Protein aggregates are transported to vacuoles by a macroautophagic mechanism in nutrient-starved plant cells

When a fusion protein of cytochrome b5 (Cyt b5) and the red fluorescent protein (RFP) are expressed in tobacco BY-2 cells, the expressed protein forms intracellular aggregates that emit red fluorescence. When such cells are grown to the stationary phase or incubated in nutrient limited medium, RFP fluorescence can be detected in the vacuolar lumen. We investigated this transport mechanism using a limited-nitrogen model. E-64 and 3-methyladenine, which inhibit autophagic processes, blocked the transport of the RFP signal to the vacuole. We next traced the autophagic process in tobacco cells using YFP fused with the tobacco Atg8 homologue (YFP-NtAtg8) and analyzed the contribution of autophagy to the vacuolar transport of the aggregates. Under limited-nitrogen conditions, the aggregates were degraded in preference to other organelles, and the autophagosomes colocalized with the aggregates at a higher frequency than with mitochondria. This is the first demonstration that selective macroautophagic degradation can occur in plant cells.     

Fluorescent reporter proteins for the tonoplast and the vacuolar lumen identify a single vacuolar compartment in Arabidopsis cells

We generated fusions between three Arabidopsis (Arabidopsis thaliana) tonoplast intrinsic proteins (TIPs; alpha-, gamma-, and delta-TIP) and yellow fluorescent protein (YFP). We also produced soluble reporters consisting of the monomeric red fluorescent protein (RFP) and either the C-terminal vacuolar sorting signal of phaseolin or the sequence-specific sorting signal of proricin. In transgenic Arabidopsis leaves, mature roots, and root tips, all TIP fusions localized to the tonoplast of the central vacuole and both of the lumenal RFP reporters were found within TIP-delimited vacuoles. In embryos from developing, mature, and germinating seeds, all three TIPs localized to the tonoplast of protein storage vacuoles. To determine the temporal TIP expression patterns and to rule out mistargeting due to overexpression, we generated plants expressing YFP fused to the complete genomic sequences of the three TIP isoforms. In transgenic Arabidopsis, gamma-TIP expression was limited to vegetative tissues, but specifically excluded from root tips, whereas alpha-TIP was exclusively expressed during seed maturation. delta-TIP was expressed in vegetative tissues, but not root tips, at a later stage than gamma-TIP. Our findings indicate that, in the Arabidopsis tissues analyzed, two different vacuolar sorting signals target soluble proteins to a single vacuolar location. Moreover, TIP isoform distribution is tissue and development specific, rather than organelle specific.     

In vivo imaging and quantitative monitoring of autophagic flux in tobacco BY-2 cells

Autophagy has been shown to play essential roles in the growth, development and survival of eukaryotic cells. However, simple methods for quantification and visualization of autophagic flux remain to be developed in living plant cells. Here, we analyzed the autophagic flux in transgenic tobacco BY-2 cell lines expressing fluorescence-tagged NtATG8a as a marker for autophagosome formation. Under sucrose-starved conditions, the number of punctate signals of YFP-NtATG8a increased, and the fluorescence intensity of the cytoplasm and nucleoplasm decreased. Conversely, these changes were not observed in BY-2 cells expressing a C-terminal glycine deletion mutant of the NtATG8a protein (NtATG8aΔG). To monitor the autophagic flux more easily, we generated a transgenic BY-2 cell line expressing NtATG8a fused to a pH-sensitive fluorescent tag, a tandem fusion of the acid-insensitive RFP and the acid-sensitive YFP. In sucrose-rich conditions, both fluorescent signals were detected in the cytoplasm and only weakly in the vacuole. In contrast, under sucrose-starved conditions, the fluorescence intensity of the cytoplasm decreased, and the RFP signal clearly increased in the vacuole, corresponding to the fusion of the autophagosome to the vacuole and translocation of ATG8 from the cytoplasm to the vacuole. Moreover, we introduce a novel simple easy way to monitor the autophagic flux non-invasively by only measuring the ratio of fluorescence of RFP and YFP in the cell suspension using a fluorescent image analyzer without microscopy. The present in vivo quantitative monitoring system for the autophagic flux offers a powerful tool for determining the physiological functions and molecular mechanisms of plant autophagy induced by environmental stimuli.     

Dynamic response of prevacuolar compartments to brefeldin a in plant cells

Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs) in the secretory pathway. Using transgenic tobacco (Nicotiana tabacum) Bright-Yellow-2 (BY-2) cells expressing membrane-anchored yellow fluorescent protein (YFP) reporters marking Golgi or PVCs, we have recently demonstrated that PVCs are mobile multivesicular bodies defined by vacuolar sorting receptor proteins. Here, we demonstrate that Golgi and PVCs have different sensitivity in response to brefeldin A (BFA) treatment in living tobacco BY-2 cells. BFA at low concentrations (5-10 microg mL(-1)) induced YFP-marked Golgi stacks to form both endoplasmic reticulum-Golgi hybrid structures and BFA-induced aggregates, but had little effect on YFP-marked PVCs in transgenic BY-2 cells at both confocal and immunogold electron microscopy levels. However, BFA at high concentrations (50-100 microg mL(-1)) caused both YFP-marked Golgi stacks and PVCs to form aggregates in a dose- and time-dependent manner. Normal Golgi or PVC signals can be recovered upon removal of BFA from the culture media. Confocal immunofluorescence and immunogold electron microscopy studies with specific organelle markers further demonstrate that the PVC aggregates are distinct, but physically associated, with Golgi aggregates in BFA-treated cells and that PVCs might lose their internal vesicle structures at high BFA concentration. In addition, vacuolar sorting receptor-marked PVCs in root-tip cells of tobacco, pea (Pisum sativum), mung bean (Vigna radiata), and Arabidopsis (Arabidopsis thaliana) upon BFA treatment are also induced to form similar aggregates. Thus, we have demonstrated that the effects of BFA are not limited to endoplasmic reticulum and Golgi, but extend to PVC in the endomembrane system, which might provide a quick tool for distinguishing Golgi from PVC for its identification and characterization, as well as a possible new tool in studying PVC-mediated protein traffic in plant cells.     

Generation of Two-color Transgenic Zebrafish Using the Green and Red Fluorescent Protein Reporter Genes gfp and rfp

Two tissue-specific promoters were used to express both green fluorescent protein (GFP) and red fluorescent protein (RFP) in transgenic zebrafish embryos. One promoter (CK), derived from a cytokeratin gene, is active specifically in skin epithelia in embryos, and the other promoter (MLC) from a muscle-specific gene encodes a myosin light chain 2 polypeptide. When the 2 promoters drove the 2 reporter genes to express in the same embryos, both genes were faithfully expressed in the respective tissues, skin or muscle. When the 2 fluorescent proteins were expressed in the same skin or muscle cells under the same promoter, GFP fluorescence appeared earlier than RFP fluorescence in both skin and muscle tissues, probably owing to a higher detection sensitivity of GFP. However, RFP appeared to be more stable as its fluorescence steadily increased during development. Finally, F₁ transgenic offspring were obtained expressing GFP in skin cells under the CK promoter and RFP in muscle cells under the MLC promoter. Our study demonstrates the feasibility of monitoring expression of multiple genes in different tissues in the same transgenic organism.     

Automated cell isolation from photodegradable hydrogel based on fluorescence image analysis

We report an automated cell-isolation system based on fluorescence image analysis of cell aggregates cultured in a photodegradable hydrogel. The system incorporates cell culture in a humidified atmosphere with controlled CO₂ concentration and temperature, image acquisition and analysis, micropatterned light exposure, and cell collection by pipetting. Cell aggregates were cultured on hydrogels, and target cells were selected by phase contrast and fluorescence image analysis. After degradation of the hydrogel by exposure to micropatterned UV light, cell aggregates were transferred to a collection vessel by robotic pipetting. We assessed the system for hydrogel degradation, recovery of target cells, and contamination by off-target cells. We demonstrated two practical applications of our method: (i) in cell aggregates from MCF-7-RFP strains in which 18.8% of cells produced red fluorescent protein (RFP), we successfully obtained 14 proliferative fluorescence-positive cell aggregates from 31-wells, and all of the isolated strains produced a higher proportion of RFP production than the original populations; (ii) after fluorescent immunostaining of human epidermal growth factor receptor 2 (HER2) in cancer cells, we successfully isolated HER2-positive cells from a mixed population of HER2-positive and -negative cells, and gene sequence analysis confirmed that the isolated cells mainly contained the target cells.     

Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer

The biochemical and biophysical properties of a red fluorescent protein from a Discosoma species (DsRed) were investigated. The recombinant DsRed expressed in E. coli showed a complex absorption spectrum that peaked at 277, 335, 487, 530, and 558 nm. Excitation at each of the absorption peaks produced a main emission peak at 583 nm, whereas a subsidiary emission peak at 500 nm appeared with excitation only at 277 or 487 nm. Incubation of E. coli or the protein at 37 degrees C facilitated the maturation of DsRed, resulting in the loss of the 500-nm peak and the enhancement of the 583-nm peak. In contrast, the 500-nm peak predominated in a mutant DsRed containing two amino acid substitutions (Y120H/K168R). Light-scattering analysis revealed that DsRed proteins expressed in E. coli and HeLa cells form a stable tetramer complex. DsRed in HeLa cells grown at 37 degrees C emitted predominantly at 583 nm. The red fluorescence was imaged using a two-photon laser (Nd:YLF, 1047 nm) as well as a one-photon laser (He:Ne, 543.5 nm). When fused to calmodulin, the red fluorescence produced an aggregation pattern only in the cytosol, which does not reflect the distribution of calmodulin. Despite the above spectral and structural complexity, fluorescence resonance energy transfer (FRET) between Aequorea green fluorescent protein (GFP) variants and DsRed was achieved. Dynamic changes in cytosolic free Ca2+ concentrations were observed with red cameleons containing yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), or Sapphire as the donor and RFP as the acceptor, using conventional microscopy and one- or two-photon excitation laser scanning microscopy. Particularly, the use of the Sapphire-DsRed pair rendered the red cameleon tolerant of acidosis occurring in hippocampal neurons, because both Sapphire and DsRed are extremely pH-resistant.     

Overexpression of a plant reticulon remodels the lumen of the cortical endoplasmic reticulum but does not perturb protein transport

We have cloned a member of the reticulon (RTN) family of Arabidopsis thaliana (RTNLB13). When fused to yellow fluorescent protein (YFP) and expressed in tobacco leaf epidermal cells, RTNLB13 is localized in the endoplasmic reticulum (ER). Coexpression of a soluble ER luminal marker reveals that YFP-tagged, myc-tagged or untagged RTNLB13 induces severe morphological changes to the lumen of the ER. We show, using fluorescence recovery after photobleaching (FRAP) analysis, that RTNLB13 overexpression greatly reduces diffusion of soluble proteins within the ER lumen, possibly by introducing constrictions into the membrane. In spite of this severe phenotype, Golgi shape, number and dynamics appear unperturbed and secretion of a reporter protein remains unaffected.     

Actin microfilaments regulate vacuolar structures and dynamics: dual observation of actin microfilaments and vacuolar membrane in living tobacco BY-2 Cells

Actin microfilaments (MFs) participate in many fundamental processes in plant growth and development. Here, we report the co-localization of the actin MF and vacuolar membrane (VM), as visualized by vital VM staining with FM4-64 in living tobacco BY-2 cells stably expressing green fluorescent protein (GFP)-fimbrin (BY-GF11). The MFs were intensively localized on the VM surface and at the periphery of the cytoplasmic strands rather than at their center. The co-localization of MFs and VMs was confirmed by the observation made using transient expression of red fluorescent protein (RFP)-fimbrin in tobacco BY-2 cells stably expressing GFP-AtVam3p (BY-GV7) and BY-2 cells stably expressing gamma-tonoplast intrinsic protein (gamma-TIP)-GFP fusion protein (BY-GG). Time-lapse imaging revealed dynamic movement of MF structures which was parallel to that of cytoplasmic strands. Disruption of MF structures disorganized cytoplasmic strand structures and produced small spherical vacuoles in the VM-accumulating region. Three-dimensional reconstructions of the vacuolar structures revealed a disconnection of these small spherical vacuoles from the large vacuoles. Real-time observations and quantitative image analyses demonstrated rapid movements of MFs and VMs near the cell cortex, which were inhibited by the general myosin ATPase inhibitor, 2,3-butanedion monoxime (BDM). Moreover, both bistheonellide A (BA) and BDM treatment inhibited the reorganization of the cytoplasmic strands and the migration of daughter cell nuclei at early G1 phase, suggesting a requirement for the acto-myosin system for vacuolar morphogenesis during cell cycle progression. These results suggest that MFs support the vacuolar structures and that the acto-myosin system plays an essential role in vacuolar morphogenesis.     

Fluorescent and bimolecular-fluorescent protein tagging of genes at their native loci in Neurospora crassa using specialized double-joint PCR plasmids

The double-joint polymerase chain reaction (DJ-PCR) is a technique that can be used to construct vectors for targeted genome integration without laborious subcloning steps. Here we report the availability of plasmids that facilitate DJ-PCR-based construction of Neurospora crassa tagging vectors. These plasmids allow the creation of green or red fluorescent protein (GFP or RFP) tagging vectors for protein localization studies, as well as split-yellow fluorescent protein (YFP) tagging vectors for bimolecular fluorescence complementation (BiFC) analyses. We have demonstrated the utility of each plasmid with the tagging of known meiotic silencing proteins. Microscopic analysis of the tagged strains indicates that SMS-2 and QIP form macromolecular complexes in the perinuclear region during meiosis.     

Garlic virus X 11-kDa protein granules move within the cytoplasm and traffic a host protein normally found in the nucleolus

The subcellular localization of the 11-kDa protein (p11) encoded by ORF3 of Garlic virus X (GarVX; genus Allexivirus, family Alphaflexiviridae) was examined by confocal microscopy. Granules with intense fluorescence were visible on the endoplasmic reticulum when p11 fused with green or red fluorescent protein (GFP or RFP) was expressed in epidermal cells of Nicotiana benthamiana. Moreover, the p11-RFP granules moved in the cytoplasm, along the cell periphery and through the cell membranes to adjacent cells. A 17-kDa protein (p17) of garlic interacting with p11 was identified by yeast two-hybridization and bimolecular fluorescence complementation assay. When p17 fused to GFP was expressed in epidermal cells of N. benthamiana, it localized to the nucleolus. However, in the presence of GarVX p11, the distribution of p17 changed to that of p11, but did not appear to affect the pattern of movement of p11.     

Protein mislocalization in plant cells using a GFP‐binding chromobody

A key challenge in cell biology is to directly link protein localization to function. The green fluorescent protein (GFP)‐binding protein, GBP, is a 13‐kDa soluble protein derived from a llama heavy chain antibody that binds with high affinity to GFP as well as to some GFP variants such as yellow fluorescent protein (YFP). A GBP fusion to the red fluorescent protein (RFP), a molecule termed a chromobody, was previously used to trace in vivo the localization of various animal antigens. In this study, we extend the use of chromobody technology to plant cells and develop several applications for the in vivo study of GFP‐tagged plant proteins. We took advantage of Agrobacterium tumefaciens‐mediated transient expression assays (agroinfiltration) and virus expression vectors (agroinfection) to express functional GBP:RFP fusion (chromobody) in the model plant Nicotiana benthamiana. We showed that the chromobody is effective in binding GFP‐ and YFP‐tagged proteins in planta. Most interestingly, GBP:RFP can be applied to interfere with the function of GFP fusion protein and to mislocalize (trap) GFP fusions to the plant cytoplasm in order to alter the phenotype mediated by the targeted proteins. Chromobody technology, therefore, represents a new alternative technique for protein interference that can directly link localization of plant proteins to in vivo function.     

Functional expression of mung bean Ca2+/H+ antiporter in yeast and its intracellular localization in the hypocotyl and tobacco cells.

The Ca2+-transport activity and intracellular localization of the translation product of cDNA for mung bean Ca2+/H+ antiporter (VCAX1) were examined. When the cDNA was expressed in Saccharomyces cerevisiae that lacked its own genes for vacuolar Ca2+-ATPase and the antiporter, VCAX1 complemented the active Ca2+ transporters, and the microsomal membranes from the transformant showed high activity of the Ca2+/H+ antiporter. Treatment of the vacuolar membranes with a cross-linking reagent resulted in a clear band of the dimer detected with antibody specific for VCAX1p. The antibody was also used for immunolocalization of the antiporter in fractions obtained by sucrose-density-gradient centrifugation of the microsomal fraction from mung bean. The immunostained band was detected in the vacuolar membrane fraction and the slightly heavy fractions that exhibited activity of the Golgi marker enzyme. A fusion protein of VCAX1p and green fluorescent protein was expressed in tobacco cells. The green fluorescence was clearly observed on the vacuolar membrane and, in some cases, in the small vesicles. The subcellular fractionation of transformed tobacco cells confirmed the vacuolar membrane localization of the fusion protein. These results confirm that VCAX1p functions in the vacuolar membrane as a Ca2+/H+ antiporter and also suggest that VCAX1p may exist in the Golgi apparatus.     

Post-translational maturation of natural and drug-induced missorted phytohemagglutinin

The bean lectin phytohemagglutinin (PHA) was expressed in transgenic suspension-cultured BY-2 tobacco cells simultaneously with another recombinant vacuolar protein, the sweet potato sporamin. In contrast to previous observations in different transgenic plant systems when expressed in BY-2 tobacco cells, phytohemagglutinin is mostly but not exclusively targeted to the vacuole. Indeed, a small amount of recombinant phytohemagglutinin is secreted into the culture medium of tobacco cells. Furthermore part of this extracellular phytohemagglutinin has no lectin activity and presents an abnormal glycosylation consistent with higher accessibility of glycans N-linked to these extracellular phytohemagglutinin forms. Phytohemagglutinin secretion occurs regardless of recombinant protein expression level. Consequently, missorting in this case is due to an abnormal phytohemagglutinin conformation or oligomerization rather than to receptor saturation. The treatment of BY-2 cells with drugs, such as monensin and wortmannin, increases even more the transport of phytohemagglutinin to the cell surface through a general inhibition of the sorting mechanisms of vacuolar proteins. The sensitivity to wortmannin is similar for the sorting of phytohemagglutinin and endogenous tobacco chitinase and β-1,3-glucanase, suggesting that phytohemagglutinin and COOH-terminal propeptide mediated vacuolar sorting share similar mechanisms. A characterization of glycans N-linked to extracellular phytohemagglutinin secreted by monensin- or wortmannin-treated transgenic tobacco cells illustrates that in contrast with monensin, wortmannin completely inhibits the sorting of vacuolar proteins without having any effect on the efficiency of Golgi processing enzymes.     

Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists

Drug discovery by high-throughput screening is a promising approach to develop new therapies for the most common lethal genetic disease, cystic fibrosis. Because disease-causing mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) protein produce epithelial cells with reduced or absent Cl(-) permeability, the goal of screening is to identify compounds that restore cell Cl(-) transport. We have developed a rapid, quantitative screening procedure for analysis of CFTR-mediated halide transport in cells with the use of a conventional fluorescence plate reader. Doubly transfected cell lines were generated that express wild-type or mutant CFTR together with a yellow fluorescent protein (YFP)-based halide sensor. CFTR function was assayed from the time course of cell fluorescence in response to extracellular addition of 100 mM I(-) followed by forskolin, resulting in decreased YFP fluorescence due to CFTR-mediated I(-) entry. Cell lines were chosen, and conditions were optimized to minimize basal halide transport to maximize assay sensitivity. In cells cultured on 96-well plastic dishes, the assay gave reproducible halide permeabilities from well to well and could reliably detect a 2% activation of CFTR-dependent halide transport produced by low concentrations of forskolin. Applications of the assay are shown, including comparative dose-dependent CFTR activation by genistein, apigenin, 8-cyclopentyl-1,3-dipropylxanthine, IBMX, 8-methoxypsoralen, and milrinone as well as activation of alternative Cl(-) channels. The fluorescence assay and cell lines should facilitate the screening of novel CFTR activators and the characterization of alternative Cl(-) channels and transporters.     

Simultaneous visualization of two protein complexes in a single plant cell using multicolor fluorescence complementation analysis

Bimolecular fluorescence complementation (BiFC) is an approach used to analyze protein–protein interaction in vivo, in which non-fluorescent N-terminal and C-terminal fragments of a fluorescent protein are reconstituted to emit fluorescence only when they are brought together by interaction of two proteins to fuse both fragments. A method for simultaneous visualization of two protein complexes by multicolor BiFC with fragments from green fluorescent protein (GFP) and its variants such as cyan and yellow fluorescent proteins (CFP and YFP) was recently reported in animal cells. In this paper we describe a new strategy for simultaneous visualization of two protein complexes in plant cells using the multicolor BiFC with fragments from CFP, GFP, YFP and a red fluorescent protein variant (DsRed-Monomer). We identified nine different BiFC complexes using fragments of CFP, GFP and YFP, and one BiFC complex using fragments of DsRed-Monomer. Fluorescence complementation did not occur by combinations between fragments of GFP variants and DsRed-Monomer. Based on these findings, we achieved simultaneous visualization of two protein complexes in a single plant cell using two colored fluorescent complementation pairs (cyan/red, green/red or yellow/red).     

The N-terminal propeptide of the precursor to sporamin acts as a vacuole-targeting signal even at the C terminus of the mature part in tobacco cells.

An asparagine-proline-isoleucine-arginine-leucine (NPIRL) and its related sequences in the N-terminal propeptides (NTPP) of several plant vacuolar proteins, including that of sporamin from sweet potato (SPO) function as vacuole-targeting determinants in a manner that is distinct from the vacuole-targeting determinant in the CTPPs of other plant vacuolar proteins. When the mutant precursor to sporamin, SPO-NTPP (in which NTPP was moved to the C terminus of the mature part), was expressed in tobacco (Nicotiana tabacum) cells, the pro-form was efficiently targeted to the vacuole and the NTPP was cleaved off. Unlike the results obtained with the wild-type precursor, substitution of the NPIRL sequence in the C-terminally located NTPP to asparagine-proline-glycine-arginine-leucine in the SPO-isoleucine-28-to-glycine mutant resulted in missorting of less than 20% of the pro-form to the medium. Unlike the vacuolar transport of SPO-NTPP, the vacuolar transport of SPO-isoleucine-28-to-glycine was strongly inhibited by 33 microM wortmannin, which is similar to the C-terminal propeptide-mediated vacuolar transport. These results suggest that the vacuole-targeting function of the NPIRL sequence is not strictly dependent on its location at the N terminus of a protein and that the C-terminally located mutant NTPP acquired some physicochemical properties of the C-terminal vacuole-targeting sequence.     

A transgenic red fluorescent protein‐expressing nude mouse for color‐coded imaging of the tumor microenvironment

The tumor microenvironment (TME) is critical for tumor growth and progression. We have previously developed color‐coded imaging of the TME using a green fluorescent protein (GFP) transgenic nude mouse as a host. However, most donor sources of cell types appropriate for study in the TME are from mice expressing GFP. Therefore, a nude mouse expressing red fluorescent protein (RFP) would be an appropriate host for transplantation of GFP‐expressing stromal cells as well as double‐labeled cancer cells expressing GFP in the nucleus and RFP in the cytoplasm, thereby creating a three‐color imaging model of the TME. The RFP nude mouse was obtained by crossing non‐transgenic nude mice with the transgenic C57/B6 mouse in which the β‐actin promoter drives RFP (DsRed2) expression in essentially all tissues. In crosses between nu/nu RFP male mice and nu/+ RFP female mice, the embryos fluoresced red. Approximately 50% of the offspring of these mice were RFP nude mice. In the RFP nude mouse, the organs all brightly expressed RFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, duodenum, the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart, and major arteries and veins. The skinned skeleton highly expressed RFP. The bone marrow and spleen cells were also RFP positive. GFP‐expressing human cancer cell lines, including HCT‐116‐GFP colon cancer and MDA‐MB‐435‐GFP breast cancer were orthotopically transplanted to the transgenic RFP nude mice. These human tumors grew extensively in the transgenic RFP nude mouse. Dual‐color fluorescence imaging enabled visualization of human tumor–host interaction. The RFP nude mouse model should greatly expand our knowledge of the TME. J. Cell. Biochem. 106: 279–284, 2009. © 2008 Wiley‐Liss, Inc.     

基于CRISPR/Cas9系统对干酪乳杆菌进行红色荧光蛋白标记

本实验利用CRISPR/Cas9系统对干酪乳杆菌(Lactobacillus casei) LC2W进行红色荧光蛋白(red fluorescent protein,RFP)标记,用于研究干酪乳杆菌在肠道内的分布和定植状况,评价其作为益生菌的功能。首先,基于本实验室已有的干酪乳杆菌CRISPR/Cas9编辑质粒pLCNICK-1628构建重组质粒pLCNICK-1628-RFP,电转入干酪乳杆菌LC2W感受态细胞中,使干酪乳杆菌基因组中的LC2W-1628基因被红色荧光蛋白基因替换,从而使干酪乳杆菌LC2W能表达出红色荧光蛋白。得到红色荧光标记的干酪乳杆菌LC2W突变株后,测定了其荧光强度-OD600标准曲线,发现RFP在干酪乳杆菌LC2W中能稳定表达。