抗体药物的植物反应器生产

大量的重组抗体被用于疾病的预防、诊断和治疗.目前,这些重组抗体绝大多数是利用鼠源细胞生产的,它的缺点是有潜在的隐患和价格昂贵.然而,植物作为反应器生产抗体药物既具有安全性又可大规模廉价生产的特点,具有广阔的前景.     

转基因植物的生态影响

自从1983年转基因植物诞生以来，至今各种类型的转基因植物进入大田实验已不计其数，很多产物已商品化。作为生物界的新品种，转基因植物在带来巨大的经济效益和社会效益的同时，也带来了潜在的风险。从生态学角度论述了转基因植物所带来的影响及采取相应的措施来加以调控和管理。     

Progress of PHA production in transgenic plants

With its completely biodegradable ability, good physical and processing properties, and biocompatability, PHA has become the most attractive alternative to take the place of chemo-plastics and eliminate environmental pollution caused by chemo-plastics. Small-scale commercial production has been realized by bacterial fermentation, but thus produced PHA is too expensive to compete with chemo-plastics. However, it is very prospective to lower biodegradable plastic price by producing PHA in transgenic plants. Presently PHB/PHBV biosynthetic genes have been transferred into plants and expressed, but it is still far away from meeting the demands of commercial production. Enhancing research on the regulation of carbon and fatty acid metabolisms, and their biochemical and molecular mechanisms is the key to achieving this aim.     

Targeting of bacterial chloramphenicol acetyltransferase to mitochondria in transgenic plants

Most mitochondrial proteins are encoded by nuclear genes and are synthesized as precursors containing a presequence at the N terminus. In yeast and in mammalian cells, the function of the presequence in mitochondrial targeting has been revealed by chimaeric gene studies. Fusion of a mitochondrial presequence to a foreign protein coding sequence enables the protein to be imported into mitochondria in vitro as well as in vivo. Whether plant mitochondrial presequences function in the same way has been unknown. We have previously isolated and characterized a nuclear gene (atp2-1) from Nicotiana plumbaginifolia that encodes the beta-subunit of the mitochondrial ATP synthase. We have constructed a chimaeric gene comprising a putative atp2-1 presequence fused to the bacterial chloramphenicol acetyltransferase (CAT) coding sequence and introduced it into the tobacco genome. We report here that a segment of 90 amino acids of the N terminus of the beta-subunit precursor is sufficient for the specific targeting of the CAT protein to mitochondria in transgenic plants. Our results demonstrate a high specificity for organelle targeting in plant cells.     

谷氨酰tRNA合成酶转基因拟南芥的生长特性分析

以不同独立株系的拟南芥谷氨酰tRNA合成酶（Arabidopsis glutamyl tRNA synthetase,简称AtGluRS）过量表达的三种纯合株系（GS3、GS4、GS6）及抑制表达的三种纯合株系（GAS2、GAS3、GAS7）为实验材料,分析其生长情况和表型.表型分析结果表明,拟南芥谷氨酰tRNA合成酶过量表达植株表现出开花晚、基叶多、茎生长快等与对照和AtGluRS抑制表达植株不同的特点.此外,用氯化钠（NaCl）模拟盐胁迫环境,对转基因植物的萌发率和生长情况进行分析,发现谷氨酰tR     

Introduction of AtNHX1 into beet improved salt-tolerance of transgenic plants

AtNHX1 gene encoding the Na ^＋/H ^＋ antiport on the vacuole membrane of Arabidopsis was transferred into small bud tips of 1-3mm in length derived from immature inflorescence cultures of six genotypes of beet （ Beta vulgaris L. ） by the infection of Agrobacterium tumefaciens and transgenic plants with improved salt-tolerance were obtained. When transgenic plants at 5-leaf stage were potted in sand and irrigated with solutions containing a range of concentrations of NaCl （171-513mM）, they showed minor symptoms of damage from salinity and better tolerance than the controls. There were considerable discrepancies of salt-tolerance between transgenic plants originated from the same genotype and also between different genotypes. After vernalization, bolting transgenic plants were enveloped with two layers of gauzes for self-pollination. T1 seedlings tolerant to 342-427mM NaCl were obtained respectively. These results revealed that it was feasible to improve salt-tolerance of beets by the introduction of AtNHX1 gene into cultured bud cells.     

Disease-tolerance of transgenic tobacco plants expressing Ah-AMP gene of Amaranthus hypochondriacus

An antimicrobial peptide gene from Amaranthus hypochondriacus, Ah-AMP, was amplified by PCR and cloned. Sequence analysis results revealed that this gene is 261 bp in length encoding a precursor polypeptide of 87 amino acid residues. Ah-AMP gene was inserted in the binary vector pBin438 to construct a plant expression vector pBinAH916. Leave explants of Nicotiana tabacum var. SR1 were transformed with Agrobacterium tumefaciens LBA4404 harboring the above expression vector. Results from PCR, Southern and Northern blot analyses confirmed that the Ah-AMP gene had been integrated into the tobacco genome and was transcribed at mRNA level. Two bacterial-resistant transgenic plants were selected by inoculating the plants with Pseudomonas solanacearum and statistic analysis of two T1 lines showed that the resistance increased by 2.24 and 1.62 grade and the disease index decreased by 49.6% and 37.3% respectively when compared with the non-transformed control plants SR1. The results from challenging the plants with inoculums of Phytophthora parasitica showed that the symptom development was delayed and disease index was significantly reduced. These results suggest that Ah-AMP gene may be a potentially valuable gene for genetic engineering of plant for disease-resistance.     

Production of transgenic calves by somatic cellnuclear transfer

Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector(pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant(Neo^r) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was cartied out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves,and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves.PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.     

Production of transgenic blastocyst of sheep by somatic cell cloning

Five samples from primary cultures of five sheep ovarian granulosa cells were transfected by pEGFP- N1 DNA. Five transgenic positive cell lines, each from one of the five samples above, were used as donor nuclei for somatic nucleus transfer. A total of 352 in vitro matured and enucleated sheep oocytes were fused electrically with transgenic granulosa cells and 329 reconstructed embryos were obtained after activation by Ionomycin/6-DMAP, and these embryos were cultured in SOFaaBSA medium for 7 d. The result shows that 312 embryos (94.8%) had gone through cleavage and among them 63 (19.1%) had developed to the blastocyst stage. Expression of GFP gene was detected in various stages of early embryonic development by sampling randomly. Blastocyst rates given by the four cells treated with 0.5% FCS starvation was 19.6% (55/280) and it had not shown difference significantly (P＞0.05) with the result obtained with another cell line that had not gone through serum starvation (16.3%, 8/49). This experiment indicates that sheep transgenic embryos up to the blastocyst stage can be produced effectively by the combination of gene transfection in somatic cells in culture and somatic cell cloning.     

Neurospora NADP-glutamate dehydrogenases and its expression in E. coli and transgenic plants

Genes of NADP-glutamate dehydrogenase (NADP-GDH) were cloned from Neurospora intermedia (Ni), N. crassa (Nc), and N. sitophila (Ns). The sequences showed a high degree of homology at the cDNA and protein level. The three GDH genes were cloned into pET30a and expressed in E. coli. The activity assay of purified GDH showed that the Ni-GDH had a higher activity and affinity to ammonia than Ns-GDH, and Nc-GDH. The K_m value of Ni-GDH ranges from 0.3 to 0.45 mmol/L. Ni-gdh gene was transformed to Nicotiana bethamiana plants. The transformed plants grew much better in low nitrogen media than the only ROKII vector transformed control.     

Generating of rice OsCENH3-GFP transgenic plants and their genetic applications

In order to investigate rice functional centromeres, OsCENH3-GFP chimeric gene was constructed and transformed into the indica rice variety, Zhongxian 3037, mediated by Agrobacturium. The integration of the exogenous genes in the transgenic plants was confirmed by PCR and Southern blotting. The transgenic plants grow normally during their whole life time, just like Zhongxian 3037. No significant defects were detected in either mitosis or meiosis of the transgenic plants. The overlapping of GFP signals and anti-CENH3 foci in both mitotic and meiotic cells from T0 and T1 generation plants indicated that GFP had been successfully fused with CENH3, so the GFP signals can well represent the CENH3 locations on each chromosome. To evaluate the applicability of the transgenic plants to other genetic studies, fluorescence in situ hybridization (FISH) using rice centromeric tandem repetitive sequence CentO as the probe was conducted on the zygotene chromosomes of pollen mother cells (PMCs). It has been revealed that the GFP signals are overlapping with CentO FISH signals, showing that CentO is one of the key elements constituting rice functional centromeres. Immunofluorescent staining using anti-α-tublin antibody and anti-PAIR2 antibody on the chromosomes during mitosis and meiosis stages of the transgenic plants further reveals that OsCENH3-GFP transgenic plants can be widely used for studying rice molecular biology, especially for tagging functional centromeres in both living cells and tissues.     

Production of transgenic rabbits, sheep and pigs by microinjection

Direct microinjection has been used to introduce foreign DNA into a number of terminally differentiated cell types as well as embryos of several species including sea urchin, Candida elegans, Xenopus, Drosophila and mice. Various genes have been successfully introduced into mice including constructs consisting of the mouse metallothionein-I (MT) promoter/regulator region fused to either the rat or human growth hormone (hGH) structural genes. Transgenic mice harbouring such genes commonly exhibit high, metal-inducible levels of the fusion messenger RNA in several organs, substantial quantities of the foreign growth hormone in serum and enhanced growth. In addition, the gene is stably incorporated into the germ line, making the phenotype heritable. Because of the scientific importance and potential economic value of transgenic livestock containing foreign genes, we initiated studies on large animals by microinjecting the fusion gene, MT-hGH, into the pronuclei or nuclei of eggs from superovulated rabbits, sheep and pigs. We report here integration of the gene in all three species and expression of the gene in transgenic rabbits and pigs.     

Generating of rice OsCENH3-GFP transgenic plants and their genetic applications

In order to investigate rice functional centromeres, OsCENH3-GFP chimeric gene was constructed and transformed into the indica rice variety, Zhongxian 3037, mediated by Agrobacturium. The integration of the exogenous genes in the transgenic plants was confirmed by PCR and Southern blotting. The transgenic plants grow normally during their whole life time, just like Zhongxian 3037. No significant defects were detected in either mitosis or meiosis of the transgenic plants. The overlapping of GFP signals and anti-CENH3 foci in both mitotic and meiotic cells from T0 and T1 generation plants indicated that GFP had been successfully fused with CENH3, so the GFP signals can well represent the CENH3 locations on each chromosome. To evaluate the applicability of the transgenic plants to other genetic studies, fluorescence in situ hybridization （FISH） using rice centromeric tandem repetitive sequence CentO as the probe was conducted on the zygotene chromosomes of pollen mother cells （PMCs）. It has been revealed that the GFP signals are overlapping with CentO FISH signals, showing that CentO is one of the key elements constituting rice functional centromeres. Immunofluorescent staining using anti-o-tublin antibody and anti-PAIR2 antibody on the chromosomes during mitosis and meiosis stages of the transgenic plants further reveals that OsCENH3-GFP transgenic plants can be widely used for studying rice molecular biology, especially for tagging functional centromeres in both living cells and tissues.