedium translocation lines, CPI 119880 and CPI 119899, showing good BYDV resistance were developed from L1 by using both CSph mutant and tissue culture. It is found that their BYDV resistance was controlled by a single dominant gene. Two cDNA probes pEleAcc3 and pPJN8 (E1-T1) were screened for detecting Th. intermedium DNA in wheat background. A specific band for the DNA of Th. intermedium and its derivatives was found in Southern hybridization. It is also possible to determine the size of the alien segment by comparing the relative density o     

STUDIES ON PREPARATION OF INACTIVATED VACCINES FROM CELL CULTURES OF MINK ENTERITIS VIRUS (MEV) AND THEIR IMMUNITY

In this paper, superhigh reproductive rate strains of MEV with titre more than HA8192* or TCID50 log9.7 10 have been achieved both by cultivation in cell lines with different susceptibility to MEV and by isolating and identifying in field by the author. The systematic tests proved that S18 and L12 strains of MEV are the best strains for vaccine preparation. In this study, the best means for the tissue cultivation of MEV and the most advanced technological process for the production and detection of serum-free cell-cultured MEV fluids with super-high HA titre in batches in large quantities have been established for the first time. Optimum conditions for MEV inactivation were determined, and safe and effective inactivated vaccines with mineral oil or A1(OH)3 gel adjuvant were successfully prepared with serum-free cell-cultured MEV fluids. Both vaccines with different adjuvants can be manufactured in batches in large quantities and have been widely used all over China since 1986. The change laws of the imm