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1
Poonlaphdecha, Poonlaphdecha Janchai. "Biosynthesis of 2-acetyl-1-pyrroline, a potent flavour compound in rice : effect of salt stress during plant growth and some clues on the biosynthetic pathway." Thesis, Montpellier 2, 2011. http://www.theses.fr/2011MON20090.
Full textAbstract:
Effet de la salinité pendant la croissance de plants de riz sur la biosynthèse de la 2-acétyl-1-pyrroline (2AP), composé aromatique clé des variétés de riz aromatique,le rôle de la 1-pyrroline dans la biosynthèse de la 2AP ainsi que l'origine du groupe acétyle ont été étudiés. La variété aromatique de Camargue (France), Aychade, a été cultivée soit dans un milieu liquide soit dans des pots en serre. Dans le milieu de culture liquide, une augmentation significative de la teneur en 2AP dans les feuilles a été observée quelle que soit la concentration en NaCl (de 1,7 à 170 mM). Une corrélation positive a été mise en évidence entre la teneur en sel et en 2AP. A de plus fortes concentrations en NaCl, il a également pu être montré qu'il y avait une relation entre la concentration en proline, un précurseur aminé majeur de la 2AP, et la teneur en 2AP. Inversement, les changements en acide -aminobutyrique (GABA), un des métabolites compétitif de la biosynthèse de la 2AP, n'ont pas été corrélés avec ceux de la 2AP. Dans les expérimentations menées en serre, la variété Aychade a été cultivée dans une solution saline à 30 mM, correspondant à une conductivité électrique de 3800 ± 400 S.cm-1, valeur considérée comme suffisante pour induire un stress salin sur le plant de riz. Cette solution saline a été appliquée à des dates et pour des durées différentes en fonction du cycle de développement de la plante. A la phase végétative, une relation positive a été observée dans les feuilles entre le traitement salin et la teneur en 2AP ainsi qu'en proline. Il est intéressant de noter que tous les grains provenant des plants traités avec la solution saline contenaient des teneurs nettement plus élevées en 2AP par rapport au contrôle. La synthèse de la 2AP la plus importante s'est produite lorsque les plants ont été soumis à un traitement salin pendant l'ensemble des phases végétative et reproductive. Cependant, dans le dernier cas, le rendement en grain était beaucoup plus faible. En supplémentant des cals de riz avec de la 1-pyrroline, il a été démontré que cette imine cyclique était un facteur limitant pour la biosynthèse de la 2AP non seulement dans la variété aromatique, mais également dans la non aromatique. Les essais conduits avec le glucose et les acides gras marqués 13C ont montré que ces molécules pourraient être précurseurs du groupe acétyle de la molécule de 2APThe effect of salinity during rice plant growth on the biosynthesis of 2-acetyl-1-pyrroline (2AP), characteristic flavour compound of fragrant rice cultivars was studied. Additionally role of 1-pyrroline in 2AP biosynthesis together with source of acetyl group were investigated. Aychade, a fragrant rice was cultivated either in a liquid medium adapted for seedlings or in pots in a greenhouse. A significant increase in 2AP levels in the leaves was observed whatever the concentration of NaCl (1.7 to 170 mM NaCl) in the culture medium. A positive correlation was found between the level of salt and that of 2AP. There was a relationship between the concentration of proline, a major amino precursor of 2AP and 2AP level at highest concentrations of NaCl. Conversely the changes in -aminobutyric acid (GABA), a competitive metabolite in 2AP biosynthesis were not correlated with those of 2AP. In greenhouse experiments, Aychade cultivar was grown under one level of salt solution, 30 mM that corresponds to EC of 3800 ±400 S.cm-1, value considered sufficient to induce salt stress in rice. Timing and duration of application of salt solution varied according to the growth stages. At vegetative phase a positive relation was observed between salt treatment and 2AP as well as proline levels in the leaves. Interestingly grains from all salt treated plants contained significantly higher levels of 2AP than those from control. The highest synthesis occurred when plants were subjected to salt treatment during whole vegetative and reproductive phases. However in the latter case crop yield decreased significantly. Through supplementation of rice callus with 1-pyrroline it was demonstrated that this cyclic imine was limiting factor for the 2AP biosynthesis not only in a fragrant variety but also in a non fragrant variety. Experiments with 13C labelled glucose and fatty acids showed that glucose and fatty acids could be precursors of acetyl group of 2AP
2
Liao, Jyun-Ling, and 廖君綾. "Effect of NaCl stress on 2-acetyl-1-pyrroline formation in Saccharomyces cerevisiae." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/14061824320788157200.
Full textAbstract:
碩士國立屏東科技大學
食品科學系
94
A popcorn-like flavor compound, 2-acetyl-1-pyrroline (2-AP), has been reported as a potent flavor component of an aromatic rice, taro, pandan and baker. Recently bakers’ yeast was recognized as a potent source of precursors for the roast-smelling odorants 2-AP in wheat bread crust. Methylglyoxal (MG), derived from dihydroxyacetone phosphate (DHAP) was proposed to be responsible for the acetyl group of 2-AP. Saccharomyces cerevisiae was treated with various concentration of NaCl. The mRNA expression level under NaCl stress were monitored by RT-PCR. The MG detoxifying genes, including aldose reductase (AR) and MG reductase (MGR) mRNA were found to increase significantly. The concentration of MG level increased with increasing NaCl concentration. In response to the increased MG level, the MG metabolyzing genes were induced in yeast cells. The MG detoxifying genes encoding ScGlyoxalase II (ScGLYⅡ), ScAR and ScMGR were cloned into expression vectors, pET28b and pET32a, respectively, and overexpressed in E. coli. The molecular mass for ScGLYⅡ, ScAR and ScMGR of the expressed recombinant proteins were 32 kDa, 55 kDa and 39 kDa, respectively. In addition, △1-pyrroline-5-carboxylate (P5C) was found to increase with increasing NaCl concentration in yeast. Gene encoding Ornithine-δ-Transferase (OAT), which converts ornithine into P5C, was cloned into pYES2A and overexpressed in INVSc1. The molecular mass of the expressed recombinant proteins were 43 kDa, and the specific activity of ScOAT was 216 U/mg. Model system composed of P5C synthesized in vitro by ScOAT and MG was set up to study the formation of 2-AP. We postulated that MG accumulated in the NaCl stressed yeast cells, P5C was synthesized in response to the toxic effect of MG, and the interaction between MG and P5C led to biosynthesis of 2-AP.
3
Zeng, Hao-Zhen, and 曾皓真. "Genes encoding enzymes related to 2-acetyl-1-pyrroline formation in Saccharomyces cerevisiae." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/23017765795320682920.
Full textAbstract:
碩士國立屏東科技大學
生物科技研究所
95
A popcorn-like flavor compound, 2-acetyl-1-pyrroline, has been reported as a potent flavor component of an aromatic rice, taro, soybean and pandan leaves. Results of tracer experiments indicated that the pyrroline source of 2-acetyl-1-pyrroline was proline, whereas the source of acetyl group was unknown. Methylglyoxal (MG), derived from dihydroxyacetone phosphate (DHAP) was proposed to be responsible for the acetyl group of 2-AP. Glycerol is common compatible osmolyte in water-stress organism. Glycerol kinase is key enzyme of the intermediate during metabolism. The concentration of MG level increased with increasing Glycerol concentration. We amplified the gene of glycerol kinase (GK), Glycerol-3-phosphate dehydrogenase (G3PDH) and Ornithine aminotransferase(OAT) from Saccharomyces cerevisiae by PCR. After confirming the sequences, we constructed the recombinant expression plasmid pYES2B-SCGK, pYES2B-SCG3PDH and pYES2A-SCOAT , and then transformed the recombinant genes into INVSC1 to express recombinant proteins. Model system composed of P5C synthesized in vitro by ScOAT and MG was set up to study the formation of 2-AP. In addition, the mRNA expression of the glycerol metabolism related genes: Glycerol kinase (GK)、Glycerol-3-phosphate dehydrogenase (G3PDH)、Glycerol dehydrogenase (GDHG)、Dihydroxyacetone kinase (DHAK) were analyzed with gene-specific primers by RT-PCR (Reverse Transcription-Polymerase Chain Reaction) in yeast treated various concentrations of NaCl and glycerol. Among the four genes tested, G3PDH overexpression of RNA was observed in NaCl treated yeast alls. The expression of G3PDH showed a dose dependent manner up to 5％ NaCl.
4
Chang, Chia-Lin, and 張佳霖. "The study of biosynthetic mechanism of 2-acetyl-1-pyrroline in aromatic rice." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/03175036645855915009.
Full textAbstract:
碩士國立屏東科技大學
生物科技研究所
96
The Contents of Abstract in This Thesis: 2-Acetyl-1-Pyrroline (2-AP) was a major flavor component in aromatic rice varieties. In our previous study, a model system was set up to investigate the formation of 2-AP and the results showed that Δ1-pyrroline-5-carboxylic acid (P5C) could react with methylglyoxal (MG) to form 2-AP. Thus, we hypothesized that P5C and MG might be precursors for 2-AP biosynthesis in aromatic rice. In aromatic rice varieties Tainung 71 and 72, the endogenous P5C and MG contents have a positive correlation with 2-AP levels. Glutamate could be converted into P5C by Δ1-pyrroline-5-carboxylate synthase (P5CS). To examine the relationship between endogeneous levels of P5C and the expression of P5CS, the level of P5CS mRNA was detected in callus of aromatic and nonaromatic rice cultivars. The expression levels of P5CS1 and P5CS2 genes were found to be significantly higher in aromatic rice than nonaromatic rice. Specific enzyme activity of P5CS also increased significantly in aromatic rice varieties. Otherwise, the expression of P5CS1 and P5CS2 gene could be stimulated by the treatment of high salt in aromatic rice varieties Tainung 72. By tracer experiment using 15N-glutamate, the result also demonstrated that the nitrogen source of 2-AP can be derived from glutamate. Based on the data collected, a formation mechanism of 2-AP in aromatic rice via the interaction between P5C and MG was proposed. Keyword: aromatic rice, 2-acetyl-1-pyrroline、△1-Pyrroline-5-Carboxylate synthase (P5CS)、△1-pyrroline-5-carboxylate (P5C)、methylglyoxal (MG)
5
Lo, Wei-Cheng, and 羅偉誠. "Effect of Stress on the Biosynthesis of 2-acetyl-1-pyrroline in Bread." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/53825157051208317980.
Full textAbstract:
碩士國立屏東科技大學
食品科學系
89
Many aroma compounds generated in wheat bread during baking process have been identified. One example is 2-acetyl-1-pyrroline, an impact compound in the wheat bread crust of Maillard reaction product with typical roasty flavor characteristic. Synthesis of the important odorant, 2-acetyl-1- pyrroline, which was often described as popcorn-like, bready and roasty, was studied. Model system composed of 1-pyrroline and various sugar and phosphorylated sugar degradation products, such as dihydroxyacetone, glyceral- dehyde, dihydroxyacetone phosphate or glycerol- dehyde phosphate were set up to study the formation mechanism of 2-acety-1-pyrroline. Volatile compounds generated were extracted and characterized by GC and GC-MS. Based on the data collected, a formation mechanism for 2-acetyl-1-pyrroline via the interaction between 1- pyrroline and dihydroxyacetone phosphate was proposed. Yeast culture was added in different salt concentration medium (0、2、4、6、8%). The results showed that under more intense stress the yeast lag phase was longer and the time of log phase was longer. Handworked dough, frozen dough and retail frozen dough were prepared. Handworked dough’s color and aroma were not obvious during baking, but GC-MS analysis indicated increased production of 2-acetyl-1-pyrroline in handworked frozen and retail frozen dough. During baking the yeast produced more 2-acetyl-1-pyrroline under stress than normal condition.
6
Jian, Ru-Yu, and 簡如玉. "Effect of salt stress on 2-acetyl-1-pyrroline formation in Bacillus subtilis var. natto." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/25272400839877098178.
Full textAbstract:
碩士國立屏東科技大學
食品科學系所
96
A popcorn-like flavor compound, 2-acetyl-1-pyrroline (2-AP), has been reported as a potent flavor component of an aromatic rice, taro, pandan and bread. The nitrogen source of pyrroline of 2-AP was proline, but the acetyl-group has not been well-characterized. On the other hand, methylglyoxal(MG), the putative precursor of 2-AP may derive from dihydroxyacetone phosphate (DHAP) through triosephosphate isomerase catalysis. It was hypothesized that 2-AP was synthesized from △1-Pyrroline-5- Carboxylate (P5C) and Methylglyoxal (MG). Therefore, in order to study the molecular mechanism of 2-AP biosynthesis, we analyzed the expression profile of MG and P5C biosynthetic enzymes and the level of MG and P5C in Bacillus subtilis var. natto under salt stress. The results showed that the level of glycerol-3-phosphate dehydrogenase (G3PDH), triosephosphate isomerase (TIM), fructose-1,6-bisphosphate aldolase (FBA), methylglyoxal synthase (MGS), Ornithne aminotransferase (OAT), proline dehydrogenase (PDH), γ-Glutamyl kinase (γ-GK) and γ-Glutamyl phosphate reductase (γ-GPR) mRNA increased significantly after 30 min of exposure at various concentration of NaCl. Otherwise, the concentrations of MG and P5C increased with increasing NaCl concentration, and when cell were subjected to 1M NaCl treatment for 20 min, accumulation of MG and P5C were reached highest (about 91.5 μmolg-1 and 0.102 μmolg-1). In vitro model system was set up to study the formation of 2-AP under salt stress. The qualitative analysis of 2-AP was performed by GC-MS. Total ion chromatogram showed the peak with retention time at 7.45 minutes corresponds to 2-AP. Therefore, we purposed MG interact with P5C to produce 2-AP.
7
Cheng, Teen-Chi, and 鄭汀琦. "Cloning and expression analysis of genes encoding 2-acetyl-1-pyrroline biosynthetic enzymes from aromatic rice." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/96674620112941066597.
Full textAbstract:
碩士國立屏東科技大學
食品科學系
91
The purpose of the research is to clone and study express the genes encoding 2-acetyl-1-pyrroline biosynthetic enzymes (glycerol kinase and dihydroxyacetone kinase) in aromatic rice (Oryza sativa cv. TN71). The glycerol kinase (GK) and dihydroxyacetone kinase (DAK) gene of Oryza sativa TN71 had been cloned by using polymerase chain reaction (PCR) cloning technology. The degenerate primers were designed based on conserved amino acid sequences in the PCR for O-GK and O-DAK gene amplification. End-to-end PCR was performed to amplify full-length GK and DAK cDNA. Rice calli were treated with different levels of proline and glycerol to study the expression of both genes, which was analysed by real time PCR and calibrated with the housekeeping actin cDNA. The results showed that both GK and DAK expression in the calli were induced by glycerol in a dose dependent manner.
8
賴怡君. "The study of biosynthetic mechanism of 2-acetyl-1-pyrroline in aromatic rice by abiotic stress." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/46135304969057940531.
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Ke, Yi-Jyun, and 柯怡君. "Cloning and Genes Expression Analyses of 2-Acetyl-1-Pyrroline Biosynthesis-Related Enzymes in Pandanus amaryllifolius." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/63061502583327533845.
Full textAbstract:
碩士國立屏東科技大學
食品科學系
94
Pandanus amaryllifolius Roxb. (Pandan) grows in Southeast Asia and is the only reported pandanus species with scented leaves. The aroma source is mainly 2-acetyl-1-pyrroline. The nitrogen source of pyrroline of 2-AP structure was proline. Proline metabolism may play a crucial role in 2-AP formation. On the other hand, methylglyoxal (MG) may derive from dihydroxyacetone phosphate (DHAP) was proposed to be responsible for 2-AP formation. This study probed into the formation mechanism of 2-AP in pandan under light or dark. In the preliminary experiment, the level of 2-AP was higher in pandan leave under light than under dark. The mRNA expression of the 2-AP synthesis related genes were analyzed with gene-specific primers by RT-PCR (Reverse Transcription-Polymerase Chain Reaction) in pandan under light and dark. MG, P5C and 2-AP contents in pandan leaves under either light or dark were examined. The levels of expression of GapC mRNA under light were the lowest. GapC exists in the cytosol and is responsible for the metabolism of GAP. The lower activity of GapC in lighted pandan leave may be attributed to the accumulation of MG. The levels of both P5C and MG were higher in pandan leave under light than that in dark were found to be parallel to the 2-AP level. A formation mechanism of 2-AP from interation between P5C and MG was proposed. In addition, the key enzymes from pandan for MG biosynthesis, including the subunit A (GapA), B (GapB) and C (GapC) of GAPDH were cloned. Overexpression of GAPDH of the recombinant proteins exhibit a molecular mass of 43.3KDa, 34.3KDa and 36.7KDa, respectively. Specific activities of GapC of the recombinant proteins purified by Ni2+ affinity column were determined as 5.11 U/mg.
10
Chuang, Hui-Su, and 莊惠夙. "Cloning and genes expression analyses of 2-Acetyl-1-Pyrroline biosynthesis-related enzymes from aromatic rice." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/65372913728821202292.
Full textAbstract:
碩士國立屏東科技大學
生物科技研究所
93
2-Acetyl-1-Pyrroline(2-AP) was the principal flavor component in taro-scented rice(Tainung 71(TNG 71)(Yihchaun Aromatic Rice) and Tainung 72(TNG72)). Results of tracer experiments indicated that the pyrroline source of 2-AP was proline, whereas the source of acetyl group was unknown. It was hypothesized that 2-AP was synthesized from △1-pyrroline-5-carboxylate (P5C) and methylglyoxal (MG). Therefore, we studied the 2-AP synthesis related genes from Oryza sativa including that the P5C synthesis related genes : △1-pyrroline-5-carboxylate synthetase(P5CS), ornithine-δ-transferase (OAT) and proline dehydrogenase (PDH); the MG synthesis related genes : glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerol kinase (GK), fructose-bisphosphate aldolase (FBA), glucose-6-phosphate isomerase(GPI) and triose-phosphate isomerase (TPI). In addition the MG metabolism related genes : glyoxalase I(GLYI), glyoxalase II(GLYII), aldose reductase (AR), betaine aldehyde Dehydrogenase (BAD) were analyzed as well. PCR was used to clone these genes from callus of TNG 67, TNG 71 and TNG 72. The mRNA expression of the 2-AP synthesis related genes were analyzed with gene-specific primers by RT-PCR (Reverse Transcription-Polymerase Chain Reaction) in both aromatic rice and non-aromatic rice. The levels of expression of OsP5CS mRNA were the highest among the P5C synthesis related genes tested. No significant variation in mRNA expression was found among the MG synthesis related genes. The levels of OsAR mRNA among the cultivars tested in this experiment were found to be in the order of TNG67>TNG 71 >TNG 72. Based on the finding that TNG 72 possesses the strongest aroma and lowest level of OsAR mRNA, a mechanism for 2-AP biosysthesis was proposed. The lack of OsAR gene expression in TNG 72 leads to the accumulation of MG. P5C is synthesized by P5CS to neutralize MG. This detoxification system in TNG 72 is responsible for the biosynthesis of 2-AP.
11
Chen, Jian-Kai, and 陳建凱. "Cloning and genes expression analyses of 2-acetyl-1-pyrroline biosynthesis-related enzymes in vegetable soybean." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/97934894949300315936.
Full textAbstract:
碩士國立屏東科技大學
食品科學系
93
2-acetyl-1-pyrroline (2AP) has been reported as a potent flavor component of aromatic rice, taro, pandan ,and green soybean. During growing to vegetable soybean, 2AP levels in the young seeds increased with increasing maturity. The nitrogen source of pyrroline of 2AP structure was praline, but the acetyl-group has not been well-characterized. On the other hand, methylglyoxal, the putative precursor of 2AP may derive from dihydroxyacetone phosphate (DHAP) through triosephosphate isomerase catalysis. In this study, the key enzymes from vegetable soybean for 2AP biosynthesis, including, the subunit A (GapA), B (GapB) and C (GapC) of glyceraldehydes-3-phosphate dehydrogenase, triosephosphate isomerase (TIM) and ornithine-δ-aminotransferase (OAT) were cloned. These genes were overexpressed in Escherichia coli or Pichia pastoris. Specific activities of GapA, GapB, GapC, TIM and OAT of the recombination proteins purified by Ni2+ affinity column were determined as 9.8, 7.9, 27.4, 46.7 and 50.8 U/mg, respectively. Expression levels of these gene encoding key enzymes between aromatic and non-aromatic vegetable soybean was campared by RT PCR. No significant variation among OAT, P5CS and PDH genes was observed. However, higher TIM and lower GapC expression in aromatic vegetable soybean was found. Therefore, we hypothesized that TIM and GapC are closely related to the 2AP biosynthesis in aromatic vegetable soybean.
12
Yen, Tzu-Yang, and 顏子揚. "Cloning and expression of the key enzymes for 2-acetyl-1-pyrroline biosynthesis in green soybean." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/58989566099136586390.
Full textAbstract:
碩士國立屏東科技大學
食品科學系
92
A popcorn-like flavor compound, 2-acetyl-1-pyrroline (2-AP), has been reported as a potent flavor component of an aromatic rice, taro and soybean. During growing to vegetable soybean, the concentration of 2-AP in the young seeds increased gradually and it reached for the maximum concentration in the edible period for edamame. 2-AP was formed in the aerial part of aromatic rice from proline as the nitrogen precursor, but the acetyl-group has not been well-characterized. We cloned the full-length gene of proline dehydrogenase (PDH), △1-pyrroline-5-carboxylate synthetase (P5CS), glycerol kinase (GK) and fructose-bisphosphate aldolase (FBA) from green soybean by RACE-PCR and registered the sequences on NCBI GenBank. These genes have been constructed into pET and pGEX System (pET-21b, pET-28b and pGEX-5X-3) and expressed in E. coli. However, the expression of these genes in E. coli resulted in the accumulation of inclusion bodies. These recombinant protein were purified by Ni2+-affinity column and the activity was lost after refolding. Only the soluble fusion protein was detected in pET28-FBA. After expression in E.coli, the proteins were purified from cell extract by Ni2+-affinity column chromatography. The purified FBA was estimated to have an specific activity of 1.355 U/mg. The others were constructed into the expression vector (pPICZαA) and expressed in Pichia pastoris. Furthermore, we also investigated the relationship between the formation of 2-AP and the expression of related genes in green soybean by Real-Time PCR. These genes will be transferred into rice calli (TNG 67) by Agrobacterium to offer the further genetic evidence for biosynthesis mechanism of 2-AP in plants.
13
SHIH, TENG CHIA, and 鄧佳詩. "Studies on the biosynthesis mechanism of 2-acetyl-1- pyrroline and its relationship with the expression of △1-pyrroline-5-carboxylate synthesis gene in fragrant rice." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/63122180633724158885.
Full textAbstract:
碩士國立屏東科技大學
食品科學系所
95
2-Acetyl-1-Pyrroline (2-AP) was a major flavor component in aromatic rice varieties Tainung 71(Yihchaun Aromatic Rice) and Tainung 72. The qualitative analysis of 2-AP was by GC-MS and found the retention time of 2-AP was approximately at 7.5 minutes. The quantity analysis of 2-AP was also performed by GC-MS. Aromatic 2-AP of Tainung 71 and Tainung 72 were 0.18 ppm and 0.34 ppm, respectively. Results of tracer experiments indicated that the pyrroline source of 2-AP was proline and glutamate, whereas the source of acetyl group was unknown. The hypothesis that 2-AP was synthesized from △1-pyrroline-5 -carboxylate (P5C) and methylglyoxal (MG) was demonstated in vitro. Proline is synthesized from glutamate via the intermediate 1-pyrroline-5-carboxylate (P5C). The intermediate P5C formation for proline synthesis is catalyzed by 1-pyrroline-5-carboxylate synthetase (P5CS). There are two kinds of P5CS which are encoded by OsP5CS1 and OsP5CS2. OsP5CS1 is a housekeeping gene, which supplies proline to the cell, and OsP5CS2 is primarily responsible for stress responses. In this study, OsP5CS1 and OsP5CS2 have been cloned from callus of non-aromatic rice (Tainung 67) and fragrant rice (Tainung 71 and 72) using PCR. The mRNA expression profiles of the OsP5CS1 and OsP5CS2 in the three cultivars were performed by RT-PCR. The results were supported by the quantition of MG and P5C by HPLC. It showed that the transcription levels of OsP5CS2 and the concentrations of MG and P5C in fragrant rice, Tainung 71 and 72, were significantly higher than those in Tainung 67. Therefore, our results indicate that the amount of 2-AP and the quantition of P5C and MG in rice callus have positive correlation.
14
Chen, Shu-sheng, and 陳士賢. "Establishment of in vitro model of 2-acetyl-1-pyrroline synthesis and expression of its biosynthesis-related enzymes." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/08719280597339865512.
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碩士國立屏東科技大學
食品科學系所
96
2-acetyl-1-pyrroline (2-AP) had been proposed as a character impact odorant causing the roasty note of bread crust and aromatic rice. The synthesis of 2-AP associated with amount of ornithine or proline in food and ornithine could reacted with carbohydrate degradation products during heating. The product of ornithine metabolism was Δ1-pyrroline-5-carboxylate (P5C) and methylglyoxal (MG) was end product of glucose degradation. We hypothesized that P5C reacted with MG result in synthesis of 2-AP. We also established the in vitro model of 2-AP synthesis successfully using ornithine and MG at 25℃, identification and quantification of 2-AP by GC/MS. The concentration of 2-AP synthesis about 0.4 μg/mL. In the time-dependent, values of 2-AP was the highest (0.43 μg/mL) in third day. In dose-dependent, the values of 2-AP synthesis decreased with increasing ornithine or MG concentrations. However, there were no relationship between maillard reaction and 2-AP biosynthesis in vivo. The reports suggested that the produ ction of 2-AP occurs via acetylation of 1-pyrroline by B. cereus. In order to study the pathway of 2-AP biosynthesis in vitro. In this study, the genes hypothesized to be involved in 2-AP biosynthesis, including glycerol-3-phosphate dehydrogenase (G3PDH), methylglypxal synthase (MGS) and acetyltransferase (MPR1), were cloned from Bacillus subtilis ssp. natto and Saccharomyces cerevisiae. These genes were overexpressed in Escherichia coli BL21 (DE3) and purified by Ni2+-affinity column. The molecular mass for MPR1, G3PDH and MGS of the expressed recombinant proteins were 26 kDa, 66 kDa and 15 kDa, respectively. The specific activity of MPR1 and G3PDH were 0.035, 0.0086 U/mg respectively.
15
Wu, Po-yi, and 吳柏毅. "The study of 2-acetyl-1-pyrroline biosynthetic mechanism in aromatic rice by RNAi-mediated silencing and Proteomic analysis." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/14205398913301545237.
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碩士國立屏東科技大學
生物科技研究所
100
2-acetyl-1-pyrroline (2-AP) was the major flavor component in aromatic rice. In aromatic rice, glutamate could be converted into P5C by △1-pyrroline-5-carboxylate synthase (P5CS), we hypothesized that P5C and MG might be precursors for 2-AP biosynthesis in aromatic rice. In previous study, we have found that the endogenous P5C and MG contents have a positive correlation with 2-AP levels in aromatic rice varieties Tainung 71 and 72. The expression levels of P5CS2 gene were found to be significantly higher in aromatic rice than non-aromatic rice. Otherwise, the biosynthesis of 2-AP and expression of P5CS2 gene could be stimulated under salt stress in aromatic rice. In this study we used the RNA-interference (RNAi) technology to investigate the role of the P5CS2 in 2-AP biosynthesis of aromatic rice. To construct transformation vector that produce double-stranded (ds) RNA expressing unit, the sense and antisense DNA fragments of P5CS2 were linked with GFP under the control of ubiquitin promoter. The dsRNA expression vector was successfully introduced into Tainung 71 callus by Agrobacterium tumefaciens-mediated transformation. Reverse transcriptase -polymerase chain reaction (RT-PCR) analysis revealed that the gene expression for P5CS2 was effectively down-regulated in RNAi transgenic rice callus. To evaluate the relationship between 2-AP and P5CS2, the concentrations of 2-AP in RNAi transgenic rice callus was analyzed by GC-MS. Our results showed that concentrations of 2-AP in non-RNAi and RNAi transgenic rice callus were estimated to be 306±37.8 ppb and 84.2±61.7ppb. The result suggested that P5CS2 plays a critical role for 2-acetyl-1-pyrroline biosynthesis in aromatic rice Tainung 71. Otherwise, the protein expression profiles were compared between aromatic and non-aromatic rice callus by proteomic techniques. Six proteins might related to aroma formation were identified with MALDI-TOF-MS. Further experimental works will be done to unveil the relationship between these proteins and aroma formation.
16
Mu, Min-Ei, and 吳銘益. "Establishment of culture system and Agrobacterium-mediated gene transformation of 2-acetyl-1-pyrroline synthesis related gene in rice." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/34490781724144348836.
Full textAbstract:
碩士國立屏東科技大學
生物科技研究所
96
The contents of abstract in this thesis:The purpose of the research was to establish the agrobacterium transformation system of 2-AP (2-acetyl-1-pyrroline) related gene. In the previous research, methylglyoxal(MG) and P5C (1-pyrroline-5-carboxylate) was supposed as the precursor of the 2-AP. In this research, the c-DNA of the gene 1-pyrroline-5 carboxylate synthetase(P5CS) and fructose bis-phosphate aldolase(FBA), extracted form the scented vegetable soybean, was cloned and transferred into the rice seedling via agrobacterium-mediated transformation in order to study the 2-AP biosynthesis. The rice embryogenic-calli culture system was established, and the transformed plant was obtained when the culture system was used as the basic for the agrobacterium transformation. The leaf of the P5CS transgenic plant turned brown was supposed as the toxic effect due to the P5C accumulation, and the FBA transformed calli over-contaminated was explained by the bacterium activation due to the accumulation of the acetyl-coA. The system established was suitable for the agrobacterium transformation of the 2AP synthesis related gene.
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Yen, Shih-Feng, and 顏士峰. "Cloning and genes expression analyses of 2-acetyl-1-pyrroline biosynthesis-related enzymes in Saccharomyces cerevisiae and Bacillus cereus." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/34482247562859178298.
Full textAbstract:
碩士國立屏東科技大學
食品科學系
93
A popcorn-like flavor compound, 2-acetyl-1-pyrroline(2-AP), has been reported as a potent flavor component of an aromatic rice, taro and pandan. Ornithine metabolism may play a crucial role in 2-AP formation. On the other hand, methylglyoxal may derive from dihydroxyacetone phosphate (DHAP) was proposed to be responsible for 2-AP formation. Important intermediate pyrroline-5-carboxylic acid (P5C) can be biosynthesized by γ-glutamyl kinase (PRO1), γ-glutamyl phosphate reductase (GPR), ornithine aminotransferase (OAT) and proline oxidase (PUT). The recombinant expression vectors pET32b-ScOAT and pET28b-BcOAT, were constructed and expressed. The molecular mass of the expressed recombinant proteins were 70 KDa and 43 KDa, respectively. These recombinant protein were purified by Ni2+-affinity column and the enzyme activities were assayed. The results indicate the specific activity of ScOAT and BcOAT were 13.89 and 268.95 U/mg, respectively. The PRO1, GPR, OAT, PUT1, triose phosphate isomerase (TIM) and glyceraldehydes-3-phoshpate dehydrogenase (GAP) mRNA expression level under stress of NaCl were monitored by RT-PCR. The results indicate the OAT mRNA was increased under salt-stress.
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Huang, Yi-Wen, and 黃意文. "Cloning and expression of the key enzymes for 2-acetyl-1-pyrroline biosynthesis in Bacillus natto and Pandanus amaryllifolius." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/15370711970642284051.
Full textAbstract:
碩士國立屏東科技大學
生物科技研究所
92
A popcorn-like flavor compound, 2-acetyl-1-pyrroline, has been reported as a potent flavor component of an aromatic rice, taro, soybean and pandan leaves. Results of tracer experiments indicated that the pyrroline source of 2-acetyl-1-pyrroline was proline, whereas the source of acetyl group was unknown. Proline and glycerol are common compatible osmolytes in water-stress organism. Proline dehydrogenase and glycerol kinase are key enzymes of these two intermediates respectively during metabolism. We amplified the gene of proline dehydrogenase (PDH) and glycerol kinase (GK) from Bacillus natto by PCR. After confirming the sequences, we constructed the recombinant expression plasmid pET21b-BNPDH and pET21b-BNGK. Otherwise, We cloned the full-length gene of fructose-bisphosphate aldolase(FBA) and glycerol kinase from Pandanus amaryllifolius by RACE-PCR and registered the sequences on NCBI GenBank. We constructed the recombinant expression vector pET28b-PaFBA and pET28b-PaGK and transformed the recombinant genes into E. coli BL21 (DE3) to express recombinant proteins. After inducing by IPTG, these proteins were purified by Ni2+-affinity column and the enzyme activities were assayed. The results indicate the specific activity of BNPDH was 28.86 U/mg, optimum pH range of 7 to 8.5 and optimum temperature of 25℃. The specific activity of GK was 0.855 U/mg. The specific activity of PaFBA was 3.762 U/mg, optimum pH range of 8.0 to 9.0 and optimum temperature range of 25℃to 50℃。However, the expression of recombinant PaGK in E. coli resulted in the accumulation of inclusion bodies.
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Wu, Chi-Ruei, and 吳其叡. "Studies on the expression of methylglyoxal metabolizing enzymes and its relation to 2-acetyl-1-pyrroline biosynthesis in soybean." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/76585038458227282490.
Full textAbstract:
碩士國立屏東科技大學
食品科學系所
95
2-acetyl-1-pyrroline (2AP) has been reported as a potent flavor component of popcorn, aromatic rice, vegetable soybean, pandan leaves. The nitrogen source of pyrroline of 2AP was proline, but the acetyl-group has not been well-characterized. According to the methylglyoxal metabolizing pathway, it has been hypothesized the methylglyoxal (MG) is the important intermediate product for acetyl-group in soybean, and concentration of MG is expected to be positively correlated to the 2AP level in soybean. MG is a kind of α-oxoaldehyde. It is synthesized either enzymatically or nonenzymatically. Exposure of cells to high concentrations of methylglyoxal, by addition of exogenousα-oxoaldehyde or inhibition of enzymatic detoxification, induces growth arrest and toxicity. In cells, methylglyoxal can be detoxified by glyoxalase system, aldose reductase, betaine aldehyde dehrdrogenase and glutathione-S-transferase. We have found that the aromatic soybean contains MG about 4.45 mM/g, which is higher than 4.09 mM/g in non-aromatic soybean. The expressions of MG metabolizing gene have been studied by RT-PCR. The variation in gene expression between aromatic and non-aromatic vegetable soybean seed has been compared. The MG detoxifying genes, including AR, BAD, Gly I, GST, were found to increase significantly in aromatic soybean. The level of 2AP in aromatic and non-aromatic soybean was quantified by GC/MASS and SPME. We purposed MG and P5C will lead to biosynthesis 2AP in aromatic soybean.
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Chen, Ching-Hsiang, and 陳青祥. "Studies on the expression of methylglyoxal metabolizing enzymes and its relation to 2-acetyl-1-pyrroline formation in Rice seedling." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/69481898801229578707.
Full textAbstract:
碩士國立屏東科技大學
生物科技研究所
94
A popcorn-like flavor compound 2-acetyl-1-pyrroline (2-AP) has been reported as a potent flavor component of an aromatic rice, taro, soybean and pandan leaves. We hypothesized that 2-AP is synthesized from methylglyoxal (MG) and △1-pyrroline-5-carboxylate (P5C). The 2-AP synthesis related genes including aldose reductase (AR), glutathione-S-transferase (GST), glyoxalase I (GLYI), glyoxalase II (GLY II) and betaine aldehyde dehydrogenase (BAD) from aromatic rice (Tainung 71(TNG 71,Yihchaun Aromatic Rice) and Tainung 72(TNG72)) and non-aromatic rice Tainung 67 (TNG 67) were cloned by PCR. The mRNA level of aroma and non-aroma rice were monitored and compared by RT-PCR. The results showed that no significant variation in gene expression of GST、GLYI、 II 、BAD were found. However, the lowest mRNA expression for AR was observed in TNG 72 compared with that of TNG 67and TNG 71. In parallel to that of gene expression , AR activity was found to be following the order of TNG 67＞TNG 71＞TNG 72.Levers of MG in all three rice varieties were quantified and found in the order of TNG 72＞TNG 71＞TNG 67. The lower expression and enzyme activity for AR in TNG 72 may be responsible for the accumulation of MG. In addition to MG, the P5C levels of all three varieties were determined as well. The results showed TNG 72＞TNG 71＞TNG 67. The 2-AP concentration were determined to be in order of TNG 72＞TNG 71＞TNG 67. The results showed that the higher P5C and MG levels may be responsible for the biosynthesis of 2-AP in TNG 72.
21
Piebiep, Goufo. "Appropriate set of pre- and post-harvest treatments for obtaining high levels of aromas and quality in fragrant rice (Oryza sativa L.)." Doctoral thesis, 2010. http://hdl.handle.net/10348/8936.
Full textAbstract:
A dissertation submitted in partial fulfillment of the requirement for the
degree of Doctor of Philosophy (Ph.D.) in CROP SCIENCE (first-level discipline),
CROP CULTIVATION AND FARMING SYSTEM (second-level discipline)Aromatic rices (Oryza sativa L.) constitute a special group of rice accessions well known for their aroma and/or superfine grain quality. For a successful development of aromatic rices, research regarding factors affecting the quality of the aroma is of economic interest to rice growers and processors. Therefore, this study was conducted as a preliminary step towards the rice quality improvement of two aromatic rice cultivars growing in South China, namely Guixiangzhan and Peizaruanxiang. The fragrance potential of two rice cultivars was investigated using headspace SPME and static headspace in conjunction with GC-MS, and under optimal conditions for the identification and quantification of the intensely popcorn-like smelling compound 2-acetyl-1-pyrroline (2-AP). About a 5-fold difference of 2-AP levels were observed among the two rices with Guixiangzhan having the highest content (3.86 µg/g) comparable to that obtained with Thai KDML 105 rice. Other compounds instead of 2-AP were assumed to contribute to the characteristic aroma of Peizaruanxiang. The two cultivars were subjected to four pre-harvest treatments (planting densities of 16, 19, 22, 28, and 37 hills/m2 , harvesting times of 10, 20, 30, 40, and 50 days after heading, ripening temperatures during the early and the late seasons, application of growth regulators consisting of gibberellic acid, paclobutrazol, 3-indole acetic acid and a mixture of paclobutrazol, proline, and zinc chloride) and three postharvest treatments (storage times of 3 and 6 months, storage temperatures of -4, 8, 20, and 30 ºC, milling degree of 85%). Results were also discussed in terms of antioxidants enzymes activities (peroxidase, superoxide dismutase, proline oxidase), yield attributes (number of panicles per hill, number of spikelets per panicle, grain-filling percentage, paddy yield, 1000-grain weight), milling quality (milled rice rate, whole rice rate, head rice rate), grain appearance (% area with chalkiness, grain vitreosity), malondialdehyde, proline, total soluble proteins, amylose and protein contents of rice samples associated with differing pre-harvest regimes. Highest 2-AP concentrations were obtained for Guixiangzhan and Peizaruanxiang with the lowest planting density of 16 hills/m2 (3.73; 0.69 µg/g), the earliest harvesting time of 10 days after heading (5.24; 0.72 µg/g), a low ripening temperature of 25 ºC (7.12; 2.42 ng/g), the shortest storage time of 3 months (2.40; 0.45 µg/g) and the coolest temperature of -4 ºC (3.42; 0.49 µg/g). After milling, 2-AP content decreased up to 1.5-fold in both Guixiangzhan and Peizaruanxiang. All treatments with growth regulators, although improved grain yield and quality, and enhanced the capacity of Peizaruanxiang and Guixiangzhan to scavenge and control the production of damaging species of active oxygen, resulted in reduced aroma content that negatively affected overall flavor in a smelling evaluation. Decreases ranged from 9 to 24% compared to the control (2.40; 0.41 µg/g). These findings indicate that manipulating pre and postharvest treatments can greatly improve the specific attributes of the domestically produced aromatic rices. It is assumed from the study that keeping aromatic rice under refrigeration, milling it at a low degree and consuming it within six months would be a practical way to preserve its desirable character as monitored by changes in the levels of 2-AP. Results from our investigation also show that altering sowing dates to allow the critical stages of seed maturation to coincide with favorable field environments (cool and dry segment of the year) as well as planting at low density and early harvesting could improve aroma content and other seed qualities. It is also recommended that the aroma quality should be carefully monitored if growth regulators are to be used. With that in mind, China could effectively increase its share of the domestic market of fragrant rices and even tap into the international market
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Cheng, Guei-Long, and 鄭貴隆. "I Rapid and Accurate Analysis of 2-Acetyl-1-pyrroline and Volatile Components in Rice Aroma Mutants by HS-SPME/GC-MSII Screening of the rice mutants with highly abundant vitamin E and γ-oryzanol." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/88803775796977364141.
Full textAbstract:
碩士國立中興大學
生命科學院碩士在職專班
105
I Fragrant rice varieties contain major aroma compounds (2-acetyl-1-pyrroline, 2-AP) popcorn-like aroma compounds. Consumer demand for fragrant rice is increasing in the international market and is valued for its better quality and leading to a high economic value. The brown rice grains of SA0420, SA1159, SA1528 from a Tainung67 (TNG67) mutation pool developed by sodium azide (NaN3) mutagenesis, and other varieties IR64, ir64A and RA1076 (a near isogenic line of SA0420 on the same background of TNG67), as well as the international fragrant rice including Jasmine85, Basmati, KDML105, TNG71 totally 11 samples. In order to accurate measure the volatile compounds in rice, the volatile compounds were collected by using the headspace solid-phase microextraction (HS-SPME) method to absorb volatiles until the state equilibrium. Gas chromatography (GC) capillary separation characteristics and temperature gradient control, the mixture separated into various components, and went through mass spectrometer (MS) for high accuracy and resolution analysis. According to the mass spectrometry diagram of different mass charge ratio and retention time, the fragments and ratios obtained by the NIST software were convert into molecular structure and content, from which the volatile compounds are analyzed and quantified. Among the 11 rice brown rice, ketones, alcohols, aldehydes, alkanes, alkenes and a total of 60 compounds were identified. Analysis of the differences in volatile of TNG67 and SA0420, SA1159, RA1076, seven volatile obtained in the vann diagram. Quantitation was conducted for the key aroma compound 2AP, using SIM mode, the linear curve R2 = 0.9902 (0.01-1 mg / kg, CV% <15). No 2AP was detected in TNG67, IR64, SA1159, the concentration in other samples showed in SA0420 (0.771), SA1528 (0.142), RA1076 (0.678), Jasmine85 (0.182), Basmati (0.444), KDML105 (0.376), TNG71 (0.120), IR64A (0.688) in mg/kg. This study established a rapid and accurate method for the identification of volatile aromatic compounds, which will help the future rice breeding for gragrance. II Rapid analysis of vitamin E and γ-mofalfol from rice mutagenesis library, screening of mutant rice with antioxidant potential. Used Ultra-Performance Liquid Chromatography (UPLC) with Triple Quadrupole Mass Spectrometry (TQMS) to enhance sample throughput and high sensitivity to detect with Multiple reaction monitoring (MRM) to establish a quantitative system for the analysis of vitamin E and γ-oryzanol. The system separates eight vitamin E and four γ-oryzanol were separated by PFP Analysis tube column, which could be detected in 10 minutes of chromatographic time. It is currently known that the shortest analysis time in the literature by high-throughput experimental methods, All the analytical compounds were linear with R2> 0.988, the linear range of vitamin E was 10 ppb ~ 160 ppb, the linear range of γ-oryzanol was 25 ppb ~ 16000 ppb, and the accuracy was 94.3% ~ 98.9%. The accuracy of intra day and inter day is within 15% of the relative standard deviation. The results were statistically calculated to show that the total vitamin E content ratio of more than 2.5 times times the sample number number 106th is more than twice times the sample 3, 16, 100, 101, 105, 107, 108. The highest 5.4 times times of the γ-rice bran alcohol ratio is the variety number 61st other than twice times the change 19, 51, 52, 53, 54, 55, 56, 57, 58, 59, 62, 117, matrix effect 2.23%. 400 kinds of analysis data to detect the difference between mutants quickly screening more efficient and economic varieties, it makes it more rapid and accurate in the development of antioxidant strains rice breeding project and functional food product, effectively shortens the analysis time and improves the efficiency of selecting new varieties, and can increase the new use of rice, and improve the pressure of domestic rice.