转烟过程防差错流程的构建与应用

针对制丝生产转烟过程中出现的问题,车间通过制定标准化的转烟流程和质量责任制度,有效规范了现场操作人员的转烟操作程序,极大限度地避免了系统或人为因素的影响,大大提高了转烟的效率和质量。

^(60)Co-γ射线辐照对转pprI基因烟草抗氧化酶活性的影响

以转pprI基因烟草植株为材料,并以同步培育的非转基因烟草植株为对照,研究了60Co-γ射线辐照对转pprI基因烟草抗氧化酶活性的影响。结果表明,经500 Gy60Co-γ辐照后,转基因和非转基因烟草的超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)3种抗氧酶活性以及可溶性蛋白质含量在开始时(0~12 h)都逐渐提高,其中POD的活性则在6 h后达最大值,而SOD和CAT活性在12 h后达到最大值,随后三者活性都逐渐降低。但在500 Gy60Co-γ辐射后相同的时间内,转基因烟草的3种抗氧化酶活性和可溶性蛋白质含量均比非转基因烟草高,SOD、POD、CAT和可溶性蛋白质含量的峰值分别是非转基因烟草峰值的1.1290、1.9690、1.5840和1.9521倍,表明pprI基因提高了烟草在500 Gy60Co-γ辐照下SOD、POD和CAT等抗氧化酶的活性水平,从而提高了转基因烟草对辐射的耐受能力。

改善切圆燃烧锅炉烟温偏差措施的数值模拟分析

针对某台超临界600MW机组切圆燃烧锅炉的烟温偏差问题,利用数值模拟技术分析了燃尽风反切和减小水平偏角辅助二次风(CFS)与一次风夹角2种措施对燃烧过程中炉膛温度场、火焰切圆、旋转动量、特征组分分布及NOx排放等方面的影响。结果表明,燃尽风反切技术能够较好地减轻烟温偏差,2种改进措施均可提升燃烧器区域烟气温度,减小火焰切圆直径,有利于稳定燃烧,减轻水冷壁结渣,但都会使NOx排放浓度增大。

Cloning of cDNA Encoding Choline Monooxygenase from Suaeda liaotungensis and Salt Tolerance of Transgenic Tobacco

Betaine is a very effective osmoprotectant found in many organisms. In high plants, betaine is synthesized by oxidation of choline in two sequential steps: choline-->betaine aldehyde-->betaine. The first step is catalyzed by choline monooxygenase (CMO). In this study, the full-length CMO cDNA (1 820 bp) was cloned from halophyte Suaeda liaotungensis Kitag by RT-PCR and RACE. It included a 123 bp 5' UTR, a 368 bp 3' UTR and a 1 329 bp open reading frame encoding a 442-amino-acid polypeptide with 77%, 72% and 74% sequence identity compared to CMOs from spinach, sugar beet and Atriplex hortensis, respectively. The CMO open reading frame (ORF) was cloned and the plant expression vector pBI121-CMO was constructed. It was transferred into tobacco ( Nicotiana tabacum L. ev. 89) via Agrobacterium mediation. PCR and Southern blotting analysis showed that the CMO gene was integrated into tobacco genome. Transgenic tobacco plants contained higher amount of betaine than that of control plants and were able to survive on MS medium containing 250 mmol/L NaCl. Relative electronic conductivity demonstrated less membrane damage in transgenic plants as in the wild type.

Aphid-resistant Transgenic Tobacco Plants Expressing Modified gna Gene

A gene sequence coding for the precursor of Galanthus nivalis agglutinin (GNA) was modified by site-directed mutagenesis to change very low usage bias codons to higher usage bias ones for improvement of the gene expression in transgenic tobacco (Nicotiana tabacum L.) plants. Results from Western blot analysis of some of the transgenic tobacco plants showed that the expression level of GNA in plants transformed with the modified gene GNA34m reached 0.25% of total soluble proteins, while that of the GNA34 gene transgenic plants was 0.17%. Since the GNA expression level increased, the aphid resistance of GNA34m transgenic plants were also enhanced significantly as judged by a 71.0% aphid population inhibition in insect bioassay of GNA34m transformed plants and 63.7% for the plants transformed with the natural GNA34 gene.

EXPRESSION OF TOMATO ANTISENSE ACC SYNTHASE GENE IN TRANSGENIC TOBACCO AND ITS ROLE IN SHOOT FORMATION

An ACC synthase cDNA isolated from tomato (Lycopersicum esculentum Mill.) fruit was constructed in antisense orientation under the transcriptional control of CaMV 35S promoter and then introduced into tobacco (Nicotiana tabacum L.) . PCR amplification demonstrated the integration of this antisense gene in tobacco genomes. Northern hybridization and reverse transcription-PCR analyses indicated the expression of this heterologous antisense gene in the transgenic tobacco tissues, which caused a decrease in the ethylene production, particularly when shoot regeneration exhibited. The ability of shoot regeneration of the transgenic plant during the culture process was enhanced remarkably as compared with that of the control. These results indicate at the molecular level that ethylene may play a regulatory role in shoot formation.

Novel Halophyte EREBP/AP2-type DNA Binding Protein Improves Salt Tolerance in Transgenic Tobacco

EREBP/AP2-type proteins are members of a large DNA binding protein (DBP) family found in plants. Some members like APETALA2 and AtDREB/CBF can regulate flower development and response to environmental stresses, respectively. To characterize transcription factors involved in plant responses to salt stress, we constructed cDNA library from salt-treated halophyte (Atriplex hortensis) and isolated a novel gene encoding EREBP/AP2-type protein from this library. This cDNA contained an ORF of 723 bp and a long 3'-Untranslated-Region (UTR) of 655 bp. The deduced amino acid sequence showed one conserved DNA binding domain of EREBP/AP2, thus the corresponding gene was named AhDREB1 with a calculated molecular mass of 26.1 kD. AhDREB1 under the control of CaMV 35S promoter was then transformed into tobacco and nine independent transgenic lines were obtained and subjected to long term salt stress. The results suggested that overexpression of AhDREB1 improved the salt tolerance in transgenic tobacco through functioning as a regulatory molecule in response to salt stress. Analysis of Arabidopsis genome in database resulted in dozens of EREBP/AP2-type homologous proteins, of which seven members showed high similarity to AhDREB1. Secondary structure analysis predicted similar arrangement of a-helix in their DNA binding domains.

Obtainment of Transgenic Tobacco Harboring phbA , phbB and phbC Genes by Twice Transformation

To avoid the long time required for conventional sexual crossing, transgenic tobacco (Nicotiana tabacum L.) plants harboring phbA gene (encoding 3_ketothiolase) were used as the target plant for the second transformation mediated by Agrobacterium tumefaciens (Smith et Townsend) Conn LBA4404 containing pZCB which was constructed by linking phbB (encoding acetoacetyl_CoA reductase), phbC (encoding PHB synthase) and ctp sequence to pBIB_HYG under the control of CaMV 35S promoter. The hygromycin resistant transformants were morphologically normal and stable integration of phbB and phbC was confirmed by PCR and PCR_Southern. Moreover, RT_PCR_DNA hybridization analysis showed that 6.67% of the transformed tobacco plants could express both phbB and phbC at transcriptional level.

Isolation of a Genomic DNA for Gastrodia Antifungal Protein and Analyses of Its Promoter in Transgenic Tobacco

A genomic DNA containing 5'-upstream region and complete open reading frame of a Gastrodia antifungal protein was isolated by screening of a genomic library from Gastrodia elata B1. To investigate the promoter activity, the 5'-flanking region - 1 157 lip upstream from the putative transcription start site was fused to the coding sequence of beta-glucuronidase (GUS) gene and transformed into Nicotiana tabacum. The strongest GUS activity was detected in the roots of transgenic tobacco, followed by stems. The leaves only showed a low GUS activity. Furthermore, the promoter established inducible expression pattern in transgenic tobacco upon fungus Trichoderma viride inoculation and jasmonic acid and salicylic acid treatments.

Increasing Accumulation Level of Foreign Protein in Transgenic Plants Through Protein Targeting

Targeting of the synthesized polypeptide in the cells is an important research field in modern cell biology. Cowpea trypsin inhibitor (cpti) gene has been modified and a fusion protein gene (sck) was produced by fusing a signal peptide sequence at cpti 5' end and an endoplasm reticulum (ER) retention signal peptide at cpti3' end respectively. The signal peptide can direct the newly synthesized polypeptide into ER, while ER retention signal can make the protein retained in the ER and its derivative protein body. ELISA test indicated that the accumulation level of foreign CpTI protein in sck transgenic tobacco (Nicotiana tabacum L.) was two times higher than cpti transgenic tobaccos and some individuals were four times higher. At the same time, sck transgenic tobacco has a high resistance to Lepidoptera pest due to the increased accumulation level of foreign CpTI protein. The strategy of foreign protein targeting can be used to increase the accumulation level of foreign protein in transgenic plants and can be widely applied to other related research field in plant genetic engineering.

Cloning of Glutamate Dehydrogenase cDNA from Chlorella sorokiniana and Analysis of Transgenic Tobacco Plants

A full_length cDNA has been cloned encoding nicotinamide adenine dinucleotide phosphate_specific glutamate dehydrogenase (NADP_GDH) from Chlorella sorokiniana with the RT_PCR method. The complete nucleotide sequence of NADP_GDH gene had 94% homology to the previously reported one . The NADP_GDH gene was constructed into a vector highly expressed in plants. The specific activity of NADP_GDH in transformants was detected, but not in the control plants. All transformed shoots on MS medium containing lower concentration of nitrogen and the transformed seedlings grown in lower concentration of nitrogen vermiculite had higher growth rate and more leaves than the control plants. Transformed leaf discs cultured on MS medium containing different nitrogen concentrations had more chlorophyll contents compared to the controls. These results suggested that exogenous NADP_GDH may enhance the absorption and utilization to ammonium in plants. The increased weight of transformed leaf discs cultured on medium supplemented with different concentrations of phosphinothricin (PPT) was more than that of control discs. 0.5 μg/mL PPT could be used as a selecting drug instead of kanamycin to develop the transformants. These results suggested that the NADP_GDH gene might be used as a new selecting gene in the future research of plant gene engineering.

Optimization of Genetic Transformation System of Tobacco K326 Mediated by Agrobacterium

[Objective]The aim was to optimize genetic transformation system in tobacco K326 mediated by Agrobacterium.[Method]The leaf of tobacco aseptic seedling was taken as explants to study the optimization of Agrobacterium-mediated genetic transformation system.[Result] The highest transformation efficiency was obtained when the explants were pre-cultured in the medium of MS ＋ 2 mg/L 6-BA ＋ 0.2 mg/L IAA for 2 d,and then infected with Agrobacterium GV3101（OD600 =0.6） for 5 min.The PCR detection proved that npt II gene had been integrated into the regenerated tobacco plants.[Conclusion]A highly efficient genetic transformation system of tobacco leaf mediated by Agrobacterium was established.

Cloning and Gentic Transformation of NbDAD1 Gene from Nicotiana benthamiana

[Objective] The aim was to clone NbDAD1 gene from Nicotiana benthami- ana and study its genetic transformation. [Method] NbDAD1 gene was isolated from N. benthamiana by using RT-PCR technology and over-expression vectors were con- structed to obtain NbDADl-overexpression resistant plants and NbDADl-overexpres- sion resistant plants carrying HA tag. [Result] The 351 bp long NbDAD1 gene was cloned from N. benthamiana; recombinant plasmids pCAMBIA1301-NbDAD1 and pCAMBIA1301-NbDAD1HAtag were constructed successfully; 50T0-generation N. ben- thamiana Hyg-resistant transgenic lines of three genotypes were obtained, including 23 positive transgenic plants. [Conclusion] This study laid the foundation for investi- gating the specific functions of NbDAD1 gene in N. benthamiana and exploring the possible functional mechanism of DAD1 protein in programmed cell death of plants.

Transformation of Two Nitrogen_fixation_related Plant Genes into Tobacco and Their Expressions

Lectins and leghemoglobins in legumes play the important roles, respectively, in recognition of host plants to their own rhizobia, and lowering the oxygen partial pressure surround the bacteroids and protecting nitrogenase from oxygen in symbiotic nitrogen_fixing nodules.In order to investigate the non_leguminous recognition of rhizobial bacteria relating to nitrogen fixation, plant expression vectors containing pea lectin gene ( pl ) and Parasponia hemoglobin gene ( phb ) have been, respectively, constructed in a plasmid and the plasmid has been introduced into tobacco ( Nicotiana tabacum L.) using Agrobacterium tumefaciens (Smith et Townsend) Conn as a vehicle for transformation. PCR and Southern blot demonstrated that the two genes were integrated into the genome of the tobacco plants. Histochemical staining for GUS activity, Western blotting,and in situ hybridization of pea lectin showed that they were expressed at translational level in the plants. These results may provide a clue for exploring whether Rhizobium leguminosarum bv. viciae could extend its host range and make the transgenic tobacco plants have the possibility of being symbiotic, or associative to nitrogen fixation.

Generating Marker-Free Transgenic Tobacco Plants by Agrobacteriummediated Transformation with Double T-DNA Binary Vector

We have developed a 'double T-DNA' binary vector system for generating selectable marker-free transgenic plants by Agrobacterium-mediated transformation. The 'double T-DNA' binary vector pDLBRBbarm which carried two independent T-DNAs, one containing a selectable marker neomycin phosphotransferase (nptII) gene and the other a bargene, was constructed. Transgenic tobacco (Nicotiana tabacum L.) plants were then produced by Agrobacterium-mediated transformation with this vector. Frequency of the primary transformants co-integrated with npt II gene and bar gene was 59.2%. Segregation of two T-DNA regions was found in 3 out of 4 T-1 lines from co-transformed T-0 plants with nptII and bar PPT-resistant and kanamycin-sensitive plants were in approximate 19.5% of the T-1 plants. The result indicated that this 'double T-DNA' vector system could be a workable approach to generate transgenic plants free from selectable marker genes. Co-transformation of nptII gene and bar gene to plants with mixtures of Agrobacterium tumefaciens strains containing single T-DNA vectors was also tested. Frequency of co-transformed plants was 20.0%-47.7% and relatively low as compared with that of 'double T-DNA' vector system.

关于锅壳式燃油气过热蒸汽锅炉的设计探讨

针对WNS型锅炉结构特点,通过对过热器的结构、布置、围护、启动保护等几个方面的讨论,介绍了一种新型的锅壳式燃油气过热蒸汽锅炉的设计方案。

Influence of Matrix Attachment Regions from Maize on Transgene Expression Level in Tobacco

The effect of matrix attachment regions (MARs) on foreign gene expression in transgenic plants was studied, The beta-glucuronidase (GUS) gene (uidA) was flanked by the MARs isolated from the genome of maize to form plant expression vector. The vectors with and without MARs were transferred into tobacco ( Nicotiana tabacum L.) through Agrobacterium-mediated transformation procedure. GUS activity assays indicated that MARs could increase expression level of uidA gene. The mean GUS activity could be increased twofold as compared to that of transformants without MARs, and the highest GUS activity of transformant could reach tenfold. The correspondence between GUS activity and mRNA accumulation was positive and indicated that MARs could improve transcription of foreign gene.

Improvement of Copper-inducible Gene Expression System for Plant

The copper-regulated gene expression system has been developed to control spacial and temporal expression of transgene in plant. It comprises two parts: (1) ace I gene encoding copper-responsive transcription factor under the control of a constitutive or organ-specific promoter, and (2) a gene of interest under the control of a chimeric promoter consisting of the CaMV 35S (-90 to +8) promoter linked to the metal responsive element (MRE) carrying activating copper-metallothionein expression (ACE1)-binding sites. Here, the effectiveness of two different ACE1-binding cis -elements which derive from 5'-regulatory region of yeast metallothionein gene was investigated in transgenic tobacco (Nicotiana tabacum L. cv. W38). The results revealed that the MRE (-210 to -126) could increase the system inducibility by 50% - 100% compared with the previously reported MRE (-148 to -105). It is potential to use the copper-inducible system to control valuable gene traits in plant biotechnology.

Involvement of cAMP in ABA Signal Transduction in Tobacco Suspension Cells

Abscisic acid (ABA) plays an important role in plant growth and developmental processes. Although some ABA signal molecules, such as cADPR, Ca2+, etc., have been reported, there. was no evidence proving the involvement of cAMP in A-B-A, signal transduction. In this present study, the constructed gene ( rd29A-GUS) was transformed into Nicotiana tabacum, and calli was induced from the transgenic plant. The suspension cells obtained from the callus grew well and uniformly. Treatment of the suspension cells with ABA led to an increase in GUS activity, indicating that these transgenic suspension cells are useful for the study of ABA signaling. Addition of nicotinamide (cADPR inhibitor) or U-73122 (phospholiphase C inhibitor) could only partially inhibit the increase of GUS activity elicited by ABA. The inhibitory effect of nicotinamide was enhanced by application of K252a (inhibitor of protein kinase). Treatment of the suspension cells with 8-Br-cAMP, a membrane-permeable analogue of cAMP, could partially replace the effect of ABA. Furthermore, intracellular addition of IBMX (phosphodiesterase inhibitor) mimicked die effect of exogenous cAMP on the deduction of expression of rd29A promoter. These results suggested that cAMP was an important messenger in ABA signal transduction in tobacco suspension cell.

转底炉烟气系统堵塞、腐蚀及防腐探索

烟气余热利用系统是转底炉生产工艺重要的组成部分,余热锅炉又是烟气余热利用系统的核心设备。对转底炉烟气特性进行了分析,重点介绍了某钢厂转底炉烟气余热利用系统工艺流程及工艺参数;对烟气余热利用系统堵塞、锅炉管腐蚀原因进行了分析,在实践基础上,提出了烟气余热利用系统防堵、防腐蚀的措施,并对余热锅炉防腐进行了有益探索。通过这些措施的实施,减轻了烟气余热利用系统的堵塞和腐蚀,并在转底炉实际生产中得到了验证,为解决转底炉烟气余热利用系统堵塞、腐蚀提供了有益的借鉴。