大豆异黄酮合成途径相关基因差异表达分析

大豆异黄酮是一种应用广泛、具有医用和保健功能的活性物质。为揭示异黄酮合成途径相关基因表达差异,本研究采用实时定量PCR技术分析相关基因在不同大豆品种、发育时期及组织部位的表达。结果发现,苯丙氨酸解氨酶基因PAL、肉桂酸羟化酶基因C4H、香豆酸辅酶A连接酶基因4CL在高异黄酮品种中豆27 R2期叶片中的表达量显著高于低异黄酮品种楚秀;查尔酮合成酶基因CHS、异黄酮合成酶基因IFS在中豆27 R8期子粒中的表达量显著高于楚秀;细胞色素还原酶基因CPR在中豆27 R7期叶片与子粒的表达量与楚秀相比显著降低。这些差异表达的基因可能是形成大豆品种异黄酮含量高低的重要原因。     

大豆籽粒发育过程中异黄酮合成相关酶基因的表达模式与异黄酮积累的相关分析

利用高效液相色谱法和实时定量PCR方法,分别测定了2个异黄酮含量显著差异的大豆品种鲁黑豆2号(LHD2)和南汇早黑豆(NHZ)在子粒发育过程中的异黄酮含量变化以及异黄酮合成相关酶基因的表达模式变化,试图分析异黄酮积累与各基因表达量变化的相关关系。结果表明在大豆子粒发育过程中,异黄酮含量逐渐升高,而不同异黄酮合成相关酶基因的表达趋势不同,CHS7、CHS8、CHR、CHI1A和IFS2的表达趋势与异黄酮积累模式基本一致,而IFS1和CHI1B1的表达趋势与异黄酮积累模式相反。IFR的表达模式在2个大豆品种中存在相反的趋势,在LHD2中与异黄酮组分积累趋势相反,而在NHZ中与异黄酮组分积累趋势相同。结果还表明,同一基因家族中不同基因在子粒发育过程中的表达量也存在差异。查尔酮合酶基因家族中CHS7和CHS8以及查尔酮异构酶基因家族的CHI1A的表达水平相对其他成员较高,异黄酮合酶基因家族中IFS2的表达量显著高于IFS1的表达量,预示这些基因家族在大豆子粒异黄酮积累过程中存在功能分化。此外,各基因表达模式与异黄酮积累的相关分析结果表明,不同基因表达模式与异黄酮积累的相关性在2个品种中也不尽相同。LHD2中CHS7、CHS8和IFS2在子粒发育过程中的表达量变化与不同异黄酮组分呈显著正相关,CHI1B1基因的表达量变化与不同异黄酮组分呈显著负相关。而在NHZ中,IFR在子粒发育过程中的表达量变化与多个异黄酮组分呈显著正相关。这预示了不同大豆品种异黄酮含量差异的潜在遗传基础。各异黄酮合成相关酶基因表达量变化的相关分析表明,在2个品种中,苯丙氨酸水解酶PAL1与4CL,4CL与CHS2以及CHS1与IFS2基因的表达量均呈现显著正相关。表明这些基因可能通过协同作用共同调控异黄酮的合成与积累。这些结果为今后利用基因工程提高大豆异黄酮含量奠定了基础。     

Lactofen induces isoflavone accumulation and glyceollin elicitation competency in soybean

Lactofen, the active ingredient of the soybean disease resistance-inducing herbicide, Cobra, induces large accumulations of isoflavone conjugates and aglycones in soybean tissues. The predominant isoflavones induced in cotyledon tissues are daidzein (and its conjugates) and formononetin and glycitein aglycones. The latter two isoflavones are usually present only at very low levels in soybean seedling tissues. In leaves, the predominant lactofen-induced isoflavones are daidzein and formononetin aglycones and the malonyl-glucosyl conjugate of genistein. Isoflavone induction also occurs in cells distal to the point of treatment, but is only weakly systemic. Lactofen also induces elicitation competency, the capacity of soybean cells to accumulate the pterocarpan phytoalexin glyceollin in response to glucan elicitors from the cell wall of the pathogen Phytophthora sojae. Comparison of the activity of a series of diphenyl ether herbicides demonstrated that while all diphenyl ethers tested induced some degree of elicitation competency, only certain ones induced isoflavone accumulation in the absence of glucan elicitor. As a group the diphenyl ethers are thought to inhibit protoporhyrinogen oxidase, eventually leading to singlet oxygen generation. Another singlet oxygen generator, rose bengal, also induced elicitation competency, but little isoflavone accumulation. It is hypothesized that diphenyl ether-induced activated oxygen species mimic some aspects of hypersensitive cell death, which leads to elicitation competency in infected tissues.     

Modification of phenolic metabolism in soybean hairy roots through down regulation of chalcone synthase or isoflavone synthase

Soybean hairy roots, transformed with the soybean chalcone synthase (CHS6) or isoflavone synthase (IFS2) genes, with dramatically decreased capacity to synthesize isoflavones were produced to determine what effects these changes would have on susceptibility to a fungal pathogen. The isoflavone and coumestrol concentrations were decreased by about 90% in most lines apparently due to gene silencing. The IFS2 transformed lines had very low IFS enzyme activity in microsomal fractions as measured by the conversion of naringenin to genistein. The CHS6 lines with decreased isoflavone concentrations had 5 to 20-fold lower CHS enzyme activities than the appropriate controls. Both IFS2 and CHS transformed lines accumulated higher concentrations of both soluble and cell wall bound phenolic acids compared to controls with higher levels found in the CHS6 lines indicating alterations in the lignin biosynthetic branch of the pathway. Induction of the soybean phytoalexin glyceollin, of which the precursor is the isoflavone daidzein, by the fungal pathogen Fusarium solani f. sp. glycines (FSG) that causes soybean sudden death syndrome (SDS) showed that the low isoflavone transformed lines did not accumulate glyceollin while the control lines did. The (iso)liquritigenin content increased upon FSG induction in the IFS2 transformed roots indicating that the pathway reactions before this point can control isoflavonoid synthesis. The lowest fungal growth rate on hairy roots was found on the FSG partially resistant control roots followed by the SDS sensitive control roots and the low isoflavone transformants. The results indicate the importance of phytoalexin synthesis in root resistance to the pathogen. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

大豆异黄酮生物合成关键酶及其代谢工程研究进展

异黄酮是一类具有C-6/C-3/C-6骨架的二次代谢产物,具有抗氧化和抗肿瘤活性。异黄酮与黄酮类物质具有相似的苯丙烷生物合成途径。天然的绝大部分异黄酮分布在豆科植物中,目前在大豆中已经发现了超过12个异黄酮(苷)。大豆异黄酮的生物合成主要涉及三个关键的酶查尔酮合酶(CHS)、查尔酮异构酶(CHI)和异黄酮合酶(IFS)。总结了大豆异黄酮的提取分离方法和生物合成途径,着重综述了CHI、CHS、IFS生物学特征和功能及异黄酮的代谢工程研究。     

A genomic approach to isoflavone biosynthesis in kudzu (<Emphasis Type="Italic">Pueraria lobata</Emphasis>)

Roots of kudzu (Pueraria lobata) are a rich source of isoflavone O- and C-glycosides. Although O-glycosylation of (iso)flavonoids has been well characterized at the molecular level, no plant isoflavonoid C-glycosyltransferase genes have yet been isolated. To address the biosynthesis of kudzu isoflavonoids, we generated 6,365 high-quality expressed sequence tags (ESTs) from a subtraction cDNA library constructed using RNA from roots that differentially accumulate puerarin. The ESTs were clustered into 722 TCs and 3,913 singletons, from which 15 family I glycosyltransferases (UGTs) were identified. Hierarchical clustering analysis of the expression patterns of these UGTs with isoflavone synthase (IFS) in a range of tissues identified UGTs with potential functions in isoflavone glycosylation. The open reading frames of these UGTs were expressed in E. coli for functional analysis, and one was shown to preferentially glycosylate isoflavones at the 7-O-position. In addition, ESTs corresponding to chalcone synthase, chalcone reductase, chalcone isomerase (CHI) and 2-hydroxyisoflavanone dehydratase were identified. Recombinant CHI proteins had high activities with both 6′-deoxy- and 6′-hydroxy chalcones, typical of Type II CHIs. Establishment of this EST database and identification of genes associated with kudzu isoflavone biosynthesis and glycosylation provide a new resource for metabolic engineering of bioactive kudzu isoflavones.     

Regulation of isoflavonoid metabolism in alfalfa

Isoflavonoids are believed to play important roles in plant-microbe interactions. During infection of alfalfa (Medicago sativa) leaves with the fungal pathogen Phoma medicaginis, rapid increases in mRNA levels and enzyme activities of isoflavone reductase, phenylalanine ammonia-lyase, chalcone synthase and other defense genes are observed within 1 to 2 hours. The phytoalexin medicarpin and its antifungal metabolite sativan increase beginning at 4 and 8 hours, respectively, along with other isoflavonoids. In contrast, during colonization of alfalfa roots by the symbiotic mycorrhizal fungus Glomus versiforme, expression of the general phenylpropanoid and flavonoid genes phenylalanine ammonia-lyase and chalcone synthase increases while mRNA levels for the phytoalexin-specific isoflavone reductase decrease. The total isoflavonoid content of colonized roots increases with time and is higher than that of uninoculated roots, but the accumulation of the antifungal medicarpin is somehow suppressed.An isoflavone reductase genomic clone has been isolated, promoter regions have been fused to the reporter gene -glucuronidase, and the promoter-reporter fusions have been transformed into tobacco and alfalfa. Using histological staining, we have studied the developmental and stress-induced expression of this phytoalexin-specific gene in whole plants at a more detailed level than other methods allow. The isoflavone reductase promoter is functional in tobacco, a plant which does not synthesize isoflavonoids. Infection of transgenic alfalfa plants by Phoma causes an increase in -glucuronidase staining, as does elicitation of transgenic alfalfa cell cultures, indicating that this promoter fusion is a good indicator of phytoalexin biosynthesis in alfalfa.Abbreviations CA4H cinnamic acid 4-hydroxylase - CHI chalcone isomerase - CHOMT chalcone O-methyltransferase - CHS chalcone synthase - 4CL 4-coumarate:CoA ligase - COMT caffeic acid O-methyltransferase - FGM malonylated glucoside of formononetin - GUS -glucuronidase - IFOH isoflavone 2-hydroxylase - IFR isoflavone reductase - IFS isoflavone synthase - IOMT isoflavone 4-O-methyltransferase - MGM medicarpin 3-O-glucoside-6-O-malonate - PAL L-phenylalanine ammonia-lyase - PTS pterocarpan synthase - VAM vesicular arbuscular mycorrhizal - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronide     

Partial reconstruction of flavonoid and isoflavonoid biosynthesis in yeast using soybean type I and type II chalcone isomerases

Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4′-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3β-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux.     

Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes

Isoflavones have drawn much attention because of their benefits to human health. These compounds, which are produced almost exclusively in legumes, have natural roles in plant defense and root nodulation. Isoflavone synthase catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway. To identify the gene encoding this enzyme, we used a yeast expression assay to screen soybean ESTs encoding cytochrome P450 proteins. We identified two soybean genes encoding isoflavone synthase, and used them to isolate homologous genes from other leguminous species including red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine, as well as from the nonleguminous sugarbeet. We expressed soybean isoflavone synthase in Arabidopsis thaliana, which led to production of the isoflavone genistein in this nonlegume plant. Identification of the isoflavone synthase gene should allow manipulation of the phenylpropanoid pathway for agronomic and nutritional purposes.     

Rapid Accumulation of Anionic Peroxidases and Phenolic Polymers in Soybean Cotyledon Tissues following Treatment with Phytophthora megasperma f. sp. Glycinea Wall Glucan

Phytophthora megasperma Drechs. f. sp. glycinea Kuan & Erwin (PMG) cell wall glucan has been extensively characterized as an elicitor of the pterocarpan phytoalexins, the glyceollins in soybean (Glycine max L.). Just recently, this glucan was shown to be a potent elicitor of conjugates of the isoflavones, daidzein and genistein as well. Here we report that PMG wall glucan also induces a rapid and massive accumulation of phenolic polymers in soybean cotyledon cells proximal to the point of elicitor application. Deposition of phenolic polymers is over then times that in wounded controls within just 4 hours of elicitor treatment and reaches a maximum by 24 hours. In the same tissues, isoflavone conjugates begin to accumulate at 8 hours and glyceollin at 12 hours. By 24 hours, the total deposition of wall bound phenolics in elicitor-treated tissues is several times greater than the peak glyceollin and isoflavone responses combined. Histochemical stains and quantitation of phenolic residues released after saponification and nitrobenzene or copper oxide oxidation suggest that the covalently linked phenolics include both lignin- and suberin-like polymers as well as simple esterified coumaric and ferulic acid monomers. Accumulations of phenolic polymers are accompanied by equally rapid and massive increases in activity of a specific group of anionic peroxidases. Although increases in peroxidase activity are not strictly limited to cells immediately adjacent to the area of elicitor treatment, the deposition of phenolic polymers is significantly less extensive in distal cells.     

Polymorphism and expression of isoflavone synthase genes from soybean cultivars

Isoflavones are synthesized by isoflavone synthases via the phenylpropanoid pathway in legumes. We have cloned two isoflavone synthase genes, IFS1 and IFS2, from a total of 18 soybean cultivars. The amino acid residues of the proteins that differed between cultivars were dispersed over the entire coding region. However, amino acid sequence variation did not occur in conserved domains such as the ERR triad region, except that one conserved amino acid was changed in the IFS2 protein of the GS12 cultivar (R374G) and the IFS1 proteins of the 99M06 and Soja99s65 cultivars (A109T, F105I). In three cultivars (99M06, 99M116, and Simheukpi), most of amino acid changes were such that the difference between the amino acid sequences of IFS1 and IFS2 was reduced. The expression profiles of three enzymes that convert naringenin to the isoflavone, genistein, chalcone isomerase (CHI), isoflavone synthase (IFS) and flavanone 3-hydroxylase (F3H) were examined. In general, IFS mRNA was more abundant in etiolated seedlings than mature plants whereas the levels of CHI and F3H mRNAs were similar in the two stages. During seed development, IFS was expressed a little later than CHI and F3H but expression of these three genes was barely detectable, if at all, during later seed hardening. In addition, we found that the levels of CHI, F3H, and IFS mRNAs were under circadian control. We also showed that IFS was induced by wounding and by application of methyl jasmonate to etiolated soybean seedlings.     

Acifluorfen-induced isoflavonoids and enzymes of their biosynthesis in mature soybean leaves : whole leaf and mesophyll responses

Mature soybean (Glycine max L. cv Harosoy 63) leaves normally contain kaempferol-3-glycosides but they accumulate no other flavonoids. Whole leaves sprayed with the diphenyl ether herbicide Acifluorfen and maintained in the light developed small necrotic lesions and accumulated isoflavone aglycones, isoflavone glucosides, and pterocarpans. Isoflavonoid accumulation was preceded by induced activity for chalcone synthase (CHS) and by increased activity for phenylalanine ammonia-lyase (PAL) and UDP-glucose:isoflavone 7-O-glucosyl transferase (IGT). PAL and CHS activity was highest between 24 and 30 hours after treatment, isoflavone aglycones and pterocarpans at 48 hours, IGT at 72 hours, and isoflavone glucosides at 96 hours.     

Disruption of the ceramide synthase LOH1 causes spontaneous cell death in Arabidopsis thaliana

The bioactive lipid ceramide is produced by the enzyme ceramide synthase, which exists in several isoforms in most eukaryotic organisms. Here, we investigated functional differences between the three ceramide synthase isoforms in Arabidopsis thaliana. The biochemical properties of the three ceramide synthases were investigated by comparing lipid profiles of yeast strains expressing LOH1, LOH2 or LOH3 with those of wild-type and loh1, loh2 and loh3 knockout plants. Expression profiles of the ceramide synthases and of the pathogenesis-related gene PR-1 were investigated by real-time PCR. Each ceramide synthase isoform showed a characteristic preference regarding acyl-CoA chain length as well as sphingoid base hydroxylation, which matches the pattern of ceramide and glucosylceramide species found in leaves. After extended culture under short-day conditions, loh1 plants showed spontaneous cell death accompanied by enhanced expression of PR-1. The levels of free trihydroxy sphingoid bases as well as ceramide and glucosylceramide species with C(16) fatty acid were significantly elevated while species with C(20) -C(28) fatty acids were reduced. These data suggest that spontaneous cell death in the loh1 line is triggered either by the accumulation of free trihydroxy sphingoid bases or ceramide species with C(16) fatty acid.