麻疯树高效再生体系的建立及纳米载体转基因技术研究
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摘要
麻疯树(Jatropha curcas L.)是一种重要的生物柴油树种,生长迅速,喜光耐旱,具有较高的经济价值和药用价值。本文以麻疯树无菌苗茎段为外植体,进行麻疯树植株再生的研究；同时,建立麻疯树悬浮细胞系,探索纳米基因载体对麻疯树遗传转化的效果,从而建立一种新的基因转化方法。主要研究结果如下：
(1)采用麻疯树无菌苗茎段为外植体,初步建立了麻疯树植株再生体系,结果表明：麻疯树茎段诱导不定芽再生的最适激素组合为：BA1.Omg/L+TDZ1.5mg/L,不定芽的平均诱导率达69.25%。不定根诱导培养中,采用生长素NAA诱导麻疯树幼苗生根,最适诱导配方为：MS+NAA1.0mg/L+蔗糖30g/L+琼脂7.2g/L,生根率达到66.67%,根系粗壮,发育良好。外植体接种于MS固体培养基和液体培养基中均有不定芽分化,但固体培养基中的外植体不定芽诱导率更高,褐化水平也较低。
(2)麻疯树悬浮细胞培养中,研究了疏松愈伤组织诱导方案及不同培养条件对麻疯树悬浮细胞生长的影响。结论如下：麻疯树疏松愈伤组织诱导的最适培养基及激素组合为：MS+2,4-D0.6mg/L+KT0.4mg/L,此培养基上诱导出的愈伤组织湿润松散,颜色鲜艳。接种愈伤组织进行悬浮培养的液体培养基最适激素组合为：NAA0.2mg/L+2,4-D1.0mg/L+BA0.5mg/L。初代、2次继代和4次继代的愈伤组织用于悬浮培养时,以初代愈伤组织较为适宜,悬浮培养系中的细胞分散度最高。培养基中添加500mg/L水解酪蛋白可以有效地促进悬浮细胞的生长,使悬浮培养系的生物量增加。悬浮细胞振荡培养过程中,摇床转速应低于120rpm,以110rpm为宜。
(3)以微乳液法制备了淀粉纳米基因载体,并对载体进行了表面修饰。结果表明：淀粉纳米载体(SNGC)制备的最优实验方案为淀粉浓度15%、搅拌速度2000rpm、油水相体积比15：1、三氯氧磷0.05%。采用多聚赖氨酸(PLL)对淀粉纳米载体(SNGC)进行表面修饰,PLL与SNGC的质量比不同,载体表面携带的电荷数量也不同。当SNGC:PLL(M:M)=2：1时,载体表面电荷数达到饱和。修饰后的PLL-SNGC为球状颗粒,大小比较一致,分散性好。多聚赖氨酸(PLL)和水溶性量子点(CdSe)两种材料修饰后的PLL-SNGC和Cs-PLL-SNGC载体均能有效的与DNA分子结合。
(4)纳米基因载体对麻疯树愈伤组织和悬浮细胞的转化结果表明：用PLL-SNGC和Cs-PLL-SNGC两种纳米基因载体溶液处理过的麻疯树愈伤组织生长良好,载体基本无细胞毒性,或者细胞毒性很小,对细胞正常的生长、分裂几乎没有影响,可以用作麻疯树遗传转化的载体。两种纳米基因载体对麻疯树愈伤组织的转化效果均不明显,但对悬浮细胞的转化具有比较明显的效果,并且载体在细胞内可以自行降解,具有良好的生物相容性。麻疯树悬浮细胞转化中,超声处理时间以10min较为适宜。
Jatropha curcas(Jatropha curcas L.) is a very important tree species of biodiesel. Jatropha curcas is characterized by rapid growth, yoshimitsu and drought tolerance, which is also has high value in Economic and Medicinal. This article studies on the plant regeneration of Jatropha curcas which is based on taking aseptic seedling stem section of planlet as explant. Meanwhile, establishing suspension cell lines of Jatropha curcas,exploring the effects of nanometer gene vector on genetic transformation of Jatropha curcas,so as to establish a new way of genetic transformation.The basic results as follows:
(1)Taking aseptic seedling stem section of planlet as explant, preliminary establishes the plant regeneration of Jatropha curcas.The result shows that:The most suitable hormone combination of adventitious bud induction by stem section of Jatropha curcas is: BA1.0mg/L+TDZ1.5mg/L, the average induction rate of adventitious bud has reached 69.25%.:In the culture of root induction, using auxin NAA, The most suitable hormone combination is:MS+NAA1.0mg/L+Sucrose 30g/L+Agar7.2g/L, and the rooting rate has reached 66.67%, thick roots and well developed. The explants has adventitious bud both in the MS solid medium and liquid medium,and in the solid medium,it has a higher induction rate of adventitious bud and a lower browning rate.
(2)In the culture of suspension cell of Jatropha curcas, the effects on the growth of suspension cell in different scheme of loose callus induction and the different culture condition. The result shows that:The most suitable hormone combination of loose callus induction is:MS+2,4-D0.6mg/L+KT0.4mg/L,in which the callus is humid, loose and colorful. The most suitable hormone combination of liquid culture callus is NAA0.2mg/L+2,4-D1.0mg/L+BA0.5mg/L.When using the callus of first generation, callus of subculture two times and callus of subculture four times, the first generation is the best one, which has the highest cell dispersion in suspension cell lines.Adding 500mg/L casein hydrolysate in medium can promote the growth of suspension cell and increase the biomass of suspension cell lines. In the culture of oscillation,the rotation speed should lower than 120rpm,and 110rpm is the most suitable.
(3)Studies on the preparation of starch nanometer gene vector and the modification in the surface of the vector, the result shows that:the optimal experimental scheme of the preparation of starch nanometer gene vector(SNGC) is:starch concentration 15%, stirring speed 2000rpm, volume ratio of oil and water 15:1,phosphorus oxychloride 0.05%.Taking modification in the surface of the starch nanometer gene vector (SNGC) by using polylysine (PLL).When the mass ratio between the PLL and the SNGC are different,the electric charge on the vector are different. When SNGC:PLL(M:M)=2:1, the electric charge on the vector are saturated. The modified PLL-SNGC are spherical particle and the size are almost uniform and well dispersive. The PLL-SNGC and Cs-PLL-SNGC vector which modified by polylysine (PLL) and water-soluble quantum dots(CdSe) both can combined with DNA molecular.
(4)Studies on nanometer gene vector on callus and suspension cell transformation of Jatropha curcas show that:The callus treated by PLL-SNGC and Cs-PLL-SNGC grow well, the vectors has no cytotoxicity or little. There is no effect on normal cell growth an division. So they can both used as vector in genetic transformation of Jatropha curcas.Eithei of two kind of vector has no significantly effect on callus transformation. But both of them has significantly effect on suspension cell transformation and can be self-degraded in cells, which have good biocompatibility. In the suspension cell transformation of Jatropha curcas,the best ultrasonic time is 10min.
(1)采用麻疯树无菌苗茎段为外植体,初步建立了麻疯树植株再生体系,结果表明：麻疯树茎段诱导不定芽再生的最适激素组合为：BA1.Omg/L+TDZ1.5mg/L,不定芽的平均诱导率达69.25%。不定根诱导培养中,采用生长素NAA诱导麻疯树幼苗生根,最适诱导配方为：MS+NAA1.0mg/L+蔗糖30g/L+琼脂7.2g/L,生根率达到66.67%,根系粗壮,发育良好。外植体接种于MS固体培养基和液体培养基中均有不定芽分化,但固体培养基中的外植体不定芽诱导率更高,褐化水平也较低。
(2)麻疯树悬浮细胞培养中,研究了疏松愈伤组织诱导方案及不同培养条件对麻疯树悬浮细胞生长的影响。结论如下：麻疯树疏松愈伤组织诱导的最适培养基及激素组合为：MS+2,4-D0.6mg/L+KT0.4mg/L,此培养基上诱导出的愈伤组织湿润松散,颜色鲜艳。接种愈伤组织进行悬浮培养的液体培养基最适激素组合为：NAA0.2mg/L+2,4-D1.0mg/L+BA0.5mg/L。初代、2次继代和4次继代的愈伤组织用于悬浮培养时,以初代愈伤组织较为适宜,悬浮培养系中的细胞分散度最高。培养基中添加500mg/L水解酪蛋白可以有效地促进悬浮细胞的生长,使悬浮培养系的生物量增加。悬浮细胞振荡培养过程中,摇床转速应低于120rpm,以110rpm为宜。
(3)以微乳液法制备了淀粉纳米基因载体,并对载体进行了表面修饰。结果表明：淀粉纳米载体(SNGC)制备的最优实验方案为淀粉浓度15%、搅拌速度2000rpm、油水相体积比15：1、三氯氧磷0.05%。采用多聚赖氨酸(PLL)对淀粉纳米载体(SNGC)进行表面修饰,PLL与SNGC的质量比不同,载体表面携带的电荷数量也不同。当SNGC:PLL(M:M)=2：1时,载体表面电荷数达到饱和。修饰后的PLL-SNGC为球状颗粒,大小比较一致,分散性好。多聚赖氨酸(PLL)和水溶性量子点(CdSe)两种材料修饰后的PLL-SNGC和Cs-PLL-SNGC载体均能有效的与DNA分子结合。
(4)纳米基因载体对麻疯树愈伤组织和悬浮细胞的转化结果表明：用PLL-SNGC和Cs-PLL-SNGC两种纳米基因载体溶液处理过的麻疯树愈伤组织生长良好,载体基本无细胞毒性,或者细胞毒性很小,对细胞正常的生长、分裂几乎没有影响,可以用作麻疯树遗传转化的载体。两种纳米基因载体对麻疯树愈伤组织的转化效果均不明显,但对悬浮细胞的转化具有比较明显的效果,并且载体在细胞内可以自行降解,具有良好的生物相容性。麻疯树悬浮细胞转化中,超声处理时间以10min较为适宜。
Jatropha curcas(Jatropha curcas L.) is a very important tree species of biodiesel. Jatropha curcas is characterized by rapid growth, yoshimitsu and drought tolerance, which is also has high value in Economic and Medicinal. This article studies on the plant regeneration of Jatropha curcas which is based on taking aseptic seedling stem section of planlet as explant. Meanwhile, establishing suspension cell lines of Jatropha curcas,exploring the effects of nanometer gene vector on genetic transformation of Jatropha curcas,so as to establish a new way of genetic transformation.The basic results as follows:
(1)Taking aseptic seedling stem section of planlet as explant, preliminary establishes the plant regeneration of Jatropha curcas.The result shows that:The most suitable hormone combination of adventitious bud induction by stem section of Jatropha curcas is: BA1.0mg/L+TDZ1.5mg/L, the average induction rate of adventitious bud has reached 69.25%.:In the culture of root induction, using auxin NAA, The most suitable hormone combination is:MS+NAA1.0mg/L+Sucrose 30g/L+Agar7.2g/L, and the rooting rate has reached 66.67%, thick roots and well developed. The explants has adventitious bud both in the MS solid medium and liquid medium,and in the solid medium,it has a higher induction rate of adventitious bud and a lower browning rate.
(2)In the culture of suspension cell of Jatropha curcas, the effects on the growth of suspension cell in different scheme of loose callus induction and the different culture condition. The result shows that:The most suitable hormone combination of loose callus induction is:MS+2,4-D0.6mg/L+KT0.4mg/L,in which the callus is humid, loose and colorful. The most suitable hormone combination of liquid culture callus is NAA0.2mg/L+2,4-D1.0mg/L+BA0.5mg/L.When using the callus of first generation, callus of subculture two times and callus of subculture four times, the first generation is the best one, which has the highest cell dispersion in suspension cell lines.Adding 500mg/L casein hydrolysate in medium can promote the growth of suspension cell and increase the biomass of suspension cell lines. In the culture of oscillation,the rotation speed should lower than 120rpm,and 110rpm is the most suitable.
(3)Studies on the preparation of starch nanometer gene vector and the modification in the surface of the vector, the result shows that:the optimal experimental scheme of the preparation of starch nanometer gene vector(SNGC) is:starch concentration 15%, stirring speed 2000rpm, volume ratio of oil and water 15:1,phosphorus oxychloride 0.05%.Taking modification in the surface of the starch nanometer gene vector (SNGC) by using polylysine (PLL).When the mass ratio between the PLL and the SNGC are different,the electric charge on the vector are different. When SNGC:PLL(M:M)=2:1, the electric charge on the vector are saturated. The modified PLL-SNGC are spherical particle and the size are almost uniform and well dispersive. The PLL-SNGC and Cs-PLL-SNGC vector which modified by polylysine (PLL) and water-soluble quantum dots(CdSe) both can combined with DNA molecular.
(4)Studies on nanometer gene vector on callus and suspension cell transformation of Jatropha curcas show that:The callus treated by PLL-SNGC and Cs-PLL-SNGC grow well, the vectors has no cytotoxicity or little. There is no effect on normal cell growth an division. So they can both used as vector in genetic transformation of Jatropha curcas.Eithei of two kind of vector has no significantly effect on callus transformation. But both of them has significantly effect on suspension cell transformation and can be self-degraded in cells, which have good biocompatibility. In the suspension cell transformation of Jatropha curcas,the best ultrasonic time is 10min.
引文
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