堤防建设重在基础处理

靖江市地处长江下游,三面环江,地理位置特殊,境内长江堤防总长120.6公里。但是,由于绝大部分江堤紧临堤后深塘,堤基稳定性较差,且白蚁危害严重,渗漏堤段较多,威胁堤防安全。为进一步提高长江堤防达标建设标准,我市于1998年9月初全面实施填塘固基工程,取得一定的成绩。主要做法有: 一、统一思想,增强对抓好填塘固基工程重要性和必要性的认识 填塘固基工程是整个长江堤防达标建设的基础性工程,无论多大的困难,多大的代价,非抓好不可。为此,我们反复宣传,从三方面统一了认识。 (一)实施填塘固基工程是长江堤防达标建设的需要。由于我市江堤内侧大都存在顺堤河。

Chloroplast Genetic Engineering in Higher Plants

Chloroplast genetic engineering, with several advantages over nuclear genetic engineering, is now regarded as an attractive new technology in basic and applied research, including deepening our understanding of plastid genome, engineering plant metabolic system, generating transplastomic plants with higher resistance to insect, disease, drought and herbicide and bioproducing of antibodies and vaccines. In this review, the principle and operating system for chloroplast genetic engineering and its application in higher plants have been discussed.

Advances in Cold Tolerance Genes and Their Application in Genetic Engineering of Plant for Cold Tolerance

Low temperature is one of the main environmental stress factors influenc- ing plant growth and development and crop yield. Cold tolerance genes and progress of their application in genetic engineering of plant for cold tolerance were reviewed comprehensively and systematically from the aspect of genes that are in- volved in biosynthesis of osmotic substances, genes coding fatty acid desaturation enzymes, antifreeze protein genes, genes coding antioxidant enzymes and so on, aiming at laying the foundation for genetic improvement of cold tolerance and breeding of plants.

Construction of a Tapetum-Specific and Tetracycline-Inducible System

A regulated gene expression system would offer the unique opportunity to study the gene physiological functions at different developmental stages. For realizing gene special expression in plant anther at given time, we constructed a new system that combined tetracycline- inducible elements with TA29 promoter, a tapetum-specific promoter of tobacco. The system was tested in transient GUS assay system by electroporation (gene gun) transformation of tobacco ( Nicotiana tabacum L. cv. Winsconsin 38) anther. In the absence of tetracycline as the inducer, no GUS activity was detected. However, strong GUS expression was observed in tapetum. tissue upon tetracycline induction, and little GUS activity was found outside the tapetum. Our results suggested that gene expression can be restricted to a specific tissue at the given time under the control of this new system, and this system would be a very useful tool for both basic plant biology research and biotechnological applications.

Creation of Male Sterile Line in Tobacco With HSP70 Anti-sense Fragment

In order to create the Male Sterile Line in tobacco, the anti-sense fragment of HSP70 gene was linked to anther specific expression promoter TA29 and the reconstructed vector was transformed into tobacco by Agrobacterium mediated transformation, and the transformants were then screened. Gus and spot blotting hybridization analysis of the transformants indicated that anti-sense fragment of HSF70 gene had been integrated into tobacco genome and expressed, thus the male sterile tobacco line was obtained. Microscope observation of anther and pollen showed that pistils of transgenic tobacco were normal, whereas anthers and pollens were fairly abortive in the same transgenic tobacco flower, comparing with pistils and stamens in control plants. The ratio of HSI：＇70 protein before and after heat shock in mitochondrial was found to be 1.39 in control tobacco plants and 1.01 in transgenic tobacco sterile lines. This is suggested that the anti-sense gene fragment of HSP70 can effectively inhibit the expression of HSP70 protein and lead to transgenic male sterility in tobacco flowers. The assay provided a new genetic engineering method for male sterility creation in plants.

Construction and Application of Plasmid pUC19-CM-D

[Objective] The aims were to construct a new suicide plasmid of Lactobacillus and gene deletion engineering bacteria of Lactobacillus with pUC19 vector. [Methods] pUC19-CM was constructed by inserting a chloramphenicol resistant gene into the multi-cloning site of pUC19,and then two homologous fragments were cloned into each side of the pUC19-CM to construct suicide plasmid pUC19-CM-D. [Results] A replacement mutant strain,whose target gene was replaced by resistant gene,could be obtained by transforming the suicide plasmid pUC19-CM-D into Lactobacillus for resistance screening. [Conclusion] The construction and application of pUC19-CM-D provided a fast and efficient means of construction of gene deletion engineering bacteria of Lactobacillus,and laid a foundation for study of gene function of Lactobacillus.

HAL1 mediate salt adaptation in Arabidopsis thaliana

The yeast HAL1 gene was introduced into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation with vacuum infiltration under the control of CaMV 35S promoter. Thirty-three individual kanamycin resistant plants were obtained from 75,000 seeds. Southern blotting analysis indicated that HAL1 gene had been integrated into all of the transgenic plants’ genomes. The copy number of HAL1 gene in transgenic plants was mostly 1 to 3 by Southern analysis. Phenotypes of transgenic plants have no differences with wild type plants. several samples of transformants were self-pollinated, and progenies from transformed and non-transformed plants (controls) were evaluated for salt tolerance and gene expression. Measurement of concentrations of intracellular K+ and Na+ showed that transgenic lines were able to retain less Na+ than that of the control under salt stress. Results from different tests indicated the expression of HAL1 gene promotes a higher level of salt tolerance in vivo in the transgenic Arabidopsis plants.

Embryonic and genetic manipulation in fish

Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were Produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An 'all-fish' gene construct CAgcGH has been made by splicing the common carp β-actin gene (CA) promoter onto the grass carp growth hormone gene (goGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate ho- mogeneous strain of valuable transgenic fish to fulfil human requirement in 21st century

Functional characterization of a potassium transporter gene NrHAK1 in Nicotiana rustica

The purpose of this study is to investigate the function of a novel potassium transporter gene (NrHAK1) isolated from Nicotiana rustica roots using yeast complement and real-time PCR technique. The complementary DNA (cDNA) of NrHAK1, 2 488 bp long, contains an open reading frame (ORF) of 2 334 bp encoding a protein of 777 amino acids (87.6 kDa) with 12 predicted transmembrane domains. The NrHAK1 protein shows a high sequence similarity to those of high-affinity potassium transporters in Mesembryanthemum, Phytolacca acinosa, Arabidopsis thaliana, and so on. We found that the NrHAK1 gene could complement the yeast-mutant defect in K+ uptake. Among several tissues surveyed, the expression level of NrHAK1 was most abundant in the root tip and was up-regulated when exposed to potassium starvation. Moreover, the transcript accumulation was significantly reduced by adding 5 mmol/L NH4+ to the solution. These results suggest that NrHAK1 plays an important role in potassium absorption in N. rustica.

QTL analysis of flag leaf in barley (Hordeum vulgare L.) for morphological traits and chlorophyll content

To understand genetic patterns of the morphological and physiological traits in flag leaf of barley, a double haploid （DH） population derived from the parents Yerong and Franklin was used to determine quantitative trait loci （QTL） controlling length, width, length/width, and chlorophyll content of flag leaves. A total of 9 QTLs showing significantly additive effect were detected in 8 intervals on 5 chromosomes. The variation of individual QTL ranged from 1.9% to 20.2%. For chlorophyll content expressed as SPAD value, 4 QTLs were identified on chromosomes 2H, 3H and 6H; for leaf length and width, 2 QTLs located on chromosomes 5H and 7H, and 2 QTLs located on chromosome 5H were detected; and for length/width, I QTL was detected on chromosome 7H. The identification of these QTLs associated with the properties of flag leaf is useful for barley improvement in breeding programs.

Magnaporthe oryzae MTP1 gene encodes a type Ⅲ transmembrane protein involved in conidiation and conidial germination

In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae. The Mtp 1 protein is 520 amino acids long and is comparable to the Ytp 1 protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1-GFP （green fluorescent protein） fusion expression results indicate that Mtp 1 is a protein located at several membranes in the cytoplasm. The functions of the MTP1 gene in the growth and development of the fungus were studied using an MTP1 gene knockout mutant. The MTP1 gene was primarily expressed at the hyphal and conidial stages and is necessary for conidiation and conidial germination, but is not required for pathogenicity. The Amtpl mutant grew more efficiently than the wild type strain on non-fermentable carbon sources, implying that the MTP1 gene has a unique role in respiratory growth and carbon source use.

Species-specific SCAR markers for authentication of Sinocalycanthus chinensis

Sinocalycanthus chmensis, an endangered species endemic to China, is cultivated as an omamental landscape tree in China. However, S. chinensis, Chimonanthus species and Calycanthusfloridus are difficult to be distinguished in seedling market because of their similar morphological characters. In this study, ISSR （inter-simple sequence repeats） were applied to detect S. chinensis from its closely related species. A unique 748-bp band was found in all accessions of S. chinensis. SCAR （sequence characterized amplified regions） markers were created by cloning and sequencing the specific band, and designing a pair of primers to amplify the band of 748 bp. Diagnostic PCRs were performed using the primer pair with the total DNAs ofS. chinensis, Chimonanthus species and C. floridus as templates, with only S. chinensis being able to be amplified. This amplification is not only rapid （results can be obtained in less than 3 h）, but is also easy to perform. Hence it is a feasible method for identifying S. chmensis in seedling market.

Proliferation of endothelial cell on polytetrafluoroethylene vascular graft materials carried VEGF gene plasmid

Objective： To investigate whether vascular endothelial growth factor （VEGF） gene plasmid carried by polytetrafluoroethylene （PTFE） vascular graft materials could transfect endothelial cells （ECs） and promote their growth. Methods： PTFE vascular graft materials carried with pCDI-hVEGF121, pCDI or pEGFP were incubated in Tris-buffer solution and the values of optical density of 260 nm at different time were plotted, then the DNA controlled release curve was made. ECs derived from human umbilical vein were seeded on the pCDI-hVEGF121/pCDI/pEGFP-PTFE materials or tissue culture plates, ECs numbers were counted and VEGF protein concentrations at different time were measured by enzyme-linked immunoadsorbent assay method. Green fluorescent protein （GFP） expression in ECs on pEGFP-PTFE materials was examined with fluorescence mi- croscopy. Results： The controlled release curve showed that the gene released from PTFE materials was rapid within 8 h, then slowed down and that the gene released continuously even after 72 h. At 24, 72 and 120 h, ECs number and proliferation rate of pCDI-hVEGFI21-PTFE materials were higher than those ofpCDI or pEGFP-PTFE materials （P〈0.05）. VEGF protein concentration of pCDI-hVEGF121-PTFE materials was higher than that of pC DI or pEGFP-PTFE materials at 6, 24, 72 and 120 h （P〈0.01）. GFP expression in ECs on the pEGFP-PTFE materials could be detected by fluorescence microscopy. Conclusion： PTFE graft can be used as a carrier of VEGF gene plasmid, VEGF gene carried by PTFE can transfect ECs and promote ECs growth.

Cloning, expression, purification, and characterization of LC-1 ScFv with GFP tag

Total RNA was isolated from the hybridoma cell line （LC- 1 ）, which secretes anti-lung adenocarcinoma monoclonal antibody, and was transferred into cDNA. Based on the FRl （framework region l） and FR4 conserved regions of LC-1 gene, the variable regions of heavy chain （Vh） and light chain （Vl） were amplified, and the Vh and modified Vl were connected to single chain Fv （ScFv） by SOE-PCR （splice overlap extension PCR）. The modified ScFv was fused with green fluorescent protein （GFP） and introduced into E. coli JM109. The fusion protein induced by lPTG （Isopropylthiogalactoside） was about 57000 on a 10% SDS-PAGE gel （10% Sds Polyacrylamide Gel Electrophoresis）, and primarily manifested as inclusion bodies. The renatured protein purified by Ni-NTA Superflow resins showed ability to bind to antigen on SPC-A-l lung adenocarcinoma. In addition, the induced host cells fluoresced bright green under 395 nm wavelength, which indicated that the expected protein with dual activity was expressed in the prokaryotic system. The ScFv with GFP tag used in this research can be applied as a new reagent to detect immunological dye, and provide a feasible way to detect adenocarcinoma in a clinical setting.

Perspectives:Gene expression in fisheries management

Functional genes and gene expression have been connected to physiological traits linked to effective production and broodstock selection in aquaculture, selective implications of commercial fish harvest, and adaptive changes reflected in non-commercial fish populations subject to human disturbance and climate change. Gene mapping using single nucleotide polymorphisms （SNPs） to identify functional genes, gene expression （analogue microarrays and real-time PCR）, and digital sequencing technologies looking at RNA transcripts present new concepts and opportunities in support of effective and sustainable fisheries. Genomic tools have been rapidly growing in aquaculture research addressing aspects of fish health, toxicology, and early development. Genomic technologies linking effects in functional genes involved in growth, maturation and life history development have been tied to selection resulting from harvest practices. Incorporating new and ever-increasing knowledge of fish gehomes is opening a different perspective on local adaptation that will prove invaluable in wild fish conservation and management. Conservation of fish stocks is rapidly incorporating research on critical adaptive responses directed at the effects of human dis- turbance and climate change through gene expression studies. Genomic studies of fish populations can be generally grouped into three broad categories： l） evolutionary genomics and biodiversity; 2） adaptive physiological responses to a changing environment; and 3） adaptive behavioral genomics and life history diversity. We review current genomic research in fisheries focusing on those that use microarrays to explore differences in gene expression among phenotypes and within or across populations, information that is critically important to the conservation of fish and their relationship to humans [Current Zoology 56 （1）： 157-174, 2010].

Genetic engineering and lignin biosynthetic regulation in forest tree species

Genetic engineering of forest tree species is regarded as a strategy to reduce worldwide pressure on natural forests, to conserve genetic resources and ameliorate stress on global climate, and to meet growing demand for forest wood and timber products. Genetic engineering approaches toward the control or management of fungal pathogens, arthropod herbivores, bacterial and viral diseases, the use of pest resistance genes, and weed competitors are being studied. Although the production of transgenic trees is relatively recent and only a few species have been successfully genetically engineered in forest tree species, very useful and valuable information is available on the application of transgenic trees. Genes involved in important agricultural traits such as herbicide resistance, insect resistance, and wood quality have been isolated and have been used to genetically engineer trees. New technologies of plant molecular biology and genomics now make it possible high-efficient genetic improvement of forest trees. Genetic engineering promises to expand greatly the potential for genetic manipulation as new genes of commercial interest are discovered and utilized. Lignification is a process essential to the nature and evolution of vascular plants that is still poorly understood, even though it has been studied for more than a century. Recent studies on mutant and transgenic plants indicate that lignification may be far more flexible than previously realized. Rines with a mutation affecting the biosynthesis of the major lignin precursor, coniferyl alcohol, show a high level of an unusual subunit, dihydroconiferyl alcohol. It is also unusual as a plant polymer in that there are no plant enzymes for its degradation. These results have significant implications regarding the tradiational definition of lignin, and highlight the need for a better understanding of the lignin precursor biosynthetic pathway. In this review, we describe the progress made recently in genetic engineering of forest tree species.

Molecular identification of Prorocentrum (Dinophyceae) species

A fragment of a large sub-unit ribosomal DNA (LrDNA) of 12 strains of Prorocentrum species was amplified by polymerase chain reaction (PCR). The PCR products were digested by 3 restriction endonucleases (Cfo I, Hae III, and RSA I) and then resolved in agarose gels. Results show that different species had different RFLP patterns, except for P. arcuatum (ME 131), which had the same pattern to P. micans (ME160 and 04). The same fragment of 19 strains of the genus was also amplified and subjected to denaturing gradient gel electropho- resis (DGGE). 11 different patterns were resolved. Different cultures of a same species had the same pattern. The results of RFLP and DGGE analyses showed that eight newly isolated epibenthic Prorocentrum species were different from each other, and also from other cultured ones examined in this study. P arcuatum (ME132) could not be differentiated from P. micans (ME160 and 04), it was probably mis-identified, since they are quite differ- ent morphologically. P. redfieldii (ME138) could also not be distinguished form P. triestinium (ME132), it should be regarded as a synonym of P. triestinium. Unexpectedly, a restriction site was found in P. micans, compared with previous sequence data.

A novel system for Agrobacterium-mediated transformation of wheat(Triticum aestivum L.) cells

A new approach for transforming the cultured cells of wheat(Triticum aestivum L. cv. Ganmai 8) was developed using Agrobacterium tumefaciens. The features of the optimum procedure were: (a) both combined synthetic signal molecules and multiple natural extracts from susceptible plants were used to pretreat the primary vigorous Agrobacterium(PVA) cells for approximately 16 h; (b) the gyratory magnetic field condition was used during cocultivation; (c) the cocultivating period and selecting condition were modified; (d) the recipient cells were at exuberant metabolism and active division while infected with Agrobacterium. Both neomycin phosphotransferase and nopaline synthase assays demonstrated the expression of NPT II and NOS genes, located on the T-DNA segment of chimaeric plasmid pGV3850::1103neo, in transformed wheat cell colonies by adopting the techniques of dot blot, ndPAGE or high voltage paper electrophoresis. Integration of the foreign genes into wheat genome was confirmed by Southern blot hybridization. Moreover, a relatively rational method was described for the estimation of transformation frequencies from cultured cell levels.

HIGH DENSITY CULTIVATION OF GENETICALLY－ENGINEERED CHO CELL LINES WITH MICROCARRIER CULTURE SYSTEMS

Genetically-engineered CHO cell lines, rβ- 13 and CLF-8B2, were cultivated with the MC- 1 microcarrier culture system. The cell density could be enhanced by increasing the concentration of microcarrier. At a microcarrier concentration of 10 mg/ml. the cell density could reach 4 to 5 × 106 cells/ml. It was shown that these cell lines would spontaneously release from the microcarrier to attach to and proliferate on fresh microcarriers. We were thus able to scale up cultivation using a simple method. i. e. by adding fresh microcarriers and medium directly into the culture system to about 2, 4 or 8 times the original volume. Using a perfusion culture system. we have successfully cultivated CLF-8B2 cells in a 2 L bioreactor for several weeks at medium perfusion rates of 0. 5 to 3working volumes. Prourokinase was stably secreted.

Engineering of Corynebacterium glutamicum to Enhance L-ornithine Production by Gene Knockout and Comparative Proteomic Analysis

Engineered Corynebacterium glutamicum was constructed for L-ornithine production by disrupting genes of argF and proB to prevent the flux away from L-ornithine.Effect of the inactivation of 2-oxoglutarate de-hydrogenase complex(ODHC) on L-ornithine production was also investigated.It was found that the inactivation of ODHC by knockout of the kgd gene enhanced L-ornithine production.The engineered C.glutamicum ATCC13032(ΔargFΔproBΔkgd) produced L-ornithine up to 4.78 g·L-1 from 0.24 g·L-1 of the wild-type strain.In order to understand the mechanism of L-ornithine production in C.glutamicum ATCC13032(ΔargFΔproBΔkgd) and find out new strategies for further enhancing L-ornithine production,the comparative proteome between the wild-type and the engineered strain was analyzed.L-Ornithine overproduction in the engineered strain was related to the up-regulation of the expression levels of enzymes involved in L-ornithine biosynthesis pathway and down-regulation of the expression levels of proteins involved in pentose phosphate pathway.The overexpression of genes in the upstream pathway of glutamate to increase the availability of endogenous glutamate may further in-crease ornithine production in the engineered C.glutamicum and the ornithine synthesis enzymes(ArgCJBD) may not be the limiting enzymes in the engineered C.glutamicum.