Positional Specificity and Fatty Acid Selectivity of Purified sn-Glycerol 3-Phosphate Acyltransferases from Chloroplasts

Soluble acyl-CoA:sn-glycerol 3-phosphate acyltransferases (EC 2.3.1.15) which are localized in chloroplasts were purified from leaves of Pisum sativum and Spinacia oleracea and obtained free from interfering activities. The purification raised the specific activities by factors of about 1,000 for pea and 200 for spinach preparations. In pea chloroplasts, acyltransferase activity occurs in two soluble forms with apparent isoelectric points of 6.3 and 6.6. For both forms, the same molecular weight of about 42,000 was determined. The enzyme from spinach chloroplasts showed a slightly higher molecular weight and a lower isoelectric point of 5.2.     

Mutants of Escherichia coli Defective in Membrane Phospholipid Synthesis: Mapping of sn-Glycerol 3-Phosphate Acyltransferase Km Mutants

plsB mutants of Escherichia coli are sn-glycerol 3-phosphate auxotrophs which owe their requirement to a K(m) defect in sn-glycerol 3-phosphate acyltransferase, the first enzyme in the phospholipid biosynthetic pathway. We have located the plsB gene at minute 69 of the E. coli genetic map, far removed from the gene defined by mutants with a temperature-sensitive sn-glycerol 3-phosphate acyltransferase. The plsB gene was cotransduced with the dctA locus, and the transduction data indicated that the clockwise gene order is asd, plsB, dctA, xyl. plsB(-) is recessive to plsB(+) and all acyltransferase K(m) mutants tested lie very close to the plsB locus. Effective supplementation of plsB mutants was shown not to require a defective glpD gene.     

Mutants of Escherichia coli Defective in Membrane Phospholipid Synthesis: Macromolecular Synthesis in an sn-Glycerol 3-Phosphate Acyltransferase Km Mutant

sn-Glycerol 3-phosphate (G3P) auxotrophs of Escherichia coli have been selected from a strain which cannot aerobically catabolize G3P. The auxotrophy resulted from loss of the biosynthetic G3P dehydrogenase (EC 1.1.1.8) or from a defective membranous G3P acyltransferase. The apparent K(m) of the acyltransferase for G3P was 11- to 14-fold higher (from about 90 mum to 1,000 to 1,250 mum) in membrane preparations from the mutants than those of the parent. All extracts prepared from revertants of the G3P dehydrogenase mutants showed G3P dehydrogenase activity, but most contained less than 10% of the wild-type level. Membrane preparations from revertants of the acyltransferase mutants had apparent K(m)'s for G3P similar to that of the parent. Strains have been derived in which the G3P requirement can be satisfied with glycerol in the presence of glucose, presumably because the glycerol kinase was desensitized to inhibition by fructose 1,6-diphosphate. Investigations on the growth and macromolecular synthesis in a G3P acyltransferase K(m) mutant revealed that upon glycerol deprivation, net phospholipid synthesis stopped immediately; growth continued for about one doubling; net ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein nearly doubled paralleling the growth curve; the rate of phospholipid synthesis assessed by labeling cells with (32)P-phosphate, (14)C-acetate, or (3)H-serine was reduced greater than 90%; the rates of RNA and DNA synthesis increased as the cells grew and then decreased as the cells stopped growing; the rate of protein synthesis showed no increase and declined more slowly than the rates of RNA and DNA synthesis when the cells stopped growing. The cells retained and gained in the capacity to synthesize phospholipids upon glycerol deprivation. These data indicate that net phospholipid synthesis is not required for continued macromolecular synthesis for about one doubling, and that the rates of these processes are not coupled during this time period.     

A Land-Plant-Specific Glycerol-3-Phosphate Acyltransferase Family in Arabidopsis: Substrate Specificity, sn-2 Preference, and Evolution

Arabidopsis (Arabidopsis thaliana) has eight glycerol-3-phosphate acyltransferase (GPAT) genes that are members of a plant-specific family with three distinct clades. Several of these GPATs are required for the synthesis of cutin or suberin. Unlike GPATs with sn-1 regiospecificity involved in membrane or storage lipid synthesis, GPAT4 and -6 are unique bifunctional enzymes with both sn-2 acyltransferase and phosphatase activity resulting in 2-monoacylglycerol products. We present enzymology, pathway organization, and evolutionary analysis of this GPAT family. Within the cutin-associated clade, GPAT8 is demonstrated as a bifunctional sn-2 acyltransferase/phosphatase. GPAT4, -6, and -8 strongly prefer C16:0 and C18:1 ω-oxidized acyl-coenzyme As (CoAs) over unmodified or longer acyl chain substrates. In contrast, suberin-associated GPAT5 can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs. These substrate specificities (1) strongly support polyester biosynthetic pathways in which acyl transfer to glycerol occurs after oxidation of the acyl group, (2) implicate GPAT specificities as one major determinant of cutin and suberin composition, and (3) argue against a role of sn-2-GPATs (Enzyme Commission 2.3.1.198) in membrane/storage lipid synthesis. Evidence is presented that GPAT7 is induced by wounding, produces suberin-like monomers when overexpressed, and likely functions in suberin biosynthesis. Within the third clade, we demonstrate that GPAT1 possesses sn-2 acyltransferase but not phosphatase activity and can utilize dicarboxylic acyl-CoA substrates. Thus, sn-2 acyltransferase activity extends to all subbranches of the Arabidopsis GPAT family. Phylogenetic analyses of this family indicate that GPAT4/6/8 arose early in land-plant evolution (bryophytes), whereas the phosphatase-minus GPAT1 to -3 and GPAT5/7 clades diverged later with the appearance of tracheophytes.sn-Glycerol-3-phosphate 1-O-acyltransferase (GPAT; Enzyme Commission [EC] 2.3.1.15) is the first enzyme in the pathway for the de novo synthesis of membrane and storage lipids. It catalyzes the transfer of an acyl group from acyl-CoA or acyl-ACP to the sn-1 position of sn-glycerol-3-phosphate (G3P). This reaction has been extensively characterized in bacteria, fungi, animals, and plants (Murata and Tasaka, 1997; Zheng and Zou, 2001; Gimeno and Cao, 2008; Zhang and Rock, 2008; Wendel et al., 2009; Chen et al., 2011a). In the Arabidopsis (Arabidopsis thaliana) genome, there are 10 genes annotated as GPATs. One of these is the soluble, plastid-localized GPAT (At1g32200) that utilizes acyl-ACP substrates and exhibits sn-1 acyl transfer regiospecificity (Nishida et al., 1993). A second enzyme is GPAT9 (At5g60620), which is localized to the endoplasmic reticulum (Gidda et al., 2009) and may be an acyl-CoA-dependent sn-1 GPAT that enables nonplastid glycerolipid synthesis. The remaining eight GPATs cluster together in a family (Zheng et al., 2003; Beisson et al., 2007; Gidda et al., 2009) that is not required for membrane or storage lipid biosynthesis; instead, several members of the family clearly affect the composition and quantity of cutin or suberin (Beisson et al., 2012).Cutin and suberin are extracellular lipid barriers deposited by certain types of plant cells. These insoluble polymers, and associated waxes, function to control water, gas, and ion fluxes and serve as physical barriers to protect plants from pathogen invasion (Kolattukudy, 2001; Schreiber, 2010; Ranathunge et al., 2011). From an evolutionary perspective, the appearance of these lipid barriers was likely a requirement for the adaptation of plants to a terrestrial environment (Rensing et al., 2008). ω-oxidized fatty acids and glycerol are usually major constituents of both polymers (Bernards, 2002; Graça and Santos, 2007; Pollard et al., 2008). The detailed structures of cutin and suberin polymers are still largely unknown (Pollard et al., 2008), but direct esterification of fatty acids to glycerol and to each other has been demonstrated in a large number of partial depolymerization studies of cutin and suberin (Graça and Santos, 2007; Graça and Lamosa, 2010). In Arabidopsis, GPAT4 and GPAT8 are required for the accumulation of C16 and C18 ω-hydroxy fatty acid (ω-OHFA) and α,ω-dicarboxylic acid (DCA) cutin monomers in stems and leaves (Li et al., 2007a). GPAT6 is required for the incorporation of the following C16 monomers: 10,16-dihydroxypalmitate (10,16-diOH C16:0-FA), hexadecane-1,16-dioic acid (C16:0-DCA), and 16-hydroxypalmitate (16-OH C16:0-FA), into flower cutin (Li-Beisson et al., 2009). (For a fatty acid, the abbreviation used is Cm:n-FA, where m is the number of carbon atoms and n is the number of double bonds. The position and number of hydroxyl groups precedes this notation. The same nomenclature is used for DCAs.) GPAT5 controls the accumulation of C22:0- and C24:0-FA, ω-OHFA, and DCA monomers in the suberin of roots and seed coats (Beisson et al., 2007). Recently, we demonstrated that GPAT4 and -6 are bifunctional enzymes that possess sn-2 acyltransferase and phosphatase activities (Yang et al., 2010) and that therefore produce sn-2 monoacylglycerols (MAGs) as the major product. GPAT5 also exhibits strong preference for sn-2 acylation but lacks phosphatase activity; thus, sn-2 lysophosphatidic acids (LPAs) are its major product (Yang et al., 2010).These observations all attest to the fact that several members of the GPAT1 to -8 family are enzymatically very distinct from the GPATs required for membrane and storage lipid biosynthesis. Indeed, they represent a new acylglycerol biosynthesis pathway that provides precursors for cutin and suberin biosynthesis. To better understand the early steps in polyester synthesis and the roles contributed by GPAT4 to -8, and to determine whether the clade of GPAT1 to -3 has distinct or similar activity, we have characterized the regiochemistry and acyl substrate specificity of GPATs representing all three clades. We show that the cutin-associated GPAT8 is a bifunctional sn-2 acyltransferase/phosphatase, while GPAT1, an isozyme with uncertain function but important for tapetum and anther development (Zheng et al., 2003; Li et al., 2012), possesses sn-2 acyltransferase activity but not phosphatase activity. An important issue in defining the pathway of cutin/suberin biosynthesis is whether to place the P450 oxidation reactions before or after the G3P acylation reactions. As discussed (Pollard et al., 2008), previous evidence has not allowed definitive determination of the alternative pathways. However, conducting a GPAT substrate specificity study, particularly with a range of ω-oxidized and unmodified acyl-CoAs can help clarify the situation. Here, we show the acyl-CoA specificities of GPAT4, -5, -6, and -8 are concordant with the compositions of their respective cutins and suberins and the resulting changes in corresponding gpat mutants and overexpression lines. Furthermore, the results provide strong evidence that acyl transfer to glycerol by GPAT occurs after ω-oxidation of acyl chains, thus increasing our limited understanding of the biochemical pathway for cutin and suberin polymer assembly.A phylogenetic analysis of Arabidopsis GPATs with vascular and nonvascular land-plant homologs provides an evolutionary view of the expansion and divergence of the gene family into three distinct clades that are associated with morphological and functional evolution and with the loss of phosphatase activity.     

Relationship between Chloroplast Membrane Fatty Acid Composition and Photosynthetic Response to a Chilling Temperature in Four Alfalfa Cultivars

The photosynthetic responses of four alfalfa (Medicago sativa L.) cultivars to 10 and 22 C air temperatures were examined and the relationship between the photosynthetic response at 10 C and the fatty acid composition of the chloroplast membranes was determined. Chilling-resistant cultivars exhibited moderate reductions in photosynthesis at 10 C, compared to 22 C, and contained a significantly greater percentage of polyunsaturated fatty acids in the chloroplast membrane and a greater double bond index than the chilling-sensitive cultivars. The chilling-sensitive cultivars exhibited severe reductions in photosynthesis at 10 C, compared to 22 C. The reduction in photosynthesis at 10 C is shown to be negatively correlated (r = −0.94) with the double bond index of the chloroplast membranes of the cultivars observed.

The results support the hypothesis that reduced photosynthesis due to chilling temperatures is influenced by the unsaturated fatty acid composition of the chloroplast membrane which affect temperature-induced phase changes in chloroplast membrane lipids.

     

Stabilizing Effect of Lipopolysaccharide on the l-Glycerol 3-Phosphate Acyltransferase of Escherichia coli Membrane

A gene, frxC, which is unique to the chloroplast genome of the liverwort Marchantia polymorpha, has sequence similarity to nifH, the product of which is an iron protein of a nitrogenase. Although frxC is expressed to produce a protein in liverwort chloroplasts, its function is not known. Using a probe of liverwort chloroplast DNA, a 10.1-kb region containing a gene cluster consisting of open reading frames (ORF278-frxC-ORF469–0RF248) was isolated from the cyanobacterium Synechocystis PCC6803. In this region, frxC and ORF469 showed sequence similarities to liverwort chloroplast frxC (83%) and immediately downstream ORF465 (74%), respectively. Synechocystis frxC showed 31% amino acid sequence identity with nifHl from Clostridium pasteurianum. Additionally, Synechocystis ORF469 showed a sequence similarity (19% identity) to C. pasteurianum nifK product, which is the β subunit of a molybdenum-iron protein of a nitrogenase complex. Conservation of the gene arrangement between liverwort and Synechocystis suggests that the liverwort chloroplast frxC-ORF465 cluster may have evolved from an ancestor common to Synechocystis, and that these two genes may have been transferred to the nuclear genome in tobacco and rice during evolution.     

Antisense-Mediated Depletion of Tomato Chloroplast Omega-3 Fatty Acid Desaturase Enhances Thermal Tolerance

A chloroplast-localized tomato （Lycopersicon esculentum Mill.） ω-3 fatty acid desaturase gene （LeFADT） was isolated and characterized with regard to its sequence, response to various temperatures, and function in antisense transgenic tomato plants. The deduced amino acid sequence had four histidine-rich regions, of which three regions were highly conserved throughout the whole ω-3 fatty acid desaturasegene family. Southern blotting analysis showed that LeFAD7was encoded by a single copy gene and had two homologous genes in the tomato genome. Northern blot showed that LeFAD7 was expressed in all organs and was especially abundant in leaf tissue. Meanwhile, expression of LeFAD7 was induced by chilling stress （4 ℃）, but was inhibited by high temperature （45 ℃）, in leaves. Transgenic tomato plants were produced by integration of the antisense LeFAD7DNA under the control of a CaMV35S promoter into the genome. Antisense transgenic plants with lower 18 ： 3 content could maintain a higher maximal photochemical efficiency （Fv/Fm） and O2 evolution rate than wild-type plants. These results suggested that silence of the LeFAD7 gene alleviated high-temperature stress. There was also a correlation between the low content of 18 ： 3 resulting from silence of the LeFAD7 gene and tolerance to high-temperature stress.     

Properties of the Plastidial Acyl-(Acyl-Carrier-Protein): Glycerol-3-Phosphate Acyltransferase from the Chilling-Sensitive Plant Squash (Cucurbita moschata)

To determine whether the plastidial acyl-(acyl-carrier-protein(ACP)): glycerol-3-phosphate acyltransferase from chilling-sensitiveplants exhibits fatty acid selectivities different from thoseof resistant plants, we characterized this enzymic activityfrom the chilling-sensitive plant Cucurbita moschata. In squashcotyledons, the glycerol-3-phosphate acyltransferase (AT) occurredas three isomeric forms: one with an isoelectric point at pH6.6 (ATI) and two at about pH 5.5 (AT2 and AT3). These isomershad approximately equal total activities in plastids. All threeforms specifically directed acyl groups to the C-l positionof glycerol-3-phosphate. However, ATI differed from the twoother isomeric forms on the basis of kinetic data determinedwith different acyl-ACPs as substrates. These kinetic differenceswere reflected in the different fatty acid selectivities ofthe acyltransferases. ATI preferably utilized oleoyl groupsin comparison to palmitoyl and stearoyl groups while AT2 andAT3 hardly discriminated between the acyl-ACP thioesters. However,the observed selectivity of ATI was significantly reduced byincreasing the pH of the reaction mixture from 7.4 to 8.0, whichis the stroma pH of illuminated chloroplasts. Consequently,the glycerol-3-phosphate acyltransferases from squash cotyledonscould account for the high proportion of saturated acyl groupsfound at the C-l position of the plastidial phosphatidylglycerolfrom this plant. (Received April 7, 1987; Accepted July 8, 1987)     

Glycerol-3-phosphate Acyltransferase (GPAT)-1, but Not GPAT4, Incorporates Newly Synthesized Fatty Acids into Triacylglycerol and Diminishes Fatty Acid Oxidation

Four glycerol-3-phosphate acyltransferase (GPAT) isoforms, each encoded by a separate gene, catalyze the initial step in glycerolipid synthesis; in liver, the major isoforms are GPAT1 and GPAT4. To determine whether each of these hepatic isoforms performs a unique function in the metabolism of fatty acid, we measured the incorporation of de novo synthesized fatty acid or exogenous fatty acid into complex lipids in primary mouse hepatocytes from control, Gpat1^−/−, and Gpat4^−/− mice. Although hepatocytes from each genotype incorporated a similar amount of exogenous fatty acid into triacylglycerol (TAG), only control and Gpat4^−/− hepatocytes were able to incorporate de novo synthesized fatty acid into TAG. When compared with controls, Gpat1^−/− hepatocytes oxidized twice as much exogenous fatty acid. To confirm these findings and to assess hepatic β-oxidation metabolites, we measured acylcarnitines in liver from mice after a 24-h fast and after a 24-h fast followed by 48 h of refeeding with a high sucrose diet to promote lipogenesis. Confirming the in vitro findings, the hepatic content of long-chain acylcarnitine in fasted Gpat1^−/− mice was 3-fold higher than in controls. When compared with control and Gpat4^−/− mice, after the fasting-refeeding protocol, Gpat1^−/− hepatic TAG was depleted, and long-chain acylcarnitine content was 3.5-fold higher. Taken together, these data demonstrate that GPAT1, but not GPAT4, is required to incorporate de novo synthesized fatty acids into TAG and to divert them away from oxidation.     

Partial Purification and Some Properties of Stearyl-Coa: Sn-Glycerol-3-Phosphate Acyltransferase from Liver Microsomes

About fourfold purification of the stearyl-CoA: sn-glycerol-3-phosphate acyltransferases (GPAT) was achieved from liver microsomes by extraction with 1M phosphate buffer (pH 7–4) followed by gel filtration. The partially purified enzyme synthesized mainly diacylgly-cerophosphate and a small amount (5%) of monoacylglycerophosphate. Patty aoyl-CoA synthetase and to some extent stearyl-CoA desaturase were copurified along with the transferases. Data obtained from molecular sieve chromatography, polyacrylamide gel electrophoresis and electron microscopy showed that GPAT activity was tightly bound to a high molecular weight protein fraction with vesicular structure.     

Glycerol-3-Phosphate Acyltransferase Contributes to Triacylglycerol Biosynthesis,Lipid Droplet Formation,and Host Invasion in Metarhizium robertsii

Enzymes involved in the triacylglycerol (TAG) biosynthesis have been well studied in the model organisms of yeasts and animals. Among these, the isoforms of glycerol-3-phosphate acyltransferase (GPAT) redundantly catalyze the first and rate-limiting step in glycerolipid synthesis. Here, we report the functions of mrGAT, a GPAT ortholog, in an insect-pathogenic fungus, Metarhizium robertsii. Unlike in yeasts and animals, a single copy of the mrGAT gene is present in the fungal genome and the gene deletion mutant is viable. Compared to the wild type and the gene-rescued mutant, the ΔmrGAT mutant demonstrated reduced abilities to produce conidia and synthesize TAG, glycerol, and total lipids. More importantly, we found that mrGAT is localized to the endoplasmic reticulum and directly linked to the formation of lipid droplets (LDs) in fungal cells. Insect bioassay results showed that mrGAT is required for full fungal virulence by aiding fungal penetration of host cuticles. Data from this study not only advance our understanding of GPAT functions in fungi but also suggest that filamentous fungi such as M. robertsii can serve as a good model to elucidate the role of the glycerol phosphate pathway in fungal physiology, particularly to determine the mechanistic connection of GPAT to LD formation.     

Activation of Chloroplast NADP-linked Glyceraldehyde-3-Phosphate Dehydrogenase by the Ferredoxin/Thioredoxin System

NADP-glyceraldehyde-3-P dehydrogenase of spinach (Spinacia oleracea) chloroplasts was activated by thioredoxin that was reduced either photochemically with ferredoxin and ferredoxin-thioredoxin reductase or chemically with dithiothreitol. The activation process that was observed with the soluble protein fraction from chloroplasts and with the purified regulatory form of the enzyme was slow relative to the rate of catalysis. The NAD-linked glyceraldehyde-3-P dehydrogenase activity that is also present in chloroplasts and in the purified enzyme preparation was not affected by reduced thioredoxin.

When activated by dithiothreitol-reduced thioredoxin, the regulatory form of NADP-glyceraldehyde-3-P dehydrogenase was partly deactivated by oxidized glutathione. The enzyme activated by photochemically reduced thioredoxin was not appreciably affected by oxidized glutathione. The results suggest that although it resembles other regulatory enzymes in its requirements for light-dependent activation by the ferredoxin/thioredoxin system, NADP-glyceraldehyde-3-P dehydrogenase differs in its mode of deactivation and in its capacity for activation by enzyme effectors independently of thioredoxin.

     

Oxidative Activity of Mitochondria Isolated from Plant Tissues Sensitive and Resistant to Chilling Injury

Arrhenius plots of the respiration rates of mitochondria isolated from chilling sensitive plant tissues (tomato and cucumber fruit, and sweet potato roots) showed a linear decrease from 25 C to about 9 to 12 C (with Q(10) values of 1.3 to 1.6), at which point there was a marked deviation with an increased slope as temperatures were reduced to 1.5 C (Q(10) of 2.2 to 6.3). The log of the respiration rate of mitochondria from chilling resistant tissues (cauliflower buds, potato tubers, and beet roots) showed a linear decrease over the entire temperature range from 25 to 1.5 C with Q(10) values of 1.7 to 1.8. Phosphorylative efficiency of mitochondria from all the tissues, as measured by ADP:O and respiratory control ratios, was not influenced by temperatures from 25 to 1.5 C. These results indicate that an immediate response of sensitive plant tissues to temperatures in the chilling range (0 to 10 C) is to depress mitochondrial respiration to an extent greater than that predicted from Q(10) values measured above 10 C. The results are also consistent with the hypothesis that a phase change occurs in the mitochondrial membrane as the result of a physical effect of temperature on some membrane component such as membrane lipids.     

The Galactolipid, Phospholipid, and Fatty Acid Composition of the Chloroplast Envelope Membranes of Vicia faba. L

The galactolipid, phospholipid, and fatty acid composition of chloroplast envelope membrane fractions isolated from leaves of Vicia faba L. has been determined. The major lipids in this fraction are: monogalactosyldiglyceride, 29%; digalactosyldiglyceride, 32%; phosphatidylcholine, 30%; and phosphatidylglycerol 9%. The lipid composition of the chloroplast envelope membranes is qualitatively similar to that of the lamellar membranes isolated from the same plastids, but the proportion of each lipid present is very different. The total galactolipid to total phospholipid ratio was 1.6: 1 in the envelope and 11.1: 1 in the lamellae. The monogalactosyldiglyceride-digalactosyl-diglyceride ratio was 0.9: 1 in the envelope and 2.4: 1 in the lamellae. Both membranes lack phosphatidylethanolamine.     

Structural Basis of Substrate Selectivity in the Glycerol-3-Phosphate: Phosphate Antiporter GlpT

Major facilitators represent the largest superfamily of secondary active transporter proteins and catalyze the transport of an enormous variety of small solute molecules across biological membranes. However, individual superfamily members, although they may be architecturally similar, exhibit strict specificity toward the substrates they transport. The structural basis of this specificity is poorly understood. A member of the major facilitator superfamily is the glycerol-3-phosphate (G3P) transporter (GlpT) from the Escherichia coli inner membrane. GlpT is an antiporter that transports G3P into the cell in exchange for inorganic phosphate (P_i). By combining large-scale molecular-dynamics simulations, mutagenesis, substrate-binding affinity, and transport activity assays on GlpT, we were able to identify key amino acid residues that confer substrate specificity upon this protein. Our studies suggest that only a few amino acid residues that line the transporter lumen act as specificity determinants. Whereas R45, K80, H165, and, to a lesser extent Y38, Y42, and Y76 contribute to recognition of both free P_i and the phosphate moiety of G3P, the residues N162, Y266, and Y393 function in recognition of only the glycerol moiety of G3P. It is the latter interactions that give the transporter a higher affinity to G3P over P_i.     

冷害对贮藏油桃膜脂脂肪酸及相关酶活性的影响

以秦光2号油桃为试材,研究了1℃和5℃贮藏温度下油桃果实的膜脂脂肪酸及相关酶活性的变化.结果表明:油桃果实的SOD、CAT和APX活性在冷害发生后迅速下降;冷害果实的相对膜透性和MDA含量显著高于对照;冷害促使油桃果实LOX活性的增强,使棕榈酸相对含量高于对照,油酸、亚麻酸相对含量低于对照,降低了膜脂脂肪酸的不饱和指数.     

简并引物在PCR扩增甘油—3—磷酸转酰酶基因中的应用

应用高简交引物,结合ＰＣＲ扩增技术从南瓜Ｃｕｃｕｒｂｉｔａ ｍｏｓｃｈａｔａ 总ＲＮＡ的逆转录产物中得到ＧＰＡＴ基因保守区。在ＰＣＲ反应中,简并引物之间的浓度配比是影响扩增效率的关键。用脱氧次黄苷替代简并引物中的高简交位中大幅度降低简并程度,显著提高扩增产物的特异性及扩增效率。