Transport of UDP-galactose in plants. Identification and functional characterization of AtUTr1, an Arabidopsis thaliana UDP-galactos/UDP-glucose transporter

The synthesis of non-cellulosic polysaccharides and glycoproteins in the plant cell Golgi apparatus requires UDP-galactose as substrate. The topology of these reactions is not known, although the orientation of a plant galactosyltransferase involved in the biosynthesis of galactomannans in fenugreek is consistent with a requirement for UDP-galactose in the lumen of the Golgi cisternae. Here we provide evidence that sealed, right-side-out Golgi vesicles isolated from pea stems transport UDP-galactose into their lumen and transfer galactose, likely to polysaccharides and other acceptors. In addition, we identified and cloned AtUTr1, a gene from Arabidopsis thaliana that encodes a multitransmembrane hydrophobic protein similar to nucleotide sugar transporters. Northern analysis showed that AtUTr1 is indeed expressed in Arabidopsis. AtUTr1 is able to complement the phenotype of MDCK ricin-resistant cells; a mammalian cell line deficient in transport of UDP-galactose into the Golgi. In vitro assays using a Golgi-enriched vesicle fraction obtained from Saccharomyces cerevisiae expressing AtUTr1-MycHis is able to transport UDP-galactose but also UDP-glucose. AtUTr1- MycHis does not transport GDP-mannose, GDP-fucose, CMP-sialic acid, UDP-glucuronic acid, or UDP-xylose when expressed in S. cerevisiae. AtUTr1 is the first transporter described that is able to transport UDP-galactose and UDP-glucose. Thus AtUTr1 may play an important role in the synthesis of glycoconjugates in Arabidopsis that contain galactose and glucose.     

Reconstitution, identification, and purification of the rat liver golgi membrane GDP-fucose transporter

Glycosylation of glycoproteins, proteoglycans, and glycolipids occurring in the Golgi apparatus requires the translocation of nucleotide sugars from the cytosol into the lumen of the Golgi. Translocation is mediated by specific nucleotide sugar transporters, integral Golgi membrane proteins that regulate the above glycosylation reactions. A defect in GDP-fucose transport into the lumen of the Golgi apparatus has been recently identified in a patient affected by leukocyte adhesion deficiency type II syndrome (Lubke, T., Marquardt, T., von Figura, K., and Korner, C. (1999) J. Biol. Chem. 274, 25986-25989). We have now identified and purified the rat liver Golgi membrane GDP-fucose transporter, a protein with an apparent molecular mass of 39 kDa, by a combination of column chromatography, native functional size determination on a glycerol gradient, and photoaffinity labeling with 8-azidoguanosine-5'-[alpha-(32)P] triphosphate, an analog of GDP-fucose. The purified transporter appears to exist as a homodimer within the Golgi membrane. When reconstituted into phosphatidylcholine liposomes, it was active in GDP-fucose transport and was specifically photolabeled with 8-azidoguanosine-5'-[alpha-(32)P]triphosphate. Transport was also stimulated 2-3-fold after preloading proteoliposomes with GMP, the putative antiporter.     

Volume enlargement and recovery of Na+-dependent amino acid transport in proteoliposomes derived from Ehrlich ascites cell membranes

Na+-dependent amino acid transport can be reconstituted from solubilized Ehrlich cell plasma membranes by addition of asolectin vesicles, gel filtration, and a freeze-thaw cycle. Removal of phosphatidic acid (approximately 10% of the total lipid) by Ba2+ precipitation reduces the efficiency of reconstitution of Na+-dependent amino acid transport by approximately 73% and decreases intravesicular volume of the proteoliposomes by approximately 43%. The loss of transport activity is not due to exclusion of specific proteins during reconstitution. The phosphatidic acid-free liposomes are less permeable and require more time to attain an equilibrium distribution of solute. Transport activity and intravesicular volume can be restored to Ba2+-precipitated asolectin proteoliposomes by addition of egg-phosphatidic acid during reconstitution. The extent of recovery of transport activity is proportional to the change in intravesicular volume and depends on the amount of phosphatidic acid present. Replacement of phosphatidic acid with 20% phosphatidylserine or phosphatidylglycerol leads to increases in intravesicular volume with little or no increase in amino acid transport. Generation of phosphatidic acid in situ by treatment of Ba2+-precipitated proteoliposomes with phospholipase D also restored transport. The observed increase in transport activity (9-fold) is accompanied by a 46% increase in intravesicular volume, presumably caused by vesicle fusion. Phosphatidic acid is also required for successful reconstitution of Na+-dependent amino acid transport from pure phosphatidylcholine:phosphatidylethanolamine (1:1) mixtures with only a small change (approximately 16%) in intravesicular volume. The results provide evidence for both indirect and direct effects of phosphatidic acid on reconstitution of Na+-dependent amino acid transport. The indirect effects occur through enlargement of intravesicular volume, large vesicles showing higher rates of transport. However, there is also evidence to indicate a specific effect of phosphatidic acid on the Na+-dependent amino acid transporter, since other acidic lipids may change intravesicular volume without a commensurate change in transport activity.     

Functional reconstitution of halorhodopsin. Properties of halorhodopsin-containing proteoliposomes

A one-step purification method for halorhodopsin was developed. Functional proteoliposomes were prepared from this preparation using cholate, which is removed by dialysis in the presence of asolectin or the polar halobacterial lipids. Light-induced outward directed transport of chloride by halorhodopsin was followed by measuring passive proton efflux in the presence of uncoupler; initial rates and extents amounted to significant fractions of values obtained for halorhodopsin-containing cell envelope vesicles. The transport activity was much higher when cholate rather than octyl glucoside was used in the reconstitution. Since CD spectra in cholate but not in octyl glucoside showed band-splitting in the visible region, suggestive of exciton interaction between halorhodopsin monomers, the reconstitution may depend on an aggregate state of the halorhodopsin. The rate constants for three thermal steps in the halorhodopsin photocycle were greatly reduced in the detergent-solubilized samples, but they increased in the proteoliposomes to values similar to those for halorhodopsin in cell envelope vesicles. Thus, the reconstitution yields halorhodopsin with both photochemical and transport activities restored. Freeze-fracture electron micrographs of the proteoliposomes showed unilammellar liposomes with numerous particles of 100-150 A diameter at the fracture faces. These should correspond to halorhodopsin aggregates, formed in the bilayer in an apparently concentration-dependent manner.     

Solubilization and reconstitution of the renal phosphate transporter

Proteins from brush-border membrane vesicles of rabbit kidney cortex were solubilized with 1% octylglucoside (protein to detergent ratio, 1:4 (w/w). The solubilized proteins (80.2 +/- 2.3% of the original brush-border proteins, n = 10, mean +/- S.E.) were reconstituted into artificial lipid vesicles or liposomes prepared from purified egg yolk phosphatidylcholine (80%) and cholesterol (20%). Transport of Pi into the proteoliposomes was measured by rapid filtration in the presence of a Na+ or a K+ gradient (out greater than in). In the presence of a Na+ gradient, the uptake of Pi was significantly faster than in the presence of a K+ gradient. Na+ dependency of Pi uptake was not observed when the liposomes were reconstituted with proteins extracted from brush-border membrane vesicles which had been previously treated with papain, a procedure that destroys Pi transport activity. Measurement of Pi uptake in media containing increasing amounts of sucrose indicated that Pi was transported into an intravesicular (osmotically sensitive) space, although about 70% of the Pi uptake appeared to be the result of adsorption or binding of Pi. However, this binding of Pi was not dependent upon the presence of Na+. Both Na+-dependent transport and the Na+-independent binding of Pi were inhibited by arsenate. The initial Na+-dependent Pi transport rate in control liposomes of 0.354 nmol Pi/mg protein per min was reduced to 0.108 and 0 nmol Pi/mg protein per min in the presence of 1 and 10 mM arsenate, respectively. Future studies on reconstitution of Pi transport systems must analyze and correct for the binding of Pi by the lipids used in the formation of the proteoliposomes.     

Reconstitution of GDP-mannose transport activity with purified Leishmania LPG2 protein in liposomes

Activated nucleotide sugars required for the synthesis of glycoconjugates within the secretory pathway of eukaryotes are provided by the action of nucleotide sugar transporters (NSTs). Typically, NSTs are studied in microsomal preparations from wild-type or mutant lines; however, in this setting it can be difficult to assess NST properties because of the presence of glycosyltransferases and other interfering activities. Here we have engineered Leishmania donovani to express high levels of an active LPG2 Golgi GDP-Man transporter bearing a C-terminal polyhistidine tag. The functional LPG2-HIS was solubilized, purified by metal affinity chromatography, and reconstituted into phosphatidylcholine-containing liposomes using polystyrene SM-2 beads. The proteoliposomes exhibited robust GDP-Man transport activity with an apparent K(m) of 6.6 mum. Transport activity was enhanced by preloading of GMP and showed specificity for multiple substrates (GDP-Ara and GDP-Fuc). In contrast to the activity in crude microsomes, transport was not dependent on the presence of divalent cations. Thus, reconstitution of transport activity using purified LPG2 protein in liposomes provides firm experimental evidence that a single polypeptide is solely required for NST activity and is able to mediate the uptake of multiple substrates. These studies are relevant to the study of NST structure and function in both protozoan parasites as well as their higher eukaryotic hosts.     

The human solute carrier gene SLC35B4 encodes a bifunctional nucleotide sugar transporter with specificity for UDP-xylose and UDP-N-acetylglucosamine

The transport of nucleotide sugars from the cytoplasm into the Golgi apparatus is mediated by specialized type III proteins, the nucleotide sugar transporters (NSTs). Transport assays carried out in vitro with Golgi vesicles from mammalian cells showed specific uptake for a total of eight nucleotide sugars. When this study was started, NSTs with transport activities for all but two nucleotide sugars (UDP-Xyl and UDP-Glc) had been cloned. Aiming at identifying these elusive NSTs, bioinformatic methods were used to display putative NST sequences in the human genome. Ten open reading frames were identified, cloned, and heterologously expressed in yeast. Transport capabilities for UDP-Glc and UDP-Xyl were determined with Golgi vesicles isolated from transformed cells. Although a potential UDP-Glc transporter could not be identified due to the high endogenous transport background, the measurement of UDP-Xyl transport was possible on a zero background. Vesicles from yeast cells expressing the human gene SLC35B4 showed specific uptake of UDP-Xyl, and subsequent testing of other nucleotide sugars revealed a second activity for UDP-GlcNAc. Expression of the epitope-tagged SLC35B4 in mammalian cells demonstrated strict Golgi localization. Because decarboxylation of UDP-GlcA is known to produce UDP-Xyl directly in the endoplasmic reticulum and Golgi lumen, our data demonstrate that two ways exist to deliver UDP-Xyl to the Golgi apparatus.     

Specific proteins are required to translocate phosphatidylcholine bidirectionally across the endoplasmic reticulum

BACKGROUND: A long-standing problem in understanding the mechanism by which the phospholipid bilayer of biological membranes is assembled concerns how phospholipids flip back and forth between the two leaflets of the bilayer. This question is important because phospholipid biosynthetic enzymes typically face the cytosol and deposit newly synthesized phospholipids in the cytosolic leaflet of biogenic membranes such as the endoplasmic reticulum (ER). These lipids must be transported across the bilayer to populate the exoplasmic leaflet for membrane growth. Transport does not occur spontaneously and it is presumed that specific membrane proteins, flippases, are responsible for phospholipid flip-flop. No biogenic membrane flippases have been identified and there is controversy as to whether proteins are involved at all, whether any membrane protein is sufficient, or whether non-bilayer arrangements of lipids support flip-flop. RESULTS: To test the hypothesis that specific proteins facilitate phospholipid flip-flop in the ER, we reconstituted transport-active proteoliposomes from detergent-solubilized ER vesicles under conditions in which protein-free liposomes containing ER lipids were inactive. Transport was measured using a synthetic, water-soluble phosphatidylcholine and was found to be sensitive to proteolysis and associated with proteins or protein-containing complexes that sedimented operationally at 3.8S. Chromatographic analyses indicated the feasibility of identifying the transporter(s) by protein purification approaches, and raised the possibility that at least two different proteins are able to facilitate transport. Calculations based on a simple reconstitution scenario suggested that the transporters represent approximately 0.2% of ER membrane proteins. CONCLUSIONS: Our results clearly show that specific proteins are required to translocate a phosphatidylcholine analogue across the ER membrane. These proteins are likely to be the flippases, which are required to translocate natural phosphatidylcholine and other phospholipids across the ER membrane. The methodology that we describe paves the way for identification of a flippase.     

Physical properties of liposomes and proteoliposomes prepared from Escherichia coli polar lipids

Reconstituted proteoliposomes serve as experimental systems for the study of membrane enzymes. Osmotic shifts and other changes in the solution environment may influence the structures and membrane properties of phospholipid vesicles (including liposomes, proteoliposomes and biological membrane vesicles) and hence the activities of membrane-associated proteins. Polar lipid extracts from Escherichia coli are commonly used in membrane protein reconstitution. The solution environment influenced the phase transition temperature and the diameter of liposomes and proteoliposomes prepared from E. coli polar lipid by extrusion. Liposomes prepared from E. coli polar lipids differed from dioleoylphosphatidylglycerol liposomes in Young's elastic modulus, yield point for solute leakage and structural response to osmotic shifts, the latter indicated by static light scattering spectroscopy. At high concentrations, NaCl caused aggregation of E. coli lipid liposomes that precluded detailed interpretation of light scattering data. Proteoliposomes and liposomes prepared from E. coli polar lipids were similar in size, yield point for solute leakage and structural response to osmotic shifts imposed with sucrose as osmolyte. These results will facilitate studies of bacterial enzymes implicated in osmosensing and of other enzymes that are reconstituted in E. coli lipid vesicles.     

Characterization of AtNST-KT1, a novel UDP-galactose transporter from Arabidopsis thaliana

Nucleotide sugar transporters (NST) mediate the transfer of nucleotide sugars from the cytosol into the lumen of the endoplasmatic reticulum and the Golgi apparatus. Because the NSTs show similarities with the plastidic phosphate translocators (pPTs), these proteins were grouped into the TPT/NST superfamily. In this study, a member of the NST-KT family, AtNST-KT1, was functionally characterized by expression of the corresponding cDNA in yeast cells and subsequent transport experiments. The histidine-tagged protein was purified by affinity chromatography and reconstituted into proteoliposomes. The substrate specificity of AtNST-KT1 was determined by measuring the import of radiolabelled nucleotide mono phosphates into liposomes preloaded with various unlabelled nucleotide sugars. This approach has the advantage that only one substrate has to be used in a radioactively labelled form while all the nucleotide sugars can be provided unlabelled. It turned out that AtNST-KT1 represents a monospecific NST transporting UMP in counterexchange with UDP-Gal but did not transport other nucleotide sugars. The AtNST-KT1 gene is ubiquitously expressed in all tissues. AtNST-KT1 is localized to Golgi membranes. Thus, AtNST-KT1 is most probably involved in the synthesis of galactose-containing glyco-conjugates in plants.     

Candida drug resistance protein 1, a major multidrug ATP binding cassette transporter of Candida albicans, translocates fluorescent phospholipids in a reconstituted system

Candida albicans drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, contributes to multidrug resistance in Candida-infected immunocompromised patients. Previous cell-based assays suggested that Cdr1p also acts as a phospholipid translocator. To investigate this, we reconstituted purified Cdr1p into sealed membrane vesicles. Comparison of the ATPase activities of sealed and permeabilized proteoliposomes indicated that Cdr1p was asymmetrically reconstituted such that approximately 70% of the molecules had their ATP binding sites accessible to the extravesicular space. Fluorescent glycerophospholipids were incorporated into the outer leaflet of the proteoliposomes, and their transport into the inner leaflet was tracked with a quenching assay using membrane-impermeant dithionite. We observed ATP-dependent transport of the fluorescent lipids into the inner leaflet of the vesicles. With approximately 6 molecules of Cdr1p per vesicle on average, the half-time to reach the maximal extent of transport was approximately 15 min. Transport was reduced in vesicles reconstituted with Cdr1p variants with impaired ATPase activity and could be competed out to different levels by a molar excess of drugs such as fluconazole and miconazole that are known to be effluxed by Cdr1p. Transport was not affected by ampicillin, a compound that is not effluxed by Cdr1p. Our results suggest a direct link between the ability of Cdr1p to translocate fluorescent phospholipids and efflux drugs. We note that only a few members of the ABC superfamily of Candida have a well-defined role as drug exporters; thus, lipid translocation mediated by Cdr1p could reflect its cellular function.     

Substrate specificity and ionic regulation of GlnPQ from Lactococcus lactis. An ATP-binding cassette transporter with four extracytoplasmic substrate-binding domains

We report on the functional characterization of GlnPQ, an ATP-binding cassette transporter with four extracytoplasmic substrate-binding domains. The first predicted transmembrane helix of GlnP was cleaved off in the mature protein and most likely serves as the signal sequence for the extracytoplasmic substrate-binding domains. Deletion analysis showed that the substrate-binding domain, in the primary sequence of GlnP nearest to the translocator domain, is used as the receptor that delivers the substrate to the translocator. Membrane reconstitution of the detergent-solubilized and purified GlnPQ complex yielded proteoliposomes that transported glutamine and glutamic acid at the expense of ATP. The transport activity of GlnPQ increased with lumenal salt concentration and internal pH, but the mechanism of ionic activation of the transporter is distinct from that of other osmoregulatory ATP-binding cassette transporters and does not depend on the presence of anionic lipids. The regulation of GlnPQ conforms to an electrostatic switch in which protein domain(s) and low molecular weight electrolytes participate.     

Identification and Characterization of a Golgi-Localized UDP-Xylose Transporter Family from Arabidopsis

Most glycosylation reactions require activated glycosyl donors in the form of nucleotide sugars to drive processes such as posttranslational modifications and polysaccharide biosynthesis. Most plant cell wall polysaccharides are biosynthesized in the Golgi apparatus from cytosolic-derived nucleotide sugars, which are actively transferred into the Golgi lumen by nucleotide sugar transporters (NSTs). An exception is UDP-xylose, which is biosynthesized in both the cytosol and the Golgi lumen by a family of UDP-xylose synthases. The NST-based transport of UDP-xylose into the Golgi lumen would appear to be redundant. However, employing a recently developed approach, we identified three UDP-xylose transporters in the Arabidopsis thaliana NST family and designated them UDP-XYLOSE TRANSPORTER1 (UXT1) to UXT3. All three transporters localize to the Golgi apparatus, and UXT1 also localizes to the endoplasmic reticulum. Mutants in UXT1 exhibit ∼30% reduction in xylose in stem cell walls. These findings support the importance of the cytosolic UDP-xylose pool and UDP-xylose transporters in cell wall biosynthesis.     

Glycosyl acceptors in intact and permeabilized normal and cystic fibrosis fibroblasts

Using cell permeabilization, a technique which allows addition of exogenously supplied radiolabeled sugar nucleotides to serve as direct glycosyl donors, oligosaccharide biosynthesis was examined in fibroblasts obtained from normal and cystic fibrosis (CF) subjects. Incubation of logarithmically growing cells with either radiolabeled leucine or xylose has indicated that there was a difference in the synthetic rate between the cell types. Protein synthesis in normal cells made permeable with 50 m?g/ml lysolecithin (LL) was demonstrated to be absent, and could not be induced to take place by adding exogenous components, including energy sources and amino acids, normally required for protein synthesis. Thus radiolabeled sugars were being added to peptide acceptors which were already present at the time of LL addition. Both permeable and intact fibroblasts were exposed to labeled UDP-xylose, UDP-galactose, and UDP-glucuronic acid, all donors of mucopolysaccharide precursors. The uptake of xylose into protein was the same for both normal and CF cells, but permeable CF fibroblasts incorporated statistically greater amounts of sugar from UDP-galactose and UDP-glucuronic acid. Intact CF cells were also labeled using these two sugar nucleotides. Trypan blue exclusion indicated CF and normal fibroblasts were equally intact. This and the fact that preincubation of CF cells with the appropriate cold sugar nucleotide eliminated the differences in incorporation between the normal and CF cells suggested that CF fibroblasts had more cell surface acceptor than the normal cells.     

Identification and characterization of GONST1, a golgi-localized GDP-mannose transporter in Arabidopsis

Transport of nucleotide sugars across the Golgi apparatus membrane is required for the luminal synthesis of a variety of plant cell surface components. We identified an Arabidopsis gene encoding a nucleotide sugar transporter (designated GONST1) that we have shown by transient gene expression to be localized to the Golgi. GONST1 complemented a GDP-mannose transport-defective yeast mutant (vrg4-2), and Golgi-rich vesicles from the complemented strain displayed increased GDP-mannose transport activity. GONST1 promoter::beta-glucuronidase studies suggested that this gene is expressed ubiquitously. The identification of a Golgi-localized nucleotide sugar transporter from plants will allow the study of the importance of this class of proteins in the synthesis of plant cell surface components such as cell wall polysaccharides.     

Fast, efficient reconstitution of the cyclooxygenases into proteoliposomes

To study the physical and catalytic properties of purified membrane proteins, it is often necessary to reconstitute them into lipid bilayers. Here, we describe a fast efficient method for the direct incorporation of cyclooxygenase-1 and -2 (COX-1 and -2) isozymes into liposomes without loss of activity. Purified COX-1 and -2 spontaneously incorporate into large unilamellar vesicles produced from a mixture of DOPC:DOPS (7:3) that has been doped with oleic acid. When incorporation was measured by comparing cyclooxygenase activity to total phospholipid in the proteoliposomes, molar reconstitution ratios of 1000:1 (phospholipid:COX) were obtained. Electron paramagnetic resonance spectroscopic spin counting analysis of proteoliposomes formed with nitroxide spin-labeled COX-2 gave a nearly identical phospholipid:COX ratio, confirming that incorporation had no effect on enzyme activity, and demonstrating that the efficiency of protein incorporation is sufficient for EPR spectroscopic analysis. The spontaneous incorporation of cyclooxygenase into intact liposomes allows only insertion into the outer leaflet for this monotopic enzyme, an orientation confirmed by immunogold staining of the proteoliposomes. This method of reconstitution into liposomes may be generally applicable to the class of monotopic integral membrane proteins typified by the cyclooxygenase isozymes.     

Translocation of UDP-N-acetylglucosamine into vesicles derived from rat liver rough endoplasmic reticulum and Golgi apparatus

A mixture of UDP-N-acetylglucosamine labeled with different radioisotopes in the uridine and glucosamine was used to show that the intact sugar nucleotide was translocated across the membrane of vesicles derived from rat liver rough endoplasmic reticulum (RER) and Golgi apparatus. Translocation was dependent on temperature, saturable at high concentrations of sugar nucleotide, and inhibited by treatment of vesicles with proteases, suggesting protein carrier mediated transport. Translocation of UDP-GlcNAc by RER-derived vesicles appeared to be specific since these vesicles were unable to translocate UDP-galactose, in contrast to those derived from the Golgi apparatus. Preliminary results suggest that the mechanism of UDP-GlcNAc translocation into RER-derived vesicles is via a coupled exchange with lumenal nucleoside monophosphate. This is similar to the recently postulated mechanism for translocation of sugar nucleotides into vesicles derived from the Golgi apparatus.     

Uridine diphosphate glucose dehydrogenase from cornea and epiphysial-plate cartilage

1. UDP-glucose dehydrogenase (EC 1.1.1.22) was extracted from epiphysial-plate cartilage of newborn pigs and from whole bovine corneas. 2. Formation of UDP-glucuronic acid was demonstrated by radioautography after separation of the sugar nucleotides by paper chromatography or t.l.c.: in these conditions a radioactive glucuronic acid spot also appears. 3. UDP-xylose prevented the formation in the incubation mixture of both UDP-glucuronic acid and free glucuronic acid. 4. In both tissues the dependence of the enzyme activity on pH and the K(m) values for UDP-glucose and NAD(+) were determined. 5. Inhibition by UDP-xylose with respect to UDP-glucose was investigated. The plots of 1/v versus 1/[UDP-glucose], and of percentage inhibition versus UDP-xylose concentration and the Hill coefficient showed that a co-operative effect existed between UDP-xylose-binding sites. 6. The physiological meaning of the different affinities of cartilage and cornea enzymes for UDP-xylose is discussed and related to the different glycosaminoglycan contents of the two connective tissues studied.     

脂质体重组和脂蛋白体在植物生物膜研究中的应用

脂质体是磷脂在一定条件下在水中形成的由脂质双分子层组成的内部为水相的闭合囊泡。在推动生物膜的研究进展中,它作为模式系统起着非常重要的作用,能用于研究膜蛋白的性质和功能;膜脂和膜蛋白的相互关系;膜的电化学性质等。近年来脂质体重组技术开始引入到植物学研究领域,用于对植物膜蛋白的研究。本文简要介绍了脂质体的制备和脂酶体重组的方法及其在植物生物膜研究中的应用。