The Production of Marker-Free Genetically Engineered Broccoli with Sense and Antisense ACC synthase 1 and ACC oxidases 1 and 2 to Extend Shelf-Life

The production of transgenic broccoli (Brassica oleracea) with increased shelf-life using an Agrobacterium rhizogenes-mediated co-transformation protocol is reported. An Agrobacterium rhizogenes Ri vector, pRi1855:GFP was constructed to allow expression of the green fluorescent protein to identify insertion of Ri T_L-DNA into plant cells. The Brassica oleracea ACC synthase 1 and ACC oxidase 1 and 2 cDNAs in sense and antisense orientations were co-transformed into GDDH33, a doubled haploid calabrese-broccoli cultivar. Transformation efficiency was 3.26%, producing 150 transgenic root lines, of which 18 were regenerated into mature plants. The floral buds from T₀ broccoli heads were assayed for post-harvest production of ethylene and chlorophyll levels. Buds from T₀ lines transformed with ACC oxidase 1 and 2 constructs produced significantly less post-harvest ethylene at 20 °C than the untransformed plants and chlorophyll loss was significantly reduced over a 96 h post-harvest period. The T₀ plants transformed with sense and antisense ACC synthase 1 had a significantly reduced 24 h post-harvest ethylene peak and delayed chlorophyll loss. A positive correlation between post-harvest bud ethylene production and chlorophyll loss was described by a regression. This demonstrates that the shelf-life of a very perishable vegetable may be increased up to 2 days at 20 °C by reducing post-harvest ethylene production.     

Cytoplasmic transduction peptide (CTP): new approach for the delivery of biomolecules into cytoplasm in vitro and in vivo

The protein transduction domain (PTD) of HIV-1 TAT has been extensively documented with regard to its membrane transduction potential, as well as its efficient delivery of biomolecules in vivo. However, the majority of PTD and PTD-conjugated molecules translocate to the nucleus rather than to the cytoplasm after transduction, due to the functional nuclear localization sequence (NLS). Here, we report a cytoplasmic transduction peptide (CTP), which was deliberately designed to ensure the efficient cytoplasmic delivery of the CTP-fused biomolecules. In comparison with PTD, CTP and its fusion partners exhibited a clear preference for cytoplasmic localization, and also markedly enhanced membrane transduction potential. Unlike the mechanism underlying PTD-mediated transduction, CTP-mediated transduction occurs independently of the lipid raft-dependent macropinocytosis pathway. The CTP-conjugated Smac/DIABLO peptide (Smac-CTP) was also shown to be much more efficient than Smac-PTD in the blockage of the antiapoptotic properties of XIAP, suggesting that cytoplasmic functional molecules can be more efficiently targeted by CTP-mediated delivery. In in vivo trafficking studies, CTP-fused beta-gal exhibited unique organ tropisms to the liver and lymph nodes when systemically injected into mice, whereas PTD-beta-gal exhibited no such tropisms. Taken together, our findings implicate CTP as a novel delivery peptide appropriate for (i) molecular targeting to cytoplasmic compartments in vitro, (ii) the development of class I-associated CTL vaccines, and (iii) special drug delivery in vivo, without causing any untoward effects on nuclear genetic material.     

The importance of valency in enhancing the import and cell routing potential of protein transduction domain-containing molecules

Protein transduction domains (PTDs) are peptides that afford the internalization of cargo macromolecules (including plasmid DNA, proteins, liposomes, and nanoparticles). In the case of polycationic peptides, the efficiency of PTDs to promote cellular uptake is directly related to their molecular mass or their polyvalent presentation. Similarly, the efficiency of routing to the nucleus increases with the number of nuclear localization signals (NLS) associated with a cargo. The quantitative enhancement, however, depends on the identity of the PTD sequence as well as the targeted cell type. Thus the choice and multivalent presentation of PTD and NLS sequences are important criteria guiding the design of macromolecules intended for specific intracellular localization. This review outlines synthetic and recombinant strategies whereby PTDs and signal sequences can be assembled into multivalent peptide dendrimers and promote the uptake and routing of their cargoes. In particular, the tetramerization domain of the tumour suppressor p53 (p53^tet) is emerging as a useful scaffold to present multiple routing and targeting moieties. Short cationic peptides fused to the 31-residue long p53^tet sequence resulted in tetramers displaying a significant enhancement (up to 1000 fold) in terms of their ability to be imported into cells and delivered to the cell nucleus in relation to their monomeric analogues. The design of future polycationic peptide dendrimers as effective delivering vehicles will need to incorporate selective cell targeting functions and provide solutions to the issue of endosomal entrapment.     

转反义PG基因番茄果实细胞结构变化的研究

经细胞学观察发现 ,转反义PG基因番茄果实在不同成熟期及存放前后 ,其果皮外面几层细胞的厚度都比未转基因的厚 1～ 5 μm ,细胞结构、细胞质和细胞核等的状态都有明显区别。尤以贮存后更为明显 ,未转基因果实的果皮细胞结构解体、细胞质凝聚、细胞核变的模糊程度都比转基因的严重。经外源乙烯处理后 ,转基因和未转基因果实的细胞结构也有相似的变化。结果表明 :反义PG基因的转入降低了PG活性 ,并且减弱了外源乙烯的作用 ,延缓了果实的衰老 ,提高了耐贮性能 ,从而起到果实保鲜作用     

Lipid-modified oligonucleotide conjugates: Insights into gene silencing,interaction with model membranes and cellular uptake mechanisms

The ability of oligonucleotides to silence specific genes or inhibit the biological activity of specific proteins has generated great interest in their use as research tools and therapeutic agents. Unfortunately, their biological applications meet the limitation of their poor cellular accessibility. Developing an appropriate delivery system for oligonucleotides is essential to achieve their efficient cellular uptake. In the present work a series of phosphorothioate lipid–oligonucleotide hybrids were synthesized introducing covalently single or double lipid tails at both 3′- and 5′-termini of an antisense oligonucleotide. Gene transfections in cultured cells showed antisense luciferase inhibition without the use of a transfecting agent for conjugates modified with the double-lipid tail at 5′-termini. The effect of the double lipid-tailed modification was further studied in detail in several model membrane systems as well as in cellular uptake experiments. During these studies the spontaneous formation of self-assembled microstructures is clearly observed. Lipidation allowed the efficient incorporation of the oligonucleotide in HeLa cells by a macropinocytosis mechanism without causing cytotoxicity in cells or altering the binding properties of the oligonucleotide conjugates. In addition, both single- and double-tailed compounds showed a similar behavior in lipid model membranes, making them useful in nucleotide-based technologies.     

Antisense downregulation of polyphenol oxidase results in enhanced disease susceptibility

Polyphenol oxidases (PPOs; EC 1.14.18.1 or EC 1.10.3.2) catalyze the oxidation of phenolics to quinones, highly reactive intermediates whose secondary reactions are responsible for much of the oxidative browning that accompanies plant senescence, wounding, and responses to pathogens. To assess the impact of PPO expression on resistance to Pseudomonas syringae pv. tomato we introduced a chimeric antisense potato PPO cDNA into tomato (Lycopersicon esculentum L.). Oxidation of caffeic acid, the dominant o-diphenolic aglycone of tomato foliage, was decreased ca. 40-fold by antisense expression of PPO. All members of the PPO gene family were downregulated: neither immunoreactive PPO nor PPO-specific mRNA were detectable in the transgenic plants. In addition, the antisense PPO construct suppressed inducible increases in PPO activity. Downregulation of PPO in antisense plants did not affect growth, development, or reproduction of greenhouse-grown plants. However, antisense PPO expression dramatically increased susceptibility to P. syringae expressing the avirulence gene avrPto in both Pto and pto backgrounds. In a compatible (pto) interaction, plants constitutively expressing an antisense PPO construct exhibited a 55-fold increase in bacterial growth, three times larger lesion area, and ten times more lesions cm^–2 than nontransformed plants. In an incompatible (Pto) interaction, antisense PPO plants exhibited 100-fold increases in bacterial growth and ten times more lesions cm^–2 than nontransformed plants. Although it is not clear whether hypersusceptibility of antisense plants is due to low constitutive PPO levels or failure to induce PPO upon infection, these findings suggest a critical role for PPO-catalyzed phenolic oxidation in limiting disease development. As a preliminary effort to understand the role of induced PPO in limiting disease development, we also examined the response of PPO promoter::-glucuronidase constructs when plants are challenged with P. syringae in both Pto and pto backgrounds. While PPO B inducibility was the same in both compatible and incompatible interactions, PPO D, E and F were induced to higher levels and with different expression patterns in incompatible interactions.     

婴幼儿呼吸道合胞病毒感染的治疗

呼吸道合胞病毒(respiratory syncytial virus RSV)是引起婴幼儿毛细支气管炎最重要的病原体。其主要临床表现为喘息。部分患儿可出现反复喘息发作而发展为哮喘。对RSV感染尚无特效治疗药物,仍然以支持对症治疗为主,目前的研究热点是中西医结合治疗,反义基因治疗,高渗盐水雾化吸入治疗等。     

反义核酸技术应用及研究进展

伴随着先进的基因克隆和基因测序技术的出现,以及DNA合成技术的迅速发展,反义核酸技术已经渗透到了生物学的各个方面,表现出了诱人的前景。综述了反义核酸技术的作用机理及其在抗病毒、抗肿瘤、细胞凋亡、信号机制、中枢神经系统以及眼科等方面的运用以及研究进展。