组培桑苗中1-脱氧野尻霉素含量影响因子的研究
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摘要
1-脱氧野尻霉素(DNJ)及其衍生物是一种高效α-葡萄糖苷酶抑制剂,具有广泛的药理作用,被用于治疗糖尿病、肥胖症、病毒感染等疾病。目前已发现多种植物和微生物具有合成和积累DNJ的特性,其中以桑叶中的含量最高。国内外有关DNJ药理作用和提取检测方法的研究较多,但有关DNJ合成调控的研究还未见报道。有鉴于此,本文探讨了不同影响因子对组培桑苗中DNJ含量的影响,旨在为以后通过组织或细胞培养生产DNJ及探讨DNJ的合成机理提供参考。主要研究结果如下:
1利用反相高效液相色谱(RP-HPLC)法测定了组培桑苗中DNJ含量,结果表明:组培桑苗中DNJ含量不仅受桑品种、组织器官的影响,还与培养时间和培养条件等因素有关。供试的5个桑品种中,农桑14号组培桑苗中DNJ含量最高达0.1302%,新桑13号含量最低为0.0701%;组培桑苗各组织器官以叶片中DNJ含量最高,芽次之,根最少。组培桑苗在培养25 d时DNJ含量最高。在MS培养基上生长的组培桑苗叶片中DNJ的含量最高达0.1043%,在N6培养基上的DNJ含量最低为0.0678%。培养基pH值5.8-6.0有利于DNJ的合成和积累。黑暗处理对组培桑苗叶片中DNJ的合成和积累具有促进作用,当黑暗处理5 d时DNJ的含量最高达0.1338%。
2探讨了培养基成分对组培桑苗叶片中DNJ含量的影响。研究表明:果糖具有促进DNJ合成和积累的作用,当果糖浓度为40 g/L时DNJ含量最高;硝态氮促进DNJ合成和积累,铵态氮抑制DNJ合成和积累;当磷酸盐浓度为0.5 mmol/L、无机盐浓度为8.175 g/L时,DNJ含量最高;烟酸、VB6对DNJ的合成和积累具有抑制作用;金属离子对组培桑苗叶片中DNJ的含量有显著影响:Fe2+对DNJ的合成和积累具有促进作用,当Fe2+浓度为0.05 mmol/L时,DNJ含量最高。当Ca2+浓度为1.5 mmol/L时,DNJ含量最高。Mg2+对DNJ的合成和积累具有抑制作用。
3研究了组培桑苗叶片中DNJ含量与培养基中生长调节剂的关系。结果表明:2,4-D具有抑制DNJ合成和积累的作用,NAA具有促进DNJ合成和积累的作用,当培养基中NAA的浓度为0.1 mg/L时,DNJ含量最高;细胞分裂素KT、6-BA具有促进DNJ合成和积累的作用;最佳激素组合为6-BA 0.5 mg/L+NAA 0.1 mg/L+KT 1.0 mg/L。
4通过测定桑品种大10不同发育阶段组织培养物(悬浮细胞以及细胞诱导愈伤组织、叶诱导绿色愈伤组织、疏松愈伤组织、茎诱导愈伤组织、根诱导愈伤组织、再生芽、再生叶片、再生根、再生苗和无菌苗等)中DNJ的含量,发现再生芽、再生叶片、再生苗和无菌苗中含有DNJ,5种愈伤组织、悬浮细胞和再生根中不含DNJ,推测DNJ可能产生于形成桑芽的发育阶段。
1-deoxynojirimycin (DNJ) and its ramifications are effectiveα- glucosidase inhibitors, and have comprehensive function of pharmacology. They are used in curing diabetes, fatness and virus infections. DNJ has been found in many kinds of plants and microbes. Compared with other plants the content of DNJ in mlberry leaves is higher. Though, there are many reports about the pharmacology function and detecting methods of DNJ, litter is known about the mechanisms for DNJ synthesis. In this study some factors associated with the contents of DNJ were investigated to provide a better understanding for the produce of DNJ by tissue or cell culture and the synthesis mechanisms of it . The main research findings are as follows.
1 The DNJ content in tissue culture mulberry seedlings was detected by using RP-HPLC method. The results showed that the DNJ content was not only influenced by mulberry varieties and tissues, but also effected by culture time and conditions etc. Among the 5 mulberry varieties, Nongsang 14 contained most DNJ production (0.1302%)and Xinsang 13 contained least DNJ (0.0701%). The leaves of all the tissue cluture mulberry seedlings showed higher DNJ content than buds and roots. The favorable culture time for the synthesize and accumulate of DNJ was 25 days. The optimal medium for the DNJ synthesis was MS-medium. When N6-medium was used, the DNJ content in tissue culture mulberry leaves was least . The optimal medium pH value was 5.8-6.0. Darkness treatments was benefit for the DNJ synthesis and the optimal darkness treatment time for DNJ synthesis was 5 days.
2 The effect of the medium compositions on the DNJ content of tissue culture mulberry seedling leaves was investigated. The results showed that adding fructose was good to the DNJ synthesis and accumulation, and the optimal fructose concertration was 40 g/L. NO3--N could enhance the DNJ content in tissue culture mulberry leaves, adversely, NH4+-N had a negative influence on the synthesis and accumulate of DNJ. When the MS-medium supplemented with 0.5 mmol/L phosphate and 8.175 g/L inorganic ions, the content of DNJ was highest. The existence of aicotinic acid and VB6 in the medium was not good to the synthesis of the DNJ. Metal ions had significant impact on the DNJ content. Proper Fe2+ and Ca2+ in the medium were propitious to the DNJ synthesis. When Fe2+ and Ca2+ concertration were 0.05 mmol/L and 1.5 mmol/L, respectively, the DNJ content was highest. The Mg2+ ions in the medium had a negative influence on the synthesis and accumulate of DNJ.
3 The effect of plant hormone on the DNJ content was studied. The results showed that NAA enhanced the DNJ content in tissue culture mulberry leaves. When NAA concent was 0.1 mg/L, DNJ content was the highest. 2,4-D in the medium had a negative influence on the DNJ synthesis. Cytokinins KT and 6-BA were favor for the DNJ synthesis. The best result was obtained when the MS-medium supplemented with is 0.5 mg/L 6-BA, 0.1 mg/L NAA and 1.0 mg/L KT.
4 The DNJ contents in the Da 10 tissue cultures of different develop phases, including suspension cells, induced callus from suspension cells, green callus, loose callus, induced callus from stem and root, regeneration buds, leaves, root and plant, aseptic plant were detected. Interestingly, DNJ was only found in regeneration buds, leaves and seedling, aseptic plant, indicating that DNJ was synthesized begining with mulberry buds phase.
1利用反相高效液相色谱(RP-HPLC)法测定了组培桑苗中DNJ含量,结果表明:组培桑苗中DNJ含量不仅受桑品种、组织器官的影响,还与培养时间和培养条件等因素有关。供试的5个桑品种中,农桑14号组培桑苗中DNJ含量最高达0.1302%,新桑13号含量最低为0.0701%;组培桑苗各组织器官以叶片中DNJ含量最高,芽次之,根最少。组培桑苗在培养25 d时DNJ含量最高。在MS培养基上生长的组培桑苗叶片中DNJ的含量最高达0.1043%,在N6培养基上的DNJ含量最低为0.0678%。培养基pH值5.8-6.0有利于DNJ的合成和积累。黑暗处理对组培桑苗叶片中DNJ的合成和积累具有促进作用,当黑暗处理5 d时DNJ的含量最高达0.1338%。
2探讨了培养基成分对组培桑苗叶片中DNJ含量的影响。研究表明:果糖具有促进DNJ合成和积累的作用,当果糖浓度为40 g/L时DNJ含量最高;硝态氮促进DNJ合成和积累,铵态氮抑制DNJ合成和积累;当磷酸盐浓度为0.5 mmol/L、无机盐浓度为8.175 g/L时,DNJ含量最高;烟酸、VB6对DNJ的合成和积累具有抑制作用;金属离子对组培桑苗叶片中DNJ的含量有显著影响:Fe2+对DNJ的合成和积累具有促进作用,当Fe2+浓度为0.05 mmol/L时,DNJ含量最高。当Ca2+浓度为1.5 mmol/L时,DNJ含量最高。Mg2+对DNJ的合成和积累具有抑制作用。
3研究了组培桑苗叶片中DNJ含量与培养基中生长调节剂的关系。结果表明:2,4-D具有抑制DNJ合成和积累的作用,NAA具有促进DNJ合成和积累的作用,当培养基中NAA的浓度为0.1 mg/L时,DNJ含量最高;细胞分裂素KT、6-BA具有促进DNJ合成和积累的作用;最佳激素组合为6-BA 0.5 mg/L+NAA 0.1 mg/L+KT 1.0 mg/L。
4通过测定桑品种大10不同发育阶段组织培养物(悬浮细胞以及细胞诱导愈伤组织、叶诱导绿色愈伤组织、疏松愈伤组织、茎诱导愈伤组织、根诱导愈伤组织、再生芽、再生叶片、再生根、再生苗和无菌苗等)中DNJ的含量,发现再生芽、再生叶片、再生苗和无菌苗中含有DNJ,5种愈伤组织、悬浮细胞和再生根中不含DNJ,推测DNJ可能产生于形成桑芽的发育阶段。
1-deoxynojirimycin (DNJ) and its ramifications are effectiveα- glucosidase inhibitors, and have comprehensive function of pharmacology. They are used in curing diabetes, fatness and virus infections. DNJ has been found in many kinds of plants and microbes. Compared with other plants the content of DNJ in mlberry leaves is higher. Though, there are many reports about the pharmacology function and detecting methods of DNJ, litter is known about the mechanisms for DNJ synthesis. In this study some factors associated with the contents of DNJ were investigated to provide a better understanding for the produce of DNJ by tissue or cell culture and the synthesis mechanisms of it . The main research findings are as follows.
1 The DNJ content in tissue culture mulberry seedlings was detected by using RP-HPLC method. The results showed that the DNJ content was not only influenced by mulberry varieties and tissues, but also effected by culture time and conditions etc. Among the 5 mulberry varieties, Nongsang 14 contained most DNJ production (0.1302%)and Xinsang 13 contained least DNJ (0.0701%). The leaves of all the tissue cluture mulberry seedlings showed higher DNJ content than buds and roots. The favorable culture time for the synthesize and accumulate of DNJ was 25 days. The optimal medium for the DNJ synthesis was MS-medium. When N6-medium was used, the DNJ content in tissue culture mulberry leaves was least . The optimal medium pH value was 5.8-6.0. Darkness treatments was benefit for the DNJ synthesis and the optimal darkness treatment time for DNJ synthesis was 5 days.
2 The effect of the medium compositions on the DNJ content of tissue culture mulberry seedling leaves was investigated. The results showed that adding fructose was good to the DNJ synthesis and accumulation, and the optimal fructose concertration was 40 g/L. NO3--N could enhance the DNJ content in tissue culture mulberry leaves, adversely, NH4+-N had a negative influence on the synthesis and accumulate of DNJ. When the MS-medium supplemented with 0.5 mmol/L phosphate and 8.175 g/L inorganic ions, the content of DNJ was highest. The existence of aicotinic acid and VB6 in the medium was not good to the synthesis of the DNJ. Metal ions had significant impact on the DNJ content. Proper Fe2+ and Ca2+ in the medium were propitious to the DNJ synthesis. When Fe2+ and Ca2+ concertration were 0.05 mmol/L and 1.5 mmol/L, respectively, the DNJ content was highest. The Mg2+ ions in the medium had a negative influence on the synthesis and accumulate of DNJ.
3 The effect of plant hormone on the DNJ content was studied. The results showed that NAA enhanced the DNJ content in tissue culture mulberry leaves. When NAA concent was 0.1 mg/L, DNJ content was the highest. 2,4-D in the medium had a negative influence on the DNJ synthesis. Cytokinins KT and 6-BA were favor for the DNJ synthesis. The best result was obtained when the MS-medium supplemented with is 0.5 mg/L 6-BA, 0.1 mg/L NAA and 1.0 mg/L KT.
4 The DNJ contents in the Da 10 tissue cultures of different develop phases, including suspension cells, induced callus from suspension cells, green callus, loose callus, induced callus from stem and root, regeneration buds, leaves, root and plant, aseptic plant were detected. Interestingly, DNJ was only found in regeneration buds, leaves and seedling, aseptic plant, indicating that DNJ was synthesized begining with mulberry buds phase.
引文
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