农杆菌介导DREB和otsA基因转化库拉索芦荟的研究与应用
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摘要
芦荟是一种新资源食品,具有药用价值、美容功效和食品营养价值。但芦荟抗旱不抗寒,不利于芦荟的大规模种植。而食用芦荟品种味苦、味感差,也制约芦荟食品产业的发展。为提高芦荟的抗寒性和改善芦荟食品的口感,本文采用农杆菌介导法将分别来自水稻和小麦的OsDREB和TaDREB基因转入芦荟,并对两种转基因芦荟,以及先期实验室转海藻糖-6-磷酸合成酶基因otsA芦荟,进行了抗逆性、海藻糖含量、多糖含量以及凝胶过滤工艺和化妆品应用等方面的研究。
1.三亲杂交法将含有水稻OsDREB的表达载体PBT导入农杆菌EHA105,通过农杆菌介导浸染库拉索芦荟(Aloe vera. L)幼叶外植体,获得98棵芦荟抗性植株,PCR、低温处理并进行电导率检测,表明转化成功。
2.利用带有小麦TaDREB基因的表达载体pBIR1,农杆菌介导转化库拉索芦荟,共筛选出58株生长良好的抗性植株。PCR检测转化率为0.22%。4℃低温处理两周,-20℃冷冻30min,发现对照植株发生严重冻害,而转基因植株生长良好,抗低温特性明显提高。对低温胁迫下培养14d的转TaDREB基因植株SOD、POD同工酶谱进行分析,表明低温胁迫下转基因植株SOD、POD活性均呈先降后升的趋势,转基因芦荟提高了体内的保护性酶活性。利用电导率法检测,转基因植株电导率平均值为0.462；明显低于对照0.685。表明转小麦TaDREB基因芦荟的抗寒性明显得到提高。
3.以转Ta DREB和Os DREB基因芦荟为材料,研究盐胁迫下转基因芦荟的多聚胺表达情况和净光合速率(Pn)、气孔导度(Gs),胞间CO2浓度(Ci)、叶绿素荧光特性(Fv/Fm),结果表明转基因芦荟的耐盐性比对照芦荟高,多聚胺在烟株缓解盐胁中可能是起了重要作用。转基因库拉索芦荟叶片在盐胁迫下的光抑制程度较小。
4.利用1-甲基咪唑、氯化羟胺、乙酸酐将海藻糖衍生化,气相色谱法对同一种糖衍生物进行分析,特征峰保留时间误差在3s内,能将D-葡萄糖、乳糖、蔗糖和海藻糖进行分离。海藻糖在3.697×10-9～28.661×10-9g范围内相关系数为0.9986。利用此方法研究不同年份(3年和5年)和半年生转otsA基因芦荟的海藻糖含量进行了测定,分别为6.905×10-7、1.103×10-6和1.614×10-6g/g,半年生otsA芦荟凝胶中海藻糖含量含量明显提高。进一步研究表明转基因芦荟叶片对皮肤表现出更优异的保湿和抗衰老功效。
5.以3年生转基因的库拉索芦荟凝胶为材料,冷处理法醇沉提取粗多糖,经IR鉴定,粗多糖为含有乙酰化β-D-吡喃甘露聚糖的混合物,质谱测定分子量测定范围在12900D和13100D。纯化后半精品经DE AE Sephadex A-25分级分离,得到中性糖和酸性糖。用Sepharose CL-6B凝胶分离中性糖,得到多糖A1和A2。用IR、MS、GC对其进行鉴定和表征分析,结果表明,中性糖为多甘露糖型的多糖。
6.为了更好的利用库拉索芦荟,对其凝胶汁过滤工艺和其在洁肤露、乳液和眼胶中的应用进行了研究,并对所研制的几种化妆品的功效性和安全性进行了评价。得到了凝胶过滤的最佳稳定工艺和以库拉索芦荟为主要功效成分的化妆品。
Aloe is a new resource of food with medicinal value, beauty efficacy and food nutritional value. As drought-resistant, but not cold-resistant, Aloe is not conducive to large-scale cultivation. The bitter tastes and poor flavor also constrain the development of Aloe food industry. To enhance the cold tolerance of Aloe vera.L and improve the food taste, The OsDREB and TaDREB gene from rice and wheat separately were transfered into Aloe vera.L mediated by Agrobacterium, and some researches in improve the Aloe vera.L resistance, trehalose, polysaccharide content and Aloe gel filtration techniques and its cosmetic applications for the two transgenic aloe, and preliminary laboratory Aloe vera.L with transfered trehalose-6-phosphate synthase gene otsA.
(1) The expression vector PBT containing the rice OsDREB gene were transferred into Agrobacterium EHA105 strain by three pro-hybridization, the aimed gene were transferred into Aloe vera. L young leaf explants mediated by Agrobacterium,98 resistant OsDREB gene plants were obtained by a series of screening protocol, and PCR, cold process treatment and conductivity detection results indicated that the OsDREB gene improved Aloe vera. L cold resistance.
(2) In this experiment, the wheat genes TaDREB in plasmid pBIR1 were transferred into Aloe vera. L young leaf explants through Agrobacterium-mediated, A total of 58 selected resistant plants that grown well were obtained by a by a series of screening protocol. The transformation rate is about 0.22% by PCR detecting. The results that the transgenic plants were growing well while the control got serious damaged after two weeks of cold treatment at 4℃,-20℃freezing treatment for 30min indicated that the TaDREB significantly increased Aloe vera. L resistance to low temperature. The isoenzyme of SOD, POD in transgenic plants which were treated by low temperature stress during 14days were analysised and the results showed that SOD, POD activity both expressed a trend of rising after an initial drop, and its indicated the transgenic aloe increased its protective activity in vivo. The average conductivity of the transgenic plants was 0.462 which is significantly lower than the 0.685 in control and it showed that Aloe vera. L with transferred of wheat TaDREB gene had improved cold resistance obviously.
(3) Polyamine(PA)content, net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO_2 concentration (Ci)and maximum photochemical efficiency of PSII(Fv/Fm) of transgenic and control Aloe vera.L were studied separately and the results indicated that the tolerance to the salt treatment(200mmol/L NaCl) of the transgenic Aloe are better than the control. PA maybe play an important role during the response to the salt treatment。Under salt stress, Pn, Gs and Fv/Fm all decreased with the salt treating time in both the wild type Aloe and the transgenic Aloe. However, the level of Fv/Fm decreased less in the transgenic Aloe compared with that in the wild type Aloe. And the result indicated that the injury of PS II is less in transgenic Aloe.
(4) In this experiment, D-glucose, lactose, sucrose and trehalose was separately derivated the by 1-methylimidazole, hydroxylamine chloride, acetic anhydride continuously, the retention time of characteristic peak of each of them is within 3 s by using gas chromatography as a means of quantitative analysis, this indicated that this method could differentiate D-glucose, lactose, sucrose and trehalose distinctly. The correlation coefficient of Trehalose is 0.9986 within the detection volume of 3.697～10~(-9)～28.661～10~(-9) g. The content of trehalose in the Aloe.vera L of 3 years,5 years old and the transgenic of trehalose synthase gene(otsA)Aloe.vera L of 6 months old was 6.905～10~(-7),1.103～10~(-6) and 1.614～10~(-6) (g/g) respectively. The results indicated that Aloe of six months transgenic of otsA gene Aloe.vera L., had significantly increased the contents of trehalose. Further studies on the leaf from transgenic Aloe.vera L showed that the transgenic aloe showed superior moisturizing and anti-aging effect on skin.
(5) Using the transgenic of otsA gene Aloe.vera L., of 3 years old as experimental materials, polysaccharide were extracted by cold processing method of alcohol precipitation and it was a mixture containing acetylatedβ-D-pyran mannan identified by IR and molecular weight range was between 12900D and 13100D detected by mass spectrometry. The preliminary purified polysaccharide chromatographic fractionation was carried out by DEAE Sephadex A-25, and neutral sugars and acidic sugars were fractioned, then neutral sugar chromatographic fractionation was carried out by Sepharose CL-6B, and A1 and A2 were fractioned. The results of identification and characterization analysis of these polysaccharides by IR, MS, GC showed that neutral sugars were the type of mannose polysaccharide.
(6) In order to make better and large use of Aloe.vera L., the juice of its gel filtration process and the gel from Aloe.vera L application in cleansers, lotions and eye glue were studied, and efficacy and safety of the developed several cosmetics were evaluated. And the more optimizing and stable gel filtration process and cosmetics with Aloe vera L., as the main effectiveness ingredient were developed respectively.
1.三亲杂交法将含有水稻OsDREB的表达载体PBT导入农杆菌EHA105,通过农杆菌介导浸染库拉索芦荟(Aloe vera. L)幼叶外植体,获得98棵芦荟抗性植株,PCR、低温处理并进行电导率检测,表明转化成功。
2.利用带有小麦TaDREB基因的表达载体pBIR1,农杆菌介导转化库拉索芦荟,共筛选出58株生长良好的抗性植株。PCR检测转化率为0.22%。4℃低温处理两周,-20℃冷冻30min,发现对照植株发生严重冻害,而转基因植株生长良好,抗低温特性明显提高。对低温胁迫下培养14d的转TaDREB基因植株SOD、POD同工酶谱进行分析,表明低温胁迫下转基因植株SOD、POD活性均呈先降后升的趋势,转基因芦荟提高了体内的保护性酶活性。利用电导率法检测,转基因植株电导率平均值为0.462；明显低于对照0.685。表明转小麦TaDREB基因芦荟的抗寒性明显得到提高。
3.以转Ta DREB和Os DREB基因芦荟为材料,研究盐胁迫下转基因芦荟的多聚胺表达情况和净光合速率(Pn)、气孔导度(Gs),胞间CO2浓度(Ci)、叶绿素荧光特性(Fv/Fm),结果表明转基因芦荟的耐盐性比对照芦荟高,多聚胺在烟株缓解盐胁中可能是起了重要作用。转基因库拉索芦荟叶片在盐胁迫下的光抑制程度较小。
4.利用1-甲基咪唑、氯化羟胺、乙酸酐将海藻糖衍生化,气相色谱法对同一种糖衍生物进行分析,特征峰保留时间误差在3s内,能将D-葡萄糖、乳糖、蔗糖和海藻糖进行分离。海藻糖在3.697×10-9～28.661×10-9g范围内相关系数为0.9986。利用此方法研究不同年份(3年和5年)和半年生转otsA基因芦荟的海藻糖含量进行了测定,分别为6.905×10-7、1.103×10-6和1.614×10-6g/g,半年生otsA芦荟凝胶中海藻糖含量含量明显提高。进一步研究表明转基因芦荟叶片对皮肤表现出更优异的保湿和抗衰老功效。
5.以3年生转基因的库拉索芦荟凝胶为材料,冷处理法醇沉提取粗多糖,经IR鉴定,粗多糖为含有乙酰化β-D-吡喃甘露聚糖的混合物,质谱测定分子量测定范围在12900D和13100D。纯化后半精品经DE AE Sephadex A-25分级分离,得到中性糖和酸性糖。用Sepharose CL-6B凝胶分离中性糖,得到多糖A1和A2。用IR、MS、GC对其进行鉴定和表征分析,结果表明,中性糖为多甘露糖型的多糖。
6.为了更好的利用库拉索芦荟,对其凝胶汁过滤工艺和其在洁肤露、乳液和眼胶中的应用进行了研究,并对所研制的几种化妆品的功效性和安全性进行了评价。得到了凝胶过滤的最佳稳定工艺和以库拉索芦荟为主要功效成分的化妆品。
Aloe is a new resource of food with medicinal value, beauty efficacy and food nutritional value. As drought-resistant, but not cold-resistant, Aloe is not conducive to large-scale cultivation. The bitter tastes and poor flavor also constrain the development of Aloe food industry. To enhance the cold tolerance of Aloe vera.L and improve the food taste, The OsDREB and TaDREB gene from rice and wheat separately were transfered into Aloe vera.L mediated by Agrobacterium, and some researches in improve the Aloe vera.L resistance, trehalose, polysaccharide content and Aloe gel filtration techniques and its cosmetic applications for the two transgenic aloe, and preliminary laboratory Aloe vera.L with transfered trehalose-6-phosphate synthase gene otsA.
(1) The expression vector PBT containing the rice OsDREB gene were transferred into Agrobacterium EHA105 strain by three pro-hybridization, the aimed gene were transferred into Aloe vera. L young leaf explants mediated by Agrobacterium,98 resistant OsDREB gene plants were obtained by a series of screening protocol, and PCR, cold process treatment and conductivity detection results indicated that the OsDREB gene improved Aloe vera. L cold resistance.
(2) In this experiment, the wheat genes TaDREB in plasmid pBIR1 were transferred into Aloe vera. L young leaf explants through Agrobacterium-mediated, A total of 58 selected resistant plants that grown well were obtained by a by a series of screening protocol. The transformation rate is about 0.22% by PCR detecting. The results that the transgenic plants were growing well while the control got serious damaged after two weeks of cold treatment at 4℃,-20℃freezing treatment for 30min indicated that the TaDREB significantly increased Aloe vera. L resistance to low temperature. The isoenzyme of SOD, POD in transgenic plants which were treated by low temperature stress during 14days were analysised and the results showed that SOD, POD activity both expressed a trend of rising after an initial drop, and its indicated the transgenic aloe increased its protective activity in vivo. The average conductivity of the transgenic plants was 0.462 which is significantly lower than the 0.685 in control and it showed that Aloe vera. L with transferred of wheat TaDREB gene had improved cold resistance obviously.
(3) Polyamine(PA)content, net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO_2 concentration (Ci)and maximum photochemical efficiency of PSII(Fv/Fm) of transgenic and control Aloe vera.L were studied separately and the results indicated that the tolerance to the salt treatment(200mmol/L NaCl) of the transgenic Aloe are better than the control. PA maybe play an important role during the response to the salt treatment。Under salt stress, Pn, Gs and Fv/Fm all decreased with the salt treating time in both the wild type Aloe and the transgenic Aloe. However, the level of Fv/Fm decreased less in the transgenic Aloe compared with that in the wild type Aloe. And the result indicated that the injury of PS II is less in transgenic Aloe.
(4) In this experiment, D-glucose, lactose, sucrose and trehalose was separately derivated the by 1-methylimidazole, hydroxylamine chloride, acetic anhydride continuously, the retention time of characteristic peak of each of them is within 3 s by using gas chromatography as a means of quantitative analysis, this indicated that this method could differentiate D-glucose, lactose, sucrose and trehalose distinctly. The correlation coefficient of Trehalose is 0.9986 within the detection volume of 3.697～10~(-9)～28.661～10~(-9) g. The content of trehalose in the Aloe.vera L of 3 years,5 years old and the transgenic of trehalose synthase gene(otsA)Aloe.vera L of 6 months old was 6.905～10~(-7),1.103～10~(-6) and 1.614～10~(-6) (g/g) respectively. The results indicated that Aloe of six months transgenic of otsA gene Aloe.vera L., had significantly increased the contents of trehalose. Further studies on the leaf from transgenic Aloe.vera L showed that the transgenic aloe showed superior moisturizing and anti-aging effect on skin.
(5) Using the transgenic of otsA gene Aloe.vera L., of 3 years old as experimental materials, polysaccharide were extracted by cold processing method of alcohol precipitation and it was a mixture containing acetylatedβ-D-pyran mannan identified by IR and molecular weight range was between 12900D and 13100D detected by mass spectrometry. The preliminary purified polysaccharide chromatographic fractionation was carried out by DEAE Sephadex A-25, and neutral sugars and acidic sugars were fractioned, then neutral sugar chromatographic fractionation was carried out by Sepharose CL-6B, and A1 and A2 were fractioned. The results of identification and characterization analysis of these polysaccharides by IR, MS, GC showed that neutral sugars were the type of mannose polysaccharide.
(6) In order to make better and large use of Aloe.vera L., the juice of its gel filtration process and the gel from Aloe.vera L application in cleansers, lotions and eye glue were studied, and efficacy and safety of the developed several cosmetics were evaluated. And the more optimizing and stable gel filtration process and cosmetics with Aloe vera L., as the main effectiveness ingredient were developed respectively.
引文
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