对烟基两场设施管护措施的思考——以常德市桃源县烟基管护为例

随着烟叶产业发展进入稳定期,产业发展重点由新建设施转移至,如何延长当前已建成的两场设施使用寿命,提高生产基础设施利用效率,桃源县立足本地实际,通过渠道化筹资、专业化管护、规范化管理、多元化发展措施,提升两场设施管护效能,确保两场设施常用常新,同时针对下阶段工作,进一步提出改进措施,以期为其他烟区两场设施管护工作提供借鉴经验。

现代烟草农业烟基建设工作的几点思考

对烟基建设工作中出现的项目规划设计和项目工程建设监理等方面的新问题开展了调研,通过认真思考和分析,提出了针对这些问题在烟基工作中应采取的相应对策。

在节能前提下优化烟基薄片纸机

介绍了抄造烟基薄片纸机各部分的用能情况,及如何运用新技术改造旧线烟片机,使之在产能提高的情况下,产品的质量提升,能耗大幅降低,实现清洁生产。

龙岩烟区烟叶生产基础设施管护模式的探讨

概述了龙岩烟区烟叶生产基础设施管护模式的背景和运行方式,介绍了"以烟草企业为主"、"以项目村为主"、"以协会为主"、"以合作社为主"4种管护模式的运行效果,并分析了4种管护模式在实践中的优势及存在问题,就如何推广管护模式提出了对策和建议:强化管护意识、多方筹措经费、完善合作社管护机制、健全管护工作考核机制、扩大管护范围、探索市场化管护方式,确保管护工作发挥持久效益。

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.

A Preliminary Study on Intensive Curing with Alcohol-based Fuels

The research investigated application effects of alcohol-based fuels in in- tensive curing. The results showed that alcohol-based fuels allow flexible adjustment of temperature and heat supplying. What＇s more, in treatment A, the cured tobac- cos are softer and brighter, with more oil content and higher proportion of first-class tobaccos. Per leaf weight increased by 0.13 g and output value per kang （a heat- able brick bed） deducting energy cost grew by 188.66 yuan. In addition, the con- tents of reducing sugar and potassium enhanced within the ranges of high-quality tobacco, and chemical components are more coordinated.

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.

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.

Flow Cytometric Evidence for Hydroxyl Radical-induced Apoptosis in Tobacco Protoplasts

Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cells have similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such as condensation and peripheral distribution of nuclei, TUNEL positive reaction, and DNA ladders were observed when tobacco protoplasts were treated with the hydroxyl radical generating system (1.0 mmol/L FeSO4/0.5 mmol/L H2O2). In animals, the loss of transmembrane potential (DeltaPsi(m)) and the exposure of phospholipid phosphatidylserine (PS) are believed to be the main apoptosis events. To test whether these significant processes take place in plants, flow cytometry was used to detect annexin V binding and changes in DeltaPsi(m). Results showed that the PS turned out from inner membrane and DeltaPsi(m) gradually decreased during the apoptosis. All these apoptotic characteristics proved that hydroxyl radicals can cause typical programmed cell death (PCD) in tobacco protoplasts and this design can be served as an effective experiment system to explore the mechanism of plant apoptosis.

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.

In Vitro Development of Tobacco Primary Endosperm Cells in Microculture

用酶解_研磨法分离出烟草 (NicotianatabacumL .)受精后胚囊和初生胚乳细胞进行微室饲养培养。培养基为Km8p附加各种其他成分 ,饲养细胞为分裂旺盛的烟草叶肉原生质体 ,在 2 5℃下静止暗培养。培养 3d后 ,初生胚乳细胞开始第一次分裂 ,继续分裂至 14d时形成大的细胞团。首次报道了双子叶植物初生胚乳细胞的离体发育。

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.

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.

Progress and Prospects of Professional Tobacco Cooperatives

Chinese agriculture is confronted with more challenges globally after en- tering WTO and it is necessary to improve agricultural cooperatives which play an important role in marketing. In the research, the significance, progress, problems of professional tobacco cooperative and the evolvement direction were explored based on existing issues of its role in monopsony markets of agricultural product and the development.

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.

Effects of Tobacco Plastid Division Genes NtFtsZ1 and NtFtsZ2 on the Division and Morphology of Chloroplasts

As an important group of plant cellular organelles, the molecular mechanism of plastid division is poorly understood. Recent studies have revealed that the homologs of ftsZ gene, an essential prokaryotic cell division gene, are involved in plastid division process of plant cells. Antisense and sense expression constructions were employed to investigate the functions of the two ftsZ genes, NtFtsZ1 and NtFtsZ2, in transgenic Nicotiana tabacum L. plants. Although antisense expression of,NtFtsZs reduced the native protein level obviously, the size and number of chloroplasts in transgenic tobacco plants had no effect. In contrast, overexpression of NtFtsZs in transgenic plants strikingly changed the number and morphology of chloroplasts. Even only 1 - 2 huge chloroplasts could be seen in the mesophyll cells of some overexpression transgenic plants. Analyses of chloroplast ultrastructures and chlorophyll content of different transgenic plants suggested that NtFtsZs gene have no direct influence on the normal development and function of chloroplasts. ne changes in chloroplast morphology must be a compensation for the change in chloroplast number. The different phenotypes of chloroplasts in antisense and sense transgenic plants implied that different members from the same ftsZ gene family may have similar function in controlling plastid division. Meanwhile, the changes of chloroplast morphology in sense transgenic plants represented the possible plastoskeleton function of ftsZ in higher plant.

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.