磷酸泛酰巯基乙胺基转移酶基因过量表达对集胞藻PCC6803脂肪酸合成的影响

磷酸泛酰巯基乙胺基转移酶（phosphopantetheinyl transferases,PPTase）是细菌脂肪酸合成中的关键酶。本研究利用同源重组手段构建集胞藻PPTase基因（slr0495）过量表达重组质粒,在集胞藻PCC6803中进行表达研究,并在DNA和RNA水平进行验证,利用气相色谱-质谱联用仪（GC-MS）检测不同条件下突变藻株脂肪酸组分及含量。结果表明：在温度为30 ℃、光照强度为50 μmol ·m^-2 · s^-1培养条件下,过量表达slr0495基因突变藻株中C12:0、C16:0和C18:0的含量分别为1. 31 mg · g^-1、8.07 mg · g^-1和1.35 mg · g^-1,比野生型藻株分别提高了23.58%、25.31%和13.45%;在温度为20 ℃、50% NaNO₃浓度、光照强度为50 μmol · m^-2 · s^-1培养条件下,与野生型相比,突变藻株中C16:0和C18:0含量分别提高了44.71%和41.51%。以上结果表明,过表达slr0495基因增加了集胞藻PCC6803中长链饱和脂肪酸的含量,并且在低温（20 ℃）和缺氮（50% NaNO₃）双重胁迫培养条件下中长链饱和脂肪酸含量得到进一步提高。本研究为探究slr0495基因功能以及在逆境中基因产生的应激反应提供了理论依据,为微藻高产多不饱和脂肪酸代谢研究奠定了基础。     

Glutathione transferase activities toward herbicides used selectively in soybean

Using extracts from suspension-cultured cells of soybean (Glycine max cv. Mandarin) as a source of active enzymes, the activities of glutathione transferases (GSTs) catalysing the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and selective herbicides were determined to be in the order CDNB≫ fomesafen>metolachlor=acifluorfen>chlorimuron-ethyl. GST activities showed a thiol dependence in a substrate-specific manner. Thus, GST activities toward acifluorfen and fomesafen were greater when homoglutathione (hGSH), the endogenously occurring thiol in soybean, was used as the co-substrate rather than glutathione (GSH). Compared with GSH, hGSH addition either reduced or had no effect on GST activities toward other substrates. In the absence of enzyme, the rates of hGSH conjugation with acifluorfen, chlorimuron-ethyl and fomesafen were negligible, suggesting that rapid hGSH conjugation in soybean must be catalysed by GSTs. GST activities were subsequently determined in 14-day-old plants of soybean and a number of annual grass and broadleaf weeds. GST activities of the plants were then related to observed sensitivities to post-emergence applications of the four herbicides. When enzyme activity was expressed on a mg^-1 protein basis, all grass weeds and Abutilon theophrasticontained considerably higher GST activity toward CDNB than soybean. With fomesafen as the substrate, GST activities were determined to be in the order soybean≫Echinochloa crus-galli>Digitaria sanguinalis>Sorghum halepense=Setaria faberi with none of the broadleaf weeds showing any activity. This order related well to the observed selectivity of fomesafen, with the exception of A. theophrasti, which was partially tolerant to the herbicide. Using metolachlor as the substrate the order of the GST activities was soybean>A. theophrasti≫S. halepense>Amaranthus retroflexus>Ipomoea hederacea, with the remaining species showing no activity. GST activities toward metolachlor correlated well with the selectivity of the herbicide toward the broadleaf weeds but not toward the grass weeds. Acifluorfen and chlorimuron-ethyl were selectively active on these species, but GST activities toward these herbicides could not be detected in crude extracts from whole plants. © 1997 SCI     

Advances in glutathione S-transferases research

Glutathione S-transferases(GSTs)are a family of soluble detoxification enzymes which use reduced glutathione(GSH)in conjugation and reduction reactions.Toxic electrophiles, including a variety of carcinogens, are substrates for the GSTs and are more easily excreted into bile or urine after conjugation or reduction.The three-dimensional structures of GSTs from several species, including humans, have been determined.GST activity has been found to present in all human tissues, and expression of the various GST isoenzymes differs in degree in the varioustissues.Glutathione S-transferases may play a role in the protection of cells against toxic electrophiles and in the resistance to cancer chemotherapeutic agants.The polymorphisms of GSTs genes are one of the important factors that exercise influence on individual susceptibility to cancer.     

Identification and expression profiles of nine glutathione S-transferase genes from the important rice phloem sap-sucker and virus vector Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae)

BACKGROUND: Glutathione S‐transferases (GSTs) have received considerable attention in insects for their roles in insecticide resistance. Laodelphax striatellus (Fallén) is a serious rice pest. L. striatellus outbreaks occur frequently throughout eastern Asia. A key problem in controlling this pest is its rapid adaptation to numerous insecticides. In this research, nine cDNAs encoding GSTs in L. striatellus were cloned and characterised. RESULTS: The cloned GSTs of L. striatellus belonged to six cytosolic classes and a microsomal subgroup. Exposure to sublethal concentrations of each of the six insecticides, DDT, chlorpyrifos, fipronil, imidacloprid, buprofezin and beta‐cypermethrin, quickly induced (6 h) up‐expression of LsGSTe1. The expression of LsGSTs2 was increased by chlorpyrifos, fipronil and beta‐cypermethrin. Furthermore, exposure of L. striatellus to fipronil, imidacloprid, buprofezin and beta‐cypermethrin increased the expression of the LsGSTm gene after 24 or 48 h. CONCLUSION: This work is the first identification of GST genes from different GST groups in Auchenorrhyncha species and their induction characteristics with insecticide types and time. The elevated expression of GST genes induced by insecticides might be related to the enhanced tolerance of this insect to insecticides and xenobiotics. Copyright © 2012 Society of Chemical Industry     

Toxicological and biochemical response to azinphos-methyl in Cydia pomonella L. (Lepidoptera: Tortricidae) among orchards from the Argentinian Patagonia

BACKGROUND: Azinphos‐methyl is the main insecticide used to control codling moth on apple and pears in Northern Patagonia. The aim of this study was to evaluate the toxicological and biochemical response of diapausing larvae of codling moth in orchards subjected to different insecticide selection pressure. RESULTS: Dose–mortality assays with azinphos‐methyl in diapausing larvae of Cydia pomonella L. showed significant differences between the LD₉₅ from a population collected in one untreated orchard (2.52 µg moth^?1) compared with that in a laboratory‐susceptible population (0.33 µg moth^?1). Toxicity to azinphos‐methyl in field populations of diapausing larvae collected during 2003–2005 was evaluated by topical application of a discriminating dose (2.5 µg moth^?1) that was obtained from larvae collected in the untreated orchard (field reference strain). Significantly lower mortality (37.71–84.21%) was observed in three out of eight field populations compared with that in the field reference strain. Most of the field populations showed higher esterase activity than that determined in both the laboratory susceptible and the field reference strains. Moreover, there was a high association between esterase activity and mortality (R² = 0.64) among the field populations. On the other hand, a poor correlation was observed between glutathione S‐transferase activity and mortality (R² = 0.33) among larvae collected from different orchards. CONCLUSIONS: All the field populations evaluated exhibited some degree of azinphos‐methyl tolerance in relation to the laboratory susceptible strain. Biochemical results demonstrated that esterases are at least one of the principal mechanisms involved in tolerance to this insecticide. Copyright © 2008 Society of Chemical Industry