植物瞬间表达系统与功能基因组学研究

结构基因组学和功能基因组学的发展使特定植物基因组和转录组序列的获取更为方便和快捷。随之而来的是对各种基因和调控序列的功能注释,探索植物生长和发育的遗传机理。表达和调控表达是遗传物质的自身语言和动态属性,因此通过植物细胞内表达来分析目标基因和序列的表达和调控行为是功能分析的主要立足点。除创造转基因植株外,近几年来植物细胞瞬间表达系统得到了广泛的使用,与基因重排、病毒诱导基因沉默和RNA干扰等新兴技术的结合使其在植物功能基因组研究中扮演了越来越重要的角色。     

Knockdown of berberine bridge enzyme by RNAi accumulates (<Emphasis Type="Italic">S</Emphasis>)-reticuline and activates a silent pathway in cultured California poppy cells

Reticuline is a key compound in the biosynthetic pathway for isoquinoline alkaloids in plants, which include morphine, codeine and berberine. We established cultured California poppy (Eschscholzia californica) cells, in which berberine bridge enzyme (BBE) was knocked down by RNA interference, to accumulate the important key intermediate reticuline. Both BBE mRNA accumulation and enzyme activity were effectively suppressed in transgenic cells. In these transgenic cells, end-products of isoquinoline alkaloid biosynthesis, such as sanguinarine, were considerably reduced and reticuline was accumulated at a maximum level of 310 μg/g-fresh weight. In addition, 1 g-fresh weight of these cells secreted significant amounts of reticuline into the medium, with a maximum level of 6 mg/20 mL culture medium. These cells also produced a methylated derivative of reticuline, laudanine, which could scarcely be detected in control cells. We discuss the potential application of RNAi technology in metabolic modification and the flexibility of plant secondary metabolism.     

Production of red-flowered plants by genetic engineering of multiple flavonoid biosynthetic genes

Orange- to red-colored flowers are difficult to produce by conventional breeding techniques in some floricultural plants. This is due to the deficiency in the formation of pelargonidin, which confers orange to red colors, in their flowers. Previous researchers have reported that brick-red colored flowers can be produced by introducing a foreign dihydroflavonol 4-reductase (DFR) with different substrate specificity in Petunia hybrida, which does not accumulate pelargonidin pigments naturally. However, because these experiments used dihydrokaempferol (DHK)-accumulated mutants as transformation hosts, this strategy cannot be applied directly to other floricultural plants. Thus in this study, we attempted to produce red-flowered plants by suppressing two endogenous genes and expressing one foreign gene using tobacco as a model plant. We used a chimeric RNAi construct for suppression of two genes (flavonol synthase [FLS] and flavonoid 3′-hydroxylase [F3′H]) and expression of the gerbera DFR gene in order to accumulate pelargonidin pigments in tobacco flowers. We successfully produced red-flowered tobacco plants containing high amounts of additional pelargonidin as confirmed by HPLC analysis. The flavonol content was reduced in the transgenic plants as expected, although complete inhibition was not achieved. Expression analysis also showed that reduction of the two-targeted genes and expression of the foreign gene occurred simultaneously. These results demonstrate that flower color modification can be achieved by multiple gene regulation without use of mutants if the vector constructs are designed resourcefully. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reversal of BCRP-mediated multidrug resistance by stable expression of small interfering RNAs

Breast cancer resistance protein (BCRP) is an ATP-binding cassette multidrug transporter that confers resistance to various anticancer drugs like Mitoxantrone. Overexpression of BCRP confers multidrug resistance (MDR) in cancer cells and is a frequent impediment to successful chemotherapy. For stable reversal of BCRP-depending MDR by RNA interference technology, a hU6-RNA gene promoter-driven expression vector encoding anti-BCRP short hairpin RNA (shRNA) molecules was constructed. By treating endogenously and exogenously expresses high levels of BCRP cells with these constructs, expression of the targeted BCRP-encoding mRNA, and transport protein was inhibited completely. Furthermore, the accumulation of mitoxantrone in the anti-BCRP shRNA-treated cells increased. And the sensitivity to mitoxantrone of anti-BCRP shRNA-treated cells is increased 14.6-fold and 2.44-fold respectively compared to their control (P < 0.05). These data indicated that stable shRNA-mediated RNAi could be tremendously effective in reversing BCRP-mediated MDR and showed promises in overcoming MDR by gene therapeutic applications.     

Redundant ecdysis regulatory functions of three nuclear receptor HR3 isoforms in the direct-developing insect Blattella germanica

In hemimetabolous insects, the molecular basis of the 20-hydroxyecdysone (20E)-triggered genetic hierarchy is practically unknown. In the cockroach Blattella germanica, we had previously characterized one isoform of the ecdysone receptor, BgEcR-A, and two isoforms of its heterodimeric partner, BgRXR-S and BgRXR-L. One of the early-late genes of the 20E-triggered genetic hierarchy, is HR3. In the present paper, we report the discovery of three isoforms of HR3 in B. germanica, that were named BgHR3-A, BgHR3-B(1) and BgHR3-B(2). Expression studies in prothoracic gland, epidermis and fat body indicate that the expression of the three isoforms coincides with the peak of circulating ecdysteroids at each nymphal instar. Experiments in vitro with fat body tissue have shown that 20E induces the expression of BgHR3 isoforms, and that incubation with 20E and the protein inhibitor cycloheximide does not inhibit the induction, which indicates that the effect of 20E on BgHR3 activation is direct. This has been further confirmed by RNAi in vivo of BgEcR-A, which has shown that this nuclear receptor is required to fully activate the expression of BgHR3. RNAi has been also used to demonstrate the functions of BgHR3 in ecdysis. Nymphs with silenced BgHR3 completed the apolysis but were unable to ecdyse (they had duplicated and superimposed the mouth parts, the hypopharinge, the tracheal system and the cuticle layers). This indicates that BgHR3 is directly involved in ecdysis. Finally, RNAi of specific isoforms has showed that they are functionally redundant, at least regarding the ecdysis process.     

Involvement of vasa homolog in germline recruitment from coelomic stem cells in budding tunicates

We investigated the mechanism by which germline cells are recruited in every asexual reproductive cycle of the budding tunicate Polyandrocarpa misakiensis using a vasa homolog (PmVas) as the germline-specific probe. A presumptive gonad of Polyandrocarpa arose as a loose cell aggregate in the ventral hemocoel of a 1-week-old developing zooid. It developed into a compact clump of cells and then separated into two lobes, each differentiating into the ovary and the testis. The ovarian tube that was formed at the bottom of the ovary embedded the oogonia and juvenile oocytes, forming the germinal epithelium. PmVas was expressed strongly by loose cell aggregates, compact clumps, and peripheral germ cells in the testis and germinal epithelium. No signals were detected in growing buds and less than 1-week-old zooids, indicating that germ cells arise de novo in developing zooids of P. misakiensis. Cells of the loose cell aggregates were 5–6 μm in diameter. They looked like undifferentiated hemoblasts in the hemocoel. To examine the involvement of PmVas in the germline recruitment at postembryonic stages, both growing buds and 1-week-old developing zooids were soaked with double-stranded PmVas RNA. The growing buds developed into fertile zooids expressing PmVas, whereas the 1-week-old zooids developed into sterile zooids that did not express PmVas. In controls (1-week-old zooids) soaked with double-stranded lacZ RNA, the gonad developed normally. These results strongly suggest that in P. misakiensis, PmVas plays a decisive role in switching from coelomic stem cells to germ cells.     

Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion

The main function of the photosynthetic process is to capture solar energy and to store it in the form of chemical 'fuels'. Increasingly, the photosynthetic machinery is being used for the production of biofuels such as bio-ethanol, biodiesel and bio-H₂. Fuel production efficiency is directly dependent on the solar photon capture and conversion efficiency of the system. Green algae (e.g. Chlamydomonas reinhardtii ) have evolved genetic strategies to assemble large light-harvesting antenna complexes (LHC) to maximize light capture under low-light conditions, with the downside that under high solar irradiance, most of the absorbed photons are wasted as fluorescence and heat to protect against photodamage. This limits the production process efficiency of mass culture. We applied RNAi technology to down-regulate the entire LHC gene family simultaneously to reduce energy losses by fluorescence and heat. The mutant Stm3LR3 had significantly reduced levels of LHCI and LHCII mRNAs and proteins while chlorophyll and pigment synthesis was functional. The grana were markedly less tightly stacked, consistent with the role of LHCII. Stm3LR3 also exhibited reduced levels of fluorescence, a higher photosynthetic quantum yield and a reduced sensitivity to photoinhibition, resulting in an increased efficiency of cell cultivation under elevated light conditions. Collectively, these properties offer three advantages in terms of algal bioreactor efficiency under natural high-light levels: (i) reduced fluorescence and LHC-dependent heat losses and thus increased photosynthetic efficiencies under high-light conditions; (ii) improved light penetration properties; and (iii) potentially reduced risk of oxidative photodamage of PSII.     

Involvement of a putative allatostatin in regulation of juvenile hormone titer and the larval development in Leptinotarsa decemlineata (Say)

Juvenile hormone III (JH III) plays primary roles in regulation of metamorphosis, reproduction and diapause in Leptinotarsa decemlineata, a notorious defoliator of potato. The neurosecretory cell-borne substance(s) negatively affects the final two steps in JH biosynthesis, catalyzed respectively by an epoxidase CYP15A1 and a juvenile hormone acid methyltransferase (JHAMT). In a few insect species other than L. decemlineata, the inhibitory substance is allatostatin (AS) neuropeptide. In this study, two putative AS genes encoding LdAS-C and LdAS-B precursors were cloned. Both LdAS-C and LdAS-B were expressed in the egg, larvae, pupae and adults, and highly expressed in the brain and the gut. Dietary introduction of double-stranded RNAs (dsRNAs) targeting LdAS-C and LdAS-B successfully knocked down respective target genes. Ingestion during 3 and 6 consecutive days of dsLdAS-C significantly increased the LdJHAMT mRNA levels by 3.8 and 9.9 fold respectively. In contrast, ingestion of dsLdAS-B only slightly increased the LdJHAMT expression level by 1.1 and 1.7 fold. Moreover, after one, two and three days' ingestion of dsLdAS-C, the relative JH levels in the hemolymph of treated larvae were 2.5, 4.2 and 1.9 fold higher than those in control beetles. Furthermore, ingestion of dsLdAS-C and dsLdAS-B significantly affected larval growth and delayed larval development. Thus, we provide a line of experimental evidence in L. decemlineata to support the concept that AS-C acts as an allatostatin and inhibit JH biosynthesis.