FAD8基因遗传转化小油桐提高幼苗抗寒性研究
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摘要
小油桐(Jatropha curcas L.)属大戟科(Euphobiaceae)落叶灌木或小乔木。其种仁含油量高,干果产量大,油脂组成符合生物柴油油脂标准,是生物柴油的理想原料。但其生长易受低温和干旱伤害。因此,研究以生物技术改良其脂肪酸组成、提高抗寒性具有重要意义。拟南芥脂肪酸脱饱和酶FAD8基因编码ω-3脂肪酸脱饱和酶(ω-3fad),催化以二烯酸底物脱饱和形成三烯酸,可增加小油桐植株不饱和脂肪酸含量,在低温下其表达量提高,可提高膜脂的流动性,增加植物的抗寒能力。本研究建立了小油桐高效遗传转化体系,利用本实验室克隆的拟南芥FAD8基因构建表达载体遗传转化小油桐,鉴定并筛选转基因小油桐植株,研究了FAD8基因脂肪酸代谢和抗寒性的影响。获得结果如下:
1、小油桐高效再生体系的建立
去壳消毒小油桐种子,沿纵轴切开胚乳,挑出幼胚接种于MS+6-BA 3mg/L+IBA 0.5mg/L的萌发培养基中培养无菌苗。以15天苗龄的无菌苗为外植体,子叶最佳分化培养基为MS+6-BA 3mg/L+IBA 0.5mg/L,分化率为82.9%;下胚轴最佳分化培养基为MS+6-BA2mg/L+IBA 0.2mg/L,分化率为23.8%;子叶柄最佳再生培养基为MS+6-BA 3mg/L+IBA0.1mg/L,分化率为32.7%。在小油桐再生培养基中添加1.0mg/L的AgNO_3能促进小油桐植株再生,并抑制褐化。将小油桐再生芽苗接种于WPM培养基中生根,生根率为29.6%。本研究中,小油桐各外植体再生率均高于以往报道,首次以子叶柄获得再生苗。
2、农杆菌介导小油桐遗传转化体系建立
采用农杆菌介导法将FAD8基因(载体为pSH-FAD8)遗传转化小油桐:以15天苗龄的小油桐无菌苗的下胚轴、子叶、子叶柄为外植体,置于含200μg/L乙酰丁香酮的农杆菌菌液中进行侵染,子叶、子叶柄侵染8min,下胚轴侵染10min,28℃暗室中共培养3天后置含头孢霉素100mg/L的MS+6-BA3mg/L+IBA0.5mg/L培养基中脱菌生长5天,再接种至筛选培养基中培养,筛选条件是子叶和子叶柄利用卡那霉素30mg/L筛选;下胚轴以卡那霉素50mg/L筛选。将以GUS染色检测出的阳性植株进行PCR、RT-PCR检测,确定转基因小油桐植株27棵。与以往报道相比,本研究中建立的遗传转化体系更加完善,并获得了转基因植株。
3、提取0.5g转基因及对照小油桐叶片脂肪酸进行反相薄层层析,结果初步显示转基因小油桐叶片脂肪酸组成成分没有发生改变。
4、抗寒生理指标检测结果表明,与对照相比转基因植株表现出抗寒性增强的生理生化特性。筛选出两个抗寒能力最强的转基因小油桐单株,经低温处理后发现,与对照相比较,其叶绿素破坏较轻,过氧化氢酶、过氧化物酶活性增加更多,丙二醛含量、脯氨酸含量也有较多增加。
Jatropha curcas of the family Euphobiaceae is a deciduous shrub or small tree,it has large output and the seeds contain high oil.the oil components meet the standard of biodiesel,so that is an ideal raw material for the biodiesel.But the Jatropha could be injuryed seriously by the low temperature and the drought.It is important to study on improving it's fatty acid composition and cold resistence through the way of biotechnology.Arabidopsisω-3 fatty acid desaturase FAD8 is an enzyme which catalyze that the dienoic acid rapidly unsaturase to produce trienoic acid in cold condition.,which can increase the content of unsaturased fatty acid in the seeds and plants,and improve the plant oil nutrition and provide plants obviously resistance in cold condition.In our research,set up better regenerated and transformed system,used agrobacteria-medidated method transformed Arabidopsis FAD8 gene into the Jatropha,identified and filtrated the transgenic plant, and studyed fatty acid metabolism and cold-resistance in the transgenic Jatropha.
1、Establishment of high efficient regeneration system of Jatropha curcas
Peeled the seeds of Jatropha curcas and sterilized them.The explants of 15 days hypocotyls、cotyledon and the petiole of cotyledon from aseptic seedings of Jatropha curcas were used to callus induce and shoots regeneration,which were growed in MS containing 6-BA 3.0mg/L and IBA0.5mg/L medium.The best cotyledon's differentiated medium is MS+6-BA 3 mg/L+ IBA 0.5 mg/L,and the differentiated rate is 82.9%.The best differentiated medium for hypocotyls's bud is MS+6-BA 2 mg/L+IBA 0.2 mg/L,and the differentiate rate is 23.8%.The best petiole of cotyledon's differentiated medium is MS+6-BA 3mg/L+IBA 0.1mg/L,and the differentiated rate is 32.7%.Adding AgNO_3 1.0mg/L into the regeneration medium of explants could promote the bud differentiation,and inhebit browning.The WPM medium is the best one in rooting of the regeneration buds,and the rooting rate is 29.6%.The differentiated rate of each explants of Jatropha curcas is higher than ever reported,and get the regeneration plants from petiole of cotyledon for the first time.
2、Establishment the transformation System of Jatropha curcas Mediated by Agrobacterium tumefaciens
Used agrobacteria-medidated method transformed the FAD8 genes(the plasmid is pSH-FAD8) into the 15 days aged Jatropha curcas hypocotyls,cotyledon and petiole of cotyledon. The explants were submerged into 0.5(OD_(600))Agrobacterium suspension contained 200μg/L AS (hypocotyls for 10 min and cotyledon and petiole of cotyledon for 8min),and placed on co-cultivation medium for 3 days dark in 28℃.Then placed them into the medium of MS+6-BA 3mg/L + IBA 0.5mg/L content cephalosporin 100mg/L for 5 days.The content of kanamycin(Kan) selection pressures were determined as 30mg/L for shoot regeneration of cotyledon and petiole of cotyledon,50mg/L for hypocotyls,the results showed that the extra gene fad8 was integrated into Jatropha curcas genomes successful confirmed by GUS staining,PCR and RT-PCR,and get 27 transgenie plants.In this study,we established more perfect ransformation System of Jatropha curcas than ever,and get the transgenic plants.
3、Analysed the fatty acid content in the transgenic and control Jatropha curcas by the reverse phase chromatography,and showed that the sort of the fatty acid in the leaf of the transgenic Jatropha curcas was not changed.
4、The test about physiological indicators of cold-resistence showed that the cold-resistance ability of the transgenic Jatropha curcas were higher than the control.Choosed two transgenic plants,and studied the cold-resisted ability:the Chlorophyll Comparison of light damage,CAT、POD、malondialdehyde and proline content all enhanced more in transgenic FAD8 gene Jatropha curcas plants.
1、小油桐高效再生体系的建立
去壳消毒小油桐种子,沿纵轴切开胚乳,挑出幼胚接种于MS+6-BA 3mg/L+IBA 0.5mg/L的萌发培养基中培养无菌苗。以15天苗龄的无菌苗为外植体,子叶最佳分化培养基为MS+6-BA 3mg/L+IBA 0.5mg/L,分化率为82.9%;下胚轴最佳分化培养基为MS+6-BA2mg/L+IBA 0.2mg/L,分化率为23.8%;子叶柄最佳再生培养基为MS+6-BA 3mg/L+IBA0.1mg/L,分化率为32.7%。在小油桐再生培养基中添加1.0mg/L的AgNO_3能促进小油桐植株再生,并抑制褐化。将小油桐再生芽苗接种于WPM培养基中生根,生根率为29.6%。本研究中,小油桐各外植体再生率均高于以往报道,首次以子叶柄获得再生苗。
2、农杆菌介导小油桐遗传转化体系建立
采用农杆菌介导法将FAD8基因(载体为pSH-FAD8)遗传转化小油桐:以15天苗龄的小油桐无菌苗的下胚轴、子叶、子叶柄为外植体,置于含200μg/L乙酰丁香酮的农杆菌菌液中进行侵染,子叶、子叶柄侵染8min,下胚轴侵染10min,28℃暗室中共培养3天后置含头孢霉素100mg/L的MS+6-BA3mg/L+IBA0.5mg/L培养基中脱菌生长5天,再接种至筛选培养基中培养,筛选条件是子叶和子叶柄利用卡那霉素30mg/L筛选;下胚轴以卡那霉素50mg/L筛选。将以GUS染色检测出的阳性植株进行PCR、RT-PCR检测,确定转基因小油桐植株27棵。与以往报道相比,本研究中建立的遗传转化体系更加完善,并获得了转基因植株。
3、提取0.5g转基因及对照小油桐叶片脂肪酸进行反相薄层层析,结果初步显示转基因小油桐叶片脂肪酸组成成分没有发生改变。
4、抗寒生理指标检测结果表明,与对照相比转基因植株表现出抗寒性增强的生理生化特性。筛选出两个抗寒能力最强的转基因小油桐单株,经低温处理后发现,与对照相比较,其叶绿素破坏较轻,过氧化氢酶、过氧化物酶活性增加更多,丙二醛含量、脯氨酸含量也有较多增加。
Jatropha curcas of the family Euphobiaceae is a deciduous shrub or small tree,it has large output and the seeds contain high oil.the oil components meet the standard of biodiesel,so that is an ideal raw material for the biodiesel.But the Jatropha could be injuryed seriously by the low temperature and the drought.It is important to study on improving it's fatty acid composition and cold resistence through the way of biotechnology.Arabidopsisω-3 fatty acid desaturase FAD8 is an enzyme which catalyze that the dienoic acid rapidly unsaturase to produce trienoic acid in cold condition.,which can increase the content of unsaturased fatty acid in the seeds and plants,and improve the plant oil nutrition and provide plants obviously resistance in cold condition.In our research,set up better regenerated and transformed system,used agrobacteria-medidated method transformed Arabidopsis FAD8 gene into the Jatropha,identified and filtrated the transgenic plant, and studyed fatty acid metabolism and cold-resistance in the transgenic Jatropha.
1、Establishment of high efficient regeneration system of Jatropha curcas
Peeled the seeds of Jatropha curcas and sterilized them.The explants of 15 days hypocotyls、cotyledon and the petiole of cotyledon from aseptic seedings of Jatropha curcas were used to callus induce and shoots regeneration,which were growed in MS containing 6-BA 3.0mg/L and IBA0.5mg/L medium.The best cotyledon's differentiated medium is MS+6-BA 3 mg/L+ IBA 0.5 mg/L,and the differentiated rate is 82.9%.The best differentiated medium for hypocotyls's bud is MS+6-BA 2 mg/L+IBA 0.2 mg/L,and the differentiate rate is 23.8%.The best petiole of cotyledon's differentiated medium is MS+6-BA 3mg/L+IBA 0.1mg/L,and the differentiated rate is 32.7%.Adding AgNO_3 1.0mg/L into the regeneration medium of explants could promote the bud differentiation,and inhebit browning.The WPM medium is the best one in rooting of the regeneration buds,and the rooting rate is 29.6%.The differentiated rate of each explants of Jatropha curcas is higher than ever reported,and get the regeneration plants from petiole of cotyledon for the first time.
2、Establishment the transformation System of Jatropha curcas Mediated by Agrobacterium tumefaciens
Used agrobacteria-medidated method transformed the FAD8 genes(the plasmid is pSH-FAD8) into the 15 days aged Jatropha curcas hypocotyls,cotyledon and petiole of cotyledon. The explants were submerged into 0.5(OD_(600))Agrobacterium suspension contained 200μg/L AS (hypocotyls for 10 min and cotyledon and petiole of cotyledon for 8min),and placed on co-cultivation medium for 3 days dark in 28℃.Then placed them into the medium of MS+6-BA 3mg/L + IBA 0.5mg/L content cephalosporin 100mg/L for 5 days.The content of kanamycin(Kan) selection pressures were determined as 30mg/L for shoot regeneration of cotyledon and petiole of cotyledon,50mg/L for hypocotyls,the results showed that the extra gene fad8 was integrated into Jatropha curcas genomes successful confirmed by GUS staining,PCR and RT-PCR,and get 27 transgenie plants.In this study,we established more perfect ransformation System of Jatropha curcas than ever,and get the transgenic plants.
3、Analysed the fatty acid content in the transgenic and control Jatropha curcas by the reverse phase chromatography,and showed that the sort of the fatty acid in the leaf of the transgenic Jatropha curcas was not changed.
4、The test about physiological indicators of cold-resistence showed that the cold-resistance ability of the transgenic Jatropha curcas were higher than the control.Choosed two transgenic plants,and studied the cold-resisted ability:the Chlorophyll Comparison of light damage,CAT、POD、malondialdehyde and proline content all enhanced more in transgenic FAD8 gene Jatropha curcas plants.
引文
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