人血清白蛋白cDNA转化橡胶树的研究
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摘要
人血清白蛋白(human serum albumin,HSA)是人血浆中最丰富的蛋白质,具有维持血液渗透压、运输营养物质等生理功能,在临床医学中应用十分广泛。而目前市售的HSA主要来源于捐血者的血浆,有限的血液资源和人血中可能存在的病原污染限制了HSA的临床使用。人们迫切期望通过新途径来开发HSA,而通过基因工程手段开发重组人血清白蛋白来替代血源白蛋白是发展前景良好的新途径。
与微生物生物反应器和动物生物反应器相比,植物生物反应器有其独特的优越性。橡胶树的乳管系统十分发达,类似于动物的乳腺系统,割胶后胶乳从乳管中排出,从而使其中的蛋白产物采收加工及分离纯化十分方便。选择合适的乳管表达基因启动子可以使外源基因在其中表达,从而把橡胶树乳管系统变成生产外源蛋白的理想生物反应器。基于这种设想,本研究对橡胶树遗传转化体系的建立进行研究,并把含有橡胶树REF启动子的HSA cDNA乳管表达载体分别通过基因枪转化法和农杆菌转化法,导入橡胶树愈伤组织中,得到了以下的结论:
(1)以未成熟的雄花、内珠被和组织培养幼苗为外植体,用不同浓度的植物生长素和细胞分裂素组合,对外植体进行诱导,获得了大量的愈伤组织。
①对未成熟的雄花来讲,其最佳的愈伤组织诱导培养基为:MS培养基+7.0%麦芽糖+4.0mg/L 2,4-D+0.2mg/L 6-BA+1.2mg/L PVP+1.0%琼脂(pH 5.8),于25±1℃,黑暗培养:继代培养基:将碳源改为7.0%蔗糖,激素浓度调至原浓度的1/10,其他条件不变。
②对橡胶树未成熟种子的愈伤组织来讲,其最佳培养基为:MS培养基+4.0mg/L 2,4-D+0.2mg/L KT+1.2mg/L PVP+7.0%麦芽糖+1.0%琼脂(pH5.8),于25±1℃黑暗培养;继代培养时的激素浓度为诱导激素浓度的1/10,其他成分和培养条件不变。
③对橡胶树组培苗来讲,其最佳的愈伤组织诱导培养基为:MS培养基+3g/L蔗糖+3g/L麦芽糖+2mg/L 2,4-D+1mg/L 6-BA+1.2mg/L PVP+1.0%琼脂(pH5.8),于25±1℃黑暗培养:继代培养的激素浓度为诱导激素浓度的1/10,其他成分和培养条件不变。
Human serum albumin (HSA) is a kind of protein abundant in human blood plasma which can keep osmotic pressure of blood and transfer nutrient from one part of the body to another. It is widely used in clinic. But now most HSA comes from blood plasma. And HSA is restricted in clinic because of shortage of blood source and the latent viruses. Then it is urgent to produce HSA by other means and recombinant HSA from genetic engineering is prosperous now.Compared with microbe bioreactor and animal bioreactor, plant bioreactor has many unique advantages. Hevea brasiliensis has strong laticifer system which is like galacto-phore in animal. Latex runs out after laticifer is cut so that it is easy for proteins contained in latex to be cured and isolated. When proper promoter is added before foreign gene, it can be expressed in laticifer system. Then laticifer system can be turned into an ideal bioreactor. Herein lies the suppose that to establish regeneration system of Hevea brasiliensis and to transfer the expression vector combining REF promoter with HSA cDNA by bombardment or agrobacteria infection. In the study, quite a few of results are got as below:(1) Immature androecia, seeds and cultivated plantlets were used as explants. They were cultured on modified MS medium with different combination of auxin and cytokinin. Then large quantities of calli came out.For immature androecia, the best callus induction medium was MS medium added to 7.0% maltose, 1.2mg/L PVP, 4.0mg/L 2,4-D and 0.2mg/L 6-BA with 1.0% agar-agar powder. The pH of the medium was 5.8. Culture the explants on medium at 25 ± 1 °C in darkness. Carbon source was changed with 7.0% sucrose and the plant hormones were turned down to 1/10 after the first culture without any change of other things.(2)For immature seed, the best induction medium was MS medium with 4.0mg/L 2,4-D,0.2mg/L KT ,1.2mg/L PVP,7.0% sucrose and 1.0% agar-agar powder with pH value of 5.8. Plant hormones were turned down to 1/10 after the first culture.(3)For cultivated plantlet, the best callus induction medium was MS medium with
与微生物生物反应器和动物生物反应器相比,植物生物反应器有其独特的优越性。橡胶树的乳管系统十分发达,类似于动物的乳腺系统,割胶后胶乳从乳管中排出,从而使其中的蛋白产物采收加工及分离纯化十分方便。选择合适的乳管表达基因启动子可以使外源基因在其中表达,从而把橡胶树乳管系统变成生产外源蛋白的理想生物反应器。基于这种设想,本研究对橡胶树遗传转化体系的建立进行研究,并把含有橡胶树REF启动子的HSA cDNA乳管表达载体分别通过基因枪转化法和农杆菌转化法,导入橡胶树愈伤组织中,得到了以下的结论:
(1)以未成熟的雄花、内珠被和组织培养幼苗为外植体,用不同浓度的植物生长素和细胞分裂素组合,对外植体进行诱导,获得了大量的愈伤组织。
①对未成熟的雄花来讲,其最佳的愈伤组织诱导培养基为:MS培养基+7.0%麦芽糖+4.0mg/L 2,4-D+0.2mg/L 6-BA+1.2mg/L PVP+1.0%琼脂(pH 5.8),于25±1℃,黑暗培养:继代培养基:将碳源改为7.0%蔗糖,激素浓度调至原浓度的1/10,其他条件不变。
②对橡胶树未成熟种子的愈伤组织来讲,其最佳培养基为:MS培养基+4.0mg/L 2,4-D+0.2mg/L KT+1.2mg/L PVP+7.0%麦芽糖+1.0%琼脂(pH5.8),于25±1℃黑暗培养;继代培养时的激素浓度为诱导激素浓度的1/10,其他成分和培养条件不变。
③对橡胶树组培苗来讲,其最佳的愈伤组织诱导培养基为:MS培养基+3g/L蔗糖+3g/L麦芽糖+2mg/L 2,4-D+1mg/L 6-BA+1.2mg/L PVP+1.0%琼脂(pH5.8),于25±1℃黑暗培养:继代培养的激素浓度为诱导激素浓度的1/10,其他成分和培养条件不变。
Human serum albumin (HSA) is a kind of protein abundant in human blood plasma which can keep osmotic pressure of blood and transfer nutrient from one part of the body to another. It is widely used in clinic. But now most HSA comes from blood plasma. And HSA is restricted in clinic because of shortage of blood source and the latent viruses. Then it is urgent to produce HSA by other means and recombinant HSA from genetic engineering is prosperous now.Compared with microbe bioreactor and animal bioreactor, plant bioreactor has many unique advantages. Hevea brasiliensis has strong laticifer system which is like galacto-phore in animal. Latex runs out after laticifer is cut so that it is easy for proteins contained in latex to be cured and isolated. When proper promoter is added before foreign gene, it can be expressed in laticifer system. Then laticifer system can be turned into an ideal bioreactor. Herein lies the suppose that to establish regeneration system of Hevea brasiliensis and to transfer the expression vector combining REF promoter with HSA cDNA by bombardment or agrobacteria infection. In the study, quite a few of results are got as below:(1) Immature androecia, seeds and cultivated plantlets were used as explants. They were cultured on modified MS medium with different combination of auxin and cytokinin. Then large quantities of calli came out.For immature androecia, the best callus induction medium was MS medium added to 7.0% maltose, 1.2mg/L PVP, 4.0mg/L 2,4-D and 0.2mg/L 6-BA with 1.0% agar-agar powder. The pH of the medium was 5.8. Culture the explants on medium at 25 ± 1 °C in darkness. Carbon source was changed with 7.0% sucrose and the plant hormones were turned down to 1/10 after the first culture without any change of other things.(2)For immature seed, the best induction medium was MS medium with 4.0mg/L 2,4-D,0.2mg/L KT ,1.2mg/L PVP,7.0% sucrose and 1.0% agar-agar powder with pH value of 5.8. Plant hormones were turned down to 1/10 after the first culture.(3)For cultivated plantlet, the best callus induction medium was MS medium with
引文
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[3] 陈正华,钱长发,岑明.三叶橡胶花药培养中体细胞与小孢子的关系.西北植物学报.1981.1(1).31-37
[4] 董去洲,贾士荣.基因枪在植物遗传转化上的应用.生物工程进展.1993.14(2).14,15-18
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[6] 傅荣昭,孙勇如,贾士荣.植物遗传转化手册.中国科学技术出版社.1994.32
[7] 郭美锦,储炬,杭海峰,庄英萍,叶勤,张嗣良.重组人血清白蛋白表达研究进展.生物工程进展.2000.20(5).39-42
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