Mn-SOD基因转化猕猴桃（Actinidia deliciosa）的研究
摘要
我国是猕猴桃的起源和分布中心，资源极其非富。猕猴桃果实风味独特，富含维生素C、糖、多种人体必需氨基酸及其他营养成分，有“果中之王”的美称。
植物的生长发育总是处于环境胁迫之中，它们严重影响着作物的产量。研究表明，提高植物体内Mn-SOD活性是增强植物抗逆性的有效途径之一。本试验通过根癌农杆菌介导将Mn-SOD基因导入美味猕猴桃，获得转化完整植株，选育具有高抗逆性的猕猴桃新品种。
以美味猕猴桃优良品种海沃德和秦美组培苗为试材，以基本培养基、碳源、6-BA和生长素为试验因子，通过L9(34)正交试验，筛选出秦美离体叶片再生的理想培养基，即MS+6-BA4.0mg/L+NAA1.0mg/L+蔗糖30g/L。通过比较TDZ、ZT、6-BA和IBA对海沃德叶片再生的影响，获得海沃德最适培养基为MS+TDZ1.0mg/L+ IBA0.15mg/L，再生频率为82.2%，再生芽数为4.75。同时，研究了接种方式、AgNO3等因素对离体叶片再生效率的影响，进一步优化了叶片再生体系，从而建立了稳定高效的猕猴桃叶片再生体系。另外，以叶片再生不定芽为试材，进行了基本培养基和不同激素组合对猕猴桃增殖和生根的研究。
在建立美味猕猴桃叶片再生体系的基础上，研究了Mn-SOD基因转化中影响叶片转化效率的多种因素，包括叶片预培养、Kan浓度、抑菌素浓度、浸染菌液以及Vir基因诱导因子等，从而确立了美味猕猴桃遗传转化体系。选取幼嫩叶平铺在分化培养基中预培养20d，然后剪成0.5cm2大小的叶块作为转化受体，用携带目的基因的农杆菌EHA105浸染10min，置于分化培养基上共培养3d，冲洗干净后，转接到选择分化培养基中。培养6~7周后，分化出抗性不定芽，及时将产生抗性芽的愈伤组织转接至抗性增殖培养基中，待抗性苗伸长到2cm后，从基部切下转接于抗性生根培养基中，3~4周后可获得转化完整植株。经GUS检测和PCR扩增，初步确认Mn-SOD基因已整合到猕猴桃植株基因组中。
China was the original and distributed resource centre of Actinidia. Kiwifruit had a nice taste with high contents of vitamin C, sugar, all kinds of amino acid necessary to human body and other nutritive components, so it was called the king of fruits.
Under nature conditions of growth and development, plants were invariably exposed to different environmental stress, which had profound effects on crop production. Many studies had examined that modification of Mn-SOD expression in transgenic plants can improve plant stress tolerance. In this study, we had introduced the Mn-SOD gene into kiwifruit to evealuate its effect on the persistence of environmental stress. The transformed kiwifruit derived from leaf explants would pave the way for its breeding via genetic engineering.
Two commercial Actinidia deliciosa cultivars “Hayward” and “Qinmei”in vitro leaves of seedlings were selected as experimental materials in this study, basal medium, carbohydrate, 6-BA and auxins as factors which had three content levels respectively, an idea medium which was MS+6-BA4.0mg/L+NAA1.0mg/L+sucrose30g/L for leaf regeneration in“Qinmei” was sifted through the crossed test of L 9(34). And leaf regeneration in “Hayward” was studied on MS medium with TDZ, ZT, 6-BA and IBA. Results showed that the optimal medium for leaf regeneration was MS+TDZ1.0mg/L+IBA 0.15mg/L, which regeneration frequency was 82.2% and its buds No.Per leaf up to 4.75. And a system for in vitro high efficient regeneration was established after some factors affecting on leaf regeneration had been studied. For the multiplication and root development of the adventitious bud from leaf disc, both basal medium and hormone concentration and composition were also examined in this study.
On the base of regeneration system from leaf explant of “Hayward” and “Qinmei”, several factors that affected genetic transformation of kiwifruit cultivars were examined, including preculture of leaf explant, selection of Kan concentrations, the suspension of strain EHA105, concentrations of Carb and Cef and Vir gene expression factors, and so on. Thus a simple and efficient genetic transformation system was developed. Selecting young leaves as explants were first precultured on the differentiation medium for 20 days, then cocultured on the differentiation medium with Agrobacterium tumefaciens EHA105 harboring a binary vector containing chimeric genes of NptⅡ, Gus report gene and Mn-SOD gene driven by CaMV35S promoter. After 3 days cocultureation, these explants were transferred to the differentiation-resistant medium to select transgenic shoots. Some regenerated Kanamycin-resistant plants were obtained by multiplying and rooting. Through examination of GUS report gene and PCR amplification of Mn-SOD gene, it was showed that that the foreign cecropin Mn-SOD gene was introduced into the regenerated plants genome.
植物的生长发育总是处于环境胁迫之中，它们严重影响着作物的产量。研究表明，提高植物体内Mn-SOD活性是增强植物抗逆性的有效途径之一。本试验通过根癌农杆菌介导将Mn-SOD基因导入美味猕猴桃，获得转化完整植株，选育具有高抗逆性的猕猴桃新品种。
以美味猕猴桃优良品种海沃德和秦美组培苗为试材，以基本培养基、碳源、6-BA和生长素为试验因子，通过L9(34)正交试验，筛选出秦美离体叶片再生的理想培养基，即MS+6-BA4.0mg/L+NAA1.0mg/L+蔗糖30g/L。通过比较TDZ、ZT、6-BA和IBA对海沃德叶片再生的影响，获得海沃德最适培养基为MS+TDZ1.0mg/L+ IBA0.15mg/L，再生频率为82.2%，再生芽数为4.75。同时，研究了接种方式、AgNO3等因素对离体叶片再生效率的影响，进一步优化了叶片再生体系，从而建立了稳定高效的猕猴桃叶片再生体系。另外，以叶片再生不定芽为试材，进行了基本培养基和不同激素组合对猕猴桃增殖和生根的研究。
在建立美味猕猴桃叶片再生体系的基础上，研究了Mn-SOD基因转化中影响叶片转化效率的多种因素，包括叶片预培养、Kan浓度、抑菌素浓度、浸染菌液以及Vir基因诱导因子等，从而确立了美味猕猴桃遗传转化体系。选取幼嫩叶平铺在分化培养基中预培养20d，然后剪成0.5cm2大小的叶块作为转化受体，用携带目的基因的农杆菌EHA105浸染10min，置于分化培养基上共培养3d，冲洗干净后，转接到选择分化培养基中。培养6~7周后，分化出抗性不定芽，及时将产生抗性芽的愈伤组织转接至抗性增殖培养基中，待抗性苗伸长到2cm后，从基部切下转接于抗性生根培养基中，3~4周后可获得转化完整植株。经GUS检测和PCR扩增，初步确认Mn-SOD基因已整合到猕猴桃植株基因组中。
China was the original and distributed resource centre of Actinidia. Kiwifruit had a nice taste with high contents of vitamin C, sugar, all kinds of amino acid necessary to human body and other nutritive components, so it was called the king of fruits.
Under nature conditions of growth and development, plants were invariably exposed to different environmental stress, which had profound effects on crop production. Many studies had examined that modification of Mn-SOD expression in transgenic plants can improve plant stress tolerance. In this study, we had introduced the Mn-SOD gene into kiwifruit to evealuate its effect on the persistence of environmental stress. The transformed kiwifruit derived from leaf explants would pave the way for its breeding via genetic engineering.
Two commercial Actinidia deliciosa cultivars “Hayward” and “Qinmei”in vitro leaves of seedlings were selected as experimental materials in this study, basal medium, carbohydrate, 6-BA and auxins as factors which had three content levels respectively, an idea medium which was MS+6-BA4.0mg/L+NAA1.0mg/L+sucrose30g/L for leaf regeneration in“Qinmei” was sifted through the crossed test of L 9(34). And leaf regeneration in “Hayward” was studied on MS medium with TDZ, ZT, 6-BA and IBA. Results showed that the optimal medium for leaf regeneration was MS+TDZ1.0mg/L+IBA 0.15mg/L, which regeneration frequency was 82.2% and its buds No.Per leaf up to 4.75. And a system for in vitro high efficient regeneration was established after some factors affecting on leaf regeneration had been studied. For the multiplication and root development of the adventitious bud from leaf disc, both basal medium and hormone concentration and composition were also examined in this study.
On the base of regeneration system from leaf explant of “Hayward” and “Qinmei”, several factors that affected genetic transformation of kiwifruit cultivars were examined, including preculture of leaf explant, selection of Kan concentrations, the suspension of strain EHA105, concentrations of Carb and Cef and Vir gene expression factors, and so on. Thus a simple and efficient genetic transformation system was developed. Selecting young leaves as explants were first precultured on the differentiation medium for 20 days, then cocultured on the differentiation medium with Agrobacterium tumefaciens EHA105 harboring a binary vector containing chimeric genes of NptⅡ, Gus report gene and Mn-SOD gene driven by CaMV35S promoter. After 3 days cocultureation, these explants were transferred to the differentiation-resistant medium to select transgenic shoots. Some regenerated Kanamycin-resistant plants were obtained by multiplying and rooting. Through examination of GUS report gene and PCR amplification of Mn-SOD gene, it was showed that that the foreign cecropin Mn-SOD gene was introduced into the regenerated plants genome.
引文
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